Alfalfa salt-tolerant gene MsgltX as well as encoding protein and application thereof

Abstract

The invention discloses an alfalfa salt-tolerant gene MsgltX as well as an encoding protein and application thereof. According to the invention, a gene MsgltX responding to salt stress is screened from alfalfa, the nucleotide sequence of the MsgltX is as shown in SEQ ID No.1, and the amino acid sequence of the encoded protein of the MsgltX is as shown in SEQ ID No.2. The MsgltX overexpressed alfalfa hairy roots are obtained by constructing a vector and a strain of the overexpressed MsgltX, infecting alfalfa leaves and performing induced differentiation and screening, so that the effect of the MsgltX on the salt tolerance of the alfalfa is further verified, and compared with a wild type, the MsgltX overexpressed alfalfa hairy roots grow longer under salt stress, the tolerance to the salt stress is higher than that of a wild plant, and the MsgltX overexpressed alfalfa hairy roots are better than those of the wild plant. The overexpression of the MsgltX gene can enhance the salt tolerance of medicago sativa, and provides theoretical and practical support for cultivating and vigorously popularizing salt-tolerant medicago sativa varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to alfalfa salt tolerance genes. MsgltX Its encoded proteins and applications. Background Technology

[0002] alfalfa ( Medicago sativa Alfalfa (L.) is known as the "King of Forage," with a crude protein content of 18-22% and a suitable calcium-to-phosphorus ratio, significantly improving livestock and poultry production performance. However, it is subject to various abiotic stresses during its growth, such as salt stress, cold stress, and drought stress, all of which severely affect its growth and yield. Therefore, developing salt-tolerant alfalfa germplasm is of great significance.

[0003] "Identification and molecular evolution of the GLX The article "Genes in 21 Plantspecies: A Focus on the Gossypium hirsutum" points out that these genes exist in all species, from lower to higher plants. GLX The presence of these genes suggests that this gene family may have significant biological importance for plants. Among 21 plant species... GLX Cis-acting element analysis of the gene showed that its promoter region was rich in elements associated with plant hormones, biotic stress, and abiotic stress, suggesting... GLX Genes can participate in a variety of life processes in plants. qRT-PCR results show that some genes in cotton... GLX Members of the alfalfa family respond to high-temperature treatment and can participate in various life processes in plants through protein-protein interactions. However, no research has yet indicated that alfalfa... MsgltX The gene has the ability to tolerate salt. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide alfalfa. MsgltX Gene.

[0005] The second object of the present invention is to provide the above-mentioned MsgltX The protein encoded by the gene.

[0006] A third objective of this invention is to provide a product comprising the above-described... MsgltX Gene-based biological materials.

[0007] The fourth object of the present invention is to provide the above. MsgltX Application of genes, encoded proteins, or biological materials in regulating salt tolerance in alfalfa.

[0008] The fifth object of the present invention is to provide the above-mentioned MsgltX Application of genes, encoded proteins, or biological materials in the creation of salt-tolerant alfalfa.

[0009] The sixth object of the present invention is to provide a method for creating salt-tolerant alfalfa.

[0010] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0011] This invention provides alfalfa MsgltX Genes, the ones mentioned MsgltX The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0012] This invention selected 358 core alfalfa germplasm accessions and conducted phenotypic-genomic association analysis, screening out a gene that responds to salt stress. MsgltX The above-mentioned genome was cloned from alfalfa genome. MsgltX The full-length CDS sequence and amino acid sequence.

[0013] Therefore, the present invention also provides the above-mentioned MsgltX The gene encodes a protein, the amino acid sequence of which is shown in SEQ ID No. 2.

[0014] The present invention also provides a method comprising the above. MsgltX Gene-derived biological materials, said biological materials including expression cassettes, expression vectors, and expression strains.

[0015] Furthermore, the expression vector is an overexpression vector.

[0016] Preferably, the overexpression vector is the pEarleyGate 100 vector.

[0017] Furthermore, the expressed strain is Agrobacterium rhizogenes strain 1193.

[0018] This invention utilizes the above-mentioned biological material to infect alfalfa leaves, and after dark culture in SH3a co-culture medium for a period of time, the samples are transferred to selection medium for screening and induced to differentiate to obtain overexpressed alfalfa leaves. MsgltX alfalfa hairy roots. Quantitative real-time PCR was used to identify successful overexpression of [the substance] in alfalfa hairy roots. MsgltX Overexpression MsgltX 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... MsgltX The 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. MsgltXGenes can enhance the salt tolerance of alfalfa, providing theoretical and practical support for the breeding and widespread promotion of salt-tolerant alfalfa varieties.

[0019] Therefore, the present invention provides the above. MsgltX Application of genes, proteins encoded by the above, or biological materials in regulating salt tolerance in alfalfa.

[0020] Furthermore, the regulation is aimed at enhancing the salt tolerance of alfalfa.

[0021] The present invention also provides the above. MsgltX Application of genes or the proteins or biological materials described above in the creation of salt-tolerant alfalfa.

[0022] This invention provides a method for creating salt-tolerant alfalfa, wherein the method involves overexpressing [a specific gene] in the hairy roots or plants of alfalfa. MsgltX Gene.

[0023] Furthermore, the method involves using the aforementioned biological material to infect alfalfa leaves, screening, and inducing the production of hairy roots or plants.

[0024] Furthermore, the screening includes screening with screening medium and identification by quantitative real-time PCR.

[0025] Preferably, the PCR primer sequences used in the quantitative real-time PCR identification are shown in SEQ ID No. 5-6.

[0026] Specifically, the present invention provides a method for creating salt-tolerant alfalfa, comprising the following steps: S1. Will MsgltX The genome was inserted into the pEarleyGate 100 vector, transformed into Agrobacterium rhizogenes strain 1193, and screened for strains containing... MsgltX Recombinant gene expression strains; S2. containing MsgltX Recombinant gene-expressing strains infect disinfected alfalfa leaves; S3. Place the infected alfalfa leaves sequentially in SH3a co-culture medium and incubate in the dark at 28°C for 2 days. 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. S5. RNA was extracted from the obtained alfalfa hairy roots and identified by quantitative real-time PCR to obtain overexpressing RNA. MsgltX The hairy roots of alfalfa.

[0027] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a salt-tolerant gene for alfalfa.MsgltX Its encoded protein and applications. Based on phenotypic and genomic association analysis, this invention screened a gene in alfalfa that responds to salt stress. MsgltX The MsgltX The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2. By constructing an overexpression... MsgltX Vectors and strains were used to infect alfalfa leaves, followed by induction of differentiation and screening to obtain overexpressing strains. MsgltX The hairy roots of alfalfa were used for further verification. MsgltX The effect of overexpression on salt tolerance in alfalfa, compared to wild type, was [increased / improved]. MsgltX 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. MsgltX 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

[0028] Figure 1 Manhattan plot for genome-wide association analysis of salt-tolerant phenotypes.

[0029] Figure 2 For overexpression MsgltX hairy roots MsgltX Results of expression level assessment.

[0030] Figure 3 For wild type and overexpression MsgltX Phenotypic results of hairy roots after NaCl treatment.

[0031] Figure 4 For wild type and overexpression MsgltX Statistical analysis results of hairy root growth after NaCl treatment. Detailed Implementation

[0032] 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.

[0033] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0034] Example 1: Gene Screening Method I. Experimental Methods 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.

[0035] II. Experimental Results Manhattan plot of genome-wide association analysis of salt tolerance phenotypes Figure 1 As shown, this is the result of filtering. MsgltX As a candidate gene associated with salt tolerance in alfalfa.

[0036] Example 2 MsgltX Gene amplification and construction of overexpression vectors I. Experimental Methods 1. MsgltX Gene amplification (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.

[0037] (2) Design alfalfa MsgltX Gene overexpression primers + vector ligation primers: F (SEQ ID No. 3): tttggagaggacacgctcgagATGGATATCAAAACCCTTTCTTTGC (XhoI restriction site and recombinant homologous fragment on vector added to overexpression primers); R (SEQ ID No. 4): ggtcttaattaactctctagaTTACTTCAAAGATGTCTGCTGCCT (Based on the overexpression primer, XbaI restriction site and recombinant homologous fragment on the vector are added).

[0038] (3) Perform PCR amplification using cDNA as a template and recover the product.

[0039] 2. Construction of overexpression vectors (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... MsgltX Overexpression vector of full-length CDS.

[0040] (2) Select positive E. coli colonies, extract plasmids, and sequence them to verify that the sequence is correct.

[0041] II. Experimental Results alfalfa MsgltX The CDS sequence of the gene is shown in SEQ ID No. 1: The amino acid sequence is shown in SEQ ID No. 2. MDIKTLSLRYIGRTATLSNSYGQDAFQTSQIDEWLEYAPVLSSGPAFENGCKYIDDYLEKRTFLVGYSLSIADLTICAGLAGAGKRWESLRKSKKYQNLARWYNSIVAEHGTALNEYIEYIGNKGSGESSATEPKNQPVVKDKVKNVNGDFSDNNKGGGKPSAEIDLPDAE VGEVRLRFAPEPSGYLHIGHAKAALLNKYFAERYKGQVILRFDDTNPEKESNKFVDNLIKDVETLGVKYDKITYTSDYFPELMELAEKLISQGKAYVDDTPDEQMRKERRAGIESKCRNHSVEENLKLWKEMIAGSERGVQCCVRGKLDMQDPNKSLRDPVYYRCNPNPHH RIEIGSKYKVYPTYDFACPFVDAREGITHALRSSEYHDRNAQYHRIQEDMGVKKVLIYEFSRLNMVYTLLSKRKLLWFVEKSKVESWDDPRFPTLQGIVRRGLKIEALIQFIVEQGASKNLNLMEWDKLWTINKKIIDPVCPRHTAVIADRRVLLTLTNGPEKSFVRIIPK HKKYEAAGNKDTTYTKRIWIDYADAESVSAGEEVTLMDWGNAIVKEVDKDQDGNVTGLSGVLHLEGSVKTTKLKLTWLPELDELVSLTLTEFDYLITKKKLEKKENFVDWLNPCTKKKILAYGDSNMRYLKRGEVLQLERKGYFRCDVPFVQPSKPIVLFAIPDGRQQTSLK In summary, this invention is the first to discover a gene in alfalfa. MsgltX They cloned its full-length CDS sequence and successfully constructed a [type of array containing...] MsgltX The gene overexpression vector was subsequently transformed into alfalfa to further validate the overexpression. MsgltX Regulation of salt tolerance in alfalfa by genes.

[0042] Example 3 Overexpression MsgltX Acquisition of alfalfa hairy roots I. Experimental Methods 1. Infection (1) The positive plasmid obtained in Example 2 was transformed into Agrobacterium rhizogenes strain 1193 and screened on resistant medium (LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampin). Positive strains were picked, cultured and preserved.

[0043] (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. (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; (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. (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. (6) Prepare disinfectant solution: 10% sodium hypochlorite + 100 μL Tween; (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. (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; (9) Use scissors that have been sterilized at high temperature to cut the leaves in half; (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. (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; (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. (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.

[0044] 2. Screening Wild-type (WT) and overexpressing hairy roots were selected. MsgltX RNA was extracted and reverse transcribed into cDNA using alfalfa actin as an internal reference primer, and then detected by qPCR. MsgltX Gene expression levels.

[0045] Table 1 qPCR primers

[0046] II. Experimental Results The results are as follows Figure 2 As shown, compared with the wild type, overexpression of hairy roots MsgltX The gene expression level was significantly higher than that of the wild type, indicating that the overexpression transgenic hairy root was successfully constructed.

[0047] Example 4 Overexpression MsgltX Salt tolerance identification of hairy roots I. Experimental Methods Wild-type (WT) and overexpressed (WT) cells with consistent and stable growth after rooting were compared. MsgltX -OE) hairy roots were treated with salt.

[0048] The salt treatment method is as follows: 1. Wild-type and overexpression hairy roots were placed vertically on salt stress medium with a 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; 2. The treated salt-stressed material was placed in a dark environment and treated at room temperature; The root elongation length was measured after 3.7 days.

[0049] II. Experimental Results The results are as follows Figure 3 and Figure 4 As shown, phenotypic observation after salt treatment revealed that overexpression MsgltX The hairy roots were significantly longer than the wild type, indicating overexpression. MsgltX Hairy roots showed significantly greater tolerance to salt stress than wild-type plants, and overexpression MsgltX Genes can enhance the salt tolerance trait of alfalfa.

[0050] 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. A type of alfalfa MsgltX Genes, characterized by, The MsgltX The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The claim 1 MsgltX The gene encodes a protein characterized by, The amino acid sequence of the encoded protein is shown in SEQ ID No.

2.

3. A device comprising the features described in claim 1 MsgltX Gene-based biomaterials, characterized in that, The biomaterials include expression cassettes, expression vectors, and expression strains.

4. The claim 1 MsgltX The application of the gene or the protein encoded by claim 2 or the biological material of claim 3 in regulating the salt tolerance of alfalfa.

5. The application according to claim 4, characterized in that, The regulation is aimed at enhancing the salt tolerance of alfalfa.

6. The claim 1 MsgltX The application of the gene or the protein encoded by claim 2 or the biological material of claim 3 in the creation of salt-tolerant alfalfa.

7. A method for creating salt-tolerant alfalfa, characterized in that, The method involves overexpression in alfalfa hairy roots or plants. MsgltX Gene.

8. The method according to claim 7, characterized in that, The method involves infecting alfalfa leaves with the biological material described in claim 3, and then screening and inducing the production of hairy roots or plants.

9. The method according to claim 8, characterized in that, The screening includes screening with screening medium and identification by quantitative real-time PCR.

10. The method according to claim 9, characterized in that, The PCR primer sequences used in the quantitative real-time PCR identification are shown in SEQ ID No. 5-6.

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