Application of MsSPL4 gene in alfalfa to improve plant stress resistance

Abstract

The present invention discloses the use of a protein or a substance for regulating the expression of a gene encoding the protein or a substance for regulating the activity or content of the protein in regulating plant stress resistance. The protein is MsSPL4 protein, and the MsSPL4 protein is any one of the following proteins: A1) a protein whose amino acid sequence is that shown in SEQ ID No. 2; A2) a protein obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in A1) and having more than 80% identity with the protein shown in A1) and having the function of regulating plant stress resistance; A3) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to alfalfa MsSPL4 protein and related biological materials and their application in improving plant stress resistance. Background Art

[0002] Alfalfa (Medicagosativa L.) is a perennial excellent forage grass of the genus Medicago in the Leguminosae family, cross-pollinated, and autotetraploid. It is rich in nutrition and has good palatability, and is known as the "king of forage grasses". In recent years, due to global climate change and the continuous decline in irrigation water quality, the degree of drought and soil salinization in my country's main alfalfa producing areas (arid and semi-arid areas north of the Yellow River Basin) has continued to intensify, resulting in reduced alfalfa production and weakened nodulation and nitrogen fixation capabilities. Soil salinization has become one of the main factors restricting the development of my country's alfalfa industry. Therefore, cultivating new salt-tolerant alfalfa varieties and improving the salt tolerance of alfalfa are important strategies for increasing alfalfa yields. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to improve the stress resistance of plants.

[0004] In order to solve the above technical problems, the present invention first provides:

[0005] The use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, wherein the protein may be MsSPL4 protein, and the MsSPL4 protein may be any of the following proteins:

[0006] A1) a protein whose amino acid sequence is SEQ ID No. 2;

[0007] A2) a protein having more than 80% identity with the protein shown in A1) and having the function of regulating plant salt tolerance, obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in A1);

[0008] A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1) or A2).

[0009] Furthermore, the protein in the above application may be derived from alfalfa.

[0010] More specifically, the protein is derived from alfalfa.

[0011] Among them, SEQ ID No. 1 consists of 143 amino acid residues.

[0012] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0013] Herein, the protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0014] Herein, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can then be obtained.

[0015] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0016] In the above application, the substance that regulates the activity or content of the protein may be a substance that regulates the expression of the gene encoding the protein.

[0017] In the above application, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0018] Furthermore, the regulating plant stress resistance in the above application may be enhancing plant stress resistance.

[0019] Furthermore, the stress resistance is salt resistance.

[0020] Specifically, in the above application, the regulating gene expression may be to improve or increase the gene expression.

[0021] Furthermore, in the application, the plant is any one of the following:

[0022] P1) dicotyledonous plants;

[0023] P2) Leguminosae;

[0024] P3) Fabaceae;

[0025] P4) Medicago;

[0026] P5) Alfalfa.

[0027] Furthermore, in the above application, the substance that improves or increases the gene expression may be a biological material related to the protein, and the biological material may be any one of the following B1) to B7):

[0028] B1) a nucleic acid molecule encoding the above protein;

[0029] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0030] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0031] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0032] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0033] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0034] B7) A transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2).

[0035] Wherein, the nucleic acid molecule in B1) can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0036] Furthermore, the nucleic acid molecule described in B1) may be the gene shown in G1) or G2) below:

[0037] G1) the coding sequence of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No. 1;

[0038] G2) the nucleotide of the coding strand is a cDNA molecule or a DNA molecule of SEQ ID No. 1;

[0039] Among them, SEQ ID No. 2 consists of 432 nucleotides, and its open reading frame (ORF) is from position 1 to position 432 from the 5′ end, encoding a protein with an amino acid sequence as shown in SEQ ID No. 2.

[0040] In the above-mentioned related biological materials, the expression cassette described in B2) refers to a DNA capable of expressing the protein MsSPL4 in a host cell, and the DNA may include not only a promoter for initiating transcription of the MsSPL4 gene, but also a terminator for terminating transcription of the MsSPL4 gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both of which can be induced by methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (China Patent No. 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, for example, Odell et al. (1985) EMBO J. 4: 3047-3053). 985)Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0041] In the above-mentioned related biological materials, the recombinant vector B3) may contain a DNA molecule encoding the protein MsSPL4 as shown in SEQ ID No.1.

[0042] A plant expression vector can be used to construct a recombinant vector containing the MsSPL4 protein encoding gene expression cassette.

[0043] Among the above-mentioned related biological materials, the recombinant microorganisms described in B4) can specifically be yeast, bacteria, algae and fungi.

[0044] Among the above-mentioned related biological materials, the plant tissue described in B6) can be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0045] Among the above-mentioned related biological materials, the transgenic plant organs described in B7) can be roots, stems, leaves, flowers, fruits and seeds of transgenic plants.

[0046] Among the above-mentioned related biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation materials.

[0047] In a second aspect, the present invention also provides a method for improving plant stress resistance, the method comprising increasing the expression level of the gene encoding the above protein in the target plant, thereby improving the stress resistance of the target plant.

[0048] The increasing the expression level of the gene encoding the protein in the target plant can be achieved by introducing the gene encoding the protein into the target plant.

[0049] In the above method, the protein encoding gene can be introduced into the target plant via a plant expression vector carrying the protein encoding gene.

[0050] The plant expression vector carrying the protein encoding gene of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant cells or tissues can be cultivated into plants.

[0051] In a specific embodiment of the present invention, the recombinant vector is a recombinant plasmid pCGW-MsSPL4. The structure of the recombinant vector pCGW-MsSPL4 is a recombinant vector obtained by replacing the fragment between the XbaI and BamHI restriction endonucleases of the pCGW vector (a small fragment between the XbaI and BamHI restriction endonucleases) with the nucleotide sequence shown in SEQ ID No.1, while keeping the other nucleotide sequences of the pCGW vector unchanged.

[0052] In the above, the stress resistance may be drought resistance and / or salt resistance.

[0053] Furthermore, in the method, the recipient plant is any one of the following:

[0054] P1) dicotyledonous plants;

[0055] P2) Leguminosae;

[0056] P3) Fabaceae;

[0057] P4) Medicago;

[0058] P5) Alfalfa.

[0059] In a third aspect, the present invention also provides the above protein, and / or biological materials related to the above protein.

[0060] The invention provides MsSPL4 protein and its encoding gene, and introduces the encoding gene of MsSPL4 protein into alfalfa to obtain MsSPL4 transgenic alfalfa plants. The transgenic plants are subjected to drought and salt stress treatment, and compared with the control, the drought and salt tolerance of the transgenic strains are enhanced.

[0061] The MsSPL4 gene and the protein encoded by it provided by the present invention play an important role in the process of plant stress resistance, have important application value in the research of improving plant stress resistance, and have broad application space and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 PCR identification of MsSPL4 transgenic tobacco resistant seedlings. Note: M, DNA ladder AL2000; 1-6, resistant seedling samples; -, wild-type tobacco DNA as negative control; +, pCGW-MsSPL4 plasmid as positive control.

[0063] Figure 2 RT-qPCR was used to detect the relative expression level of MsSPL4 in MsSPL4-overexpressing tobacco grown for one month.

[0064] Figure 3 The overall growth appearance of transgenic tobacco overexpressing MsSPL4 and wild-type tobacco grown for 2 months.

[0065] Figure 4 The heights of transgenic tobacco plants overexpressing MsSPL4 and wild-type tobacco plants grown for 2 months.

[0066] Figure 5 The internode lengths of transgenic tobacco overexpressing MsSPL4 and wild-type tobacco grown for 2 months.

[0067] Figure 6 The number of leaves of transgenic tobacco overexpressing MsSPL4 and wild-type tobacco grown for 2 months.

[0068] Figure 7 The length-to-width ratio of leaves of transgenic tobacco overexpressing MsSPL4 and wild-type tobacco grown for 2 months.

[0069] Figure 8 The T1 seedlings overexpressing MsSPL4 were selected by hygromycin.

[0070] Fig. 9 Preliminary evaluation of salt tolerance in MsSPL4 overexpressing tobacco

[0071] Fig.10 PCR identification of MsSPL4-overexpressing alfalfa resistant seedlings. Note: M, DNA ladder AL2000; 1-22, resistant seedling samples; -, wild-type alfalfa DNA as negative control; +, pCGW-MsSPL4 plasmid as positive control.

[0072] Fig.11 PCR identification of alfalfa resistant seedlings with suppressed expression of MsSPL4. Note: M, DNA ladder AL2000; 1-21, resistant seedling samples; -, wild-type alfalfa DNA as negative control; +, pCGW-MsSPL4-SRDX plasmid as positive control.

[0073] Fig.12 RT-qPCR was used to detect the relative expression level of MsSPL4 in MsSPL4-overexpressing alfalfa grown for one month.

[0074] Fig.13 RT-qPCR was used to detect the relative expression level of MsSPL4 in MsSPL4-repressed alfalfa grown for one month.

[0075] Fig.14 The overall growth appearance of transgenic alfalfa overexpressing and suppressed MsSPL4 and wild-type alfalfa grown for 2 months.

[0076] Fig.15 Appearance of shoots of transgenic alfalfa overexpressing and suppressing MsSPL4 and wild-type alfalfa grown for 2 months.

[0077] Fig.16 Leaf appearance of transgenic alfalfa overexpressing and suppressing MsSPL4 and wild-type alfalfa grown for 2 months.

[0078] Fig.17 The average leaf numbers on the branches of MsSPL4-overexpressing and MsSPL4-repressed alfalfa grown for 2 months.

[0079] Fig.18 The length-to-width ratio of leaves of alfalfa overexpressing and knockdown expressing MsSPL4 grown for 2 months.

[0080] Fig.19 The heights of alfalfa plants overexpressing and suppressing MsSPL4 after 2 months of growth.

[0081] Fig. 20 Internode length of alfalfa overexpressing and suppressing MsSPL4 after 2 months of growth.

[0082] Fig.21 Evaluation of salt tolerance of transgenic plants overexpressing and inhibiting the expression of MsSPL4; (a), (b), and (c) show the plant morphology after salt treatment for 0 days, 15 days, and 20 days, respectively.

[0083] Fig. 22 The relative water content of leaves of MsSPL4 transgenic alfalfa and wild-type alfalfa plants treated with salt for 0, 15, and 20 days,

[0084] Fig.23 Leaf electrolyte permeability of MsSPL4 transgenic alfalfa and wild-type alfalfa plants treated with salt for 0, 15, and 20 days.

[0085] Fig.24 Proline content in leaves of MsSPL4 transgenic alfalfa and wild-type alfalfa plants treated with salt for 0, 15, and 20 days. DETAILED DESCRIPTION

[0086] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0087] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0088] The basic formula of Hoagland nutrient solution is:

[0089] Table 1 Hoagland nutrient solution components

[0090]

[0091]

[0092] The following implementation used the one-way analysis of variance procedure in IBM SPSS Statistics 24.0 software to perform analysis of variance and multiple comparisons on the data. English letters indicate the degree of significance of the difference. There was no significant difference between treatments with at least one identical letter at the 0.05 level, and there was a significant difference between treatments without the same letter at the 0.05 level.

[0093] Example 1. Cloning of MsSPL4 gene and construction of expression vector

[0094] The coding sequence of the MsSPL4 gene is SEQ ID No. 1, which is derived from alfalfa and encodes a protein MsSPL4 with an amino acid sequence of SEQ ID No. 2.

[0095] >MsSPL4 nucleotide sequence (SEQ ID No.1)

[0096] ATGGATGGAAGTTGGGGTGAGGGAAAGAGAATCTATGAGTACAGAGAAGAAGATGAGTATGAAGTAGAAGTAGAAGAAGAGGAAGATGTGAGTTATGGATGATGGGAAGAAGAAAAGGGTGGTGACTGATCATCTTTACAACAGGAAAGGGTCAAAAGCTGGAGGATCAGTGACACCTTTCTTGTCAAGTTGATAACTGTAATGCTGATCTA AGTGTTGCTAAGCAGTACCATAAGCGCCATAAGGTTTGTGAATACCATTCTAAGGCTCATTCCGTACTCATTTCAGAGCTTCAACAAAGGTTTTGCCAGCAATGTAGCAGGTTTCATGAGGTATCAGAATTTGATGACTTGAAAAGGAGTTGTAGGAGGCGTTTGGCTGGACATAACGAGAGGCGCCGCAAAAGCACGTCTGAATATCACTGA

[0097] >MsSPL4 protein sequence (SEQ ID No.2)

[0098] MDGSWGEGKRIYEYREEDEYEVEVEEEEDVSYGDDGKKKRVVTDHLYNRKGSKAGGSVTPSCQVDNCNADLSVAKQYHKRHKVCEYHSKAHSVLISELQQRFCQQCSRFHEVSEFDDLKRSCRRRLAGHNERRRKSTSEYH*

[0099] RNA was extracted from alfalfa (Zhongmu No. 1) and reverse transcribed to obtain cDNA.

[0100] The cloning primers covering the entire cDNA sequence were designed based on the third-generation database data as follows:

[0101] MsSPL4-XbaI-F:5'- TCTAGA ATGGATGGAAGTTGGGGTG-3' (SEQ ID No. 3, the underlined region is the Xba I recognition site);

[0102] MsSPL4-BamHI-R:5'- GGATCC TTAGTGATATTCAGACGTGC-3' (SEQ ID No. 4, the underlined region is the BamH I recognition site);

[0103] MsSPL4-SRDX-HindIII-R:

[0104] 5'- AAGCTT TTACGCAAAGCCCAGGCGCAGTTCCAGATCCAGATCCAGGTGATATTCAGACGTGCTTTTGCG-3′ (SEQ ID No. 5, the underlined region is the Hind III recognition site).

[0105] Using cDNA as a template, using MsSPL4-XbaI-F and MsSPL4-BamHI-R as primers to perform PCR, the cloned PCR product contains a DNA molecule of the MsSPL4 coding region sequence with restriction endonucleases recognition sites at both ends. The PCR product and the pCGW vector are double-digested with restriction endonucleases Xba I and BamH I, respectively, and the obtained restriction endonucleases are connected. The obtained connection products are transferred into Escherichia coli DH5α strains, and positive clones are picked for sequencing. The sequencing results show that the structure of the recombinant vector is to replace the fragment between the restriction endonuclease Xba I and BamH I recognition sites of the pCGW vector (a small fragment between the restriction endonuclease recognition sites of Xba I and BamH I) with the MsSPL4 gene whose nucleotide sequence is SEQ ID No.1, and the recombinant vector obtained by keeping the other nucleotide sequences of the vector pCGW unchanged is named pCGW-MsSPL4, that is, the overexpression vector of the MsSPL4 gene.

[0106] Using cDNA as a template, MsSPL4-XbaI-F and MsSPL4-SRDX-HindIII-R as primers for PCR, the cloned PCR product contains a DNA molecule of the MsSPL4 coding region sequence and the SRDX sequence with restriction endonucleases at both ends. The PCR product and the pCGW vector are double-digested with restriction endonucleases Xba I and Hind III, respectively, and the obtained restriction endonucleases are connected. The obtained connection products are transferred into the Escherichia coli DH5α strain, and the positive clones are picked for sequencing. The sequencing results show that the structure of the recombinant vector is to replace the fragment between the restriction endonuclease XbaI and Hind III recognition sites of the pCGW vector (a small fragment between the restriction endonuclease recognition sites of Xba I and BamH I) with the MsSPL4 gene whose nucleotide sequence is SEQ ID No. 1, and the recombinant vector obtained by keeping the other nucleotide sequences of the vector pCGW unchanged is named pCGW-MsSPL4-SRDX, which is the inhibition expression vector of the MsSPL4 gene.

[0107] Example 2. Acquisition and phenotypic analysis of tobacco plants overexpressing MsSPL4

[0108] 2.1 Agrobacterium-mediated genetic transformation

[0109] The constructed pCGW-MsSPL4 overexpression and MsSPL4-SRDX inhibition expression vectors were transformed into Agrobacterium tumefaciens EHA105 by heat shock transformation method, and positive clones were screened in YEP medium containing 100 mg / L kanamycin and 50 mg / L rifampicin. The vector was successfully transformed into EHA105 Agrobacterium through bacterial liquid PCR verification.

[0110] The tobacco transformation was carried out using the Agrobacterium-mediated genetic transformation method published in the literature. The recipient plant used was tobacco "Coker176" and the transformation site was the mature leaves of sterile seedlings. The method reference is:

[0111] Then, the genetic transformation was carried out using the Agrobacterium-mediated genetic transformation method published in the previous method literature of our laboratory. The recipient plant used was the alfalfa variety Zhongmu No. 1, and the transformation site was the mature leaves. The method reference is: [1] Feng Jiaxin, Zhang Xing, Chi Lixin, Fan Wenqiang, Yue Sijun, Shi Jing, Zheng Rui. Analysis of genetic transformation of Lycium barbarum LbMYB12 gene in tobacco [J / OL]. Molecular Plant Breeding: 1-14.

[0112] Through hygromycin screening, 6 hygromycin-resistant tobacco plants with the MsSPL4 gene were obtained, namely the T0 generation MsSPL4 overexpressing tobacco, and the plants were numbered 1 (OE1), 2 (OE2), 3 (OE3), 4 (OE4), 5 (OE5), and 6 (OE6).

[0113] 2.2 PCR detection of MsSPL4 overexpression in tobacco

[0114] In order to detect whether the vector fragment containing the target gene is integrated into the plant genome, the CTAB method was used to extract the genomic DNA of the young leaves of the wild-type and MsSPL4-transgenic hygromycin-resistant plants. The transgenic resistant tobacco DNA was used as a template, the wild-type "Coker176" tobacco plant was used as a negative control (-), and the plant expression vector pCGW-MsSPL4 was used as a positive control (+). The specific fragment of the MsSPL4 gene was amplified using 35S-F: 5′-CGCACAATCCCACTATCCTTC-3′ and MsSPL4-BamHI-R as primers. The identification results are shown in Figure 2. Figure 1 As shown, the transgenic positive plants amplified a band with the same size as the positive control (+), and the negative control (WT) did not amplify the target band, indicating that the vector fragment has been integrated into the plant genome. PCR confirmed that 6 tobacco plants with the MsSPL4 gene were obtained.

[0115] 2.3 qRT-PCR detection of MsSPL4 overexpression in tobacco

[0116] In order to detect the expression of the target gene in the transgenic plants, the expression of the target gene in the transgenic plants was detected and analyzed by qRT-PCR. The cDNA of the transgenic PCR-positive plants was used as a template, and the housekeeping gene NtActin was used as an internal reference gene to detect the relative expression of the target gene in the transgenic plants. The results showed that OE1 and OE2 in the MsSPL4 gene-positive tobacco were low-fold overexpression plants, while OE4 and OE5 were high-fold overexpression tobacco ( Figure 2 ).

[0117] Quantitation primers:

[0118] MsSPL4-qRT-F:5'-ATGGATGGAAGTTGGGGTGAGGG-3'(SEQ ID No.6)

[0119] MsSPL4-qRT-R:5'-TCAGTCACCACCCTTTTCTTCTTCC-3'(SEQ ID No.7)

[0120] Internal reference primer:

[0121] NtActin-F: 5'-CTGAGGTCCTTTTCCAACCA-3' (SEQ ID No. 8)

[0122] NtActin-R: 5'-TACCCGGGAACATGGTAGAG-3' (SEQ ID No. 9)

[0123] 2.4 Analysis of the growth and appearance of MsSPL4 overexpressing tobacco

[0124] The T0 generation seeds of overexpressing tobacco were collected and screened with 15 mg / L hygromycin to obtain transgenic T1 generation tobacco to ensure that the growth period of wild-type tobacco and transgenic tobacco was completely consistent. The offspring of high-expressing plants (OE4 and OE5) were selected for preliminary phenotype observation.

[0125] For the phenotypic determination of tobacco grown for 2 months, the data were subjected to analysis of variance and multiple comparisons using the one-way analysis of variance procedure in IBM SPSS Statistics 24.0 software. English letters indicate the degree of significance of the difference. There was no significant difference between treatments with at least one identical letter at the 0.05 level, and there was a significant difference between treatments without the same letter at the 0.05 level.

[0126] The overall growth status of wild-type and MsSPL4 overexpressing tobacco plants is shown in Figure 2. Figure 3 The results showed that compared with the wild type, the height of MsSPL4 overexpressing tobacco plants was significantly reduced ( Figure 4 ), and its internode length was significantly shortened ( Figure 5), and the number of leaves per plant ( Figure 6 ) and blade aspect ratio ( Figure 7 ) had no significant difference.

[0127] 2.5 Detection of salt tolerance in tobacco plants with MsSPL4 overexpression

[0128] The T1 generation of MsSPL4-OE tobacco was screened on MS medium containing 15 mg / L hygromycin. On the 4th day after germination, wild-type seedlings that germinated at the same time as the T1 generation seeds of MsSPL4-OE tobacco on MS medium without hygromycin and had the same growth were selected and transferred into culture bottles containing different concentrations of NaCl (0 mM NaCl, 150 mM NaCl, 200 mM NaCl) to observe the growth of the plants at different stages ( Figure 8 ).

[0129] After culturing for 18 days under 0mM NaCl conditions, it can be observed that the plant height of MsSPL4-OE tobacco plants is lower than that of the wild type, while under 150mM and 200mM NaCl treatments, tobacco seedlings are severely inhibited, and the phenotypic differences between the wild type and transgenic alfalfa are small. At 48 days of cultivation, there was no significant difference in plant height between the wild type and MsSPL4-OE plants under 0mM NaCl treatment. Under 150mM NaCl treatment, the leaves at the base of the wild type plants turned white, and the transgenic plants grew well. Under 200mM NaCl treatment, the bottom leaves of both transgenic and wild type plants showed obvious damage. There was no difference in growth status until 68 days. Under 0mM NaCl conditions, there was no significant difference in tobacco plants. Under 150mM and 200mM NaCl treatments, the plant height of the wild type plants was lower than that of the MsSPL4-OE plants ( Fig. 9 ). In summary, the initial growth rate of MsSPL4-OE plants was slower than that of the wild type. Under salt stress conditions, MsSPL-OE plants had higher tolerance to salt stress, so their later growth was better than that of the wild type.

[0130] Example 3: Acquisition and phenotypic analysis of MsSPL4 transgenic alfalfa

[0131] 3.1 Agrobacterium-mediated genetic transformation

[0132] Genetic transformation was performed using the Agrobacterium-mediated genetic transformation method previously published by our laboratory. The recipient plant used was the alfalfa variety Zhongmu No. 1, the transformation site was mature leaves, and the transformation vector was the pCGW-MsSPL4 overexpression and pCGW-MsSPL4-SRDX suppression expression vector. The references for the method are: [2] Li Juan, Zhang Wanjun, Wang Tao. Study on the establishment of a high-frequency plant regeneration system for alfalfa [J]. Acta Grasslanda Sinica, 2014, 22(04): 834-839; [3] Wang Kexin; Liu Yanrong; Teng Fengkui; Cen Huifang; Yan Jianping; Lin Shiwen; Li Dayong; Zhang Wanjun*; Heterogeneous expression of Osa-MIR156bc increases abiotic stress resistance and forage quality of alfalfa, The Crop Journal, 2021:

[0133] https: / / doi.org / 10.1016 / j.cj.2020.11.009.

[0134] Through hygromycin screening, 18 MsSPL4 overexpression plants and 20 MsSPL4 suppression expression plants were obtained, which were T0 generation transgenic plants.

[0135] 3.2 PCR detection of MsSPL4 transgenic alfalfa

[0136] In order to detect whether the vector fragment containing the target gene is integrated into the plant genome, the genomic DNA of young leaves of wild-type and MsSPL4 transgenic plants with hygromycin resistance was extracted by CTAB method. The transgenic resistant plant DNA was used as template, the wild-type alfalfa Zhongmu 1 variety plant (WT49, i.e., the recipient plant) was used as negative control, and the plant expression vectors pCGW-MsSPL4 and pCGW-MsSPL4-SRDX were used as positive controls (+), respectively. The 35S-F:5′

[0137] -CGCACAATCCCACTATCCTTC-3′ (SEQ ID No. 10) and MsSPL4-BamHI-R were used as primers to amplify the specific fragment of MsSPL4 gene.

[0138] The results of MsSPL4 overexpression in alfalfa were as follows: Fig.10 As shown, MsSPL4 inhibits the expression of alfalfa identification results as shown Fig.11As shown, the transgenic positive plants amplified a band with the same size as the positive control (+), and the negative control (WT) did not amplify the target band, indicating that the vector fragment has been integrated into the plant genome. Through PCR detection, 18 transgenic MsSPL4 overexpression plants were obtained, which were marked as OE plants according to the serial number, such as plant No. 2 was marked as OE2; 20 expression suppression plants were marked as SR plants according to the serial number, such as plant No. 2 was marked as SR2.

[0139] 3.3 qRT-PCR detection of MsSPL4 transgenic plants

[0140] In order to detect the expression of the target gene in the transgenic plants, qRT-PCR was used to detect and analyze the expression of the target gene in the transgenic plants. The cDNA of the transgenic PCR-positive plant was used as a template, and the housekeeping gene Actin was used as an internal reference gene to detect the relative expression of the target gene in the transgenic plants.

[0141] The results showed that the expression levels of MsSPL4 in MsSPL4 overexpressing plants OE11, OE25, OE28, OE2, OE4, OE27, and OE13 were higher than those in alfalfa wild-type plants WT49 ( Fig.12 ), the expression levels of SR3, SR25, SR22, SR2, SR4, SR8, SR7, SR24, SR5, SR11, and SR6 in the MsSPL4-suppressed plants were higher than those in the wild-type alfalfa WT49 ( Fig.13 ).

[0142] Quantitation primers:

[0143] MsSPL4-qRT-F:5'-ATGGATGGAAGTTGGGGTGAGGG-3'(SEQ ID No.6)

[0144] MsSPL4-qRT-R:5'-TCAGTCACCACCCTTTTCTTCTTCC-3'(SEQ ID No.7)

[0145] Internal reference primer:

[0146] actin-F: 5'-CAAAAGATGGCAGATGCTGAGGAT-3' (SEQ ID No. 11)

[0147] actin-R: 5'-CATGACACCAGTATGACGAGGTCG-3' (SEQ ID No. 12)

[0148] 3.4 Analysis of the appearance of MsSPL4 transgenic plants during growth

[0149] The T0 transgenic plants were propagated by cuttings, and the line number was the same as the mother plant. Through the previous expression level detection, this study selected MsSPL4 overexpression and suppression expression alfalfa plants OE2, OE4, SR6, SR7, and SR8 with different MsSPL4 expression levels for in-depth research.

[0150] The phenotypes of the plants grown for 2 months were measured, and the data were subjected to analysis of variance and multiple comparisons using the one-way analysis of variance program in IBM SPSS Statistics 24.0 software. English letters indicate the degree of significance of the difference. There was no significant difference between treatments with at least one identical letter at the 0.05 level, and there was a significant difference between treatments without the same letter at the 0.05 level.

[0151] The growth status of wild-type and transgenic plants Fig.14 As shown, the branches look like Fig.15 As shown, the leaf shape is Fig.16 The results showed that the number of leaves on the branches of MsSPL4-SRDX plants ( Fig.17 ) and blade aspect ratio ( Fig.18 ) was significantly lower than that of wild-type and MsSPL4-OE plants. With the increase of MsSPL4 expression, the height of alfalfa plants decreased ( Fig.19 ), but there was no significant change in internode length ( Fig. 20 ).

[0152] 3.5 Analysis of salt tolerance of MsSPL4 transgenic alfalfa

[0153] The wild-type strain (WT49) and T0 transgenic strains OE2, OE4, SR6, SR7, and SR8 were propagated by cuttings. After the plants grew for 4 months, plants with good growth conditions were selected. Four bottles of each strain, one plant in each bottle, were used for salt treatment experiments. At the beginning of the experiment, 50 mL of NaCl solution was irrigated every day. 50 / 100 / 150 mM NaCl was irrigated on the 1st to 3rd day, and 200 mM NaCl was irrigated every day from the 4th day. This was counted as the first day of salt treatment, and the total treatment was 20 days. Phenotypic statistics of overexpressed MsSPL4, suppressed expression of MsSPL4, and wild-type alfalfa were performed before the start of salt treatment (0d) and on the 15th and 20th days of 200 mM NaCl treatment, and leaves at the same position were selected for physiological index detection.

[0154] The results are as follows Fig.21 As shown, before salt treatment, the plants grew uniformly and well ( Fig.21After 15 days of treatment with 200 mM NaCl, the leaves in the middle and lower parts of the MsSPL4-inhibited plants (SR6, SR7, and SR8) were obviously dried up, while the leaves in the MsSPL4-overexpressed plants (OE2 and OE4) and the wild type were only damaged at the bottom of the plants ( Fig.21 After 20 days of treatment, the damage degree of the overexpression plants was significantly lower than that of the MsSPL4-SRDX plants and the wild type (WT49) alfalfa ( Fig.21 (c)).

[0155] In order to quantitatively evaluate the salt tolerance of transgenic plants and wild-type plants, we measured the physiological indicators of different plants under salt stress. Before salt treatment, there were no significant differences in leaf relative water content, electrolyte permeability, and proline content between wild-type plants and transgenic plants. After 15 days of salt treatment, the relative water content of leaves was significantly reduced compared with that at 0 days (P<0.05), and the water content in MsSPL4-SRDX alfalfa plants decreased the most, about 50% lower than before treatment. There was no significant difference in the relative water content between wild-type and MsSPL4-OE plants (P>0.05), which were 73% and 75%-76%, respectively. After 20 days of salt treatment, the relative water content of MsSPL4-SRDX plants and wild-type plants was significantly lower than that of MsSPL4-OE plants (P<0.05) ( Fig. 22 ). The results of the leaf electrolyte permeability test showed that the electrolyte permeability increased in both transgenic and wild-type plants at 15 and 20 days after salt treatment, and the increase in electrolyte permeability in MsSPL4 overexpression plants was significantly lower than that in wild-type and MsSPL4 suppression expression plants (P<0.05)( Fig.23 ). Leaf proline content detection found that after 15 days of salt treatment, the proline content in all plants increased. At this time, the proline accumulation in MsSPL4-SRDX plants was significantly higher than that in wild-type and MsSPL4-OE plants, indicating that the MsSPL4-inhibited expression plants were more severely affected by salt stress. There was no significant difference in proline content after 20 days of salt treatment and 15 days of treatment. At this time, the regulation of proline on salt stress may have reached its upper limit ( Fig.24 The above physiological indexes showed that overexpression of MsSPL4 gene significantly increased the salt tolerance of alfalfa.

[0156] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for improving plant salt tolerance, characterized in that: The method comprises increasing the expression amount of a gene encoding a MsSPL4 protein in a plant, thereby increasing the salt tolerance of the plant; the plant is alfalfa or tobacco; and the amino acid sequence of the MsSPL4 protein is SEQ ID No.

2.

2. The method according to claim 1, characterized in that The nucleotide sequence of the MsSPL4 protein encoding gene is SEQ ID No.1.

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