Drought stress related alfalfa gene and use thereof

Abstract

The application relates to the biotechnology field and discloses a drought stress related alfalfa gene and application thereof, and the MsCAS15 gene is cloned from alfalfa, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the encoded dehydrin is shown as SEQ ID NO. 2. The protein is rich in glycine and lysine and has the functions of protecting cell membranes and biological macromolecules. The application obtains a transgenic plant overexpressing the MsCAS15 by constructing a recombinant expression vector containing the gene and using an agrobacterium-mediated method. The experimental results show that overexpression of the gene enhances the drought resistance of the plant, and the specific performance is that the seed germination rate of the transgenic plant is higher than that of the wild type under osmotic stress, and under the natural drought condition of soil, the wilting degree of the transgenic plant leaves is reduced, and the growth state is better than that of the wild type. The application provides an effective gene resource for cultivating new drought-resistant crop varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a drought-stress-related alfalfa gene and its applications. Background Technology

[0002] Currently, alfalfa, a widely cultivated perennial legume forage crop globally, plays a vital role in animal husbandry and ecological restoration due to its high protein content and ecological restoration functions. However, with the intensification of climate change, major alfalfa producing areas in Northwest and North my country face severe water shortage challenges. Uneven annual rainfall distribution and frequent seasonal droughts have led to water stress that restricts the normal growth and development of alfalfa. This environmental pressure directly results in large fluctuations in forage yield and a decline in quality, becoming a core bottleneck restricting the high-quality development of the industry.

[0003] For drought resistance genetic improvement, current technologies mainly involve molecular breeding through targeted discovery of stress-related genes in plants. Researchers have cloned genes such as ABF and WRKY from model plants like Arabidopsis and soybean, and identified key factors such as MsNTF2 and MsP5CS in alfalfa. Among these, cold acclimatization-specific proteins (CAS), as members of the dehydration protein family, are rich in glycine and lysine in their structure. When exposed to low temperatures or drought, these proteins can accumulate intracellularly, helping plants cope with abiotic stresses through mechanisms such as stabilizing cell membrane structure, protecting biomolecules, and participating in signal transduction.

[0004] Although the mechanism of dehydration proteins in plant stress resistance is well understood, and studies have been conducted on heterologous expression using genes from the closely related species *Alfalfa truncatum*, research on endogenous CAS proteins in alfalfa itself remains lacking. Existing genetic resources lack endogenous alfalfa-specific genes with highly efficient drought resistance, limiting the precision and compatibility of molecular breeding. When faced with dehydration caused by osmotic stress, existing varieties often suffer from poor seed germination and low emergence rates due to insufficient protection from dehydration proteins. Simultaneously, plants cannot effectively maintain cell turgor pressure and membrane integrity during the seedling and mature stages, easily leading to leaf wilting or even death, making it difficult to meet the stable and high-yield planting requirements of arid regions.

[0005] Therefore, this invention provides a drought stress-related alfalfa gene and its application to address the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a drought stress-related alfalfa gene and its application, solving the problem of the lack of efficient and specific key endogenous gene resources in existing alfalfa drought-resistant molecular breeding, which limits the breeding and improvement of new stress-resistant alfalfa varieties in arid and semi-arid regions, and makes it difficult to solve the problems of plant growth inhibition and yield reduction caused by drought stress.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a drought-stress-related alfalfa gene, employing the following technical solution:

[0009] A drought-stress-associated alfalfa gene contains nucleotide sequences selected from the following group:

[0010] (a) The nucleotide sequence shown in SEQ ID NO.1;

[0011] (b) The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2;

[0012] (c) The nucleotide sequence shown in SEQ ID NO.3;

[0013] (d) A nucleotide sequence that hybridizes under strict conditions with the nucleotide sequence defined in (a) or (c) and encodes a dehydrated protein that enhances plant drought resistance;

[0014] (e) has more than 90% identity with the nucleotide sequence defined in (a) or (c) and encodes a nucleotide sequence that encodes a dehydrated protein that enhances plant drought resistance.

[0015] By adopting the above technical solution, the present invention can achieve the following effects:

[0016] The MsCAS15 gene cloned in this invention encodes a cold acclimatization-specific protein (CAS), which belongs to the dehydrins family. It possesses typical dehydrins structural characteristics, is rich in glycine (Gly) and lysine (Lys), and can play a protective role in plants when subjected to water deficit. Its mechanism of action in enhancing plant drought resistance is specifically manifested in the following process:

[0017] Osmotic regulation and water retention: Highly hydrophilic amino acid residues (such as glycine and lysine) in the MsCAS15 protein sequence can bind intracellular water molecules, increase the viscosity and water retention capacity of the cytoplasm, and reduce water loss from cells under drought conditions.

[0018] Membrane structure stability: Under dehydration conditions, the lipid bilayer structure of the cell membrane is prone to phase transition or rupture. The MsCAS15 protein can bind to cell membrane phospholipids through electrostatic or hydrophobic interactions, maintaining the integrity and fluidity of the membrane structure and preventing electrolyte leakage.

[0019] Biomolecular protection: As a molecular chaperone, this protein can prevent intracellular functional proteins (such as enzymes) and nucleic acids from denaturing or irreversibly aggregating due to dehydration, thereby maintaining normal physiological metabolic functions of the cell.

[0020] Experimental data showed that the seed germination of plants overexpressing this gene was less inhibited than that of wild-type plants under osmotic stress simulating drought with 300mM mannitol. Under natural soil drought conditions, the plant leaves remained in an expanded state without severe wilting and wrinkling, confirming that this gene has significant drought resistance function.

[0021] Preferably, the protein encoded by the alfalfa gene has the amino acid sequence shown in SEQ ID NO.2, or is a protein derived from SEQ ID NO.2 by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.2, and has the function of improving plant drought resistance. This ensures that even when non-critical variations occur in the protein sequence, its core structural domain can still be used to exert its stress resistance function.

[0022] Preferably, the present invention also provides a recombinant expression vector containing the above-mentioned drought stress-related alfalfa gene.

[0023] More preferably, the recombinant expression vector is constructed using a plant binary expression vector backbone (such as pCAMBIA3301), and the alfalfa gene is expressed by the CaMV 35S promoter.

[0024] By adopting the above-mentioned preferred scheme, constitutive strong expression of the MsCAS15 gene can be achieved throughout the entire growth period of the plant and in various tissues using the CaMV 35S promoter, ensuring that the plant has accumulated a sufficient amount of MsCAS15 protein for defense when encountering sudden drought stress. The selection markers (such as the bar gene) carried by the pCAMBIA3301 vector facilitate the subsequent resistance selection of transformed plants.

[0025] Preferably, the present invention also provides a recombinant bacterium or transgenic plant cell containing the above-mentioned gene or recombinant expression vector. This provides the necessary biomaterial basis for gene amplification, preservation, and plant genetic transformation.

[0026] Secondly, this invention provides the application of the aforementioned drought-stress-related alfalfa genes, recombinant expression vectors, recombinant bacteria, or transgenic plant cells in improving plant drought resistance, employing the following technical solutions:

[0027] This application is specifically achieved by constructing transgenic plants, and the method for constructing transgenic plants includes the following steps:

[0028] S1. The alfalfa gene is constructed into a plant expression vector to obtain a recombinant expression vector;

[0029] S2. Transform the recombinant expression vector into Agrobacterium;

[0030] S3. Using Agrobacterium containing a recombinant expression vector to infect the target plant, transgenic positive plants were obtained through screening.

[0031] By adopting the above technical solution, the present invention can achieve the following beneficial effects:

[0032] This application provides an effective way to improve crop drought resistance using genetic engineering. Introducing the MsCAS15 gene into target plants and overexpressing it can enhance the plant's drought resistance throughout its entire growth cycle. Specifically, the effects are as follows:

[0033] Germination period stress resistance: In arid or high osmotic pressure environments, the water absorption and germination ability of transgenic plant seeds is superior to that of wild types, improving the emergence rate and uniformity.

[0034] Resistance during seedling and mature stages: When water supply is insufficient, transgenic plants can maintain good leaf turgor pressure, delay leaf senescence and death, ensure continuous photosynthesis, thereby maintaining plant growth and reducing biomass loss caused by drought.

[0035] Preferably, in step S3, the screening is carried out using a herbicide, namely glufosinate; the target plant is a dicotyledonous plant, specifically including alfalfa or Arabidopsis thaliana.

[0036] Preferably, in step S2, the Agrobacterium is Agrobacterium tumefaciens GV3101; in step S3, the infection method is either inflorescence immersion or leaf disc transformation.

[0037] By adopting the above-mentioned optimized scheme, a highly efficient transformation system for alfalfa and the model plant Arabidopsis thaliana was identified. Glufosinate screening combined with the bar gene on the pCAMBIA3301 vector can rapidly and accurately eliminate false-positive plants, obtain homozygous transgenic lines, and shorten the breeding cycle.

[0038] This invention provides a drought-stress-related alfalfa gene and its application. It has the following beneficial effects:

[0039] 1. This invention has identified and cloned the endogenous gene MsCAS15 in alfalfa. The protein encoded by this gene exhibits typical dehydrated protein structural characteristics and is rich in glycine and lysine residues. When plants suffer from water deficit, this protein protects intracellular macromolecules from dehydration damage and maintains normal cellular physiological metabolism by binding water molecules and stabilizing cell membrane structure. The discovery of this gene enriches the genetic resources for molecular breeding of alfalfa for stress resistance and provides genetic material with a clearly defined function for cultivating new drought-resistant varieties.

[0040] 2. Transgenic plants constructed using the genes provided in this invention enhance the germination ability of plant seeds under osmotic stress. Experimental results show that, under simulated drought conditions containing 300 mM mannitol, the seeds of transgenic plants overexpressing the MsCAS15 gene have a higher germination rate than wild-type plants. This characteristic helps to solve the problem of difficult seedling emergence caused by insufficient soil moisture after sowing in arid regions, and improves the seedling survival rate of crops under adverse conditions.

[0041] 3. This invention enhances the plant's tolerance to soil drought during the vegetative growth stage. By enhancing the expression of the MsCAS15 gene in the plant, the transgenic plants maintained better tissue turgor pressure and leaf morphology during natural drought treatment after watering was stopped. Compared to the wilting and wrinkling of leaves exhibited by wild-type plants, the leaves of the transgenic plants remained relatively unfolded, and the degree of growth inhibition was less, confirming that this gene can effectively delay drought symptoms and improve the crop's survival ability under water-deficient conditions. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the MsCAS15 amplification fragment of the present invention;

[0043] Figure 2 This is a schematic diagram showing the alignment of the amino acid sequences of alfalfa, cloverleaf, and Arabidopsis thaliana of the present invention with homologous class I (A), class II (B), and class III (C) CAS (in the figure, MS.gene067857.t1 is MsCAS15).

[0044] Figure 3 This is a schematic diagram illustrating the detection of the relative expression level of MsCAS15 overexpressing Arabidopsis thaliana using qRT-PCR according to the present invention;

[0045] Figure 4 This is a schematic diagram showing the seed germination of wild-type (WT) Arabidopsis thaliana and MsCAS15 overexpression lines on 1 / 2 MS medium according to the present invention.

[0046] Figure 5 This is a schematic diagram showing the seed germination of wild-type (WT) Arabidopsis thaliana and MsCAS15 overexpression lines of the present invention on 1 / 2 MS medium containing 300 mM mannitol.

[0047] Figure 6 This is a schematic diagram showing the germination rate of the wild-type (WT) Arabidopsis thaliana and the MsCAS15 overexpression line of the present invention on 1 / 2MS medium;

[0048] Figure 7 This is a statistical diagram showing the germination rate of the wild-type (WT) Arabidopsis thaliana and the MsCAS15 overexpression line of the present invention on 1 / 2 MS medium containing 300 mM mannitol.

[0049] Figure 8 This is a schematic diagram of the phenotype of wild-type (WT) Arabidopsis thaliana seedlings and overexpressing MsCAS15 line seedlings under natural drought conditions in soil. Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] The plant materials used in the embodiments of this invention include: Arabidopsis thaliana wild type Col-0 ecotype; and wild type alfalfa (Medicago sativa) Zhongmu No. 1.

[0052] The strains and vectors used in the embodiments of this invention include: Escherichia coli DH5α competent cells, Agrobacterium tumefaciens GV3101 competent cells; cloning vectors (such as the pEASY-T5 series) and plant binary expression vector pCAMBIA3301.

[0053] Unless otherwise specified, all other biochemical reagents and kits used in the embodiments of this invention are commercially available products; the experimental procedures (such as RNA extraction, reverse transcription, PCR amplification, vector construction, transformation, etc.) are performed in accordance with the kit instructions or conventional molecular biology experimental manuals (such as "Molecular Cloning: A Laboratory Manual").

[0054] Preparation Examples 1-3:

[0055] Preparation Example 1:

[0056] Cloning of the full-length CDS sequence of the alfalfa MsCAS15 gene:

[0057] This preparation example aims to clone the coding region sequence of the MsCAS15 gene from alfalfa. First, total RNA was extracted from alfalfa (Zhongmu 1) leaves, and the RNA concentration and purity were determined using a UV spectrophotometer. Its integrity was then checked by agarose gel electrophoresis. Subsequently, the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit, which served as a template for subsequent PCR amplification.

[0058] Next, based on the alfalfa reference genome sequence, specific amplification primers were designed for the full-length 5'UTR and 3'UTR regions of the MsCAS15 gene. The upstream primer MsCAS15F sequence was 5'-AAAACAAAACAAAGAAGAAACAC-3', and the downstream primer MsCAS15R sequence was 5'-TACAAGCACTCGAATTACCAAC-3'. Using the obtained cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase. The PCR reaction system (50 μL) contained: 2 μL of cDNA, 0.25 μL of high-fidelity DNA polymerase, and 10× Buffer (Mg... 2+ The reagents were: 5 μL of pre-denaturation (plus), 4 μL of dNTP Mixture, 2 μL each of upstream and downstream primers, and 34.75 μL of ddH2O. The PCR reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 1 min, for 25 cycles; and finally, 72℃ extension for 5 min, and storage at 4℃.

[0059] After PCR amplification, the product was detected by agarose gel electrophoresis, and the gel was excised and purified. The purified gene fragment was mixed with a cloning vector (e.g., pEASY-T5) and ligated at 25°C for 30 min. The ligation product was transformed into *E. coli* DH5α competent cells (after ice bath, 42°C heat shock, and recovery), plated on LB agar containing kanamycin (Kan), and incubated overnight at 37°C. Single colonies were picked and identified by colony-specific PCR using universal primers M13F / R. Positive clones were sequenced, and the sequencing results showed that the MsCAS15 gene cDNA sequence, as shown in SEQ ID NO.3, was successfully cloned, with a full length of 1046 bp. Sequence analysis revealed that the cDNA sequence contained the MsCAS15 gene coding region (CDS) sequence, as shown in SEQ ID NO.1, with a full length of 411 bp. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The plasmid with the correct sequence was extracted, named pEASYT5-MsCAS15, and the bacterial strain was preserved.

[0060] Preparation Example 2:

[0061] Construction of the plant overexpression vector pCAMBIA3301-MsCAS15:

[0062] This preparation example utilizes seamless cloning technology to construct a plant overexpression vector. First, the plant binary expression vector pCAMBIA3301 was double-digested using restriction endonucleases NcoI and BglII. The reaction system contained 1-3 μg of plasmid template, 1 μL each of NcoI and BglII, and the corresponding buffer solutions. After the digestion products were detected by agarose gel electrophoresis, the linearized vector fragments were recovered from the gel, and their concentration was determined.

[0063] Using the pEASYT5-MsCAS15 plasmid extracted in Preparation Example 1 as a template, the MsCAS15 gene fragment was amplified using seamless cloning primers with homologous arms.

[0064] The upstream primer 3301-MsCAS15-F sequence is as follows:

[0065] 5'-GAACACGGGGGACTCTTGACCATGGCAGGAATCATGAACAA-3'

[0066] The sequence of the downstream primer 3301-MsCAS15-R is as follows:

[0067] 5'-CTCCTTTACTAGTCAGATCTACCATCTAATCACTGTCACTGCTGC-3'

[0068] The PCR reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 3 min, and final extension at 72℃ for 5 min, then stored at 4℃. The amplified target band was then excised and recovered from the gel.

[0069] Subsequently, using a seamless cloning kit, the recovered PCR product was mixed with the linearized pCAMBIA3301 vector according to the manufacturer's instructions, and ligation was performed using 2×Seamless Master Mix. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing kanamycin (50 mg / L) for selection. Single clones were picked and identified by colony PCR using the universal vector primer 35SF and the seamless cloning primer. After sequencing verification of the sequence of positive clones, the plasmid was extracted, which was the successfully constructed overexpression vector pCAMBIA3301-MsCAS15.

[0070] Preparation Example 3:

[0071] Preparation of recombinant Agrobacterium:

[0072] In this preparation example, the constructed plant expression vector was introduced into Agrobacterium for subsequent plant transformation. The recombinant plasmid pCAMBIA3301-MsCAS15 obtained in Preparation Example 2 was transformed into Agrobacterium GV3101 competent cells using a freeze-thaw method. The transformed Agrobacterium was plated on YEB solid medium containing kanamycin and rifampin and cultured. Single colonies were picked for PCR verification. The positively verified recombinant Agrobacterium culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C for later use.

[0073] Example:

[0074] This embodiment provides a drought stress-related alfalfa gene and its application, specifically involving a method for obtaining and purifying transgenic Arabidopsis thaliana lines overexpressing MsCAS15, including the following steps:

[0075] First, preparations were made for the planting and transformation of Arabidopsis thaliana plants. Wild-type Arabidopsis thaliana (Col-0 ecotype) seeds were placed in 75% ethanol and shaken for 30 seconds. After removing the ethanol, 5% sodium hypochlorite solution was added and shaken at room temperature for 10 minutes. The seeds were rinsed 5-6 times with sterile water and vernalized at 4°C in the dark for 2-3 days. The seeds were then sown on 1 / 2 MS medium and germinated under 22°C, 16h light / 8h dark conditions. When the seedlings had two true leaves, they were transplanted into the soil, and after thinning, one healthy seedling was retained per pot. When the Arabidopsis thaliana bolted, the top of the main stem was removed to promote lateral branching. Simultaneously, an inoculum solution was prepared. Agrobacterium containing the pCAMBIA3301-MsCAS15 vector obtained in Example 3 was inoculated into 5 mL of YEB liquid medium containing kanamycin and rifampin and incubated overnight at 28°C with shaking for activation. Add 2 mL of activated bacterial culture to 200 mL of YEB liquid medium containing the same antibiotic for expansion culture until the OD600 reaches between 0.6 and 0.8. Centrifuge the bacterial culture in a 50 mL centrifuge tube at 5000 rpm for 10 min, discard the supernatant, and add an equal volume of pre-prepared transformation suspension (formulation: 5 g sucrose, 20 μL Silwet L-77, and bring the volume to 100 mL ddH2O) to resuspend the bacterial cells.

[0076] Next, genetic transformation was carried out using the inflorescence immersion method. Arabidopsis thaliana plants with abundant buds and a few open flowers on lateral branches were selected, and fully opened flowers and pods were removed. The bacterial suspension was poured into a container, and the Arabidopsis inflorescences were completely immersed in the suspension for 5-10 minutes. After removal, they were placed flat on a tray, covered with plastic wrap to maintain moisture, and incubated in the dark for 24 hours, followed by incubation under normal light. To improve transformation efficiency, the infection was repeated once after a 7-day interval. Once the Arabidopsis siliques matured, individual plants were harvested for seed production, yielding T0 generation seeds.

[0077] Finally, the positive transgenic plants were screened and purified. T0 generation seeds were sterilized and vernalized, then sown on 1 / 2 MS medium containing 4 mg / L glufosinate (PPT) for screening to obtain resistant transgenic seedlings. When the seedlings had four true leaves, they were transplanted into soil for cultivation. Once the seeds of the positive plants matured, individual plants were harvested to obtain T1 generation seeds, which were then sown on 1 / 2 MS medium containing 4 mg / L PPT for further screening. The segregation ratio was calculated, and positive seedlings meeting a 3:1 segregation ratio were transplanted and harvested to obtain T2 generation seeds. T2 generation seeds were then sown on 1 / 2 MS medium containing 4 mg / L PPT for further screening until homozygous lines that survived completely on antibiotic-containing medium without phenotypic segregation were selected. These seeds were then harvested as T3 generation homozygous seeds. Through this process, homozygous transgenic Arabidopsis lines were finally obtained, named OE55, OE77, and OE85, for subsequent functional verification.

[0078] Comparative example:

[0079] This comparative example provides an Arabidopsis thaliana plant for use as a control. The difference between this comparative example and the example is that the wild-type Arabidopsis thaliana (Col-0 ecotype) preserved in our laboratory is used. This plant has not been infected with Agrobacterium tumefaciens or subjected to subsequent transgenic screening operations, i.e., the pCAMBIA3301-MsCAS15 recombinant vector has not been introduced. The other planting conditions and growth environment are consistent with those of the example.

[0080] Test Examples 1-4:

[0081] Test Example 1: Bioinformatics Analysis of the MsCAS15 Gene

[0082] This test case uses bioinformatics techniques to perform sequence characterization and homology analysis on the protein encoded by the cloned MsCAS15 gene. The specific process and results are as follows:

[0083] Analytical Methods: The MsCAS15 amino acid sequence of alfalfa obtained in Preparation Example 1 was used as the query sequence, and a BLASTP search was performed in the Phytozome and EnsemblPlant databases. The screening criteria were set as sequence identity greater than 90% and an E value less than 10^-10. Homologous protein sequences meeting the above criteria were collected, including members of the LEA, RAB, and Dehydrin families. Homologous protein sequences from Medicago truncatula and Arabidopsis thaliana were selected, and multiple sequence alignment was performed using MEGA software. The sequence alignment results were processed and output using the ESPript 3.0 online tool.

[0084] Analysis results: Multiple sequence alignment results are as follows Figure 2As shown. Structural analysis reveals that the secondary structure of MsCAS15 and related homologous proteins is mainly composed of α-helices. In terms of amino acid composition, the N-terminal region is rich in glycine (Gly), while the C-terminal region contains repeating sequences rich in lysine (Lys).

[0085] Conserved motif analysis of different subgroups revealed:

[0086] The N-terminus of the ClassI subfamily contains the conserved sequence QN(Q)××G×Q;

[0087] Class II subfamily includes the conserved sequences D(E)E(H)××N(S) and G××××L;

[0088] The Class III subfamily includes the conserved sequences MAG××NKIG×ALH×GG and K(N)KEG(D)EHKG(K)E.

[0089] Figure 2 The red-marked regions in the image show the highly conserved amino acid sequences between MsCAS15 and homologous class I, II, and III CAS proteins.

[0090] Conclusion: Sequence alignment and domain analysis confirmed that the MsCAS15 protein possesses typical structural features of the dehydrin family. The high proportion of glycine and lysine residues in its sequence, along with specific conserved domains (such as the N-terminal glycine-rich region and the C-terminal lysine repeat region), constitute the structural basis for its stress-resistance function. These structural features are typically associated with plant cell membrane stabilization and protection of nucleic acids and proteins from damage under stress. As a dehydrin with typical CAS characteristics, MsCAS15 structurally possesses the molecular potential to respond to drought stress and participate in plant drought resistance regulation.

[0091] Test Example 2: Molecular Identification of Transgenic Plants

[0092] This test case aims to validate the transgenic Arabidopsis plants obtained in the previous examples at both the genomic and transcriptional levels, in order to screen and identify positive lines that successfully overexpress the MsCAS15 gene. The specific experimental steps and results are as follows:

[0093] Experimental methods:

[0094] Genomic DNA level identification (PCR): Leaves of T0 generation Arabidopsis thaliana plants obtained through resistance selection in the examples were collected, and genomic DNA was extracted as a template. PCR amplification and detection were performed using specific primers.

[0095] The primer sequences are as follows:

[0096] Upstream primer MsCAS15F: 5'-AAAACAAAACAAAGAAGAAACAC-3'

[0097] Downstream primer MsCAS15R: 5'-TACAAGCACTCGAATTACCAAC-3'

[0098] The PCR reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 25 cycles; final extension at 72℃ for 5 min, and storage at 4℃. The reaction products were detected by agarose gel electrophoresis.

[0099] Transcriptional level identification (qRT-PCR): Total RNA was extracted from leaves of plants that tested positive by PCR and reverse transcribed to synthesize cDNA. Real-time quantitative PCR was performed using a CFX384 Real-Time PCR instrument.

[0100] Internal reference gene: Arabidopsis thaliana AtActin.

[0101] The quantitative primer sequences are as follows:

[0102] qPCRMsCAS15F:GATTGGTGATGCCCTTCACGG

[0103] qPCRMsCAS15R:CGGCAAATCCCTCCTGTGC

[0104] Reaction system (20 μL): 10 μL TBGreenPremix, 1 μL each of forward and reverse primers (0.2 μM) (adjust according to total volume), 2 μL cDNA, add water to 20 μL.

[0105] Reaction program: 95℃ for 30s; (95℃ for 5s, 60℃ for 30s) × 40 cycles.

[0106] Data processing: using 2 −ΔΔCt The relative expression level was calculated and normalized using a log2 transformation. Three technical replicates were set up for each line.

[0107] Experimental results:

[0108] Genomic PCR amplification results showed that specific bands of the expected size were amplified in the tested plants (corresponding to...). Figure 1 The size of the target gene fragment shown indicates that the exogenous MsCAS15 gene has been successfully integrated into the Arabidopsis genome.

[0109] qRT-PCR test results are as follows Figure 3As shown in the figure, the transcription level of the MsCAS15 gene was significantly increased in the transgenic lines compared to the control group (wild-type WT). Based on the relative expression data, three independent lines with high expression levels were selected and named OE55, OE77, and OE85, respectively. These three lines exhibited stable high expression characteristics in subsequent passaging and screening, and were used for subsequent drought resistance testing.

[0110] Conclusion: Molecular identification results confirmed that the MsCAS15 gene was successfully integrated into the Arabidopsis genome via Agrobacterium-mediated genetic transformation and achieved efficient transcriptional expression driven by the 35S promoter. The high expression levels in the OE55, OE77, and OE85 lines indicate that the exogenous gene can be stably transcribed, providing a material basis for the accumulation of MsCAS15 protein in the plant, thus enabling the plants to meet the molecular requirements for verifying drought resistance.

[0111] Test Example 3: Seed germination test under simulated drought stress

[0112] This test case uses mannitol to simulate osmotic stress caused by drought. By comparing the performance of transgenic Arabidopsis thaliana lines obtained in the comparative example with that of wild-type Arabidopsis thaliana during the germination stage, the function of the MsCAS15 gene is verified.

[0113] Experimental steps:

[0114] Material preparation and sterilization: Three T3 generation homozygous transgenic Arabidopsis thaliana lines (OE55, OE77, OE85) obtained in the examples and wild-type Arabidopsis thaliana (WT) seeds for the comparative example were selected. The seeds were immersed in sodium hypochlorite solution for 10 min for sterilization, and then rinsed 5 times with sterile water to remove residual reagents.

[0115] The experimental setup for flat plate laying and treatment included a control group and a stress treatment group.

[0116] Control group: Disinfected seeds were sown on 1 / 2 MS solid medium without osmotic regulator.

[0117] Treatment group: Sterilized seeds were sown on 1 / 2 MS solid medium supplemented with 300 mM mannitol to simulate drought stress.

[0118] Culture conditions: After sowing, the plates were placed in a 4℃ dark environment for 2 days for low-temperature vernalization treatment to break dormancy and promote uniform germination. Then the plates were transferred to a constant temperature and light incubator for cultivation, with the following conditions: temperature 24℃ (day) / 20℃ (night), photoperiod 16h light / 8h dark.

[0119] Data analysis: Observations were conducted continuously for 7 days after the plants were moved into the light incubator. The number of germinating seeds for each strain was counted daily, and the germination rate was calculated. Three biological replicates were set up for each treatment group, and the average value was used for analysis.

[0120] Experimental data and results:

[0121] Experimental results are attached in conjunction with the instruction manual. Figure 4 - Appendix Figure 7 As shown:

[0122] Under control conditions (1 / 2 MS) on normal culture medium without mannitol supplementation, there was no significant difference in seed germination between wild-type (WT) and the three transgenic lines (OE55, OE77, OE85). The germination rate of seeds from all lines increased synchronously over time from day 1 to day 4, reaching 100% germination by day 4, indicating that the insertion of the exogenous gene did not negatively affect the germination of Arabidopsis thaliana under normal conditions.

[0123] Under simulated drought stress (1 / 2 MS + 300 mM mannitol): osmotic stress caused by 300 mM mannitol delayed the germination process of all Arabidopsis seeds. Under these conditions, transgenic lines showed a significantly superior germination advantage compared to wild-type lines.

[0124] Germination rate: The seeds of transgenic lines (OE55, OE77, OE85) initiate germination earlier and at a faster rate than those of wild-type lines. For example... Figure 6 and Figure 7 As shown, during the first 5 days of stress treatment, the germination rate curve of the transgenic lines remained above that of the wild type.

[0125] Final germination rate: As the culture time was extended, the germination rate of each strain tended to be consistent after the 6th day.

[0126] Conclusion: The MsCAS15 gene belongs to the dehydration protein family. Overexpression of this gene enhances the tolerance of transgenic Arabidopsis to osmotic stress during seed germination. Under drought conditions simulated by 300 mM mannitol, the accumulation of MsCAS15 protein effectively alleviated the inhibitory effect of water deficit on seed germination, enabling transgenic plants to complete the germination process faster than wild-type plants. This result demonstrates that the MsCAS15 gene has a positive regulatory function on drought resistance during plant germination.

[0127] Test Example 4: Seedling Growth Test under Natural Soil Drought Stress

[0128] This test case uses the natural drought method in soil pots to compare and analyze the drought resistance performance of the transgenic Arabidopsis thaliana lines obtained in the example and the wild-type Arabidopsis thaliana in the seedling stage, and verifies the stress resistance function of the MsCAS15 gene in the plant growth stage.

[0129] Experimental steps:

[0130] Seedling preparation and transplanting: The three T3 generation homozygous transgenic Arabidopsis lines (OE55, OE77, OE85) obtained in the examples and the wild-type Arabidopsis (WT) seeds of the comparative example were sterilized and sown on 1 / 2 MS solid medium. After culturing in an artificial climate chamber for 7 days, robust seedlings with uniform growth and two true leaves were selected for transplanting.

[0131] Soil cultivation conditions: Select uniformly sized plastic flower pots, and fill each pot with an equal total weight of mixed nutrient soil (vermiculite: nutrient soil = 1:2). Transplant the selected seedlings into the pots, planting one seedling per pot, and place them in a light incubator for normal management. The cultivation conditions are 22℃, 16 hours of light / 8 hours of darkness. During the cultivation period, water regularly and in measured amounts to maintain uniform soil moisture in all pots.

[0132] Drought stress treatment: After three weeks of normal growth in the soil, the plants entered the seedling stage of vigorous growth. At this time, all watering was stopped, and natural drought treatment was carried out. Water was withheld for 14 days, during which time plant morphological changes were observed and recorded.

[0133] Experimental results:

[0134] Experimental results are attached in conjunction with the instruction manual. Figure 8 As shown:

[0135] Overall growth status: After 14 days of natural drought treatment, the growth rate of all plants was inhibited due to water deficiency, which was reflected in the smaller rosette leaf area compared with normally growing plants.

[0136] Phenotypic differences: The phenotypic differences between the control group (WT) and the example (transgenic lines) under drought stress were significant.

[0137] Comparative Example (WT): Wild-type plants showed more severe dehydration symptoms, with rosette leaves showing obvious wilting and wrinkling, leaves becoming thinner, leaf area significantly reduced, and some leaves showing signs of drying out.

[0138] Examples (OE55, OE77, OE85): Although the transgenic lines were also affected by drought, their overall growth was significantly better than that of the wild type. Their rosette leaves remained relatively spread out, with less leaf wrinkling, maintaining a larger leaf area and better tissue turgor pressure.

[0139] Conclusion: The protein encoded by the MsCAS15 gene possesses dehydration protein properties. Under water stress caused by natural soil drought, overexpression of the MsCAS15 gene enabled transgenic Arabidopsis plants to accumulate more dehydration proteins. These proteins maintain cellular physiological metabolic functions by protecting the stability of intracellular macromolecules and membrane structures, thereby mitigating tissue damage caused by drought. Experiments confirmed that MsCAS15 expression effectively alleviated leaf wrinkling and wilting caused by water shortage and enhanced the drought resistance of the MsCAS15 gene in alfalfa seedlings.

[0140] Appendix:

[0141] SEQ ID NO.1 (MsCAS15 CDS sequence):

[0142] ATGGCAGGAATCATGAACAAGATTGGTGATGCCCTTCACGGAGGAGGAGACAAGAAAGAGGGAGAGCACAAAGGAGAACAACATGGGCATGTAGGAGGAGAACATCATGGTGAGTACAAAGGAGAACAACATGGATTTGTTGGAGGACATGCTGGTGACCACAAAGGAGAACAACATGGTTTTGTAGGAGGACATGGGGGTGATT ACAAAGGAGAGCAACATGGATTTGGTCATGGAGACCACAAGGAGGGATACCATGGGGAAGAGCACAAGGAGGGATTTGCCGACAAGATCAAGGACAAAATTCATGGTGAAGGTGCAGATGGTGAAAAGAAAAAGAAGAAGGAGAAGAAGAAACATGGAGAGGGTCATGAACATGGCCATGATAGCAGCAGCAGTGACAGTGATTAG

[0143] SEQ ID NO.2 (MsCAS15 amino acid sequence):

[0144] MAGIMNKIGDALHGGGDKKEGEHKGEQHGHVGGEHHGEYKGEQHGFVGGHAGDHKGEQHGFVGGHGGDYKGEQHGFGHGDHKEGYHGEEHKEGFADKIKDKIHGEGADGEKKKKKEKKKHGEGH

[0145] SEQ ID NO.3 (MsCAS15 cDNA sequence):

[0146]

[0147] (5'UTR) sequence:

[0148] GGCGAAAACTTTCAAGGATTCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAACGGAATAAGTGCGACACGGAAATGTTGAATACTCATTTTAGCTTCCTTTAGCTCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAAGTCGAAAGCCTCCGGTCGGAGGCTTTTGACTTTCTGCTATGGACAGGAAACAGCTATGACCATCGTCAGTATTGACTTCAGGCAGACGCGACATCGACTTCGCGTGTCGCCCTTAAAACAAAACAAAGAAGAAACACATTCATTTAGAAACAAATAGAAATACAAT

[0149] (3'UTR) sequence:

[0150] ATCTTAATTTCACTGCTTCATCATGTTGAGAGGTGAATGGAGTCATATGTGACGGCAATCTCCTATGTTTTAATTTCTTGTTGTAATATCTACGTATGTGTGTGGAAAAAAATACGTGTTCGTAGTTGGTAATTCGAGTGCTTGTAAAGGGCGACACGCGAATACGATAACAAGCATCAGACGAGTCGTA

[0151] Forward primer (MsCAS15F):

[0152] 5’-AAAACAAAACAAAGAAGAAACAC-3’

[0153] Reverse primer (MsCAS15R):

[0154] 5’-TACAAGCACTCGAATTACCAAC-3’

Claims

1. A drought stress-related alfalfa gene, characterized in that, Nucleotide sequences selected from the following group: (a) The nucleotide sequence shown in SEQ ID NO.1; (b) The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; (c) The nucleotide sequence shown in SEQ ID NO.3; (d) A nucleotide sequence that hybridizes under strict conditions with the nucleotide sequence defined in (a) or (c) and encodes a dehydrated protein that enhances plant drought resistance; (e) has more than 90% identity with the nucleotide sequence defined in (a) or (c) and encodes a nucleotide sequence that encodes a dehydrated protein that enhances plant drought resistance.

2. The drought stress-related alfalfa gene according to claim 1, characterized in that, The protein encoded by the alfalfa gene has the amino acid sequence shown in SEQ ID NO.2, or is a protein derived from SEQ ID NO.2 with one or more amino acid residues substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO.2, and has the function of improving plant drought resistance.

3. A recombinant expression vector, characterized in that, It contains the drought stress-related alfalfa gene as described in claim 1 or 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector was constructed using a plant binary expression vector backbone; Preferably, the plant binary expression vector is pCAMBIA3301; Preferably, the alfalfa gene is expressed by the CaMV 35S promoter.

5. A recombinant bacterium or transgenic plant cell containing a drought-stress-related alfalfa gene as described in claim 1 or 2, or a recombinant expression vector as described in claim 3 or 4.

6. The application of the drought stress-related alfalfa gene as described in claim 1 or 2, the recombinant expression vector as described in claim 3 or 4, or the recombinant bacteria or transgenic plant cells as described in claim 5 in improving plant drought resistance.

7. The application according to claim 6, characterized in that, The improvement of plant drought resistance is specifically manifested in the following ways: To increase the germination rate of plant seeds under drought or osmotic stress conditions; Under drought or osmotic stress conditions, it reduces the degree of wilting of leaves in seedlings or mature plants and maintains plant growth.

8. The application according to claim 6, characterized in that, This is achieved by constructing transgenic plants, and the method for constructing transgenic plants includes the following steps: S1. The alfalfa gene is constructed into a plant expression vector to obtain a recombinant expression vector; S2. Transform the recombinant expression vector into Agrobacterium; S3. Using Agrobacterium containing a recombinant expression vector to infect the target plant, transgenic positive plants were obtained through screening.

9. The application according to claim 8, characterized in that, In step S3, the screening is carried out using a herbicide, preferably glufosinate-ammonium; The target plant is a dicotyledonous plant, preferably a legume or cruciferous plant, and more preferably alfalfa or Arabidopsis thaliana.

10. The application according to claim 8, characterized in that, In step S2, the Agrobacterium is Agrobacterium tumefaciens GV3101; In step S3, the infection method is either inflorescence immersion or leaf disc transformation.

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