Medicago sativa gene MsELIP1 and application thereof in salt tolerance

Abstract

The invention belongs to the technical field of gene engineering and salt-tolerant plant cultivation, and particularly relates to an alfalfa gene MsELIP1 and application thereof in salt tolerance. According to the invention, the structure and the evolutionary relationship of an early-stage photoinduced protein family of medicago sativa are analyzed, preliminary expression analysis is carried out on family genes, a representative member MsELIP1 gene is cloned, and the important effect of the MsELIP1 gene in the process of coping with salt stress by plants is clarified. The MsELIP1 not only can improve the germination rate of plants under salt stress and promote root growth and biomass accumulation, but also can effectively regulate ion balance in plant cells and reduce the content of sodium ions, which shows that the MsELIP1 plays a positive regulation role in salt tolerance of alfalfa and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and salt-tolerant plant cultivation, and specifically relates to a gene encoding alfalfa MsELIP1 and its application in salt tolerance. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Light is an indispensable environmental factor in plant growth and development, but excessive light, especially intense light, can cause photostress and photooxidative damage. Early light-induced proteins (ELIPs), a crucial component of plant photoprotection mechanisms, have garnered widespread attention since their discovery. In-depth research on ELIPs will help uncover the molecular mechanisms by which plants adapt to changing light environments and is crucial for improving plant stress resistance and optimizing agricultural production. ELIPs belong to the chlorophyll a / b binding protein superfamily. They typically possess a typical three-transmembrane helix domain, with the N- and C-termini located on the stroma side of the thylakoid membrane. Within the transmembrane helices lie conserved amino acid residues that bind to chlorophyll and carotenoids.

[0004] Research on early light-induced proteins (ELIPs) in plants has made significant progress in recent years. Their structural characteristics, gene expression regulation, physiological functions, and research findings in different plant species have provided a wealth of information for a deeper understanding of plant light adaptation mechanisms. However, few ELIPs have been reported in alfalfa. Therefore, further analysis and identification of ELIPs in alfalfa are necessary to explore their relationship with stress resistance and provide essential support for sustainable agricultural development. Summary of the Invention

[0005] In view of the above prior art, the present invention aims to provide a kind of alfalfa gene MsELIP1 Specifically, the present invention analyzes the structure and evolutionary relationship of the early light-induced protein family of alfalfa, performs preliminary expression analysis on the family genes, and clones representative members. MsELIP1 Genes, identified MsELIP1 This gene plays an important role in plant response to salt stress. It not only improves plant germination rates under salt stress, promoting root growth and biomass accumulation, but also effectively regulates ion balance within plant cells, reducing sodium ion content. This suggests that MsELIP1 plays a positive regulatory role in salt tolerance in alfalfa. Based on these research findings, the present invention was completed.

[0006] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows: The first aspect of the present invention provides a gene encoding an early light-induced protein of alfalfa, which is named MsELIP1 , the alfalfa early light-induced protein encoding gene has: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but differing in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes the same functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

[0007] The second aspect of the present invention provides an alfalfa early light-induced protein, wherein the alfalfa early light-induced protein is encoded by the alfalfa early light-induced protein encoding gene.

[0008] The third aspect of the present invention provides a recombinant expression vector comprising the alfalfa early light-induced protein encoding gene.

[0009] The fourth aspect of the present invention provides a transgenic cell line, a host bacteria or a transgenic plant containing the alfalfa early light-induced protein encoding gene or the recombinant expression vector.

[0010] A fifth aspect of the present invention provides the use of the alfalfa early light-induced protein encoding gene, alfalfa early light-induced protein, recombinant expression vector, transgenic cell line, host bacteria or transgenic plant in any one or more of the following: (c1) Regulate ion balance in plant cells; (c2) regulating plant root growth, leaf area and biomass accumulation; (c3) regulating the germination rate of plants under salt stress; (c4) regulating plant salt tolerance; (c5) Screening and / or breeding of salt-tolerant plants.

[0011] A sixth aspect of the present invention provides a method for improving salt tolerance of plants, the method comprising: promoting the expression of the alfalfa early light-induced protein encoding gene in the plant.

[0012] Beneficial technical effects of one or more of the above technical solutions: The above technical scheme identified the early light-induced protein family genes that respond to salt stress in alfalfa for the first time. MsELIP1 , and overexpression was achieved by Agrobacterium transformation MsELIP1 The results of experiments have shown that the gene encoding the early light-induced protein in alfalfa MsELIP1 The results show that the expression of MsELIP1 can effectively improve the salt tolerance of plants, indicating that MsELIP1 plays a positive regulatory role in plant salt tolerance. The above technical solution provides a basis for screening and verifying functional salt-tolerance genes, and then screening or breeding salt-tolerant plant varieties, especially salt-tolerant alfalfa varieties, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0014] Figure 1 This is the phylogenetic tree analysis and motif analysis of the alfalfa ELIP gene in Example 1 of the present invention; the left side is the phylogenetic tree of alfalfa, and the right side is the gene structure diagram of the corresponding gene, with yellow boxes representing exons, black lines representing introns, and green boxes representing non-coding regions.

[0015] Figure 2 Alfalfa in Example 1 of the present invention MsELIPs Relative expression analysis under salt stress conditions; A, B, C, D, E, F: changes in the expression levels of MsG0180005555.01.T01, MsG0180005556.01.T01, MsG0180005558.01.T01, MsG0180005558.01.T02, and MsG0180005545.01.T01 under 200 mM NaCl treatment, respectively; standard errors were from three biological replicates. Data were analyzed using one-way ANOVA and Duncan test. Different lowercase letters indicate significant differences ( P <0.05).

[0016] Figure 3 Alfalfa in Example 1 of the present invention MsELIP1 Gene cloning.

[0017] Figure 4 This is the subcellular localization of MsELIP1-GFP in Example 1 of the present invention.

[0018] Figure 5 This is the genetic transformation of alfalfa in Example 2 of the present invention; A: co-cultivation; B, C: callus induction; D, E: bud induction; F, G: rooting induction; H, I: tissue culture seedlings.

[0019] Figure 6This is the positive identification of transgenic alfalfa in Example 2 of the present invention; A: 35S:: MsELIP1 DNA level identification of some transgenic lines. 1-14 are transgenic alfalfa lines. M: DNA Marker. Plasmid is a control plasmid containing the corresponding target gene. Empty vector: transgenic alfalfa lines carrying only the vector. B: 35S:: MsELIP1 Characterization of RNA levels in Medicago sativa.

[0020] Figure 7 The differences in growth and plant height of transgenic positive seedlings under control and salt treatment in Example 2 of the present invention are shown in Figures A and C. The images and statistical analysis graphs are shown before salt treatment, and the images and statistical analysis graphs are shown after salt treatment.

[0021] Figure 8 The empty strains and MsELIP1 Differences in root length and individual leaf area among overexpression lines.

[0022] Figure 9 The empty strains and MsELIP1 Differences in fresh weight and photosynthetic indices among the overexpression lines. A is fresh weight, B is chlorophyll content, C is net photosynthetic rate, D is transpiration rate, E is stomatal conductance, and F is intercellular CO2 concentration.

[0023] Figure 10 This is the positive identification of transgenic Arabidopsis thaliana in Example 3 of the present invention; A: 35S:: MsELIP1 Positive identification at the DNA level; M: DNA Marker; Numbers indicate different lines; B: Identification of transgenic Arabidopsis at the transcriptional level.

[0024] Figure 11 The germination rate of overexpressed Arabidopsis thaliana under different treatments in Example 3 of the present invention is measured; A: germination rate of the control group; B: germination rate of Arabidopsis thaliana under 100 mM NaCl treatment.

[0025] Figure 12 The fresh weight and root length of overexpressed Arabidopsis thaliana under different treatments in Example 3 of the present invention are measured; A: actual development of Arabidopsis thaliana in the control group and under 100 mM NaCl treatment; B: statistical graph of fresh weight of Arabidopsis thaliana in the control group and under 100 mM NaCl treatment; C: statistical graph of root length of Arabidopsis thaliana in the control group and under 100 mM NaCl treatment. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] The present invention will now be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the reagent company. Reagents and consumables used in the following examples are commercially available unless otherwise specified.

[0029] In a typical embodiment of the present invention, a gene encoding an early light-induced protein of alfalfa is provided, which is named MsELIP1 , the alfalfa early light-induced protein encoding gene has: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but differing in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes the same functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

[0030] In another specific embodiment of the present invention, an alfalfa early light-induced protein is provided. The alfalfa early light-induced protein is encoded by the alfalfa early light-induced protein encoding gene.

[0031] The alfalfa early light-induced protein has any one of the amino acid sequences (b1) to (b3): (b1) the amino acid sequence shown in SEQ ID NO. 2; (b2) a protein derived from the amino acid sequence shown in SEQ ID NO. 2, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function as the amino acid sequence shown in SEQ ID NO. 2; (b3) proteins encoded by other genes that have an amino acid sequence similarity of more than 90% with that of SEQ ID NO. 2 and have the activity of the protein of SEQ ID NO. 2; In another embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the alfalfa early light-induced protein encoding gene; Furthermore, the recombinant expression vector is obtained by effectively connecting the above-mentioned alfalfa early light-induced protein encoding gene to an expression vector. The expression vector can be any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome, and further a plasmid. The plasmid can be a pCAMBIA series vector. In a specific embodiment of the present invention, the plasmid is pCAMBIA1300.

[0032] In another embodiment of the present invention, a transgenic cell line, host bacteria or transgenic plant containing the alfalfa early light-induced protein encoding gene or the recombinant expression vector is provided.

[0033] The transgenic cell line may be isolated, in vitro, in culture, or preferably, part of a plant.

[0034] The host bacteria may be eukaryotic bacteria (such as fungi, further such as yeast) or prokaryotic bacteria (such as bacteria, further such as Escherichia coli).

[0035] In the present invention, the transgenic plant can be a crop, further an economic crop, such as alfalfa.

[0036] In another embodiment of the present invention, there is provided the use of the alfalfa early light-induced protein encoding gene, alfalfa early light-induced protein, recombinant expression vector, transgenic cell line, host bacteria or transgenic plant in any one or more of the following: (c1) Regulate ion balance in plant cells; (c2) regulating plant root growth, leaf area and biomass accumulation; (c3) regulating the germination rate of plants under salt stress; (c4) regulating plant salt tolerance; (c5) Screening and / or breeding of salt-tolerant plants.

[0037] In another embodiment of the present invention, a method for improving plant salt tolerance is provided, the method comprising: transferring the alfalfa early light-induced protein encoding gene or recombinant expression vector into the plant.

[0038] Specifically, the specific method for transferring the recombinant expression vector into plants can be Agrobacterium transformation, specifically, transferring the recombinant expression vector into Agrobacterium, and using the Agrobacterium to infect plants.

[0039] The plant may be a crop, further an economic crop, such as alfalfa.

[0040] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are generally carried out under conventional conditions.

[0041] Example 1 1. Experimental Materials The seeds of Zhongmu No. 1 used in the experiment were from a variety preserved by the Dongying Academy of Agricultural Sciences in Shandong Province and stored in this laboratory.

[0042] Experimental methods 2.1 Plant cultivation and treatment Wild-type seeds of alfalfa (Zhongmu No. 1) were broken with sandpaper to remove hard cores, placed on moistened filter paper, and vernalized at 4°C for 2 days in an artificial climate incubator (16 h light, 24°C / 8 h dark, 20°C, relative humidity 60%) for germination. After the cotyledons opened, the seeds were transferred to 1 / 2 Hogland nutrient solution for growth, and the nutrient solution was changed weekly.

[0043] Genetic tissues, including roots, stems, and leaves, were collected from 6-week-old alfalfa plants. Leaves from 4-week-old alfalfa plants were collected at 0, 4, 8, 12, 24, 48, and 72 h after treatment with NaCl (200 mM).

[0044] 2.2 Bioinformatics analysis Ensembl Plants (http: / / plants.ensembl.org / index.html) was used to extract species genome annotation information and related protein amino acid sequences. Multiple sequence alignment was performed using DNAMAN (Version 7). Phylogenetic trees were constructed using the neighbor-joining method of MEGA (Version 7.0) with a bootstrap value of 1000, and joint mapping was performed using TBtools.

[0045] 2.3 Extraction of plant total RNA and cDNA synthesis RNA was extracted according to the Promega Plant Total RNA Extraction Kit instructions. The integrity of the extracted RNA was assessed by 0.8% agarose gel electrophoresis. RNA was reverse transcribed to synthesize cDNA using the Evo M-MLV RT Mix Kit with GDNA Clean for qPCR Version 2.

[0046] 2.4 Cloning of MsELIP1 Total RNA was extracted from 4-week-old alfalfa plants and cDNA was obtained by reverse transcription. Amplification primers were designed based on the alfalfa MsELIP1 gene sequence from the Ensembl Plants database. The specific reaction system is as follows:

[0047] Use 1% agarose gel electrophoresis to detect whether the target band is amplified successfully. If amplified successfully, use a gel recovery kit to recover and purify the target band. For specific operations, refer to the instructions.

[0048] 2.5 Construction and transformation of cloning vectors 1. Mix the connection system according to the table below and place in a metal bath at 25°C for 5 minutes.

[0049] Total RNA was extracted from 4-week-old alfalfa and cDNA was obtained by reverse transcription. Amplification primers were designed based on the alfalfa MsELIP1 gene sequence in the Ensembl Plants database.

[0050] Specific reaction systems such as:

[0051] 2. Take 500 μL of competent E. coli, add 3 μL of ligation product when it has just melted, gently pipette to mix, and place on ice. Incubate at 42°C for 30 min, heat shock at 42°C for 30 s, and immediately place on ice for 2 min.

[0052] 3. Add 500 μL LB liquid medium to the transformation product of step 2, pipette and mix, and place in a constant temperature shaker at 37°C. Shake the culture at 200 rpm for 1 h.

[0053] 4. Take the activated bacterial solution from the previous step and apply it on a plate containing kanamycin and rifampicin (Kan, 50 mg·L -1 , Rif, 50mg·L -1 ) antibiotic-containing LB solid medium, seal the culture dish with sealing film, and place it upside down in a 37°C constant temperature incubator for 12-18 h.

[0054] 5. Use a pipette tip to pick up a single colony and place it in 500 μL LB (containing 50 mg·L -1 Kan) liquid medium, shaken at 37°C and 200 rpm until the bacterial solution becomes turbid.

[0055] Colony PCR was performed using the universal primers 1300-F / R on the cloning vector. Positive clones were selected and sent for sequencing (Qingke Biotechnology Co., Ltd.). 50% glycerol was added to the bacterial suspension with the correct sequence at a ratio of 1:1 to preserve the bacteria. The suspension was then stored in a -80°C ultra-low temperature freezer until use. The specific reaction system for colony PCR was as follows:

[0056] 2.6 Construction of plant expression vectors 1. Using LB (Kan, 50 mg·L -1 , Rif, 50 mg·L -1 ) liquid culture medium, activate the bacterial solution containing pCAMBIA1300-MsELIP1 at a ratio of 1:100, and shake at 37°C, 200 rpm for 8-10 h to an OD600 value of 0.6-0.8.

[0057] 2. Refer to the instructions of the Plasmid Rapid Extraction Kit (OMEGA) to extract the target gene and pCAMBIA1300 vector plasmid.

[0058] 3. Referring to the Smarter Clone kit, design linker primers 1300-MsELIP F / R, R. Using the five plasmids mentioned above as templates, amplify the target fragments by PCR with 15-25 bp overlap regions at both ends of the linearized vector.

[0059] 4. Linearize the pCAMBIA1300 vector plasmid using the restriction endonuclease SalI. Incubate with seamless cloning enzyme at 50°C for 15 minutes, then transform E. coli. Select a culture containing the correct sequence and extract the plasmid to obtain the 35S::MsELIP1-GFP fusion expression vector.

[0060] 2.7 MsELIP1 Differential expression of Four-week-old alfalfa was used as the material to quantitatively analyze the expression of MtANN2 in the aboveground and aerial parts of alfalfa.

[0061] 2.8 Subcellular localization of MsELIP1 1. Inoculate the 35S::MsELIP1-GFP fusion expression plasmid and the 35S::GFP empty bacterial suspension at a dilution ratio of 1:100 into 50 mL of kanamycin (50 mg·L -1 ) and rifampicin (50 mg·L -1) in LB liquid culture medium to OD600 = 0.6-0.8, and then reactivated at a ratio of 1:100 to a bacterial liquid OD600 = 0.8-1.0.

[0062] 2. Centrifuge the bacterial suspension (5000 rpm, 5 min) and resuspend the bacteria in buffer (10 mM MgCl2, 5 mM MES, 100 μM acetosyringone, pH = 5.3) to OD600 = 0.8-1.0. Mix P19 and target gene bacterial suspension in a 1:1 ratio.

[0063] 3. The mixture was allowed to stand in the dark at room temperature for 3 h, and injected into 4-week-old tobacco ( Nicotiana benthamiana ) epidermis, and then the plants were placed in a plant growth climate chamber for 48-72 hours, and fluorescence was observed using a confocal fluorescence microscope (Leica TCS SP8). MsELIP1 Quantitative analysis of expression was performed.

[0064] 3. Experimental Results 3.1 Bioinformatics analysis of MsELIP1 Based on the conserved domain PF00010 feature file of the ELIP family in the Pfam database, six candidate ELIP gene sequences were retrieved from the Zhongmu No.1 transcriptome database using the AtELIP1 and AtELIP2 sequences as references. The conserved motifs of the six Cinnamomum camphora ELIP genes were analyzed using Tbtools and MEME programs. Figure 1 All alfalfa bHLHs contain the ELIP protein domain, but the locations in the sequence are different; ELIP members from the same subfamily have similar motif types and numbers, but also have different motifs.

[0065] According to the ELIP genome annotation information, the six alfalfa ELIP family members are distributed on the same chromosome.

[0066] 3.2 MsELIP1 Expression analysis and cloning of For 6 different branches MsELIPGenes (MsG0180005555.01.T01, MsG0180005556.01.T01, MsG0180005558.01.T01, MsG0180005558.01.T02, MsG0180005545.01.T01) were treated with 200mM NaCl in alfalfa (4-week-old alfalfa Zhongmu No.1 wild type) and the expression levels of the representative genes in the leaves of the treated materials were analyzed. The results are shown in Figure 2. Figure 2 Compared with the control, the above results showed that all 6 genes from different groups were induced by NaCl treatment (within 72 h), and MsG0180005558.01.T02 had a relatively weak response to salt. Among them, MsG0180005555.01.T01 was upregulated more than the other 5 genes, and its expression level continued to increase from 8 h and maintained at 4.1-4.5 times that of the control, indicating that this gene was induced by salt.

[0067] Therefore, MsG0180005555.01.T01 was selected for subsequent functional analysis and the gene was named MsELIP1 .

[0068] Amplified from wild-type cDNA of Medicago sativa Zhongmu No.1 MsELIP1 The open reading frame of Figure 3 As shown, the electrophoresis bands were consistent with the target fragment size, and sequencing confirmed MsELIP1 Already cloned into the intermediate vector.

[0069] 3.3 MsELIP1 subcellular localization To investigate the localization of MsELIP1 in cells, the control 35S::GFP plasmid and the 35S::MsELIP1-GFP recombinant plasmid were transiently expressed in Nicotiana benthamiana. Figure 4 As shown, the control expression signal is distributed in the nucleus, cell membrane, and part of the cytoplasm of tobacco epidermal cells. The green fluorescence signal of the recombinant protein MsELIP1-GFP is mainly located in the cell nucleus.

[0070] Example 2 1. Experimental Materials The seeds of Zhongmu No. 1 used in the experiment were from a variety preserved by the Dongying Academy of Agricultural Sciences in Shandong Province and stored in this laboratory.

[0071] 2. Experimental Methods 2.1 Agrobacterium-mediated transformation of alfalfa cotyledonary nodes 1. Activate Agrobacterium and take the target gene MsELIP1Agrobacterium containing plasmid was inoculated into 50 mL LB (containing 50 mg·L -1 Rifampicin and 50 mg·L -1 The cells were cultured in liquid medium containing kanamycin and shaken at 28°C until the OD600 value reached 0.6-0.8.

[0072] 2. When the secondary activation bacterial solution reaches an OD600 value of 0.6-0.8, centrifuge at 4000 rpm for 10 min and discard the supernatant.

[0073] 3. Preparation of infection solution: The preparation method of SH3a resuspension is shown in the following table.

[0074] Sterilize the prepared SH3a resuspension in a high-pressure steam sterilizer at 121°C for 20 minutes and let it cool. Determine the concentration of the SH3a resuspension. When OD600 is ≈ 0.3-0.4, it is the appropriate resuspension concentration. Resuspend the bacterial block with SH3a resuspension in a clean bench and vortex to make the final concentration of the bacterial suspension uniformly distributed between 0.6-0.8. 4. Place Zhongmu No. 1 seeds in a sterile bottle and rinse three times with double-distilled water. Rinse with 75% ethanol for 5 minutes, discard the ethanol, and rinse three to five times with double-distilled water to ensure the ethanol is removed. In a laminar flow hood, add 20% sodium hypochlorite to the sterile bottle and rinse for 20 minutes, discard the sodium hypochlorite, and rinse three to four times with double-distilled water in the laminar flow hood to ensure the sodium hypochlorite is removed. Plant the seeds on Ms medium in a laminar flow hood and culture in a plant tissue culture room for 7 to 9 days. Tear off alfalfa cotyledonary nodes and create a wound by pinching the edges with tweezers.

[0075] 5. Place the treated cotyledonary node in the resuspension solution, completely submerging the explant. Place the sterile bottle in a shaker at 95-100 rpm for 20 minutes. In a laminar flow hood, blot the soaked explant dry with filter paper and transfer it to the co-culture medium, with the leaf facing upward and the underside and wound edge touching the medium surface. Seal the medium, wrap it in tin foil, and place it in a tissue culture chamber (24°C, 24-hour light cycle, 24-hour dark cycle) in a dark-proof chamber for 3 days.

[0076] 6. Wash the leaves with ddH2O 5-7 times until the washing solution is clear, and transfer to a microporous plate containing hygromycin (5 mg·L -1 ), cephalosporins (200 mg·L -1 )、Timentin (200 mg·L -1 ) in a solid culture medium containing SH3a resistance, and culture in an artificial incubator in the dark for 6-8 weeks. Replace the subculture medium every 14 days and discard the leaves or tissues that grow bacteria.

[0077] 7. Transfer the fluffy, granular callus into MSBK (containing hygromycin 5 mg·L -1 , cephalosporin 200mg·L -1 , Timentin 200 mg·L -1 ) culture medium, restore normal light and culture for about 2 weeks to induce budding.

[0078] 8. Transfer the callus tissue to SH9a solid medium (containing hygromycin 2.5 mg·L -1 , cephalosporin 200 mg·L -1 , Timentin 200 mg·L -1 ) induce root differentiation, grow straight and strong root system, and move into soil for growth (nutrient soil: vermiculite = 1:1).

[0079] 2.2 Positive identification of transgenic alfalfa Leaves of transgenic plants were taken to extract genomic DNA and RNA (the method referred to the plant genomic DNA extraction kit, Kangwei Century, and the RNA extraction kit instructions, Vazyme), and positive identification was performed at the DNA and RNA levels.

[0080] 2.3 Overexpression MsELIP1 Analysis of salt tolerance of alfalfa The positive seedlings with rooting in hydroponics were transferred to soil (nutrient soil: vermiculite = 1:1) and grown in an artificial climate chamber for 5 days. -1 ) treatment (normal watering for the control), watering once every 3 days, 20 mL each time, and observe the phenotype and measure the plant biomass and photosynthetic indices after 14 days of treatment.

[0081] 3. Experimental Results 3.1 Agrobacterium-mediated transformation of alfalfa cotyledonary nodes Medicago sativa (Zhongmu No. 1) was genetically transformed with the target gene and the pCAMBIA1300 empty vector (control) by Agrobacterium-mediated leaf disc infection ( Figure 5 ).

[0082] 3.2 Positive identification of transgenic alfalfa Overexpression MsELIP 20 positive seedlings of alfalfa were obtained and their expression levels were identified by qRT-PCR technology. Figure 6 , the transgenic lines OE1, OE2, and OE3 with higher expression levels were selected for hydroponic rooting to ensure the consistency of genetic background. After rooting, they were moved to nutrient soil for subsequent functional verification.

[0083] 3.3 MsELIP1 Overexpression functional verification Cultivation of transgenic empty alfalfa and transgenic MsELIP1 There was no significant difference in the growth of alfalfa. Soil salt (200 mM NaCl) treatment was performed. Figure 7 As described above, after 14 days of salt treatment, the overexpression MsELIP1 (OE1, OE2, OE3) plant height was significantly higher.

[0084] At the same time, if Figure 8 As shown, under 200 mM NaCl treatment, overexpression MsELIP1 (OE1, OE2, OE3) The root length was significantly longer than that of the empty vector strain, and the leaf area was also slightly larger than that of the empty vector strain.

[0085] The results are as follows Figure 9 As shown, overexpression MsELIP1 The fresh weight of (OE1, OE2, OE3) was higher than that of the empty-loaded strain. Under normal conditions, the net photosynthetic rate of the transgenic empty-loaded alfalfa was lower than that of the empty-loaded strain, but the stomatal conductance and intercellular CO2 concentration were higher than those of the empty-loaded strain. After salt treatment, the overexpression MsELIP 1 (OE1, OE2, OE3) had higher net photosynthetic rate, transpiration rate, and stomatal conductance than those of the unloaded strain, and there was no significant difference in intercellular CO2 concentration.

[0086] Example 3 1. Experimental Materials 1300 empty Arabidopsis, overexpression MsELIP1 Arabidopsis thaliana.

[0087] 2. Experimental Methods 2.1 Genetic transformation of Arabidopsis thaliana and positive identification 1. Transfer the DNA containing pCAMBIA1300- MsELIP1 Agrobacterium carrying the plasmid and pCAMBIA1300 were inoculated into LB liquid medium at a ratio of 1:100 at 28°C and 200 rpm until the OD600 value reached 0.6-0.8. The bacterial solution was activated for the second time until it reached the logarithmic phase.

[0088] 2. Centrifuge at 5000 rpm for 10 min at room temperature, discard the supernatant, add suspension solution (5% sucrose, 0.05% Silwet L-77) in proportion, and resuspend until the OD600 value of the infection solution is 0.2-0.4.

[0089] 3. Using the inflorescence infection method, remove the opened flowers and pods from four-week-old wild-type Arabidopsis plants. Immerse the closed inflorescences in the infection solution for 1 minute. After infection, incubate in the dark for 24 hours and then transfer to a plant climate chamber. Repeat the infection 5-8 days later to increase the positive transformation rate.

[0090] 4. Harvest mature seeds and plant them on 1 / 2 MS solid medium (containing 10 mg·L -1 Hyg) to screen positive strains.

[0091] 5. Move the transgenic Arabidopsis thaliana (T0) plants that have grown cotyledons and true roots into pots (nutrient soil: vermiculite = 1:1) and place them in a plant growth climate chamber for growth.

[0092] 6. Harvest mature seeds of Arabidopsis thaliana (T0) and plant them in 1 / 2 MS solid medium (containing 10 mg·L -1 Hyg) to screen positive lines. Individual transgenic Arabidopsis thaliana (T1) plants that had developed cotyledons and true roots were transplanted into pots (nutrient soil: vermiculite = 1:1) and grown in a plant growth chamber. Three generations were harvested to obtain T3 generation seeds. Mature Arabidopsis thaliana (T3) seeds were harvested and genomic DNA was extracted from T3 generation transgenic plants. Positive DNA identification was performed at the DNA level using the 35S upstream primer and the designed vector downstream primer 1300R (Appendix A-2).

[0093] 7. Analyze the expression level of target genes in transgenic Arabidopsis by qRT-PCR.

[0094] 2.2 Overexpression MsELIP1 Arabidopsis germination rate determination Empty Arabidopsis, overexpression MsELIP1 T3 generation Arabidopsis seeds (sterilized with 75% ethanol for 10 min and washed with ddH2O 5-7 times) were vernalized at 4°C for 2 days and plated on 1 / 2 MS solid medium containing 0 mM and 200 mM NaCl. The germination rate was calculated daily for one week (40 seeds per dish, with three biological replicates).

[0095] 2.3 Overexpression MsELIP1 Analysis of salt tolerance in Arabidopsis seedlings Arabidopsis seeds were germinated on 1 / 2 MS solid medium. When the roots grew to 2-3 cm in length, seedlings with consistent growth were selected and transferred to 1 / 2 MS solid medium with 0 mM and 100 mM NaCl concentrations for vertical culture for 14 days. The root growth indicators of Arabidopsis were counted. Three biological replicates were set up in the experiment.

[0096] 3. Results and Analysis 3.1 Positive identification of transgenic Arabidopsis The transgenic Arabidopsis thaliana with hygromycin resistance was verified at the DNA level, and a total of overexpression MsELIP1 8 strains of Arabidopsis thaliana ( Figure 10 A). RNA of positive strains was extracted, β-actin was used as the internal reference gene, AtactinF / R was designed, and the overexpressed MtANN2 was quantitatively analyzed ( Figure 10 B), indicating that the table MsELIP1 Highly expressed in Arabidopsis thaliana.

[0097] 3.2 Overexpression MsELIP1 Analysis of germination rate of Arabidopsis thaliana For overexpression MsELIP1 Transgenic Arabidopsis thaliana was analyzed, and the statistical results are shown in Figure 11 As shown in the figure, under 0 mM NaCl treatment, the empty and transgenic lines (OE-4, OE-8, OE-7, and OE-2) showed similar germination trends. Under salt treatment, the germination initiation time of the above Arabidopsis lines was delayed, but the germination rate of the transgenic lines was significantly improved.

[0098] 3.3 Overexpression MsELIP1 Analysis of salt tolerance in Arabidopsis seedlings For overexpression MsELIP1 Transgenic Arabidopsis thaliana was analyzed, and the statistical results are shown in Figure 12 As shown in the figure, under 0 mM NaCl treatment, the empty-load and transgenic lines showed similar fresh weight and root length. Under salt treatment, the fresh weight and root length of the above Arabidopsis lines decreased, indicating inhibition; however, the degree of inhibition of fresh weight and root length of the transgenic lines was significantly lower than that of the empty-load group.

[0099] Alfalfa MsELIP1 Genetic transformation, overexpression and functional analysis of the gene in Arabidopsis thaliana have clarified MsELIP1 The gene plays an important role in plants' response to salt stress. It not only improves the germination rate of plants under salt stress, promotes root growth and biomass accumulation, but also effectively regulates the ion balance in plant cells and reduces the sodium ion content.

[0100] The nucleotide and amino acid sequences involved in the present invention are: MsELIP1 Nucleotide sequence: (SEQ ID NO. 1) MsELIP1 amino acid sequence: MAVSSCQSIMSSSMTSISSRPRVNQFNNIPSVYMPSFRRNASLKVRSMAEEGQKEQPKVPVDPITPPSAPTPPPQPQPTYTRSPKMSTKFSDLMAFGGPAPERINGRLAMIGFVAAMGVEIANGQGLFDQISGGGIPWFLGTSVLLSLASLIPFFQGVSVESKSKGVMSSDAELWNGRIAMLGLIALAFTEYVKGTALV* (SEQ ID NO.2) The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A gene encoding an early light-induced protein in alfalfa, named MsELIP1 , characterized in that, The alfalfa early light-induced protein encoding gene has: (a1) the nucleotide sequence shown in SEQ ID NO. 1; (a2) a nucleotide sequence encoding a protein having the same amino acid sequence as the nucleotide sequence shown in (a1), but differing in sequence due to the degeneracy of the genetic code; (a3) a nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (a1) or (a2) and encodes the same functional protein; (a4) A nucleotide sequence complementary to any one of (a1) to (a3).

2. An alfalfa early light-induced protein, characterized in that The alfalfa early light-induced protein is encoded by the alfalfa early light-induced protein encoding gene according to claim 1.

3. The alfalfa early light-induced protein according to claim 2, wherein The alfalfa early light-induced protein has any one of the amino acid sequences (b1) to (b3): (b1) the amino acid sequence shown in SEQ ID NO. 2; (b2) a protein derived from the amino acid sequence shown in SEQ ID NO. 2, wherein one or more amino acid residues are substituted and / or deleted and / or added and which has the same function as the amino acid sequence shown in SEQ ID NO. 2; (b3) Proteins encoded by other genes that have an amino acid sequence composition that is more than 90% similar to that of SEQ ID NO. 2 and have the activity of the protein of SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the alfalfa early light-induced protein encoding gene according to claim 1; Furthermore, the recombinant expression vector is obtained by effectively connecting the alfalfa early light-induced protein encoding gene to an expression vector, and the expression vector is any one or more of a viral vector, a plasmid, a phagemid, a cosmid, or an artificial chromosome, and further is a plasmid, and the plasmid is a pCAMBIA series vector, and further, the plasmid is pCAMBIA1300.

5. A transgenic cell line, host bacteria or transgenic plant containing the gene encoding the alfalfa early light-induced protein according to claim 1 or the recombinant expression vector according to claim 4.

6. Furthermore, the transgenic plant is a crop, further an economic crop, including alfalfa.

7. Use of the gene encoding the alfalfa early light-induced protein of claim 1, the alfalfa early light-induced protein of any one of claims 2-3, the recombinant expression vector of claim 4, the transgenic cell line, host bacteria, or transgenic plant of claim 5 in any one or more of the following: (c1) Regulate ion balance in plant cells; (c2) regulating plant root growth, leaf area and biomass accumulation; (c3) regulating the germination rate of plants under salt stress; (c4) regulating plant salt tolerance; (c5) Screening and / or breeding of salt-tolerant plants.

8. The use according to claim 6, characterized in that The plant roots are hairy roots.

9. A method for reducing salt tolerance of plants, characterized in that: The method comprises: transferring the alfalfa early light-induced protein encoding gene according to claim 1 or the recombinant expression vector according to claim 4 into a plant, thereby reducing the fresh weight, dry weight and number of hairy roots of the plant.

10. The method according to claim 8, wherein The specific method for transferring the recombinant expression vector into plants is the Agrobacterium transformation method, specifically, transferring the recombinant expression vector into Agrobacterium, using the Agrobacterium to infect plant cotyledons, and culturing to obtain plant hairy roots.

11. A method for improving plant salt tolerance, characterized in that: The method comprises: inhibiting the expression of the gene encoding the alfalfa early light-induced protein according to claim 1 in a plant.

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