Application of TaSMAX1 protein and coding gene thereof in promoting salt tolerance in wheat seedling stage

Abstract

The invention discloses application of a TaSMAX1 protein and a coding gene thereof in promoting salt tolerance in a wheat seedling stage. The invention belongs to the technical field of biology, and particularly relates to application of TaSMAX1 protein and a coding gene thereof in promoting salt tolerance in a wheat seedling stage, the protein is SEQ ID NO: 2, and the coding gene is 58-3123 sites of SEQ ID NO: 1. Experiments prove that compared with a receptor plant Fielder, the survival rate of a TaSMAX1 protein overexpressed strain under salt treatment is remarkably improved, it is proved that the overexpressed TaSMAX1 protein can enhance the salt tolerance of wheat in the seedling stage, gene resources are provided for cultivating high-yield and high-quality wheat varieties, and the TaSMAX1 protein overexpressed strain has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of TaSMAX1 protein and its encoding gene in promoting salt tolerance in wheat seedling stage. Background Art

[0002] Wheat, a globally important food crop, is second only to corn and rice in terms of cultivated area and annual yield in my country. Increasing wheat yield is crucial for ensuring food security in my country and globally. However, global climate change, coupled with frequent extreme weather events and environmental stresses such as soil salinization, poses a serious threat to wheat production and a significant challenge to global food security. Wheat is extremely sensitive to salt, and high salt levels inhibit its growth, resulting in its yield being frequently impacted by salt stress. Saline-alkali land, representing a reserve of arable land in my country, covers approximately 100 million hectares, representing approximately 5% of the country's usable land area. Therefore, developing and utilizing saline-alkali land resources and using modern molecular breeding methods to precisely, rapidly, and efficiently improve wheat salt tolerance are crucial for ensuring national food security. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to improve the salt tolerance of wheat. To this end, the present invention provides 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. The protein is TaSMAX1 protein, which is any of the following: a1) a protein having the amino acid sequence of SEQ ID NO: 2, a2) a protein having the same function as the amino acid sequence of SEQ ID NO: 2, wherein one or more amino acid residues are substituted and / or deleted and / or added. a3) A protein having an amino acid sequence identity of at least 80% with that specified in a1) or a2) and having the same function, a4) A fusion protein obtained by ligating a tag to the end of any of the proteins defined in a1) to a3); The application is any of the following: b1) Regulating plant salt tolerance, b2) Preparation of products for regulating plant salt tolerance, b3) Cultivating plants with altered salt tolerance, b4) preparing products for breeding plants with altered salt tolerance, b5) Plant breeding, b6) Preparation of products for use in plant breeding.

[0004] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0005] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0006] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0007] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaSMAX1 protein of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that are 75% or more identical to the nucleotide sequence of the isolated TaSMAX1 protein of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the TaSMAX1 protein and possess the function of the TaSMAX1 protein.

[0008] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0009] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, the blastp program can be used with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0010] 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.

[0011] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0012] In the above application, the protein is derived from wheat ( Triticum aestivum L.).

[0013] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein TaSMAX1.

[0014] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: c1) regulation at the transcription level of the coding gene, c2) regulation after transcription of the coding gene, c3) regulation of RNA transport of the coding gene, c4) regulation of translation of the coding gene, c5) regulation of mRNA degradation of the coding gene, and c6) post-translational regulation of the gene.

[0015] In the present invention, the regulation may be upregulation, enhancement or improvement.

[0016] In the above applications, the substance that regulates gene expression or the substance that regulates the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following: d1) a nucleic acid molecule encoding the protein described above; d2) an expression cassette containing the nucleic acid molecule described in d1); d3) a recombinant vector containing the nucleic acid molecule described in d1), or a recombinant vector containing the expression cassette described in d2); d4) a recombinant microorganism containing the nucleic acid molecule described in d1), or a recombinant microorganism containing the expression cassette described in d2), or a recombinant microorganism containing the recombinant vector described in d3); d5) a transgenic plant cell line containing the nucleic acid molecule described in d1), or a transgenic plant cell line containing the expression cassette described in d2); d6) transgenic plant tissue containing the nucleic acid molecule described in d1), or transgenic plant tissue containing the expression cassette described in d2), d7) a transgenic plant organ containing the nucleic acid molecule described in d1), or a transgenic plant organ containing the expression cassette described in d2); In the above application, the nucleic acid molecule in d1) can be any of the following DNA molecules, e1) The nucleotide sequence is the DNA molecule shown in SEQ IE NO: 3, e2) The coding region sequence is the DNA molecule shown in SEQ ID NO: 1 at positions 58-3123 in the sequence listing, e3) a DNA molecule that is 90% or more identical to the nucleotide sequence defined in e1) or e2), is derived from wheat and encodes the protein described above, e4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in e1) or e2) and encodes the protein described above.

[0017] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0018] The vectors described herein are well known to those skilled in the art, and include, but are not limited to, plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector may be PC186.

[0019] Existing plant expression vectors can be used to construct TaSMAX1 Recombinant expression vectors for genes. Such plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment. Such plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. Examples include, but are not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (e.g., the rouge synthase Nos gene) and the 3' transcribed untranslated regions of plant genes (e.g., the soybean storage protein gene), all of which have similar functions.

[0020] use TaSMAX1When constructing a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter and the ubiquitin promoter of corn. These promoters can be used alone or in combination with other plant promoters. In addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene.

[0021] To facilitate identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include, but are not limited to, genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene or luciferase gene), antibiotic resistance markers (such as gentamicin and kanamycin), or chemical resistance marker genes (such as herbicide resistance genes). For safety reasons, it is possible to omit any selectable marker genes and directly screen for transformed plants using stress.

[0022] Utilize any vector that can guide the expression of foreign genes in plants to TaSMAX1 By introducing genes or gene fragments into plant cells or recipient plants, transgenic cell lines and transgenic plants with altered salt tolerance can be obtained. TaSMAX1 Gene expression vectors can be transformed into plant cells or tissues through conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

[0023] The present invention also provides a method for cultivating plants with improved salt tolerance, the method comprising step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the protein described above in the target plant, or / and, enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to cultivate plants with improved salt tolerance.

[0024] The present invention also provides a method for regulating wheat salt tolerance, which includes step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein mentioned above, so as to positively regulate the salt tolerance of wheat.

[0025] In the above method, increasing the expression level and / or activity of the gene encoding the protein TaSMAX1 in the target plant may be achieved by overexpressing the gene to enhance the activity of the gene encoding the protein TaSMAX1 in the genome of the target plant.

[0026] The present invention provides a method for producing plants with enhanced salt tolerance, comprising overexpressing the gene encoding the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in target plants, thereby obtaining plants with enhanced salt tolerance and ultimately increasing yield under salt stress.

[0027] In one embodiment of the present invention, the method may comprise the steps of: f1) constructing a recombinant expression vector for enhancing, increasing or upregulating the gene encoding the protein mentioned above; f2) Transforming the recombinant expression vector constructed in step f1) into a recipient plant (such as a crop or wheat) to obtain a plant having better salt tolerance than the recipient plant.

[0028] In the present invention, the plant breeding index may include plant salt tolerance. The purpose of the plant breeding may include cultivating plants with improved salt tolerance.

[0029] In the above application or method, the plant may be any of the following plants: N1) monocotyledonous plants or dicotyledonous plants, N2) plants of the order Poaceae, N3) plants of the family Poaceae, N4) plants of the genus Triticum, and N5) wheat.

[0030] The present invention also provides a product, which may be the protein or biological material described above.

[0031] The present invention provides a wheat salt-tolerant gene TaSMAX1 The cloning method of TaSMAX1 was used to reveal that it is an important protein regulating salt tolerance in wheat. TaSMAX1 The expression of can significantly enhance the salt tolerance of wheat. TaSMAX1 It can increase the survival rate of wheat after salt treatment, indicating that TaSMAX1 can improve the salt tolerance of wheat at the seedling stage. These findings provide genetic resources for breeding high-yield and high-quality wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 for TaSMAX1 The full-length cDNA of the target gene is cloned; Lane M is D2000 plus DNA Ladder; Lanes 1 and 2 are TaSMAX1 The cDNA is full-length and its size is 3139 bp.

[0033] Figure 2 for TaSMAX1 Identification results of T0 generation of transgenic lines.

[0034] Figure 3 for TaSMAX1 Detection of gene expression levels in overexpressed materials.

[0035] Figure 4 for TaSMAX1 Phenotypes of overexpressing materials at the seedling stage under normal conditions and after salt stress treatment. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0037] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0038] The quantitative experiments in the following examples were performed three times unless otherwise specified, and the results were averaged.

[0039] The wheat material "Fielder" in the following examples was donated by Professor Li Genying from the Crop Research Institute of Shandong Academy of Agricultural Sciences and has been recorded in: Zhang, S., Zhang, R., Gao, J., Song, G., Li, J., Li, W., Qi, Y., Li, Y., & Li, G. (2021). CRISPR / Cas9-mediated genome editing for wheatgrain quality improvement. Plant Biotechnology Journal, 19(9): 1684–1686. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0040] The Escherichia coli strain DH5α in the following examples was purchased from Beijing Quanshijin Biotechnology Co., Ltd., China.

[0041] In the following examples, Agrobacterium strain EHA105 was purchased from Beijing Biomed Gene Technology Co., Ltd., China.

[0042] The vector pBM27 used in the following examples was purchased from Beijing Biomed Biotechnology Co., Ltd., China.

[0043] The PC186 carrier used in the following examples was a gift from Professor Fu Daolin of the College of Agriculture, Shandong Agricultural University. The biomaterial is available to the public from the applicant as described in the following document, page 11, right column, fourth line from the bottom. The biomaterial is intended for use solely in repeating the experiments described in the present invention and is not intended for other purposes: Hao Q, Wang W, Han X, Wu J, Lyu B, Chen F, Caplan A, Li C, Wu J, Wang W, Xu Q, Fu D. (2018) Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum inbarley. Molecular Plant Pathology. 19(8):1995–2010.

[0044] The data in the following examples were processed using SPSS 19.0 statistical software, and the experimental results were expressed as mean ± standard deviation using Student's t Test results, * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.

[0045] Example 1 TaSMAX1 Gene cloning 1. Total RNA extraction from wheat leaves RNA extraction using Trizol (1) Take 150 mg-200 mg of Fielder seedling leaves, place them in an RNase-free mortar pre-cooled in liquid nitrogen, quickly grind the material into a uniform powder in liquid nitrogen, and add it to a 2.0 mL RNase-free centrifuge tube; (2) Add 1 mL of Trizol to the centrifuge tube, vortex and place at room temperature for 5 minutes; (3) Add 200 μL of chloroform, shake vigorously for 30 seconds, and then let it stand at room temperature for 5 minutes; (4) Centrifuge at 12,000 rpm, 4°C for 15 min and transfer the supernatant (550-650 μL) to a new 1.5 mL RNase-free centrifuge tube. (5) Add an equal volume of isopropanol to the supernatant, mix thoroughly by inversion, let stand at room temperature for 5-10 min, centrifuge at 12,000 rpm, 4°C for 15 min, pour off the supernatant and retain the precipitate; (6) Add 1 mL of 75% alcohol to wash the precipitate, centrifuge at 12,000 rpm, 4°C for 5 min, and discard the supernatant; (7) Repeat step (6), use the gun tip to absorb the alcohol and then place it on the clean bench to dry; (8) Add 50 μL of RNase-free ddH2O to dissolve RNA. Dissolve RNA at 50°C for 5-10 min to remove polysaccharides and polyphenols. (9) Centrifuge at 12,000 rpm, 4°C for 5-10 min, aspirate the supernatant into a new 1.5 mL RNase-free centrifuge tube, measure the concentration or store at -80°C for later use.

[0046] 2. cDNA Synthesis Reverse transcription was performed using the Novozymes HiScript® III RT SuperMix for qPCR (+gDNA wiper) kit, performed entirely on ice. The reaction system consisted of 1.5 μg of RNA template, 4 μL of 4× gDNA wiper mix, and RNase-free ddH2O to a final volume of 16 μL. Mix by pipetting. The PCR instrument was maintained at 42°C for 2 min.

[0047] The reverse transcription system consists of 16 μL of the product from the previous step and 4 μL of 5× HiScript III qRT SuperMix. Mix thoroughly by pipetting. Perform the reverse transcription reaction in a PCR instrument as follows: 50°C for 15 minutes, 85°C for 5 seconds. Immediately place on ice after the reaction. Dilute with 180 μL of ddH2O and store at -20°C.

[0048] 3. TaSMAX1 Gene cloning The wheat leaf cDNA obtained in the above step was used as a template, and primers were designed based on the UTR region of the gene to amplify TaSMAX1 gene, and the primer sequences were as follows: TaSMAX1-F: 5′-AGAATTCGTGCCCTTTGATC-3′, TaSMAX1-R: 5′-CTTTCTCAAATGCCCTCTACA-3′.

[0049] The PCR amplification system was 50 μL and included the following components: 2× PCR Buffer 25 μL, dNTPs (2 mM) 10 μL, cDNA 1 μL, KOD Fx 1 μL, Primer-F (10 μM) 1.5 μL, Primer-R (10 μM) 1.5 μL, and ddH2O 10 μL.

[0050] PCR reaction program: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 56℃ annealing for 30 sec, 68℃ extension for 3 min 30 sec, 35 cycles; 68℃ extension for 10 min, 12℃ end reaction. The amplified products were obtained, including TaSMAX1 The coding region sequence of the gene was separated by 1% agarose gel electrophoresis, and a band of about 3100 bp was seen ( Figure 1 ), where lane M is D2000 plus DNA Ladder; lanes 1 and 2 are TaSMAX1 The full-length cDNA is 3149 bp in size, and the specific nucleotide sequence is SEQ ID NO: 1. The target fragment is quickly cut out under a UV gel cutting instrument and chopped into pieces and placed in a clean centrifuge tube for gel recovery.

[0051] TaSMAX1 The coding sequence (CDS) of the gene in wheat material Fielder is SEQ ID NO: 1, positions 58-3123, and the encoded amino acid sequence is SEQ ID NO: 2. TaSMAX1 Protein. The genomic sequence encoding the TaSMAX1 protein in wheat genomic DNA is shown in SEQ ID NO: 3 in the sequence listing. Positions 1-1211 of SEQ ID NO: 3 are the first exon, positions 2764-3028 are the second exon, and positions 3117-4706 are the third exon.

[0052] 4. TaSMAX1 Ligation of pBM27 vector Using the product recovered from the previous step as a template, amplify with the following primers (pBM27-TaSMAX1-F: 5'-CACCATGAGGGCGGATCTCAGCA-3', pBM27-TaSMAX1-R: 5'-CATTCCATCGATGGCAATCG-3'; "CACC" in pBM27-TaSMAX1-F represents the pBM27 vector linker sequence). Use the same amplification system as in the previous step, add 50 ng of template, and make up to 50 μL with water. Follow the same PCR procedure as in the previous step. Separate the reaction by 1% agarose gel electrophoresis, and excise the desired band.

[0053] The gel-recovered product was ligated to the pBM27 vector using the pBM27 cloning kit. The reaction system was as follows: 100 ng of gel-recovered product, 1 μL of pBM27 Vector, 1 μL of 10× Toposmart, and ddH2O added to 10 μL. After addition, the mixture was gently flicked and centrifuged. The PCR instrument was controlled to react at 25°C for 30 min. After the reaction, the competent E. coli DH5α was transformed. After positive clones were identified and shaken, the plasmid was extracted and sent to the company for sequencing. The correctly sequenced plasmid was named pBM27- TaSMAX1 And stored at -20℃.

[0054] Recombinant vector pBM27- TaSMAX1 The structure of the pBM27 vector is described as follows: the fragment between 5'-CCCTT-3' and 5'-AAGGG-3' of the pBM27 vector is replaced with the 58th to 3123rd position of SEQ ID NO: 1, while keeping the other nucleotides of the pBM27 vector unchanged. TaSMAX1 Containing attL1- TaSMAX1 -attL2.

[0055] Example 2 TaSMAX1 Construction of overexpression vector Vector pBM27- TaSMAX1 After LR reaction, TaSMAX1 Connected to PC186 vector. The reaction system is pBM27- TaSMAX1 150 ng of protein, 150 ng of PC186, 1 μL of LR enzyme, and ddH2O were added to 5 μL. After overnight reaction at 25°C, the cells were transformed into E. coli DH5α competent cells. After single clone identification, the cells were shaken and the plasmid was extracted and sent to the company for sequencing. The correctly sequenced plasmid was named PC186- TaSMAX1 .

[0056] Recombinant vector PC186- TaSMAX1 The structure of the recombinant vector PC186- TaSMAX1 The fragment between attR1 site and attR2 site of PC186 vector is replaced with attB1- TaSMAX1 -attB2, the recombinant expression vector is obtained by keeping the other nucleotides of PC186 vector unchanged. TaSMAX1 Containing the 58th to 3123rd positions of SEQ ID NO: 1 in the sequence list TaSMAX1 The gene CDS sequence, Ubi promoter and NOS terminator can express TaSMAX1 protein, and the expression of the protein is driven by the Ubi promoter.

[0057] Example 3 TaSMAX1Gene overexpression in wheat significantly enhances salt tolerance 1. TaSMAX1 Obtaining overexpression transgenic plants will build the correct PC186- TaSMAX1 The vector was transformed into Agrobacterium competent EHA105, and single clones were picked for positive clone identification. The correctly identified positive clones were transferred into the callus tissue of the wheat material Fielder through Agrobacterium-mediated genetic transformation. Positive strains were screened using the Bar selection marker carried by the vector, and then moved into pots and cultured normally in the greenhouse (16 h light, 8 h dark), which were the T0 generation plants.

[0058] 2. TaSMAX1 Identification of overexpression positive plants 1) PCR identification Wheat leaf DNA was extracted using the CTAB method. 1 cm × 3 cm wheat leaves were cut and placed in a 2.0 mL centrifuge tube with steel balls. They were quickly frozen in liquid nitrogen for 2 min and then crushed into powder using a high-throughput grinder. Immediately after taking out, 600 μL of CTAB preheated at 65°C was added, and the mixture was thoroughly mixed and then placed in a 65°C water bath for 45 min. During this period, the mixture was inverted every 15 min to mix thoroughly. After taking out the centrifuge tube, 200 μL of chloroform was added, and the mixture was repeatedly inverted until thoroughly mixed. The tube was centrifuged at 12,000 rpm for 10 min at room temperature. 600 μL of the supernatant was carefully transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was thoroughly mixed and then centrifuged at 12,000 rpm for 10 min at room temperature. The supernatant was poured out, and 1 mL of 70% ethanol was added to wash the precipitate. The precipitate was centrifuged at 12,000 rpm for 10 min and the washing was repeated once. The residual ethanol was aspirated with a pipette tip, and the tube was opened and inverted in a clean bench to dry. 60 μL of DNA was dissolved in ddH2O, and the DNA concentration and quality were detected using a Nano Drop 2000 UV spectrophotometer. The DNA was diluted to 50 ng / μL with ddH2O and used as a standby.

[0059] Transgenic positive plants were identified by PCR. DNA was amplified using two pairs of primers (Primer 1: Primer-F1: 5'-ACTCGGTGGGGATGTTATCA -3', Primer-R1: 5'-GCCAAATGTTTGAACGATCGG-3'; Primer 2: Primer-F2: 5'-ACCAGGAAGCCCAGTGAAA-3', Primer-R2: 5'-GCCAAATGTTTGAACGATCGG-3'). The PCR system was as follows: 2×M5 HiPer plus Taq HiFi PCR Mix 7.5 μL, DNA 1 μL, Primer-F (10 μM) 0.5 μL, Primer-R (10 μM) 0.5 μL, and ddH2O 5.5 μL.

[0060] PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 56℃ annealing for 30 sec, 72℃ extension for 1 min (2 kb / min), 35 cycles; 72℃ extension for 10 min, 12℃ termination. PCR products were separated by 1.5% agarose gel electrophoresis. The results are as follows: Figure 2 As shown, lane M is D2000 plus DNA Ladder; lanes 1 and 2 are negative control Fielder, lanes 3 and 4 are OETaSMAX1 The upper and lower panels represent two different pairs of primers (primer 1 in the upper panel and primer 2 in the lower panel). The product size in the upper panel is 2028 bp, and the product size in the lower panel is 1536 bp. Positive plants were selected and selfed to obtain the T2 generation for subsequent phenotypic analysis.

[0061] 2) RNA level identification Extraction with Trizol TaSMAX1 The leaf RNA of the T2 generation of transgenic overexpression materials was reverse transcribed to obtain cDNA and the expression level of the target gene was analyzed by RT-qPCR. The primers used were TaSMAX1-qF1: 5'-GCGTCGTCCTCTGAGGGT-3', TaSMAX1-qR1: 5'-AGATCTTACACCTGAGGTAT-3'. The internal reference gene was TaActin The primers were TaActin-F: 5′-ACCTTCAGTTGCCCAGCAAT-3′, TaActin-R: 5′-CAGAGTCGAGCACAATACCAGTTG-3′.

[0062] The results are as follows Figure 3 As shown, compared with the transgenic recipient material Fielder, TaSMAX1In the overexpression lines TaSMAX1 The gene expression level was increased by about 14 times, indicating that the overexpression transgenic plant was successfully constructed. The obtained overexpression strain was named OETaSMAX1 .

[0063] 3. TaSMAX1 Overexpression enhances salt tolerance in wheat seedlings The above steps obtained TaSMAX1 Overexpression materials OETaSMAX1 The T2 generation homozygous seeds and transgenic recipient material Fielder were subjected to salt stress. OETaSMAX1 The seedlings of the materials were fixed with planting cotton and placed on hydroponic racks. After being cultured with 1 / 2 Hoagland nutrient solution for one week (three-leaf stage), salt treatment was started, using 250 mM NaCl treatment (NaCl was added to 1 / 2 Hoagland nutrient solution to a final concentration of 250 mM). The control group was cultured with 1 / 2 Hoagland nutrient solution (the nutrient solution and salt treatment solution were replaced every 3 days). After one week of treatment (long day, 16 h light, 8 h dark, 22°C), the materials showed obvious differences. The materials were photographed, and the treatment was continued for another week before rehydration. The phenotypes were observed and the survival rate was calculated 5 days after rehydration.

[0064] The results are as follows Figure 4 As shown, where A is Fielder, TaSMAX1 Overexpression materials OETaSMAX1 Phenotypes of seedlings under normal culture conditions and one-week salt treatment; B is the survival rate of seedlings after rehydration for 5 days after two weeks of salt stress treatment. More than 20 individual plants of different strains were measured, and the experiment was repeated three times. The values are mean ± SD, ** P <0.01, (Student's t Test) Compared with Fielder, after salt treatment TaSMAX1 The survival rate of the overexpression line was 91.54%, which was significantly higher than that of Fielder (18.15%). TaSMAX1 Enhanced salt tolerance of wheat.

[0065] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. 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, characterized in that: The protein is TaSMAX1 protein, which is any one of the following: a1) a protein having the amino acid sequence of SEQ ID NO: 2, a2) a protein having the same function as the amino acid sequence of SEQ ID NO: 2, wherein one or more amino acid residues are substituted and / or deleted and / or added. a3) A protein having an amino acid sequence identity of at least 80% with that specified in a1) or a2) and having the same function, a4) A fusion protein obtained by ligating a tag to the end of any of the proteins defined in a1) to a3); The application is any of the following: b1) Regulating plant salt tolerance, b2) Preparation of products for regulating plant salt tolerance, b3) Cultivating plants with altered salt tolerance, b4) preparing products for breeding plants with altered salt tolerance, b5) Plant breeding, b6) Preparation of products for use in plant breeding.

2. The use according to claim 1, characterized in that The protein is derived from wheat.

3. The use according to claim 1 or 2, characterized in that The regulation is at least one of the following 6 types of regulation: c1) regulation at the transcriptional level of the coding gene, c2) post-transcriptional regulation of the coding gene, c3) regulation of RNA transport of the coding gene, c4) regulating the translation of the coding gene, c5) regulating the degradation of the mRNA encoding the gene, c6) post-translational regulation of the gene.

4. The use according to claim 1 or 2, characterized in that The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein of claim 1 or 2, and the biological material is any one of the following: d1) a nucleic acid molecule encoding the protein according to claim 1 or 2; d2) an expression cassette containing the nucleic acid molecule described in d1); d3) a recombinant vector containing the nucleic acid molecule described in d1), or a recombinant vector containing the expression cassette described in d2); d4) a recombinant microorganism containing the nucleic acid molecule described in d1), or a recombinant microorganism containing the expression cassette described in d2), or a recombinant microorganism containing the recombinant vector described in d3); d5) a transgenic plant cell line containing the nucleic acid molecule described in d1), or a transgenic plant cell line containing the expression cassette described in d2); d6) transgenic plant tissue containing the nucleic acid molecule described in d1), or transgenic plant tissue containing the expression cassette described in d2), d7) a transgenic plant organ containing the nucleic acid molecule described in d1), or a transgenic plant organ containing the expression cassette described in d2); d1) The nucleic acid molecule is any one of the following: e1) The nucleotide sequence is the DNA molecule shown in SEQ IE NO: 3, e2) The coding region sequence is the DNA molecule shown in SEQ ID NO: 1 at positions 58-3123 in the sequence listing, e3) a DNA molecule that is 90% or more identical to the nucleotide sequence defined in e1) or e2), is derived from wheat and encodes the protein described above, e4) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in e1) or e2) and encodes the protein of claim 1 or 2.

5. A method for cultivating plants with improved salt tolerance, characterized in that: The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to cultivate plants with improved salt tolerance.

6. A method for improving plant salt tolerance, characterized in that: The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2 to improve the salt tolerance of the plant.

7. The method according to claim 5 or 6, characterized in that The regulation is at least one of the following 6 types of regulation: e1) Regulation at the transcriptional level of the coding gene, e2) Regulation after transcription of the coding gene, e3) regulation of RNA transport of the coding gene, e4) regulation of the translation of the coding gene, e5) regulating the degradation of the mRNA encoding the gene, e6) Post-translational regulation of the genes.

8. The method according to claim 5, 6 or 7, characterized in that: The method comprises the following steps: f1) constructing a recombinant expression vector for enhancing, increasing or upregulating the gene encoding the protein of claim 1 or 2; f2) Transforming the recombinant expression vector constructed in step f1) into a recipient plant (such as a crop or wheat) to obtain a plant having better salt tolerance than the recipient plant.

9. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any of the following plants: N1) Monocots or dicots; N2) Gramineae; N3) Grasses; N4) Triticum; N5) Wheat.

10. The product is characterized in that The product is the protein described in claim 1 or the biomaterial described in claim 4.

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