Application of TaSMAX1 protein and its coding gene in promoting salt tolerance of wheat seedlings

Abstract

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

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of TaSMAX1 protein and its coding gene in promoting salt tolerance of wheat seedlings. BACKGROUND

[0002] Wheat is a very important food crop in the world. Its planting area and annual yield in China are only inferior to corn and rice. Improving wheat yield is of great significance to guarantee food security in China and even the world. However, global climate change has led to frequent extreme weather, and environmental pressures such as soil salinization have posed a serious threat to wheat production and brought great challenges to global food security. Wheat is extremely sensitive to salt, and high salt can inhibit its growth, so its yield is often affected by high salt stress. Saline-alkali land is a reserve of cultivated land in China, with a total area of about 100 million mu, accounting for about 5% of the total available land area in China. Therefore, developing and utilizing saline-alkali land resources and using modern molecular breeding methods to accurately, quickly and efficiently improve the salt tolerance of wheat are of great significance to guarantee national food security. SUMMARY

[0003] The technical problem to be solved by the present application is how to improve the salt tolerance of wheat. To this end, the present application provides the application of a protein or a substance for regulating the expression of a coding gene of the protein or a substance for regulating the activity or content of the protein, wherein the protein is TaSMAX1 protein, and the protein is any one of the following:

[0004] a1) a protein with an amino acid sequence of SEQ ID NO: 2,

[0005] a2) a protein with an amino acid sequence shown in SEQ ID NO: 2 after substitution, deletion and / or addition of one or more amino acid residues and having the same function,

[0006] a3) a protein with an amino acid sequence identical to a1) or a2) by more than 80% and having the same function,

[0007] a4) a fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to a3);

[0008] The application is any one of the following:

[0009] b1) regulating the salt tolerance of plants,

[0010] b2) preparing a product for regulating the salt tolerance of plants,

[0011] b3) cultivating plants with changed salt tolerance,

[0012] b4) preparing a product for cultivating plants with changed salt tolerance,

[0013] b5) plant breeding,

[0014] b6) preparing a product for plant breeding.

[0015] In order to facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0016] The above-mentioned protein can be artificially synthesized, or a gene encoding the same can be synthesized first and then expressed biologically.

[0017] The tag protein includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0018] Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein TaSMAX1 isolated in the present application, as long as they encode the protein TaSMAX1 and have the function of the protein TaSMAX1, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0019] The above-mentioned 75% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0020] Herein, the identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence can be determined using the homology search site on the internet, such as the BLAST page of the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search in the Advanced BLAST 2.1, and then the value of the identity (%) can be obtained.

[0021] Herein, the more than 80% identity can 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.

[0022] Herein, the more than 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0023] In the above use, the protein is derived from wheat (Triticum aestivum L.). Triticum aestivum L.).

[0024] Herein, the substance that modulates the activity and / or the content of the protein can be a substance that modulates the expression of a gene that encodes the protein TaSMAX1.

[0025] In the above, the substance that modulates the expression of a gene can be a substance that performs at least one of the following 6 kinds of modulation: c1) modulation at the transcription level of the encoding gene, c2) modulation after the transcription of the encoding gene, c3) modulation of the RNA transport of the encoding gene, c4) modulation of the translation of the encoding gene, c5) modulation of the mRNA degradation of the encoding gene, and c6) post-translational modulation of the gene.

[0026] In the present application, the modulation can be up-regulation or enhancement or increase.

[0027] In the above use, the substance that modulates the expression of a gene or the substance that modulates the activity or the content of the protein can be a biological material related to the aforementioned protein, and the biological material can be any one of the following:

[0028] d1) a nucleic acid molecule that encodes the aforementioned protein;

[0029] d2) an expression cassette that contains the nucleic acid molecule of d1);

[0030] d3) a recombinant vector that contains the nucleic acid molecule of d1), or a recombinant vector that contains the expression cassette of d2);

[0031] d4) a recombinant microorganism that contains the nucleic acid molecule of d1), or a recombinant microorganism that contains the expression cassette of d2), or a recombinant microorganism that contains the recombinant vector of d3);

[0032] d5) a transgenic plant cell line that contains the nucleic acid molecule of d1), or a transgenic plant cell line that contains the expression cassette of d2);

[0033] d6) Transgenic plant tissue containing the nucleic acid molecules described in d1), or transgenic plant tissue containing the expression cassette described in d2),

[0034] d7) Transgenic plant organs containing the nucleic acid molecules described in d1), or transgenic plant organs containing the expression cassette described in d2);

[0035] In the above applications, the nucleic acid molecule described in d1) can be any of the following DNA molecules:

[0036] e1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 3.

[0037] e2) The coding region sequence is the DNA molecule shown in positions 58-3123 of SEQ ID NO: 1 in the sequence listing.

[0038] e3) has 90% or more identity with the nucleotide sequence defined by e1) or e2), and is a DNA molecule derived from wheat that encodes the protein described above.

[0039] e4) A DNA molecule that hybridizes under strict conditions to a nucleotide sequence defined by e1) or e2) and encodes the protein described above.

[0040] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0041] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the PC186 vector.

[0042] Existing plant expression vectors can be used to construct structures containing... TaSMAX1 Recombinant gene expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0043] use TaSMAX1In constructing the recombinant plant expression vector, any one of the enhancer promoters or constitutive promoters can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter, the ubiquitin promoter of corn, which can be used alone or in combination with other plant promoters; in addition, when the plant expression vector of the present application is used, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be the ATG initiation codon or the adjacent region initiation codon, but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the initiation codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0044] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can express enzymes or luminescent compounds that can produce color changes in plants (GUS genes, luciferase genes, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical reagent marker genes (such as anti-herbicide genes), etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0045] The gene or gene fragment provided by the present application is introduced into plant cells or recipient plants using any vector that can guide the expression of foreign genes in plants, so as to obtain transgenic cell lines and transgenic plants with altered salt tolerance. TaSMAX1 The expression vector carrying the gene can be transformed into plant cells or tissues by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc. Conventional biological methods, and the transformed plant tissues are cultivated into plants. TaSMAX1

[0046] The present application also provides a method for cultivating plants with improved salt tolerance, which comprises the step P of enhancing, improving or up-regulating the activity and / or content of the protein described above in the target plant, or / and, enhancing, improving or up-regulating the expression amount of the gene encoding the protein, to cultivate plants with improved salt tolerance.

[0047] The present application also provides a method for regulating the salt tolerance of wheat, which comprises the step P of enhancing, improving or up-regulating the activity and / or content of the protein described above in the target plant, or / and, enhancing, improving or up-regulating the expression amount of the gene encoding the protein, to positively regulate the salt tolerance of wheat.

[0048] ​In the above method, the increasing expression amount and / or activity of the gene encoding the protein TaSMAX1 in the target plant can be increasing the activity of the gene encoding the protein TaSMAX1 in the genome of the target plant by overexpression.

[0049] The present application provides a method for obtaining a plant with enhanced salt tolerance, which comprises overexpressing the expression of the gene encoding the above protein and / or the content and / or activity of the above protein in a target plant, so as to obtain a plant with increased salt tolerance and finally increased yield under salt stress.

[0050] In one embodiment of the present application, the method can comprise the following steps:

[0051] f1) constructing a recombinant expression vector for enhancing, increasing or up-regulating the gene encoding the above protein;

[0052] f2) transforming the recombinant expression vector constructed in step f1) into a recipient plant (such as a crop or wheat) to obtain a plant with better salt tolerance than the recipient plant.

[0053] In the present application, the index of plant breeding can include plant salt tolerance. The purpose of plant breeding can include breeding plants with improved salt tolerance.

[0054] In the above application or method, the plant can be any of the following: N1) a monocotyledon or a dicotyledon, N2) a plant of Poales, N3) a plant of Poaceae, N4) a plant of Triticum, and N5) wheat.

[0055] The present application also provides a product, which can be the above protein or biological material.

[0056] The present application provides a cloning method for a wheat salt tolerance gene TaSMAX1 TaSMAX1 is an important protein for regulating wheat salt tolerance, and increasing the expression amount can significantly enhance the salt tolerance of wheat. TaSMAX1 The present application provides a cloning method for a wheat salt tolerance gene TaSMAX1 The present application provides a cloning method for a wheat salt tolerance gene BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The cloning of the full-length cDNA of the target gene is shown in the figure. TaSMAX1 The full-length cDNA is shown in lanes 1 and 2, and its size is 3139 bp. TaSMAX1 The full-length cDNA is shown in lanes 1 and 2, and its size is 3139 bp.

[0058] TaSMAX1 The cloning of the full-length cDNA of the target gene is shown in the figure. Figure 2Transgenic line T0 identification results.

[0059] TaSMAX1 For Figure 3 Overexpression of material gene expression level detection.

[0060] TaSMAX1 For Figure 4 Overexpression of material seedling stage under normal conditions, after salt stress treatment of phenotype. DETAILED DESCRIPTION

[0061] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0062] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0063] In the quantitative experiments in the following examples, unless otherwise specified, three repeated experiments were set up, and the results were averaged.

[0064] The wheat material "Fielder" in the following examples was a gift from Mr. Li Genying of Crop Research Institute, Shandong Agricultural Academy of 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 wheat grain quality improvement. Plant Biotechnology Journal, 19(9): 1684-1686, which is publicly available from the applicant. This biological material is only used for repeating the experiments of the application, and cannot be used for other purposes.

[0065] In the following examples, the E. coli strain DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd., China.

[0066] In the following examples, the Agrobacterium strain EHA105 was purchased from Beijing Bomeide Gene Technology Co., Ltd., China.

[0067] In the following examples, the vector pBM27 was purchased from Beijing Bomeide Biotechnology Co., Ltd., China.

[0068] The vector PC186 in the following examples was a gift from Professor Fu Daolin, College of Agronomy, Shandong Agricultural University. It has been described on page 11, right column, last 4th line of the following document, which is publicly available from the applicant, and which is only for the purpose of repeating the experiments of the present invention and not for any other use: 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 TaSMAX1 powdery mildew in barley. Molecular Plant Pathology. 19(8): 1995-2010.

[0069] The following examples use SPSS 19.0 statistical software to process the data, and the experimental results are expressed as mean ± standard deviation, and Student's t t-test is used, and the results of * and ** represent significant differences at the 0.05 and 0.01 levels, respectively.

[0070] Example 1, Fusarium graminearum Cloning of the gene

[0071] 1. Extraction of total RNA from wheat leaves

[0072] Extraction of RNA using Trizol method

[0073] (1) Take 150 mg-200 mg of Fielder material seedling leaves and place them in an RNase free mortar pre-cooled in liquid nitrogen. After grinding the material into a uniform powder in liquid nitrogen, add it to a 2.0 mL RNase free centrifuge tube;

[0074] (2) Add 1 mL Trizol to the centrifuge tube, vortex, and then let it stand at room temperature for 5 min;

[0075] (3) Add 200 μL of chloroform, shake vigorously for 30 s, and then let it stand at room temperature for 5 min;

[0076] (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;

[0077] (5) Add the same volume of isopropanol as the supernatant, mix well by inverting, and then stand at room temperature for 5-10 min, centrifuge at 12,000 rpm and 4°C for 15 min, and pour out the supernatant while retaining the precipitate;

[0078] (6) Add 1 mL of 75% alcohol to wash the precipitate, centrifuge at 12,000 rpm and 4°C for 5 min, and discard the supernatant;

[0079] (7) Repeat step (6), and then place the gun head in a clean bench to dry after the alcohol is absorbed;

[0080] (8) Add 50 μL of RNase-free ddH2O to dissolve the RNA, and remove polysaccharides and polyphenols at 50°C for 5-10 min;

[0081] (9) Centrifuge at 12,000 rpm and 4°C for 5-10 min, and then aspirate the supernatant into a new 1.5 mL RNase-free centrifuge tube, measure the concentration, or store at -80°C for standby.

[0082] 2. cDNA synthesis

[0083] The HiScript® III RT SuperMix for qPCR (+gDNA wiper) kit from Novozyme was used for reverse transcription, and the whole process was operated on ice. The reaction system was as follows: the gDNA removal reaction system included 1.5 μg of RNA template, 4 μL of 4×gDNA wiperMix, and RNase-free ddH2O was supplemented to 16 μL. After mixing with a pipette gun, the PCR instrument was controlled at 42°C for 2 min.

[0084] The reverse transcription system included 16 μL of the product of the reaction system in the previous step, 4 μL of 5×HiScript III qRT SuperMix, and after mixing with a pipette gun, the following reverse transcription reaction program was performed in a PCR instrument: 50°C for 15 min, 85°C for 5 sec, and then immediately placed on ice after the reaction was completed. After dilution with 180 μL of ddH2O, it was ready for use and stored at -20°C.

[0085] 3、 TaSMAX1 Gene cloning

[0086] The cDNA of the wheat leaf obtained in the previous step was used as a template, and the primers were designed according to the UTR region of the gene to amplify TaSMAX1 the gene. The primer sequences were as follows: TaSMAX1-F: 5'-AGAATTCGTGCCCTTTGATC-3', and TaSMAX1-R: 5'-CTTTCTCAAATGCCCTCTACA-3'.

[0087] PCR amplification system is 50 microliters, including the following components: 2 x 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, ddH2O 10 μL.

[0088] PCR reaction program: 94℃ pre-denaturation 5 min; 98℃ denaturation 10 sec, 56℃ annealing 30 sec, 68℃ extension 3 min 30 sec, 35 cycles; 68℃ extension 10 min, 12℃ end reaction. The amplified product, including the coding region sequence of TaSMAX1 , is separated by 1% agarose gel electrophoresis, and a band of about 3100 bp can be seen TaSMAX1 , wherein M lane is D2000 plus DNA Ladder; lanes 1 and 2 are Figure 1 cDNA full-length, which is 3149 bp in size, and the specific nucleotide sequence is SEQ ID NO: 1. The band of the target fragment is quickly cut under the ultraviolet gel instrument and cut into a clean centrifuge tube for gel recovery.

[0089] TaSMAX1 The coding sequence (CDS) of the gene in the wheat material Fielder is 58-3123 of SEQ ID NO: 1, and the encoded amino acid sequence is SEQ ID NO: 2 TaSMAX1 Protein. The genomic sequence encoding the TaSMAX1 protein in the genomic DNA of wheat is shown in SEQ ID NO: 3 of the sequence listing. The first exon is 1-1211 of SEQ ID NO: 3, the second exon is 2764-3028, and the third exon is 3117-4706.

[0090] 4、 TaSMAX1 Linking pBM27 vector

[0091] The above step gel recovery product is used as a template for the following primer amplification (pBM27-TaSMAX1-F: 5'-CACCATGAGGGCGGATCTCAGCA-3', pBM27-TaSMAX1-R: 5'-CATTCCATCGATGGCAATCG-3', "CACC" in pBM27-TaSMAX1-F is the pBM27 vector linker sequence), and the amplification system is the same as the above step. Add 50 ng of template, and finally add water to 50 microliter system. The PCR reaction program is the same as the above step, and after the reaction, 1% agarose gel electrophoresis is used for separation, and the target band is cut and recovered.

[0092] The gel-recovered product was ligated into the pBM27 vector using a pBM27 cloning kit. The reaction mixture was as follows: 100 ng gel-recovered product, 1 μL pBM27 Vector, 1 μL 10×Toposmart, and ddH2O to a final volume of 10 μL. After addition, the mixture was gently swirl-mixed and centrifuged. The reaction was carried out at 25°C for 30 min using a PCR instrument. After the reaction, the cells were transformed into competent E. coli DH5α cells. Positive clones were identified and cultured, and plasmids were extracted and sent to the company for sequencing. The correctly sequenced plasmid was named pBM27- TaSMAX1 Store at -20℃.

[0093] Recombinant vector pBM27- TaSMAX1 The structure is described as follows: It is a recombinant vector obtained by replacing the fragment between 5'-CCCTT-3' and 5'-AAGGG-3' of the pBM27 vector with positions 58-3123 of SEQ ID NO: 1, while keeping the other nucleotides of the pBM27 vector unchanged. Recombinant vector pBM27- TaSMAX1 Contains attL1- TaSMAX1 -attL2.

[0094] Example 2 TaSMAX1 Construction of overexpression vectors

[0095] vector pBM27- TaSMAX1 via LR reaction TaSMAX1 Connected to the PC186 support. The reaction system is pBM27- TaSMAX1 150 ng of PC186, 150 ng of LR enzyme, and ddH2O to a final volume of 5 μL were added. The mixture was incubated overnight at 25°C and then transformed into *E. coli* DH5α competent cells. Single clones were picked for identification, followed by culture and plasmid extraction. The plasmid was sent to the company for sequencing, and the correctly sequenced plasmid was named PC186-. TaSMAX1 .

[0096] Recombinant vector PC186- TaSMAX1 The structure is described as follows: After the LR reaction, the recombinant vector PC186- TaSMAX1 This involves replacing the fragment between the attR1 and attR2 sites in the PC186 vector with attB1- TaSMAX1 -attB2, a recombinant expression vector obtained by keeping other nucleotides of the PC186 vector unchanged. PC186- TaSMAX1 Contains bits 58-3123 of SEQ ID NO: 1 in the sequence list. TaSMAX1 The gene's CDS sequence, Ubi promoter, and NOS terminator enable the expression of the TaSMAX1 protein, whose expression is driven by the Ubi promoter.

[0097] Example 3, TaSMAX1 Overexpression of the gene in wheat significantly enhances the salt tolerance of wheat

[0098] 1、 TaSMAX1 Obtaining of overexpression transgenic plants

[0099] Construction of correct PC186- TaSMAX1 The vector was transformed into Agrobacterium competent EHA105, single colonies were picked for positive clone identification, and the correctly identified positive clones were transformed into the callus of wheat material Fielder by Agrobacterium-mediated genetic transformation method, and positive plant screening was performed by the Bar screening marker carried by the vector, then it was moved to a flowerpot and normally cultured in a greenhouse (16 h light, 8 h darkness), which was the T0 generation plant.

[0100] 2、 TaSMAX1 Identification of overexpression positive plants

[0101] 1) PCR identification

[0102] CTAB method was used to extract wheat leaf DNA, 1 cm x 3 cm wheat leaves were cut and placed in a 2.0 mL centrifuge tube with steel balls, quickly frozen in liquid nitrogen for 2 min, then ground into powder with a high-throughput grinder, and immediately added with 600 μL of preheated CTAB at 65°C, mixed well, then incubated at 65°C for 45 min, and inverted and mixed every 15 min during the incubation; after centrifugation, 200 μL of chloroform was added, inverted and mixed until well mixed, then centrifuged at 12000 rpm at room temperature for 10 min; 600 μL of supernatant was carefully transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added, mixed well, then centrifuged at 12000 rpm at room temperature for 10 min; the supernatant was discarded, 1 mL of 70% ethanol was added to wash the precipitate, centrifuged at 12000 rpm for 10 min, and repeated once; the residual ethanol was aspirated with a gun head, and the tube was inverted on the clean bench to dry; 60 μL of ddH2O was added to dissolve the DNA, and the DNA concentration and quality were detected by a micro UV spectrophotometer Nano Drop 2000, and the DNA was diluted with ddH2O to 50 ng / μL for standby.

[0103] The transgenic positive plants were identified by PCR. 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') were used to amplify the DNA. 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, ddH2O 5.5 μL.

[0104] PCR reaction procedure: 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℃ to terminate the reaction. PCR products were separated by 1.5% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown, lane M is the D2000 plus DNA Ladder; lanes 1 and 2 are negative control Fielder, and lanes 3 and 4 are... OETaSMAX1 The top and bottom images represent two different pairs of primers for identification (primer 1 is used in the top image; primer 2 is used in the bottom image). The product size in the top image is 2028 bp, and the product size in the bottom image is 1536 bp. Positive plants were selected and self-crossed to obtain the T2 generation for subsequent phenotypic analysis.

[0105] 2) RNA level identification

[0106] Extraction using the Trizol method TaSMAX1 RNA from leaves of the T2 generation of transgenic overexpression material was reverse transcribed to obtain cDNA, and the expression level of the target gene was analyzed by RT-qRCR. The primers used were TaSMAX1-qF1: 5'-GCGTCGTCCTCTGAGGGT-3' and TaSMAX1-qR1: 5'-AGATCTTACACCTGAGGTAT-3'. The internal reference gene was... TaActin The primers are TaActin-F: 5'-ACCTTCAGTTGCCCAGCAAT-3' and TaActin-R: 5'-CAGAGTCGAGCACAATACCAGTTG-3'.

[0107] The results are as follows Figure 3As shown, compared to the transgenic receptor material Fielder, TaSMAX1 In overexpression lines TaSMAX1 The gene expression level was upregulated by approximately 14-fold, indicating that the overexpression transgenic plant was successfully constructed. The obtained overexpression line was named... OETaSMAX1 .

[0108] 3. TaSMAX1 Overexpression enhances salt tolerance in wheat seedlings

[0109] The results obtained in the above steps TaSMAX1 Overexpression materials OETaSMAX1 T2 generation homozygous seeds and transgenic recipient material Fielder were subjected to salt stress treatment. Fielder seeds of uniform size and one week after germination were harvested. OETaSMAX1 After the seedlings were fixed with planting cotton, they were placed on a hydroponic rack and cultured in 1 / 2 Hoagland nutrient solution for one week (three-leaf stage). Salt treatment was then initiated using 250 mM NaCl (NaCl was added to the 1 / 2 Hoagland nutrient solution to make the final concentration 250 mM). The control group was cultured in 1 / 2 Hoagland nutrient solution (the nutrient solution and salt treatment solution were changed every 3 days). After one week of treatment (long day, 16 h light, 8 h darkness, 22℃), the differences in the materials were obvious, and photos were taken. After another week of treatment, the materials were rehydrated. Five days after rehydration, the phenotypes were observed and the survival rate was calculated.

[0110] 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 after one week of salt treatment; B represents the survival rate of seedlings 5 ​​days after rehydration following two weeks of salt stress treatment. More than 20 individual plants from different lines were measured, and the experiment was repeated three times. Values ​​are expressed as mean ± SD. P <0.01, (Student's t (Test) Compared to Fielder, after salt treatment TaSMAX1 The survival rate of the overexpression lines was 91.54%, significantly higher than that of Fielder (18.15%). Therefore, compared with the control Fielder, overexpression in wheat is a more effective treatment. TaSMAX1 It enhances the salt tolerance of wheat.

[0111] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. Use of a substance which up-regulates or enhances or increases the expression of a gene encoding a protein or a substance which up-regulates or enhances or increases the activity or content of a protein, characterised in that, The protein is TaSMAX1 protein, which is a protein with an amino acid sequence of SEQ ID NO: 2, The application is any one of the following: b1) increasing salt tolerance of plants, b2) preparing a product for increasing salt tolerance of plants, b3) breeding plants with enhanced salt tolerance, b4) preparing a product for breeding plants with enhanced salt tolerance, The plant is wheat.

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

3. Use according to claim 1 or 2, characterized in that, The substance is a biological material related to the protein in claim 1 or 2, and the biological material is any one of the following: d1) a nucleic acid molecule encoding the protein in claim 1 or 2; d2) an expression cassette containing the nucleic acid molecule in d1); d3) a recombinant vector containing the nucleic acid molecule in d1), or a recombinant vector containing the expression cassette in d2); d4) a recombinant microorganism containing the nucleic acid molecule in d1), or a recombinant microorganism containing the expression cassette in d2), or a recombinant microorganism containing the recombinant vector in d3); d5) a transgenic plant cell line containing the nucleic acid molecule in d1), or a transgenic plant cell line containing the expression cassette in d2); d6) a transgenic plant tissue containing the nucleic acid molecule in d1), or a transgenic plant tissue containing the expression cassette in d2), d7) a transgenic plant organ containing the nucleic acid molecule in d1), or a transgenic plant organ containing the expression cassette in d2); The nucleic acid molecule in d1) is any one of the following: e1) a DNA molecule with a nucleotide sequence shown in SEQ ID NO: 3, e2) a DNA molecule with a coding region sequence shown in positions 58-3123 of SEQ ID NO: 1 in the sequence listing.

4. A method for breeding a plant with improved salt tolerance, characterized in that, The method comprises step P, which is enhancing, increasing or up-regulating the activity and / or content of the protein in claim 1 or 2 in the target plant, or / and, enhancing, increasing or up-regulating the expression amount of the gene encoding the protein in claim 1 or 2, to breed plants with enhanced salt tolerance; The plant is wheat.

5. A method for increasing salt tolerance in plants, characterized by, The method comprises step P, which is enhancing, increasing or up-regulating the activity and / or content of the protein in claim 1 or 2 in the target plant, or / and, enhancing, increasing or up-regulating the expression amount of the gene encoding the protein in claim 1 or 2, to increase salt tolerance of plants; The plant is wheat.

6. The method according to claim 4 or 5, characterized in that, The method comprises the following steps: f1) constructing a recombinant expression vector for enhancing, increasing or up-regulating the gene encoding the protein in claim 1 or 2; f2) transforming the recombinant expression vector constructed in step f1) into a recipient plant to obtain a plant with better salt tolerance than the recipient plant.

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