Salt-tolerant gene mskup1 of alfalfa and application thereof

Abstract

This invention discloses a salt-tolerant gene in alfalfa. MsKUP1 Its applications. This invention, based on phenotypic and genomic association analysis, screened a gene in alfalfa that responds to salt stress. MsKUP1 The MsKUP1 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. This was achieved by constructing an overexpression... MsKUP1 Vectors and strains were used to infect alfalfa leaves, followed by induction of differentiation and screening to obtain overexpressing strains. MsKUP1 The hairy roots of alfalfa were used for further verification. MsKUP1 The effect of overexpression on salt tolerance in alfalfa, compared to wild type, was [increased / improved]. MsKUP1 The hairy roots of alfalfa under salt stress grow longer and are more tolerant of salt stress than wild plants, indicating that overexpression of [specific expression] is key. MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsKUP1 MsK Genes can enhance the salt tolerance of alfalfa, providing theoretical and practical support for the breeding and widespread promotion of salt-tolerant alfalfa varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a salt-tolerant gene for alfalfa. MsKUP1 And its applications. Background Technology

[0002] Soil salinity is a prominent factor contributing to land degradation worldwide and has become an increasingly serious global problem. It is a key abiotic stress that adversely affects plant growth and development, limits crop productivity, and exacerbates global food shortages.

[0003] alfalfa ( Medicago sativa Alfalfa (Alfalfa spp.), also known as purple alfalfa, is a perennial herb belonging to the genus Alfalfa in the legume family (Fabaceae). It is one of the earliest cultivated and most widely distributed legume forage crops in the world, and one of the most important forage crops globally. Due to its high yield and palatability to livestock, it is crucial to the dairy industry. Although alfalfa exhibits moderate tolerance to soil salinization compared to other crops, making it a dominant species in saline-alkali lands, high concentrations of saline-alkali soil can still inhibit its growth and lead to yield losses. Therefore, discovering new salt-tolerant genes in alfalfa can provide important genetic resources for breeding new salt-tolerant alfalfa varieties. Breeding new alfalfa varieties carrying salt-tolerant genes is also an effective way to restore the ecology of saline-alkali lands and promote agricultural production.

[0004] The research progress on the role of the HAK / KUP / KT family of potassium ion transporters in plant salt tolerance points out that potassium can participate in plant growth and development through various mechanisms, playing an important role in alleviating abiotic stresses such as salt. Although plant salt tolerance can be regulated by modulating potassium absorption and transport, the current technologies are not publicly available. MsKUP1 Its role in improving plant salt tolerance. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide alfalfa. MsKUP1 Gene.

[0006] The second object of the present invention is to provide the above-mentioned MsKUP1 Nucleotides of a gene.

[0007] A third objective of this invention is to provide an expression cassette, expression vector, and expression strain containing the aforementioned nucleotides.

[0008] The fourth object of the present invention is to provide the above. MsKUP1 Application of genes, expression cassettes, expression vectors, or expression strains in regulating salt tolerance in alfalfa.

[0009] The fifth object of the present invention is to provide the above-mentioned MsKUP1 Application of genes, expression cassettes, expression vectors, or expression strains in the creation of salt-tolerant alfalfa.

[0010] The sixth object of the present invention is to provide a method for creating salt-tolerant alfalfa.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides alfalfa MsKUP1 Genes, the ones mentioned MsKUP1 The amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0013] This invention selected 358 core alfalfa germplasm accessions and conducted phenotypic-genomic association analysis, screening out a gene that responds to salt stress. MsKUP1 And cloned from the alfalfa genome MsKUP1 The full-length CDS sequence and amino acid sequence.

[0014] Therefore, the present invention also provides an encoding of the above. MsKUP1 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0015] The present invention also provides an expression cassette comprising the above-described nucleotides.

[0016] The present invention also provides an expression vector comprising the above-described nucleotides or the above-described expression cassette.

[0017] Furthermore, the vector is an overexpression vector.

[0018] Preferably, the overexpression vector is the pEarleyGate 100 vector.

[0019] The present invention also provides an expression strain, wherein the expression strain comprises the above-described nucleotides or the above-described expression cassette or the above-described expression vector.

[0020] Preferably, the recombinant bacteria is Agrobacterium rhizogenes strain 1193.

[0021] This invention constructs overexpression MsKUP1 The vector and strain were further infected with alfalfa leaves, then induced to differentiate and screened to obtain overexpressing strains. MsKUP1 alfalfa hairy roots. Quantitative real-time PCR was used to identify successful overexpression of [the substance] in alfalfa hairy roots. MsKUP1 Overexpression MsKUP1 Both alfalfa hairy roots and wild-type hairy roots with the same growth status were placed vertically in a salt-stressed culture medium. Root elongation was measured after 7 days of culture. The results showed that overexpression... MsKUP1The alfalfa plant with the overexpression of this gene has longer hairy roots than the wild type and is more tolerant of salt stress than the wild plant, indicating that it has longer hairy roots. MsKUP1 Genes can enhance the salt tolerance of alfalfa, providing theoretical and practical support for the breeding and widespread promotion of salt-tolerant alfalfa varieties.

[0022] Therefore, the present invention provides the above. MsKUP1 Application of genes, expression cassettes, expression vectors, or expression strains in regulating salt tolerance in alfalfa.

[0023] Furthermore, the regulation aims to enhance the salt tolerance trait of alfalfa.

[0024] The present invention also provides the above. MsKUP1 Application of genes, expression cassettes, expression vectors, or expression strains in the creation of salt-tolerant alfalfa.

[0025] This invention also provides a method for creating salt-tolerant alfalfa, the method being to overexpress [a specific substance] in the hairy roots or plants of alfalfa. MsKUP1 Gene.

[0026] Furthermore, the method involves using the aforementioned expression strain to infect alfalfa leaves, screening for and inducing the production of hairy roots or plants.

[0027] Furthermore, the screening includes screening with screening medium and identification by quantitative real-time PCR.

[0028] Preferably, the primer sequences for the fluorescence quantitative PCR identification are shown in SEQ ID No. 5-6.

[0029] Specifically, the present invention provides a method for creating salt-tolerant alfalfa, comprising the following steps:

[0030] S1. Will MsKUP1 The genome was inserted into the pEarleyGate 100 vector and transformed into Agrobacterium rhizogenes strain 1193 to obtain a genome containing... MsKUP1 Recombinant strains of genes;

[0031] S2. containing MsKUP1 Recombinant strains of the gene infect disinfected alfalfa leaves;

[0032] S3. Place the infected alfalfa leaves sequentially in SH3a co-culture medium and incubate in the dark at 28°C for 2 days.

[0033] S4. After dark culture, alfalfa leaves were transferred to 1 / 2 MS selection medium and cultured at 22°C. Subculture was performed every 14 days to obtain alfalfa hairy roots.

[0034] S5. RNA was extracted from the obtained alfalfa hairy roots and identified by quantitative real-time PCR.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention provides a salt-tolerant gene for alfalfa. MsKUP1 Its applications. This invention, based on phenotypic and genomic association analysis, screened a gene in alfalfa that responds to salt stress. MsKUP1 The MsKUP1 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. 2. This was achieved by constructing an overexpression... MsKUP1 Vectors and strains were used to infect alfalfa leaves, followed by induction of differentiation and screening to obtain overexpressing strains. MsKUP1 The hairy roots of alfalfa were used for further verification. MsKUP1 The effect of overexpression on salt tolerance in alfalfa, compared to wild type, was [increased / improved]. MsKUP1 The hairy roots of alfalfa under salt stress grew longer and showed greater tolerance to salt stress than wild plants, indicating that overexpression of [specific gene expression] was key to this phenomenon. MsKUP1 Genes can enhance the salt tolerance of alfalfa, providing theoretical and practical support for the breeding and widespread promotion of salt-tolerant alfalfa varieties. Attached Figure Description

[0037] Figure 1 Manhattan plot for genome-wide association analysis of salt-tolerant phenotypes.

[0038] Figure 2 For overexpression MsKUP1 hairy roots MsKUP1 Results of expression level assessment.

[0039] Figure 3 For wild type and overexpression MsKUP1 Phenotypic results of hairy roots after NaCl treatment.

[0040] Figure 4 For wild type and overexpression MsKUP1 Statistical analysis results of hairy root growth after NaCl treatment. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0043] Example 1: Gene Screening Method

[0044] I. Experimental Methods

[0045] Thirty-eight core alfalfa germplasms were selected for phenotypic-genomic association analysis. First, salt stress tolerance phenotypic data were obtained through salt stress treatment: the ratio of average regeneration under stress to that under non-stress conditions was used as an indicator of salt tolerance. Then, genomic DNA was extracted from each sample, and whole-genome resequencing was performed using the Illumina platform to obtain high-quality sequencing data. The sequencing data were aligned to the alfalfa reference genome, SNP variants were identified and filtered, and a genotype matrix containing millions of high-quality SNPs was constructed. Combining phenotypic data and SNP genotypes, genome-wide association analysis (GWAS) was used to screen for SNP loci significantly associated with salt tolerance. Finally, functional annotation was performed on the significantly associated SNPs to locate their corresponding genes, thereby identifying candidate genes associated with salt tolerance.

[0046] II. Experimental Results

[0047] Manhattan plot of genome-wide association analysis of salt tolerance phenotypes Figure 1 As shown, this is the result of filtering. MsKUP1 As a candidate gene associated with salt tolerance in alfalfa.

[0048] Example 2 MsKUP1 Gene amplification and construction of overexpression vectors

[0049] I. Experimental Methods

[0050] 1. MsKUP1 Gene amplification

[0051] (1) Take healthy alfalfa plants, extract alfalfa RNA using the TIANGEN kit, and then reverse transcribe the RNA using the TIANGEN reverse transcription kit to obtain cDNA. Use the cDNA as a template and amplify it by PCR using overexpression primers.

[0052] (2) Design alfalfa MsKUP1 Gene overexpression primers + vector ligation primers:

[0053] F (SEQ ID No. 3): TTTGGAGAGGACACGCTCGAGATGAATCCTTCTCAGCAATTTG (with XhoI restriction site and recombinant homologous fragment on the vector added to the overexpression primer);

[0054] R (SEQ ID No. 4): GGTCTTAATTAACTCTCTAGATTAGACATGGTATACCATACCAAC (Based on the overexpression primer, XbaI restriction site and recombinant homologous fragment on the vector are added).

[0055] (3) Perform PCR amplification using cDNA as a template and recover the product.

[0056] 2. Construction of overexpression vectors

[0057] (1) Using the pEarleyGate 100 vector, digestion with XhoI and XbaI restriction endonucleases was performed, and the vector backbone was recovered and ligated with the PCR-recovered product. The ligation was then performed into Turbo Escherichia coli, and screening was conducted on an antibiotic-resistant medium (LB solid medium containing 50 mg / L kanamycin) to construct a vector containing... MsKUP1 Overexpression vector of full-length CDS.

[0058] (2) Select positive E. coli colonies, extract plasmids, and sequence them to verify that the sequence is correct.

[0059] II. Experimental Results

[0060] alfalfa MsKUP1 The CDS sequence of the gene is shown in SEQ ID No. 1:

[0061]

[0062] The amino acid sequence is shown in SEQ ID No.2:

[0063] MNPSQQFVEQGISHQNLKRTSCANVLTLAYQSLGVVYGDLSTSPLYVYKTSFSGKLSLKEDDEEIFGVLSFIFWTFTIIALFKYVFIVMSADDNGEGGTFALYSLLCRHARLSILPNQQPTDENLSAYSTEDSADTWQSSLLKLFFEKHPRFQKGLLIFVLLGTCMTIGDGVITPAISVFSAVSGVQVKINQLHDNYVVIVSCIILVGLFSIQHHGTHRVAFMFAPVVAAWLLCISGIGIYNIFRWNRQVYRALSPVYMFRFLKTTGIEGWLSLSGVVLSITGVETMYADMGHFSALSIKIAFTCLVYPCLILAYMGEAAFLSKHHYDIERSFYKAIPEAVFWPVFIVATFAAVVGSQAVISATFSIISQCCALNCFPRVKIVHTSSKIYGQIYVPEVNWILMCLCLSVTIGLRDTTMMGHAYGLAITTVMFVTTCLMTLVIIIVWKQGIIKALTCFLLFGSIELLYISASVCKIPEGGWIPISLSFIFMAIMFTWNYGTMKKHQFDVENKVSMSKMLSLGPCLGMVRVPGIGLIFSNLASGIPAIFGHFITNLPAFHQVLVFVCAKSVQVPYVSENERLVISRIGPKEFYMFRCIVRYGYKDMQQENYNFDNKLVSAIIQFIETKESVPEQTNELTIDDGRNINVEDLGASQHTLKLNWSHSENNCLPFSCHGQQLRDESYKVESLQILKAKESGVTYIVGHSYAEAKKSSSILKKFGIDVVYAFLSKNCREPDIMLEVAHTSLLEVGMVYHV

[0064] Example 3 Overexpression MsKUP1 Obtaining of Medicago sativa hairy roots with overexpressed gene

[0065] I. Experimental method

[0066] 1. Infection

[0067] (1) The positive plasmid obtained in Example 2 was transformed into Agrobacterium rhizogenes strain 1193, and screened on LB solid medium (containing 50 mg / L kanamycin and 50 mg / L rifampin). Positive strains were selected, cultured, and preserved.

[0068] (2) Add 300 μL of Agrobacterium strain to a 50 mL centrifuge tube, use 50 mL LB (containing 50 μL kanamycin and 25 μL rifampin), and incubate overnight at 28°C and 220 rpm.

[0069] (3) Measure the OD value of the bacterial solution to be 0.6-0.8, pour the bacterial solution into a 50 mL centrifuge tube, and centrifuge at 3500 rpm for 15 min;

[0070] (4) Discard the supernatant, add 50 mL of infection solution to resuspend the bacterial cells, then add 50 μL of acetylsalicylic acid to the infection solution and shake incubate for 2 h.

[0071] (5) Pick the leaves of the large potted alfalfa seedlings that are 2-3 years old, remove the petioles, and rinse the picked leaves with gauze under the tap.

[0072] (6) Prepare disinfectant solution: 10% sodium hypochlorite + 100 μL Tween;

[0073] (7) Soak the leaves in disinfectant for 5-8 minutes. During the soaking process, shake constantly to ensure even disinfection. Observe the condition of the leaves. Disinfection is complete when there is damage on the leaves.

[0074] (8) After disinfection, rinse with sterile water more than 5 times, pick out the leaves one by one with tweezers, place them on filter paper to absorb the moisture;

[0075] (9) Use scissors that have been sterilized at high temperature to cut the leaves in half;

[0076] (10) Take out the inoculum, adjust the OD value to 0.2-0.4 with the inoculum, add 50 μL of acetylsuccinone, place the leaves in the inoculum, and shake incubate for 30 min.

[0077] (11) Use filter paper to absorb the bacterial solution from the leaves, and place them in SH3a co-culture medium in sequence, and incubate in the dark at 28°C for 2 days;

[0078] (12) After two days, the leaves were placed on 1 / 2 MS screening medium after the bacterial solution was dried with filter paper. The leaves were sealed with breathable tape and cultured at 22°C. Subculture was performed every 14 days.

[0079] (13) Use 1.5 mL small centrifuge tubes. When sampling, take about 2-3 cm of root from each tube. After testing the roots, select the positive root series for subsequent experiments.

[0080] 2. Screening

[0081] Wild-type (WT) and overexpressing hairy roots were selected. MsKUP1 RNA was extracted and reverse transcribed into cDNA using alfalfa actin as an internal reference primer, and then detected by qPCR. MsKUP1 Gene expression levels.

[0082] Table 1 qPCR primers

[0083]

[0084] II. Experimental Results

[0085] The results are as follows Figure 2 As shown, compared with the wild type, overexpression of hairy roots MsKUP1 The gene expression level was significantly higher than that of the wild type, indicating that the overexpression of hairy roots was successfully constructed.

[0086] Example 4 Overexpression MsKUP1 Salt tolerance identification of hairy roots

[0087] I. Experimental Methods

[0088] Wild-type (WT) and overexpressed (WT) cells with consistent and stable growth after rooting were compared. MsKUP1 -OE) hairy roots were treated with salt.

[0089] The salt treatment method is as follows:

[0090] 1. Salt treatment

[0091] Wild-type and overexpressed hairy roots were placed vertically on salt stress medium with a medium gradient of 1 / 2 MS + 0 mM NaCl, 1 / 2 MS + 100 mM NaCl and 1 / 2 MS + 150 mM NaCl, with 3 dishes for each treatment.

[0092] 2. The treated salt-stressed material was placed in a dark environment and treated at room temperature;

[0093] The root elongation length was measured after 3.7 days.

[0094] II. Experimental Results

[0095] The results are as follows Figure 3 and Figure 4 As shown, phenotypic observation after salt treatment revealed that overexpression MsKUP1 The alfalfa plants with the overexpressed gene have significantly longer hairy roots than the wild type and exhibit significantly greater tolerance to salt stress than wild plants, indicating that the gene is overexpressed. MsKUP1 Genes can enhance the salt tolerance of alfalfa, providing theoretical and practical support for the breeding and widespread promotion of salt-tolerant alfalfa varieties.

[0096] In summary, this invention has screened out a salt tolerance in alfalfa. MsKUP1 Genes provide important genetic resources for breeding new salt-tolerant alfalfa varieties.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression of alfalfa MsKUP1 Genes and including alfalfa MsKUP1 The application of gene expression cassettes, expression vectors, or expression strains in regulating salt tolerance of alfalfa hairy roots, characterized by: The MsKUP1 The amino acid sequence encoded by the gene is shown in SEQ ID No. 2; the regulation is to enhance the salt tolerance trait of alfalfa.

2. Overexpression of alfalfa MsKUP1 Genes and including alfalfa MsKUP1 The application of gene expression cassettes, expression vectors, or expression strains in the cultivation of salt-tolerant alfalfa, characterized in that... The MsKUP1 The amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

3. A method for cultivating salt-tolerant alfalfa, characterized in that, The method involves overexpression in alfalfa. MsKUP1 Genes; the stated MsKUP1 The amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

4. The method according to claim 3, characterized in that, The method involves using alfalfa. MsKUP1 Gene-expressing strains were infected with alfalfa leaves, and the resulting hairy roots or plants were selected and induced.

5. The application according to any one of claims 1 to 2 or the method according to claim 3, characterized in that, The MsKUP1 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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