CN112322600A - Alfalfa salt-tolerant gene MsSnRK2.3 and encoding protein and application thereof - Google Patents
Alfalfa salt-tolerant gene MsSnRK2.3 and encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses an alfalfa salt-tolerant gene MsSnRK2.3 and a coding protein and application thereof, and mainly relates to the technical field of plant genetic engineering. The nucleotide sequence is shown in SEQ ID NO. 13. The nucleotide sequence of the protein coded by the gene MsSnRK2.3 is shown in SEQ ID NO. 15. The invention has the beneficial effects that: the MsSnRK2.3 gene overexpression vector can be used for genetic improvement of forest grass plants so as to improve the salt stress resistance of the forest grass plants.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to an alfalfa salt-tolerant gene MsSnRK2.3 and a coding protein and application thereof.
Background
Alfalfa (Medicago Sativa L.) is the earliest and most widely used pasture in the world as a leguminous pasture with excellent quality, and is called as the king of pasture and the queen of feed. The alfalfa has high yield, rich nutritive value, long service life and strong palatability, and plays an important role in soil improvement, water and soil conservation and ecological environment protection. However, the yield and quality of alfalfa is severely limited by high salt stress. With the development of biotechnology, plant improvement by means of plant genetic engineering has become an important approach for modern breeding. The function of MsZAT10 gene transferred into tobacco shows that MsZAT10 gene can improve the tolerance of tobacco to low temperature and salt stress (Sun Asian male, et al, 2019). SoSnRK2.1 gene in ABA, drought (PEG) + ABA, drought (PEG), NaCl, low temperature (4 ℃) and H2O2The gene shows the tendency of induced expression under exogenous stress, may participate in the regulation of drought, high salt, low temperature and other stress processes, and plays an important role in the stress resistance of sugarcaneApplied (Tanshen Qiliang et al, 2013).
Harsh environmental conditions, especially the salinity of the soil, greatly limit plant growth and crop yield. In order to be able to adapt to the changing environmental conditions outside, plants need to sense stress and respond through a series of defense-related metabolic mechanisms. Protein kinases and phosphorylation play crucial roles in signal transduction pathways in recognizing and transmitting stress signals to different parts of the cell. SnRK2 is a serine/threonine protein kinase with a protein size of about 40 kDa. All SnRK2 amino acid sequences can be divided into two regions: the nitrogen end is a highly conserved kinase region, and the similarity with SnRK1, SNF1 and AMPK reaches 42-46%; the carbon terminal, with an acidic amino acid arm, is polyglutamic acid or polyaspartic acid. Phosphatidic acid is an important signal lipid in various stress-induced signal transduction pathways. The research finds that SnRK2.4 and SnRK2.10 are probably gathered on cell membranes by combining with phosphatidic acid, so that the plants have the characteristic of salt resistance and promote the growth and the morphogenesis of roots under the salt stress. Further studies demonstrated that snrk2.4 and snrk2.10 bind phosphatidic acid through other anionic phosphates and further participate in the reconstitution of cell membranes under salt stress in response to salt stress at the root of arabidopsis thaliana. Members in the SnRK2 gene family respond to multiple stresses and participate in regulating and controlling the stress resistance of plants, however, the research on the SnRK2 gene related to the stress resistance in alfalfa has not been reported yet.
Disclosure of Invention
The invention aims to provide an alfalfa salt-tolerant gene MsSnRK2.3 and a coding protein and application thereof, and the MsSnRK2.3 gene overexpression vector can be used for genetic improvement of forest grass plants so as to improve the salt stress resistance of the forest grass plants.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the nucleotide sequence of the alfalfa salt-tolerant gene MsSnRK2.3 is shown in SEQ ID NO. 13.
Furthermore, the nucleotide sequence of the open reading frame is shown as SEQ ID NO. 14.
As another aspect of the invention, the alfalfa salt-tolerant gene MsSnRK2.3 encodes a protein, and the nucleotide sequence of the protein is shown as SEQ ID NO. 15.
As another aspect of the invention, the use of the alfalfa salt gene MsSnRK2.3 of claim 1 for improving the salt stress resistance of plants.
Further, when the gene MsSnRK2.3 is specifically applied, a plant expression vector containing the gene MsSnRK2.3 is introduced into plant cells or seeds by an agrobacterium tumefaciens bacterial liquid medium method, and a transgenic plant with salt stress resistance higher than that of a wild plant is obtained by overexpression.
Further, the plant is selected from alfalfa or tobacco.
As another aspect of the invention, a method for obtaining a transgenic plant by overexpressing the gene mssnrk2.3, characterized in that: introducing a plant expression vector containing the gene MsSnRK2.3 into plant cells or seeds to enable the gene to be over-expressed, thereby obtaining a transgenic plant with higher salt stress resistance than a wild plant.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a full-length sequence of alfalfa salt-tolerant gene MsSnRK2.3, wherein the nucleotide sequence of the gene is shown as SEQ ID No.13, the open reading frame sequence of the gene is shown as SEQ ID No.14, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 15. The MsSnRK2.3 gene structure characteristic analysis provided by the invention shows that: the total length of the MsSnRK2.3 gene is 1346bp, the open reading frame length is 1092bp, and the gene codes 363 amino acids.
The invention discloses a cloning vector containing the alfalfa salt-tolerant gene MsSnRK2.3, further constructs an overexpression vector pCAMBIA2300-MsSnRK2.3 containing the alfalfa salt-tolerant gene MsSnRK2.3, transforms agrobacterium LBA4404, transforms the alfalfa salt-tolerant gene MsSnRK2.3 into tobacco by an agrobacterium-mediated method, and screens to obtain a transgenic overexpression plant. Under the salt stress treatment, the germination rate and the growth vigor of the transgenic plant are superior to those of the control, and simultaneously, the transgenic plant shows stronger salt tolerance compared with the control, which indicates that the gene participates in the process of resisting the salt stress of the plant. The MsSnRK2.3 gene disclosed by the invention provides important resources for the cultivation of a new alfalfa strain, and lays a theoretical foundation for the production practice of a plant molecule breeding system.
Drawings
FIG. 1 is an electrophoresis diagram of gene MsSnRK2.3 obtained by RT-PCR method, wherein M is 2000bp Marker, and 1 is PCR product.
Figure 2 is the protein domain of mssnrk2.3.
Figure 3 is a protein homology tree of mssnrk2.3 with other species.
FIG. 4 shows the tissue expression pattern of the MsSnRK2.3 gene in alfalfa roots, stems, leaves, flowers, fruits and the like.
FIG. 5 shows the expression pattern of MsSnRK2.3 gene after 6h, 24h and 48h of salt stress.
FIG. 6 shows a tobacco rooted shoot regenerated by Agrobacterium tumefaciens mediated transformation of MsSnRK2.3.
FIG. 7 shows the PCR detection of NPT II gene of transgenic tobacco genomic DNA, wherein M is 2000bpMarker, WT is control, and 1, 2, 3 are tobacco lines of transgenic MsSnRK2.3 gene.
FIG. 8 shows the RT-PCR detection of transgenic tobacco, in which M is 2000bp Marker, WT is control, and 1, 2, and 3 are tobacco lines transformed with MsSnRK2.3 gene.
FIG. 9 shows the growth of transgenic tobacco seeds under salt stress (NaCl 200mM), wherein WT is the control and L1 is the MsSnRK2.3 transgenic tobacco line.
FIG. 10 shows the germination of transgenic tobacco seeds under salt stress (NaCl 300mM), wherein WT is the control and L1, L2 and L3 are the MsSnRK2.3 transgenic tobacco lines.
FIG. 11 shows the effect of salt stress at different concentrations on the relative conductivity of transgenic tobacco, wherein WT is a control and L1, L2 and L3 are MsSnRK2.3 transgenic tobacco lines.
FIG. 12 shows the effect of salt stress at different concentrations on MDA content in transgenic tobacco, wherein WT is a control and L1, L2 and L3 are tobacco lines transgenic for MsSnRK2.3 gene.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the present application.
Example 1: obtaining of alfalfa stress response gene MsSnRK2.3
(1) Extraction of RNA: the alfalfa RNA was extracted by the TRIzol reagent (Invitrogen) method.
(2) And (3) cDNA synthesis: first strand cDNA synthesis was performed using Reverse Transcriptase XL (AMV) (TaKaRa).
(3) Designing a primer: the text file of the known alfalfa MtSRK2I sequence from the Tribulus terrestris was searched and downloaded in the NCBI database, using this sequence, primers were designed using PrimerPremier 5.0, the primers were:
MsSnRK2.3F：5′-GCCGTTTTCTCTCCCATAAC-3′(SEQ ID NO.1)；
MsSnRK2.3R：5′-CCTCCAAGAAGCACGCACAC-3′(SEQ ID NO.2)。
(4) and (3) PCR reaction: polymerase Chain Reaction (PCR) reagents and conditions were:
the following reagents were mixed together:
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the following cycle is then entered: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, 30 cycles; finally, extension is carried out for 10min at 72 ℃.
(5) The PCR product was recovered as a band and ligated to the EcoRV site in the pMD18-T (TaKaRa) vector.
(6) The recombinant plasmid was transformed into E.coli DH 5. alpha. and positive clones were selected for sequencing (Shanghai Boshang).
(7) Finally, the full length of the MsSnRK2.3 gene is obtained through identification. The total length of the MsSnRK2.3 gene is 1346bp, the open reading frame length is 1092bp, and the gene codes 363 amino acids. In SMART analysis in EMBL, as shown in FIG. 1, the MsSnRK2.3 protein was found to possess the conserved domain of CBL. The MsSnRK2.3 protein amino acid sequence and the MtSRK2I protein amino acid sequence were analyzed by DNAMAN software, and as shown in FIG. 2, the amino acid homology was found to be 99% with MtSRK 2I.
Example 2 expression patterns of the MsSnRK2.3 Gene in different tissues of Medicago sativa
In order to explore the expression modes of the MsSnRK2.3 gene in different tissues of alfalfa, total RNA of roots, stems, leaves, flowers and fruits of alfalfa is extracted and subjected to reverse transcription to form cDNA, the cDNA is used for real-time fluorescent quantitative PCR analysis, and the expression conditions of the MsSnRK2.3 gene in different tissues of alfalfa are detected.
Designing a fluorescent quantitative expression primer according to the full-length cDNA sequence of the MsSnRK2.3 gene, wherein the target sequence is 94bp in length, and the primer is as follows:
YGSnRKF：5′-TCAACAACTCATATCTGGGGTCA-3′(SEQ ID NO.3)；
YGSnRKR：5′-CGGGCTTCCATCCAACA-3′(SEQ ID NO.4)。
the alfalfa ACTIN2 gene (JQ028730) is used as an internal reference gene, a fluorescent quantitative expression primer is designed, the length of a target sequence is 197bp, and the primer is as follows:
ACTINF：5′-CCCACTGGATGTCTGTAGGTT-3′(SEQ ID NO.5)；
ACTINR：5′-AGAATTAAGTAGCAGCGCAAA-3′(SEQ ID NO.6)。
as shown in FIG. 4, the results show that the expression level of the MsSnRK2.3 gene is lower in alfalfa root and leaves, higher in flowers and fruits, and lowest in stems, which is only 1/20 of the expression level of fruits, the expression level of the MsSnRK2.3 gene is 6.7 times of the expression level of roots in fruits, and the expression level of the MsSnRK2.3 gene is 3.5 times of the expression level of roots in flowers.
Example 3 expression Pattern of MsSnRK2.3 Gene under alfalfa salt stress
In order to explore the expression modes of the MsSnRK2.3 gene in alfalfa salt stress in different time periods, alfalfa seedlings are treated by 200mM NaCl for 6h, 24h and 48h, untreated seedlings are used as a control, total RNA of the treated seedlings is extracted respectively after the treatment is finished, and the total RNA is subjected to reverse transcription to form cDNA for real-time fluorescent quantitative PCR analysis. The primers were the same as in example 2.
As shown in FIG. 5, the results show that the expression level of the MsSnRK2.3 gene is increased by NaCl induction, the expression level of the MsSnRK2.3 gene reaches the highest value after 6h of salt stress and is 3.1 times of that of the control, and the expression level of the MsSnRK2.3 gene after 24h and 48h of salt stress is 2.5 times and 2.6 times of that of the control respectively.
Example 4 construction of MsSnRK2.3 Gene overexpression vector
(1) Designing a primer: the gene MsSnRK2.3 sequence is used for designing an over-expression primer, and the primer is as follows:
SnRKEF：5′-GGGGTACCGCCGTTTTCTCTCCCATAAC-3′(SEQ ID NO.7)；
SnRKER：5′-GCGTCGACCCTCCAAGAAGCACGCACAC-3′(SEQ ID NO.8)。
(2) and (3) PCR reaction: the PCR reaction reagents and conditions were the same as in example 1 (4).
(3) The PCR product was recovered as a band and ligated to the EcoRV site in the pMD18-T (TaKaRa) vector.
(4) The recombinant plasmid was transformed into E.coli DH 5. alpha. and positive clones were selected for confirmation by sequencing.
(5) Carrying out enzyme digestion reaction on the pCAMBIA2300 vector by KpnI and SalI, and recovering for later use; extracting a plasmid containing the MsSnRK2.3 gene, carrying out enzyme digestion by KpnI and SalI, recovering the plasmid, and then carrying out ligation reaction with the pCAMBIA2300 vector recovered by enzyme digestion.
(6) The ligation product was transformed into E.coli DH 5. alpha. and positive clones were selected for confirmation by sequencing.
(7) Extracting the connected pCAMBIA2300-MsSnRK2.3 plasmid, transforming Agrobacterium tumefaciens LBA4404, screening positive clones, and sequencing and confirming.
Example 5 Agrobacterium tumefaciens-mediated transformation of tobacco
Agrobacterium tumefaciens LBA4404 containing the pCAMBIA2300-MsSnRK2.3 plasmid was cultured with shaking at 28 ℃ to the logarithmic growth phase (OD 600. RTM.0.6) in YEP liquid medium containing 50mg/L of Rif and antibiotics. Cutting tobacco (NC89) sterile seedling leaf into 0.5cm pieces2The small blocks are immersed into agrobacterium tumefaciens bacterial liquid diluted by 3 times by using a liquid culture medium, and the immersion dyeing is carried out for 5-10 min. Taking out the leaves, blotting the bacteria solution with sterile paper, placing on a solid culture medium containing 3.0% sucrose and 0.7% agar, co-culturing at 26 deg.C in dark for 2d,then transferred to a selection medium containing 500mg/L of cefuroxime axetil. After three successive screenings, small shoots were visible at the tobacco leaf disc edge. Cut off with a scalpel, and transfer the cut-off to a culture medium with 200mg/L of cefamycin to induce the continuous growth of the seedlings. After about one week, the plantlets grow to 3-4 cm high, and are transferred to a rooting culture medium for strengthening the plantlets. And transplanting the plantlets into the flowerpot when the plantlets grow to be 5-6 cm high, as shown in figure 6.
Example 6 detection of exogenous genes in transgenic tobacco genomic DNA
(1) Tobacco leaf was taken, put into a 1.5mL centrifuge tube, frozen in liquid nitrogen and immediately ground, 500. mu.L of DNA extraction buffer (200mM pH7.5 Tris-Cl, 250mM NaCl, 25mM EDTA, 0.5% SDS) was added, and mixed by shaking.
(2) Placing in water bath at 60 deg.C for 2min, taking out, adding 500 μ L phenol, shaking, mixing, centrifuging at 12000rpm, and standing for 10 min.
(3) The supernatant was transferred to another new 1.5mL centrifuge tube, 500. mu.L phenol/chloroform (1:1) was added, mixed well with shaking at 12000rpm, and centrifuged for 10 min.
(4) The supernatant was transferred to another new 1.5mL centrifuge tube, 500. mu.L chloroform was added, shaken and mixed well, 12000rpm, and centrifuged for 10 min.
(5) The supernatant was transferred to another new 1.5mL centrifuge tube, 30. mu.L of 3M NaAc (pH5.4) and 350. mu.L of isopropanol were added, mixed by inversion, and centrifuged at 12000rpm for 10 min. The mixture is not too violent when inverted and mixed, and the fragmentation of the genomic DNA is prevented.
(6) Pouring out the supernatant, washing the precipitate with 70% ethanol at 12000rpm, centrifuging for 1min, drying the precipitate, and adding 50 μ L ddH2And (4) re-dissolving the O.
(7) The gene NPT II sequence is used for designing a primer, and the primer is as follows:
NPTF：5′-AGCTCTTCAGCAATATCACGGGTAGC-3′(SEQ ID NO.9)；
NPTR：5′-GTGGAGAGGCTATTCGGCTATGACTG-3′(SEQ ID NO.10)。
(8) and (3) PCR reaction: polymerase Chain Reaction (PCR) reagents and conditions were:
the following reagents were mixed together:
the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the following cycle is then entered: denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃.
(9) The results of electrophoresis are shown in FIG. 7, where the insertion of NPT II was detected in the transgenic lines, but not in the control, demonstrating the successful insertion of the gene MsSnRK2.3 into the tobacco genome.
Example 7 RT-PCR detection of transgenic tobacco MsSnRK2.3 Gene
(1) RNA extraction and cDNA Synthesis were as in example 1.
(2) Primers were designed using PrimerPremier 5.0 and were:
SnRKNF：5′-ATGGATCGAGCTGCGATGACGG-3′(SEQ ID NO.11)；
SnRKNR：5′-TTAGTACATAGCATACACTATC-3′(SEQ ID NO.12)。
(3) the PCR reaction reagents and conditions were the same as in example 1 (4).
(4) The electrophoresis results are shown in FIG. 8, and the gene MsSnRK2.3 was detected in the transgenic line, but not in the control, which demonstrated successful insertion and stable overexpression of the gene MsSnRK2.3 in tobacco.
Example 8 functional analysis of transgenic plants
And after the obtained positive transgenic tobacco blooms and fruits, collecting transgenic seeds for a single plant, and performing salt stress treatment analysis on the seeds and seedlings.
(1) Growth analysis of transgenic tobacco under salt stress
Transgenic tobacco and non-transgenic tobacco (WT) were sown on 1/2MS medium containing 200mM NaCl and the seed growth was observed after 30 days. As shown in FIG. 9, the results show that the transgenic tobacco plants can still grow normally under the salt stress of 200mM NaCl, the growth vigor of the transgenic tobacco plants is obviously better than that of the non-transgenic tobacco plants, and the biomass is also obviously greater than that of the non-transgenic plants, which indicates that the MsSnRK2.3 gene can improve the resistance of the plants to the salt stress.
(2) Germination of transgenic tobacco seeds under salt stress
Transgenic tobacco (lines L1, L2, L3) and non-transgenic tobacco (WT) were sown on 1/2MS medium containing 300mM NaCl and observed for seed germination. As shown in FIG. 10, the results show that under the salt stress of 300mM NaCl, the transgenic tobacco plants can germinate mostly although the growth of the transgenic tobacco plants is partially inhibited, and only a few seeds of the control plants can germinate, which indicates that the MsSnRK2.3 gene can enhance the capability of the plants to resist the salt stress.
(3) Effect of salt stress on relative conductivity of transgenic tobacco
One of the important factors for plants to maintain normal physiological functions is the selective permeability of the cytoplasmic membrane, the damage to plants being reflected by the magnitude of the relative conductivity values. The less the damage to the cell, the less the plant cell membrane is damaged and the lower the relative conductivity. As shown in FIG. 11, the relative conductivities of the control (WT) and the transgenic plants (lines L1, L2 and L3) were improved to different degrees under the influence of different concentrations of NaCl in tobacco, but the relative conductivity of the transgenic lines was always lower than that of the control under the stress of NaCl salt with the same concentration. This indicates that salt stress has already caused significant damage to the membrane structure of the control tobacco lamina, rendering the membrane system functionally hindered; compared with the control, the transgenic tobacco strain shows higher membrane stability and is less damaged. The MsSnRK2.3 gene is proved to improve the resistance of plants to salt stress.
(4) Effect of salt stress on transgenic tobacco MDA content
MDA is one of the major products of membrane lipid peroxidation, and if accumulated excessively in plants, it may have toxic effects on cells, so that its content is often used to reflect the degree of stress damage to plants. As shown in FIG. 12, the content of MDA in the leaf of the control tobacco (WT) increased rapidly with the increase of NaCl concentration, and the increase rate was significantly higher than that of the transgenic plants (lines L1, L2, L3), and the content of MDA in the transgenic lines was always lower than that of the control under the stress of NaCl salt with the same concentration. The MsSnRK2.3 gene can enhance the capability of plants to resist salt stress.
Sequence listing
<110> scientific research institute of forestry in Shandong province
<120> alfalfa salt-tolerant gene MsSnRK2.3, and coding protein and application thereof
<141> 2020-10-22
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tgatatggac gatgaatttg atgacttgga atatgaatcc gagcttgata tagatagcag 1140
tggggagata gtgtatgcta tgtactaatt tgatcattac cagtacttga aatacaacat 1200
gttaaaagga gagcattctt aatattgggc tatatagaat tttcagcctc aagagaaatg 1260
tgtggtgcat gcattttctg tagattatag tatgaacaac acaaagaagt acatgttaat 1320
ttttatgtgt gcgtgcttct tggagg 1346
<210> 14
<211> 1092
<212> DNA
<213> SnRK2.3 ORF
<400> 14
atggatcgag ctgcgatgac ggtcggaccg ggtatggatt taccgatcat gcacgacagt 60
gatcggtacg atctggttcg tgatatcggg tccggaaatt tcggtattgc taggttgatg 120
caagataaac aaaccaaaga gcttgttgct gttaaatata tcgaacgtgg tgataagatt 180
gatgaaaatg tgaagagaga aattattaat cacaggtctc tgagacatcc taacattgtt 240
aggttcaagg aggtcatttt aacacctact catcttgcta ttgtaatgga gtatgcatct 300
ggaggagaac ttttcgagcg aatcagtaat gctggccatt tttctgagga cgaggctcgt 360
ttcttctttc aacaactcat atctggggtc agctactgcc atgcaatgca agtatgtcac 420
cgggacctga aattggaaaa tactttgttg gatggaagcc cgactcctcg tctgaagata 480
tgtgattttg gttactccaa gtcttcagtg cttcattcac aaccgaagtc aactgtggga 540
actccggcat atattgctcc agaagtatta ctgagacaag agtatgatgg aaagattgca 600
gatgtttggt cttgtggggt aaccttatat gtgatgctag tgggatcgta tccctttgaa 660
gatcctaacg agccaaaaga tttccggaag acaatacaga gagtactaag tgtccagtat 720
tccattccag acaatgttca gataactcca gagtgtcgcc accttatctc aaggatcttt 780
gtttttgacc ctgcagagag aattaccatg cccgaaatct ggaaacataa atggtttctg 840
aagaatcttc ccatggacct gatggatgag aagataatgg gtaaccaatt tgaagagcct 900
gaacaaccca tgcagagcat tgatgcaatc atgcagataa tttcagaagc tactatacca 960
gcagctggaa cctgttcttt agaccagttt atggcagata acattgatat ggacgatgaa 1020
tttgatgact tggaatatga atccgagctt gatatagata gcagtgggga gatagtgtat 1080
gctatgtact aa 1092
<210> 15
<211> 363
<212> PRT
<213> Medicago sativa
<400> 15
Met Asp Arg Ala Ala Met Thr Val Gly Pro Gly Met Asp Leu Pro Ile
1 5 10 15
Met His Asp Ser Asp Arg Tyr Asp Leu Val Arg Asp Ile Gly Ser Gly
20 25 30
Asn Phe Gly Ile Ala Arg Leu Met Gln Asp Lys Gln Thr Lys Glu Leu
35 40 45
Val Ala Val Lys Tyr Ile Glu Arg Gly Asp Lys Ile Asp Glu Asn Val
50 55 60
Lys Arg Glu Ile Ile Asn His Arg Ser Leu Arg His Pro Asn Ile Val
65 70 75 80
Arg Phe Lys Glu Val Ile Leu Thr Pro Thr His Leu Ala Ile Val Met
85 90 95
Glu Tyr Ala Ser Gly Gly Glu Leu Phe Glu Arg Ile Ser Asn Ala Gly
100 105 110
His Phe Ser Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser
115 120 125
Gly Val Ser Tyr Cys His Ala Met Gln Val Cys His Arg Asp Leu Lys
130 135 140
Leu Glu Asn Thr Leu Leu Asp Gly Ser Pro Thr Pro Arg Leu Lys Ile
145 150 155 160
Cys Asp Phe Gly Tyr Ser Lys Ser Ser Val Leu His Ser Gln Pro Lys
165 170 175
Ser Thr Val Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Leu Arg
180 185 190
Gln Glu Tyr Asp Gly Lys Ile Ala Asp Val Trp Ser Cys Gly Val Thr
195 200 205
Leu Tyr Val Met Leu Val Gly Ser Tyr Pro Phe Glu Asp Pro Asn Glu
210 215 220
Pro Lys Asp Phe Arg Lys Thr Ile Gln Arg Val Leu Ser Val Gln Tyr
225 230 235 240
Ser Ile Pro Asp Asn Val Gln Ile Thr Pro Glu Cys Arg His Leu Ile
245 250 255
Ser Arg Ile Phe Val Phe Asp Pro Ala Glu Arg Ile Thr Met Pro Glu
260 265 270
Ile Trp Lys His Lys Trp Phe Leu Lys Asn Leu Pro Met Asp Leu Met
275 280 285
Asp Glu Lys Ile Met Gly Asn Gln Phe Glu Glu Pro Glu Gln Pro Met
290 295 300
Gln Ser Ile Asp Ala Ile Met Gln Ile Ile Ser Glu Ala Thr Ile Pro
305 310 315 320
Ala Ala Gly Thr Cys Ser Leu Asp Gln Phe Met Ala Asp Asn Ile Asp
325 330 335
Met Asp Asp Glu Phe Asp Asp Leu Glu Tyr Glu Ser Glu Leu Asp Ile
340 345 350
Asp Ser Ser Gly Glu Ile Val Tyr Ala Met Tyr
355 360
Claims (7)
1. The alfalfa salt-tolerant gene MsSnRK2.3 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 13.
2. The alfalfa salt-tolerant gene MsSnRK2.3 of claim 1, wherein the nucleotide sequence of the open reading frame is shown in SEQ ID No. 14.
3. The protein coded by the alfalfa salt-tolerant gene MsSnRK2.3 is characterized in that the nucleotide sequence of the protein is shown in SEQ ID NO. 15.
4. The application of the alfalfa salt gene MsSnRK2.3 of claim 1 to improving the salt stress resistance of plants.
5. The application of claim 1, wherein in the specific application, a plant expression vector containing the gene MsSnRK2.3 is introduced into plant cells or seeds by an Agrobacterium tumefaciens bacterial liquid medium method, and a transgenic plant with salt stress resistance higher than that of a wild plant is obtained by overexpression.
6. Use according to claim 1, wherein said plant is selected from alfalfa or tobacco.
7. A method for obtaining a transgenic plant by overexpressing the gene mssnrk2.3, characterized in that: introducing a plant expression vector containing the gene MsSnRK2.3 into plant cells or seeds to enable the gene to be over-expressed, thereby obtaining a transgenic plant with higher salt stress resistance than a wild plant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789313A (en) * | 2021-09-30 | 2021-12-14 | 南通大学 | Lagerstroemia indica salt-tolerant gene LiCIPK24 and application thereof |
| CN114891805A (en) * | 2022-07-01 | 2022-08-12 | 中国农业科学院北京畜牧兽医研究所 | MsHMG-Y gene and encoding protein and application thereof |
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| CN104140462A (en) * | 2013-05-07 | 2014-11-12 | 中国农业大学 | Plant salt tolerance related protein GhSnRK2-6, and coding gene and applications thereof |
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| CN104140462A (en) * | 2013-05-07 | 2014-11-12 | 中国农业大学 | Plant salt tolerance related protein GhSnRK2-6, and coding gene and applications thereof |
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| SUN C等: "Medicago sativa serine_threonine protein kinase 3 (SnRK2.3) mRNA", 《GENBANK》 * |
| 曾文婕等: "植物SnRKs基因家族的研究进展", 《分子植物育种》 * |
| 苗丽丽等: "植物多功能调控因子SnRK2研究进展", 《麦类作物学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789313A (en) * | 2021-09-30 | 2021-12-14 | 南通大学 | Lagerstroemia indica salt-tolerant gene LiCIPK24 and application thereof |
| CN113789313B (en) * | 2021-09-30 | 2022-05-13 | 南通大学 | Lagerstroemia indica salt-tolerant gene LiCIPK24 and application thereof |
| CN114891805A (en) * | 2022-07-01 | 2022-08-12 | 中国农业科学院北京畜牧兽医研究所 | MsHMG-Y gene and encoding protein and application thereof |
| CN114891805B (en) * | 2022-07-01 | 2023-08-01 | 中国农业科学院北京畜牧兽医研究所 | MsHMG-Y gene and encoding protein and application thereof |
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