Alfalfa salt-tolerant protein, gene as well as preparation method and application of alfalfa salt-tolerant protein

Abstract

The invention discloses a medicago sativa salt-tolerant protein, a gene and a preparation method and application thereof, and relates to the field of biological agriculture, the protein is medicago sativa pectin methylesterase MsPME2 or a variant protein with the same enzyme function as the medicago sativa pectin methylesterase MsPME2, the gene encodes the protein, and the preparation is obtained based on cDNA amplification. The gene is transferred into medicago sativa to improve the salt tolerance, the invention provides the gene of the salt-tolerant medicago sativa variety and the application method of the gene, and the gene can be used for breeding the salt-tolerant medicago sativa.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological agriculture, and particularly relates to a salt-tolerant alfalfa protein, a gene and a preparation method and application thereof. BACKGROUND

[0002] Alfalfa (Medicago sativa L.) is a perennial legume plant, which is widely used as high-quality forage, vegetables and health product raw materials due to its high protein, vitamins and minerals, rapid growth and large biomass. In view of the ecological conditions in different regions, developing new varieties with strong adaptability and matching cultivation techniques has become the key to the current alfalfa industry development, which provides opportunities for genetic improvement of the crop and also brings new challenges.

[0003] The plant cell wall, as an important protective barrier of the cell, can selectively block harmful ions through its structural characteristics. The cell wall polysaccharide components (such as cellulose, hemicellulose and pectin) are rich in negative groups such as carboxyl groups, which can adsorb cations through ion exchange or chelation. Among them, pectin methylesterase (PME) regulates the charge distribution and spatial conformation of pectin by catalyzing the demethylation of pectin, and then affects the mechanical properties of the cell wall. According to the structural differences, PME can be divided into two types: type I (PME type) contains only a catalytic domain, and the N-terminal leader sequence is short or missing; type II (ProPME type) has both PME catalytic domain and PMEI inhibitory domain, and has a longer N-terminal leader region. Natural pectin is usually in a highly methylated state, and the carboxyl groups (COO-) exposed after demethylation can form "egg box" cross-linking structure with Ca 2+ and other divalent cations, significantly enhancing the rigidity of the cell wall. Studies have shown that PME genes can positively regulate the salt tolerance of rice, rapeseed, Arabidopsis and other plants. At present, there is no report on the sequence of salt-tolerant PME coding genes in alfalfa and their specific molecular mechanisms in salt response.

[0004] Therefore, the skilled in the art is committed to developing a salt-tolerant alfalfa variety and a breeding method thereof. SUMMARY

[0005] In view of the above defects of the prior art, the technical problem to be solved by the present application is to develop a salt-tolerant alfalfa variety and a breeding method thereof.

[0006] In order to achieve the above-mentioned purpose, the application provides a salt-tolerant protein of Medicago sativa, characterized in that the protein is Medicago sativa pectin methylesterase MsPME2, or a protein having the same enzyme function as MsPME2 and having a variant amino acid sequence of the amino acid sequence of MsPME2, i.e. a MsPME2 variant protein, the amino acid sequence of MsPME2 being shown as SEQ ID NO. 2.

[0007] In a preferred embodiment of the application, the MsPME2 variant protein is a protein having the amino acid sequence shown as SEQ ID NO. 2 with one or more amino acids being substituted, deleted or added and having the characteristics of the MsPME2 protein of Medicago sativa.

[0008] In another preferred embodiment of the application, the sequence of the MsPME2 variant protein is a sequence obtained by deleting, inserting and / or substituting 1-50 amino acids of the amino acid sequence shown as SEQ ID NO. 2, or adding 1-20 amino acids at the C-terminal and / or N-terminal end.

[0009] In another preferred embodiment of the application, the sequence of the MsPME2 variant protein is a sequence obtained by substituting 1-10 amino acids of the amino acid sequence shown as SEQ ID NO. 2 with amino acids having similar or similar properties.

[0010] The application also protects a gene of the above-mentioned salt-tolerant protein of Medicago sativa, characterized in that the sequence of the gene is shown as SEQ ID NO. 1, or the sequence of the gene has at least 90% homology with the nucleic acid shown as SEQ ID NO. 1, or the gene has a sequence capable of hybridizing with the nucleic acid shown as SEQ ID NO. 1.

[0011] In a preferred embodiment of the application, the sequence of the gene is specifically a sequence obtained by deleting, inserting and / or substituting 1-150 nucleotides of the nucleic acid sequence shown as SEQ ID NO. 1, or adding 60 nucleotides at the 5'- and / or 3'-end.

[0012] The application also includes a preparation method of the above-mentioned salt-tolerant protein MsPME2 gene of Medicago sativa, characterized by comprising the following steps:

[0013] Step 1, taking leaf tissues of Medicago sativa treated with salt for extracting RNA;

[0014] Step 2, extracting total RNA of the leaf tissues in step 1, identifying the integrity of the RNA by gel electrophoresis, measuring the purity and concentration of the RNA by spectrophotometer, and synthesizing cDNA by reverse transcription of the RNA;

[0015] Step 3, full-length cloning of the gene: according to the full-length sequence of the gene of the MsPME2 protein of Medicago sativa, specific primers are respectively designed from the start codon and the stop codon

[0016] SEQ ID NO. 3 ORF-F: 5'-ATGGCTACCCAAGAAA-3'

[0017] SEQ ID NO. 4 ORF-R: 5'-CTAAAGGCCTTCGATGAA-3'

[0018] The full-length coding sequence of the MsPME2 protein of Medicago sativa is obtained by PCR with the cDNA of Medicago sativa as a template.

[0019] In a preferred embodiment of the present application, the following steps are included:

[0020] Step 1, constructing a plant expression vector containing the gene;

[0021] Step 2, transforming the expression vector into Medicago sativa;

[0022] Step 3, screening transgenic positive lines.

[0023] In another preferred embodiment of the present application, the step 1 specifically includes:

[0024] Specific primers are respectively designed from the start codon and the stop codon

[0025] SEQ ID NO. 5 PME1-F:

[0026] 5'-GCTTGGATCCTCGAGCTGCAGATGGCTACCCAAGAAA-3';

[0027] SEQ ID NO. 6 PME1-R:

[0028] 5'-GCCCTTGCTCACCATACTAGTAAGGCCTTCGATGAAGGC-3', and a Bam HI and a Spe I enzyme digestion site is respectively introduced at both sides of the full-length sequence of the gene, the cDNA of Medicago sativa is used as a template for PCR amplification, the PCR product, i.e. the target gene fragment, is connected to a pMD18-T Simple vector, a single colony bacterial plaque is picked for PCR verification, and the positive clone bacterial liquid plasmid is extracted, i.e. the target gene fragment plasmid;

[0029] The plasmid of the target gene fragment and the PHB binary transformation vector are double digested by Bam HI and Spe I, the pHB-Flag vector after digestion is recovered, the target gene fragment is connected with the pHB-Flag vector by using T4 ligase, and the vector is transformed into Agrobacterium GV3101;

[0030] In another preferred embodiment of the present application, the step 2 specifically comprises:

[0031] Step 2.1, pre-shaking Agrobacterium: the final positive monoclonal in step 1 is picked into YEP liquid medium containing 50 mg / L Kan, 50 mg / L gentamicin and 25 mg / L Rif, and shaken at 28°C and 200 rpm for 24 hours;

[0032] Step 2.2, expanding Agrobacterium: the pre-shaken Agrobacterium is expanded at 1:100 into YEP medium containing the same resistance, and cultured at 28°C and 200 rpm for 13-16 hours until the absorbance OD 600 reaches 0.6-1.5, and the bacteria are collected at 18°C and 3500 rpm for 15 minutes;

[0033] Step 2.3, transforming alfalfa: the overexpression connected with the target gene sequence is transformed into Agrobacterium GV3101, and the positive monoclonal strain is obtained by performing bacterial liquid PCR and sequencing; the monoclonal is picked into a centrifuge tube containing 10 mL of LB solution containing antibiotics, shaken at 28°C and 200 rpm / min for 24 hours, 1 mL of bacterial liquid is taken and injected into a centrifuge tube containing 50 mL of LB solution containing antibiotics, shaken at 28°C and 200 rpm / min for 12 hours; then the Agrobacterium is collected and resuspended with SM4 liquid medium, adjusted to OD600 of 0.2-0.4 to obtain the infection resuspension;

[0034] Sterile seedling leaf treatment and transformation: the tender leaves are selected from the sterile seedlings of alfalfa, a wound is made on the surface of the leaves with a sterile forceps, then the leaves are immersed in the resuspension, vacuum treated for 10 minutes, ultrasonically broken for 2 minutes, vacuum treated again for 10 minutes, the treated leaves are taken out, evenly laid on sterile filter paper to dry, and then transferred to SM4 solid medium for culture;

[0035] Screening and callus induction: after the leaves are cultured in SM4 solid medium for 5 days, they are transferred to SM4 screening medium added with corresponding antibiotics for screening culture, fresh screening medium is replaced after 1 month of culture, and the screening is continued until callus formation, the formed callus is transferred to MSBK screening medium containing corresponding antibiotics for subsequent culture, the MSBK screening medium is replaced every 2 weeks until the differentiation of leaves, the brown necrotic tissue is cut off and transferred to MSR medium for vegetative growth, and after the seedlings grow high, they are transferred to a glass bottle containing MSR medium, and after the plants grow mature, they are transplanted to nutrient soil.

[0036] Technical effects

[0037] 1. The alfalfa genetic transformation and function verification of MsPME2 are carried out, and a theoretical basis is provided for alfalfa salt-tolerant new variety breeding.

[0038] 2. The alfalfa is subjected to salt stress for different time periods, the expression amount of MsPME2 is determined, and the expression of alfalfa MsPME2 in response to salt stress is clarified.

[0039] 3. The alfalfa MsPME2 gene is obtained, and the subsequent function verification research is facilitated.

[0040] Alfalfa is a commonly used forage crop and is widely applied and has great market demand. The coding sequence of the cell wall modification protein MsPME2 with obvious response to salt stress in alfalfa is cloned for the first time, the expression pattern of the MsPME2 gene is analyzed by using a fluorescent real-time quantitative PCR method, the location of the MsPME2 protein is analyzed by using tobacco leaf epidermal cells, the spatial and temporal expression of the MsPME2 gene is regulated by using genetic engineering technology, and a theoretical basis is provided for improving alfalfa salt-tolerant new variety breeding, and the application value is great.

[0041] The concept, specific structure and generated technical effects of the present application will be further described in combination with the drawings, so that the purpose, features and effects of the present application can be fully understood. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1Nucleotide sequence and amino acid homology comparison (DNAMAN) results of Medicago sativa MsPME2 gene and Medicago truncatula PME2 gene and MsPME2 domain schematic diagram for a preferred embodiment of the present application.

[0043] Figure 2 MsPME2 gene in tobacco leaf epidermal cell localization map for a preferred embodiment of the present application;

[0044] Figure 3 MsPME2 gene expression change schematic diagram in the process of salt stress for a preferred embodiment of the present application;

[0045] Figure 4 PCR and qRT-PCR identification schematic diagram of transgenic Medicago sativa for a preferred embodiment of the present application;

[0046] Figure 5 Wild type and MsPME2 transgenic Medicago sativa phenotype observation diagram under salt stress for a preferred embodiment of the present application;

[0047] Figure 6 Salt stress effect on wild type and MsPME2 transgenic Medicago sativa growth and physiology schematic diagram for a preferred embodiment of the present application;

[0048] Figure 7 Wild type and MsPME2 transgenic Medicago sativa root Na + , K + and Na + / K + schematic diagram under salt stress for a preferred embodiment of the present application.

[0049] Figure 8 Wild type and MsPME2 transgenic Medicago sativa stem and leaf Na + , K + and Na + / K + schematic diagram under salt stress for a preferred embodiment of the present application. DETAILED DESCRIPTION

[0050] The following reference to the drawings of the specification introduces a plurality of preferred embodiments of the present application, so that its technical content is more clear and convenient to understand. The present application can be embodied by many different forms of embodiments, the protection scope of the present application is not limited to the examples mentioned in the text.

[0051] The application provides a Medicago sativa MsPME2 protein with a salt stress response function and a protection function, and the protein is a protein composed of an amino acid sequence as shown in SEQ ID NO. 2, or an amino acid sequence as shown in SEQ ID NO. 2 with one or more amino acids being substituted, deleted or added and having the Medicago sativa MsPME2 protein function.

[0052] The protein has the MsPME2 protein characteristics, and the expression amount of the protein is different in different salt stress stages.

[0053] Preferably, the protein is a sequence obtained by deleting, inserting or substituting 1-50 amino acids of the amino acid sequence as shown in SEQ ID NO. 2, or adding 1-20 amino acids at the C terminal and / or N terminal.

[0054] Further preferably, the protein is a sequence obtained by substituting 1-10 amino acids in the amino acid sequence as shown in SEQ ID NO. 2 with amino acids with similar or similar properties.

[0055] In another aspect, the application provides a nucleic acid sequence encoding the above protein.

[0056] Preferably, the nucleic acid sequence is specifically (a) a base sequence as shown in SEQ ID NO. 1, 1-1662; or (b) a sequence with at least 90% homology with the nucleic acid as shown in SEQ ID NO. 1, 1-1662; or (c) a sequence capable of hybridizing with the nucleic acid as shown in SEQ ID NO. 1, 1-1662.

[0057] Preferably, the nucleic acid sequence is specifically a sequence obtained by deleting, inserting and / or substituting 1-150 nucleotides of the nucleic acid sequence as shown in SEQ ID NO. 1, 1-1662, or adding 60 nucleotides at the 5' and / or 3'-end.

[0058] In the application, the "isolated DNA" and "purified DNA" mean that the DNA or fragment has been separated from the sequences on both sides in the natural state, and also means that the DNA or fragment has been separated from the components of the nucleic acid in the natural state, and has been separated from the proteins accompanied in the cell.

[0059] In the present application, the term "a gene encoding alfalfa pectin methylesterase" refers to a nucleotide sequence encoding a polypeptide having the activity of alfalfa protein, such as the nucleotide sequence from 1 to 1662 of SEQ ID NO. 1 and its degenerate sequences. The degenerate sequences refer to sequences obtained by replacing one or more codons in the nucleotide sequence from 1 to 1662 of SEQ ID NO. 1 with degenerate codons encoding the same amino acid. Due to the degeneracy of codons, degenerate sequences having as low as about 70% homology with the nucleotide sequence from 1 to 1662 of SEQ ID NO. 1 can also encode the sequence of SEQ ID NO. 2. The term also includes nucleotide sequences having at least 70% homology with the nucleotide sequence of SEQ ID NO. 1.

[0060] The term also includes variants of the sequence of SEQ ID NO. 1 that can encode the same function of native alfalfa pectin methylesterase MsPME2. These variants include, but are not limited to, deletions, insertions and / or substitutions typically of 1 to 90 nucleotides, and additions of up to 60 nucleotides at the 5' and / or 3' end.

[0061] In the present application, the term "alfalfa MsPME2 pectin methylesterase" refers to a polypeptide having the sequence of SEQ ID NO. 2 that has the activity of alfalfa MsPME2 pectin methylesterase. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as native alfalfa MsPME2 pectin methylesterase. These variants include, but are not limited to, deletions, insertions and / or substitutions typically of 1 to 50 amino acids, or additions of up to 20 amino acids at the C-terminus and / or N-terminus. For example, in the art, substitutions with similar or identical properties are usually made to maintain the function of the protein. For another example, additions of one or several amino acids at the C-terminus and / or N-terminus usually do not change the function of the protein. The term also includes active fragments and active derivatives of alfalfa MsPME2 protein.

[0062] Variants of alfalfa MsPME2 protein of the present application include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, polypeptides or proteins that can hybridize to DNA of MsPME2 protein under high or low stringency conditions, and polypeptides or proteins obtained using antisera of alfalfa MsPME2 protein.

[0063] Variants of alfalfa MsPME2 protein of the present application include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, polypeptides or proteins that can hybridize to DNA of MsPME2 protein under high or low stringency conditions, and polypeptides or proteins obtained using antisera of alfalfa MsPME2 protein.

[0064] In the present application, "conserved variant polypeptide of Medicago sativa MsPME2 protein" refers to a polypeptide in which up to 10 amino acids are replaced by amino acids of similar or similar properties compared with the amino acid sequence shown in SEQ ID NO. 2.

[0065] The present application also includes analogs of Medicago sativa MsPME2 protein or polypeptide. The differences between these analogs and the polypeptide of Medicago sativa MsPME2 protein can be differences in amino acid sequence, differences in modified forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, and by site-directed mutagenesis or other known molecular biology techniques. The analogs also include analogs with residues other than natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present application are not limited to the representative polypeptides listed above.

[0066] Modified (usually not changing the primary structure) forms include chemical derivatives of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during synthesis and processing of the polypeptide or further processing steps. Such modifications can be accomplished by exposing the polypeptide to enzymes that perform glycosylation (such as mammalian glycosylation enzymes or deglycosylation enzymes). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or to optimize their solubility.

[0067] In the present application, the expression pattern of Medicago sativa MsPME2 gene product can be analyzed by real-time fluorescent quantitative PCR method, i.e. the presence or absence and quantity of mRNA transcript of Medicago sativa MsPME2 gene in cells.

[0068] The detection method of the present application for detecting whether there is a nucleotide sequence related to Medicago sativa MsPME2 gene in a sample includes hybridizing the probe described above with the sample, and then detecting whether the probe has been bound. The sample is the product of PCR amplification, in which the PCR amplification primers correspond to the coding sequence of the nucleotide sequence related to Medicago sativa MsPME2 gene and can be located on both sides or in the middle of the coding sequence. The length of the primers is generally 15-50 nucleotides.

[0069] In addition, based on nucleic acid homology or expression protein homology, the Medicago sativa MsPME2 gene related homologous genes or homologous proteins can be screened according to the Medicago sativa MsPME2 nucleotide sequence and amino acid sequence of the present application.

[0070] The full-length sequence of the Medicago sativa MsPME2 related nucleotide or its fragment can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the disclosed nucleotide sequence, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template for amplification to obtain the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified in each amplification are spliced together in the correct order.

[0071] When the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually to clone it into a vector, then transfer it into cells, and then separate the relevant sequence from the proliferated host cells by conventional methods.

[0072] In addition, mutations can be introduced into the protein sequence of the application by chemical synthesis.

[0073] In addition to being produced by recombination, fragments of the protein of the application can also be produced by directly synthesizing peptides using solid-phase technology. For example, the peptide can be automatically synthesized using a peptide synthesizer model 431A of Applied Biosystems (Foster City, CA). Each fragment of the protein of the application can be chemically synthesized separately, and then chemically linked to produce a full-length molecule.

[0074] Using the Medicago sativa MsPME2 protein of the application, substances related to the Medicago sativa MsPME2 protein, or inhibitors and antagonists, etc. can be screened by various conventional screening methods.

[0075] Figure 1 A is a nucleotide sequence alignment chart of Medicago sativa and Medicago truncatula, Figure 1 B is an amino acid sequence alignment chart of Medicago sativa and Medicago truncatula, Figure 1 C is the conserved domain of Medicago sativa MsPME2.

[0076] Example 1 Cloning of Medicago sativa MsPME2 gene

[0077] 1. Obtaining plant material

[0078] Leaf tissue of salt-treated Medicago sativa is used for RNA extraction;

[0079] 2. Extraction of RNA

[0080] Total RNA was extracted by TransZol Up Plant Total RNA Extraction Kit, and the integrity of RNA was identified by gel electrophoresis, and the purity and concentration of RNA were determined by spectrophotometer (Thermo Scientific Nanodrop 1000);

[0081] 3. Full-length cloning of the gene

[0082] According to the nucleic acid sequence and protein function annotation results of Medicago truncatula 'A17'_Chr7g0233801, the full-length of Medicago sativa MsPME2 gene was obtained.

[0083] According to the nucleic acid sequence results of 'A17'_Chr7g0233801 and the ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) prediction of NCBI, the ORF reading frame of Medicago sativa MsPME2 gene was found. According to the obtained sequence, specific primers SEQ ID NO. 3 ORF-F (5'-ATGGCTACCCAAGAAA-3') and SEQ ID NO. 4 ORF-R (5'-CTAAAGGCCTTCGATGAA-3') were designed from the start codon and stop codon respectively, and the full-length coding sequence of 1662bp of Medicago sativa MsPME2 protein (SEQ ID NO. 1) was amplified by PCR with Medicago sativa cDNA as the template.

[0084] Example 2 Sequence information and homology analysis of Medicago sativa MsPME2 gene

[0085] The full-length open reading frame sequence of Medicago sativa MsPME2 of the application is 1662bp, and the detailed sequence is shown in SEQ ID NO. 1. According to the open reading frame sequence, the amino acid sequence of Medicago sativa MsPME2 protein is deduced, which has 553 amino acid residues, a molecular weight of 60.44kDa, and an isoelectric point (pI) of 8.73. The detailed sequence is shown in SEQ ID NO. 2.

[0086] The sequencing results were compared with the database (GenBank) on the NCBI website by BLAST (http: / / blast.ncbi.nlm.nih.gov / ), and it was found that the nucleic acid sequence and the encoded protein had high homology with the known PME genes of Medicago truncatula and Trifolium pratense L., and it was preliminarily considered to be a PME gene.

[0087] Example 3 Subcellular localization analysis of Medicago sativa MsPME2 in tobacco leaves

[0088] The identified Agrobacterium rhizogenes GV3101 strain containing pHB-MsPME2-YFP plasmid was inoculated in 5 mL LB (containing Kan 50 mg / L) and cultured at 28 degrees Celsius with shaking until OD 600 was about 0.5; 1 mL of the bacterial solution was added to 50 mL of LB liquid medium and cultured at 28 degrees Celsius with shaking until OD 600 was about 0.5; 5 mL of the bacterial solution was centrifuged at 4000 rpm / min for 10 min; the bacterial body was suspended in MS liquid medium to OD 600 was about 0.6, and AS (final concentration 0.2 mM) and MES (final concentration 10 mM) were added, and the mixture was placed at room temperature for 3 hrs; the P19 and MS suspended bacterial solution were mixed at a volume ratio of 1:1; the tobacco leaves were injected, and placed in the dark for 48 hrs; the Agrobacterium-infected tobacco leaves were placed under a laser confocal microscope for microscopic observation. The results showed that the Medicago sativa MsPME2 protein was located on the cell wall, as shown in Figure 2 .

[0089] Example 4 Expression difference of Medicago sativa MsPME2 gene under salt stress

[0090] 1. Material acquisition: Medicago sativa was treated with NaCl, and samples were taken at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h. The samples were wrapped with tin-platinum paper and placed in liquid nitrogen, and then transferred to a -80 degrees Celsius ultra-low temperature freezer for storage;

[0091] 2. RNA extraction, RNA integrity, purity, and concentration determination, and cDNA acquisition refer to Example 1;

[0092] 3. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in each tissue; according to the obtained Medicago sativa MsPME2 gene sequence, specific primers for quantitative analysis of MsPME2 gene in Real-time PCR were designed, primer SEQ ID NO. 7 qPME2-F (5'-CCACACACATTCAAAAAGCC-3'), primer SEQ ID NO. 8 qPME2-R (5'-TGCATCTTGCTGTGAGTCAG-3'), the reference gene was elongation factor EF-α gene, and the primers were SEQ ID NO. 9 EF-F (5'-GCACCAGTGCTCGATTGC-3') and SEQ ID NO. 10 EF-R (5'-TCGCCTGTCAATCTTGGTAACAA-3');

[0093] 4. Standard curves for the target gene and internal reference gene: The standard cDNA solution was serially diluted with ddH2O. Then, using the diluted cDNA solution as templates, Real-time PCR amplification was performed with specific primers for the target gene and internal reference gene, respectively. After the reaction, melting curves and standard curves were plotted. The melting curves were analyzed to determine whether a single peak was obtained for the target gene and internal reference gene, in order to determine whether a single PCR amplification product could be obtained using the primers. The appropriate dilution factor of the template cDNA was determined by the standard curves.

[0094] 5. Real-time quantitative analysis of the target gene in the sample: Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were amplified with specific primers for quantitative analysis. The real-time PCR reaction was performed on a BIO-RAD CFX real-time quantitative analyzer. The reaction system was 20 μL. The reaction adopted a three-step method: denaturation at 94℃ for 20 s, followed by 40 cycles: 94℃ for 15 s; 55℃ for 15 s; 72℃ for 25 s. After each amplification, a melting curve was plotted to verify the specificity of the amplified products.

[0095] 6. Use 2 -△△Ct Relative quantitative analysis showed that under salt stress, the expression level of MsPME2 in roots and leaves of alfalfa increased. Figure 3 ).

[0096] Example 5 alfalfa MsPME2 gene transformation alfalfa

[0097] 1. Constructing plant expression vectors

[0098] Design specific primers for the start and stop codons, respectively. (SEQ ID NO. 5)

[0099] PME1-F (5'-GCTTGGATCCTCGAGCTGCAGATGGCTACCCAAGAAA-3') and SEQ ID NO.6 PME1-R (5'-GCCCTTGCTCACCATACTAGTAAGGCCTTCGATGAAGGC-3') were modified by introducing Bam HI and Spe I restriction sites on both sides of the full-length gene sequence. PCR amplification was performed using alfalfa cDNA as a template. The PCR products were recovered and ligated into the pMD18-T Simple vector. Single colonies were picked for PCR verification. Plasmids were extracted from positive clones. The target fragment plasmid and the PHB binary transformation vector were double-digested with Bam HI and Spe I. The digested pHB-Flag vector was recovered and...

[0100] MsPME2 fragment, ligate with T4 ligase at 16℃ overnight, and transform the vector into Agrobacterium GV3101. Select a non-conserved region of about 300 bp in the full-length sequence of the PME gene, and connect the non-conserved region fragment to the pTOPO intermediate vector by homologous recombination (enzyme digestion sites are BamHI and EcoRI), and then use LR enzyme to connect the target fragment of the intermediate vector to the Phellsgate12 interference vector.

[0101] 2. Transform Medicago sativa

[0102] (1) Pre-shake Agrobacterium: pick positive single clones into 5 ml of YEP liquid medium containing 50 mg / L Kan, 50 mg / L gentamicin, and 25 mg / L Rif, and shake at 28℃, 200 rpm for 24 h;

[0103] (2) Expand Agrobacterium: dilute the pre-shaken Agrobacterium liquid 1:100 into the same resistant YEP medium, and culture at 28℃, 200 rpm for 13-16 h, until the absorbance OD 600 reaches about 0.6-1.5, collect the bacteria at 18℃, 3500 rpm, and 15 min;

[0104] (3) Transformation of alfalfa: Overexpression and interference vectors linking the target gene sequence were transformed into Agrobacterium GV3101. Fluid PCR and sequencing were performed to obtain positive single clones. Single clones were picked and placed in centrifuge tubes containing 10 mL of LB solution with antibiotics. The cells were incubated at 28°C and 200 rpm for 24 hours (small shake). 1 mL of the bacterial suspension was injected into a centrifuge tube containing 50 mL of LB solution with antibiotics. The cells were incubated at 28°C and 200 rpm for 12 hours (large shake). Simultaneously, SM4 liquid and solid culture media were prepared. After large shake, the Agrobacterium was collected and resuspended in SM4 liquid culture medium, adjusting the OD600 to 0.2-0.4. The infection resuspension was obtained. Treatment and transformation of sterile seedling leaves: Young leaves were selected from sterile alfalfa seedlings. Wounds were made on the leaf surface using sterile forceps, and the leaves were then immersed in the resuspension. Vacuum treatment was performed for 10 minutes, followed by ultrasonic disruption for 2 minutes, and then vacuum treatment for another 10 minutes. The treated leaves were removed, evenly spread on sterile filter paper, and air-dried before being transferred to SM4 solid medium for culture. Screening and callus induction: After 5 days of culture in SM4 solid medium, the leaves were transferred to SM4 selection medium supplemented with the appropriate antibiotic for selection culture. After one month of culture, the selection medium was replaced with fresh medium, and selection continued until callus formation. The formed callus was transferred to MSBK selection medium containing the appropriate antibiotic for further culture. The MSBK selection medium was changed every two weeks until leaf differentiation occurred. The brown necrotic tissue was then removed and transferred to MSR medium for vegetative growth. Once the seedlings had grown taller, they were transferred to glass bottles containing MSR medium. After the plants matured, they were transplanted into nutrient soil.

[0105] 3. Screening of transgenic positive lines

[0106] Add 5 mg / L hygromycin or kanamycin to SM4 selection medium to screen alfalfa positive plants. Design specific primers, standard curves for the target gene and internal reference gene, and real-time fluorescence quantitative analysis of the target gene in the test samples as described in Example 3; use 2 -△△Ct Relative quantitative analysis (method) Figure 4 The results showed that the expression level of MsPME2-overexpressing transgenic alfalfa was increased, while the expression level of MsPME2-interfering transgenic alfalfa was decreased, indicating that the MsPME2 gene had been successfully transferred into alfalfa. Figure 4 ).

[0107] Example 6 Salt stress analysis of MsPME2 transgenic plants

[0108] Wild-type and genetically modified alfalfa cuttings were propagated for 30 days, then watered with a 1 / 2 Hoagland nutrient solution containing 200 mM NaCl for 15 days. (Photos were taken.) Figure 5Plant height and root length were measured with a ruler, and the data were collected and weighed. Simultaneously, malondialdehyde content and relative electrolyte permeability were determined. Figure 6 The results showed that under salt stress, the plant height, root length, aboveground and belowground biomass of MsPME2-OE, MsPME2-RNAi, and wild-type plants were significantly reduced, while malondialdehyde content and relative electrolyte permeability increased after salt treatment, indicating that all plants suffered from salt stress, but the degree of damage differed significantly. Under salt treatment, the plant height of MsPME2-OE plants was significantly higher than that of the wild-type, while the plant height of MsPME2-RNAi plants was significantly lower than that of the wild-type. Figure 6 A). Under normal growth conditions, the root length of MsPME2-OE plants was significantly longer than that of the wild type, while the root length of MsPME2-RNAi plants was not significantly different from that of the wild type. Under salt treatment, the root length of MsPME2-OE plants was not significantly different from that of the wild type, while the root length of MsPME2-RNAi plants was significantly shorter than that of the wild type. Figure 6 B). Under normal growth conditions, the root weight of MsPME2-OE plants was not significantly different from that of the wild type, while the root weight of the wild type was significantly higher than that of MsPME2-RNAi plants; under salt treatment, the root weight of MsPME2-OE plants was significantly higher than that of the wild type, while the root weight of MsPME2-RNAi plants was not significantly different from that of the wild type. Figure 6 C). Under normal growth conditions, the aboveground biomass of MsPME2-OE plants was significantly higher than that of the wild type, and the aboveground biomass of the wild type was...

[0109] There was no significant difference between MsPME2-RNAi plants and wild-type plants; however, the aboveground biomass of MsPME2-OE plants was significantly higher than that of wild-type plants under salt treatment, while the aboveground biomass of wild-type plants was significantly higher than that of MsPME2-RNAi plants. Figure 6 D). The above results indicate that overexpression of MsPME2 can attenuate growth inhibition caused by salt stress, while silencing expression...

[0110] MsPME2 exacerbated the growth inhibition caused by salt stress. Under normal growth conditions, there were no significant differences in relative electrolyte leakage and malondialdehyde (MDA) content between MsPME2-OE, MsPME2-RNAi, and wild-type plants. However, under salt treatment, the relative electrolyte leakage and MDA content of MsPME2-OE plants were significantly lower than those of the wild-type, while the MDA content of the wild-type was significantly lower.

[0111] MsPME2-RNAi showed no significant difference in relative electrolyte permeability compared to MsPME2-RNAi plants. Figure 6 E, F). The above results indicate that MsPME2 can positively regulate the salt tolerance of alfalfa.

[0112] Example 7 Analysis of Na+, K+ content and Na+ / K+ ratio in roots, stems and leaves of MsPME2 transgenic alfalfa under salt stress

[0113] After 30 days of cutting, wild type and transgenic alfalfa were irrigated with 1 / 2 Hoagland nutrient solution containing 200 mM NaCl for 15 days, dried at 60 °C to constant weight, weighed, and placed in digestion tubes, 1 mL of HNO3 and 1 mL of H2O2 were added for digestion, after digestion at 120 °C for 2 h, the digested liquid was diluted to 10 mL. 2 mL of supernatant was taken and filtered with a 0.45 μm filter head, and the element content was determined by ICP-AES. The results showed that the root Na + content of MsPME2-OE, MsPME2-RNAi and wild type had no significant difference under normal growth environment, but the root Na + content of MsPME2-OE was significantly lower than that of wild type under salt stress, and the root Na + content of MsPME2-RNAi was significantly higher than that of wild type Figure 7 A). Whether under salt stress or not, the root K + content of MsPME2-OE was higher than that of wild type, and the K + content of MsPME2-RNAi was significantly higher than that of wild type and MsPME2-OE under control treatment, and was significantly lower than that of wild type and MsPME2-OE under salt stress Figure 7 B). Under salt stress, the root Na + / K + of MsPME2-OE was significantly lower than that of wild type, and the root Na + / K + of MsPME2-RNAi was significantly higher than that of wild type Figure 7 C), indicating that overexpression of MsPME2 under salt stress can reduce Na + and increase K + absorption and accumulation. The root Na + content of MsPME2-OE, MsPME2-RNAi and wild type had no significant difference under normal growth environment, but under salt stress

[0114] the root Na + content of MsPME2-OE was significantly lower than that of wild type, and the root Na + content of MsPME2-RNAi was significantly higher than that of wild type Figure 7 A). Whether under salt stress or not, the root K + content of MsPME2-OE was higher than that of wild type, and the K+ The content was significantly higher in the control treatment than in the wild-type and MsPME2-OE plants, and significantly lower in the salt stress treatment than in the wild-type and MsPME2-OE plants. Figure 7 B). Under salt stress, the Na content in the roots of MsPME2-OE plants... + / K + Significantly lower than wild type, while MsPME2-RNAi's Na... + / K + Significantly higher than wild type ( Figure 7 C) This indicates that overexpression of MsPME2 under salt treatment can reduce Na2+. + Increase K + The absorption and accumulation of.

[0115] Under normal growth conditions, MsPME2-OE, MsPME2-RNAi, and wild-type stem and leaf Na + There was no significant difference in content, but under salt treatment, the Na content in the stems and leaves of MsPME2-OE plants was significantly higher. + The content was significantly lower than that of the wild type, with decreases of 15.9% and 87.6% respectively. Meanwhile, the Na content in the stems and leaves of MsPME2-RNAi plants was significantly lower. + The content was significantly higher than that of the wild type, with increases of 41.8% and 96.1% respectively. Figure 8 AB). Figure 4 Compared to the wild type, the stems and leaves of MsPME2-OE plants treated with salt showed lower potassium levels. + The content increased significantly, while the K content in the leaves of MsPME2-RNAi plants was significantly increased. + The content decreased significantly, while the difference was not significant in the stem. Figure 8 C,D). Na in the stems and leaves of MsPME2-OE plants + / K + Compared with the wild type, the Na+ levels in the stems and leaves of MsPME2-RNAi plants were significantly lower under salt treatment, with reductions of 32.9% and 89.2% respectively. + / K + All were higher than the wild type. These results indicate overexpression.

[0116] MsPME2 reduced Na+ in alfalfa stems and leaves under salt treatment. + Content and Na + / K + This promoted K + Absorption.

[0117] The preferred embodiments of the present application have been described above in detail. It should be understood that modifications and variations to the preferred embodiments could be made by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that there be included within the scope of the application, all such modifications and variations as would be apparent to those skilled in the art upon reading this disclosure. It is intended to obtain for the inventors such patent rights as are available for any patent granted on the present application.

Claims

1. A salt-tolerant protein from alfalfa, characterized in that, The protein is alfalfa pectin methyl esterase MsPME2, or a protein with the same enzymatic function as MsPME2 but with a variant amino acid sequence of MsPME2, i.e., a variant MsPME2 protein. The amino acid sequence of MsPME2 is shown in SEQ ID NO.

2.

2. The protein as described in claim 1, characterized in that, The MsPME2 variant protein is a protein whose amino acid sequence shown in SEQ ID NO.2 has been substituted, deleted, or added with one or more amino acids and has the characteristics of the MsPME2 protein of alfalfa.

3. The protein as described in claim 2, characterized in that, The sequence of the MsPME2 variant protein is the amino acid sequence shown in SEQ ID NO.2, obtained by deleting, inserting and / or substituting 1 to 50 amino acids, or by adding 1 to 20 amino acids at the C-terminus and / or N-terminus.

4. The protein as described in claim 3, characterized in that, The sequence of the MsPME2 variant protein is formed by replacing 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO.2 with amino acids of similar or related properties.

5. A gene encoding the salt-tolerant protein of alfalfa as described in any one of claims 1-4, characterized in that, The gene sequence is as shown in SEQ ID NO.1, or the gene sequence has at least 90% homology with the nucleic acid shown in SEQ ID NO.1; or the gene has a sequence that can hybridize with the nucleic acid shown in SEQ ID NO.

1.

6. The gene as described in claim 5, characterized in that, The specific sequence of the gene is the deletion, insertion and / or substitution of 1 to 150 nucleotides in the nucleic acid sequence shown in SEQ ID NO.1, or a sequence formed by adding up to 60 nucleotides at the 5'- and / or 3'- ends.

7. A method for preparing the MsPME2 gene, a salt-tolerant protein from alfalfa, characterized in that, Includes the following steps: Step 1: Take leaf tissue from salt-treated alfalfa for RNA extraction; Step 2: Extract total RNA from the leaf tissue in Step 1, identify the integrity of RNA by gel electrophoresis, determine the purity and concentration of RNA by spectrophotometer, and reverse transcribe the RNA to synthesize cDNA. Step 3: Full-length cloning of the gene: Based on the full-length gene sequence of the MsPME2 protein from alfalfa, specific primers were designed at the start and stop codons, respectively. SEQ ID NO.3ORF-F: 5'-ATGGCTACCCAAGAAA-3' SEQ ID NO.4ORF-R: 5'-CTAAAGGCCTTCGATGAA-3' Using alfalfa cDNA as a template, PCR was performed to amplify the full-length coding sequence of the MsPME2 protein from alfalfa.

8. The application of the alfalfa salt-tolerance protein gene as described in any one of claims 5-6, characterized in that, Includes the following steps: Step 1: Construct a plant expression vector containing the gene; Step 2: Transfer the expression vector into alfalfa; Step 3: Screen for transgenic positive lines.

9. The application as described in claim 8, characterized in that, Step 1 specifically includes: Design specific primers at the start codon and stop codon respectively. SEQ ID NO.5PME1-F: 5'-GCTTGGATCCTCGAGCTTGCAGATGGCTACCCAAGAAA-3'; SEQ ID NO.6PME1-R: The gene was divided into 5'-GCCCTTGCTCACCATACTAGTAAGGCCTTCGATGAAGGC-3', with Bam HI and Spe I restriction sites introduced on both sides of the full-length gene sequence. PCR amplification was performed using alfalfa cDNA as a template. The PCR product, i.e. the target gene fragment, was recovered and ligated into the pMD18-T Simple vector. Single colony plaques were picked for PCR verification. The plasmid of the positive clone was extracted, which is the target gene fragment plasmid. The target gene fragment plasmid and the PHB binary transformation vector were digested with Bam HI and Spe I. The digested pHB-Flag vector and the target gene fragment were recovered and ligated with T4 ligase. The vector was then transformed into Agrobacterium GV3101.

10. The application as described in claim 9, characterized in that, Step 2 specifically includes: Step 2.1, Pre-shaking Agrobacterium: Pick the final positive single clones from Step 1 into YEP liquid medium containing 50 mg / L Kan, 50 mg / L Gentamicin, and 25 mg / L Rif, and shake at 28°C and 200 rpm for 24 h. Step 2.2, Agrobacterium expansion: Expand the pre-shaken Agrobacterium culture at a 1:100 ratio into YEP medium containing the same resistance, incubate at 28℃ and 200 rpm for 13-16 hours, until the absorbance OD reaches a certain level. 600 To reach a concentration of 0.6-1.5, collect bacteria at 18℃, 3500rpm, for 15 minutes. Step 2.3, Transformation of Alfalfa: Overexpression of the target gene sequence was transformed into Agrobacterium GV3101. Fluid PCR and sequencing were performed to obtain positive single clones. Single clones were picked and placed in a centrifuge tube containing 10 mL of LB broth containing antibiotics. The culture was incubated at 28°C and 200 rpm for 24 hours. 1 mL of the bacterial culture was injected into a centrifuge tube containing 50 mL of LB broth containing antibiotics. The culture was incubated at 28°C and 200 rpm for 12 hours. Agrobacterium was then collected and resuspended in SM4 liquid medium, adjusting the OD600 to 0.2-0.4 to obtain the infection resuspension. Aseptic seedling leaf treatment and transformation: Select tender leaves from alfalfa aseptic seedlings, make wounds on the leaf surface with sterile forceps, then immerse them in resuspension and vacuum treat for 10 minutes, then sonicate for 2 minutes, vacuum treat again for 10 minutes, take out the treated leaves, spread them evenly on sterile filter paper to dry, and then transfer them to SM4 solid medium for culture. Screening and callus induction: After culturing leaves in SM4 solid medium for 5 days, they were transferred to SM4 selection medium supplemented with the corresponding antibiotics for screening culture. After 1 month of culture, the selection medium was replaced with fresh selection medium, and screening continued until callus formation. The formed callus was transferred to MSBK selection medium containing the corresponding antibiotics for further culture. The MSBK selection medium was replaced every 2 weeks until leaf differentiation occurred. The brown necrotic tissue was removed and transferred to MSR medium for vegetative growth. After the seedlings grew taller, they were transferred to glass bottles containing MSR medium. After the plants matured, they were transplanted into nutrient soil.

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