Plant salt tolerance related transcription factor TaVOZ1 as well as coding gene and application thereof

Abstract

The invention provides a plant salt tolerance related transcription factor TaVOZ1 as well as a coding gene and application thereof, and belongs to the technical field of functional genes. The TaVOZ1 is related to plant salt tolerance and has an amino acid sequence as shown in SEQ ID No. 1, and a gene for coding the protein has a nucleotide sequence as shown in SEQ ID No. 2. The plant salt tolerance can be regulated by regulating the content or activity of the protein or the expression quantity of the gene, for example, when the gene is over-expressed, the plant salt tolerance is enhanced, and when the gene is knocked down, the salt tolerance is weakened. The TaVOZ1 disclosed by the invention can be combined with 5 '-CTTCT-3'or a reverse complementary sequence 5'-AAGAAG-3 'in a downstream target gene promoter to activate the expression of a stress response gene so as to improve the salt tolerance of a plant, so that the TaVOZ1 can be used for culturing a salt-tolerant wheat variety.

Description

Technical Field

[0001] The invention belongs to the technical field of functional genes, and in particular relates to a plant salt tolerance-related transcription factor TaVOZ1 and a coding gene and application thereof. Background Art

[0002] Soil salinization is one of the most common abiotic stresses, which seriously affects agricultural production worldwide. Wheat (Triticum aestivum L.) is one of the main sources of carbohydrates and proteins for humans.

[0003] Excess cations in saline soils (such as Na + ) or anions (such as Cl - ) will lead to reduced absorption of water and nutrients by wheat, resulting in osmotic stress and ion stress, affecting wheat's physiological and biochemical functions such as photosynthesis and protein synthesis, thereby reducing wheat yield and quality. Although wheat is the food crop with the largest planting area in the world, wheat is relatively sensitive to salt stress. Therefore, improving wheat salt tolerance and cultivating salt-tolerant wheat varieties are particularly important to ensure my country's food security and sustainable agricultural development. However, crop salt tolerance is a complex quantitative trait controlled by multiple genes. Traditional breeding methods can no longer meet the needs of genetic improvement of crop salt tolerance. It is urgent to tap new gene resources through molecular breeding methods to improve wheat salt tolerance in a targeted manner. Therefore, mining salt-tolerant genes in wheat is of great significance for cultivating salt-tolerant wheat varieties and increasing wheat yields. Summary of the invention

[0004] The present invention provides a plant salt tolerance related transcription factor TaVOZ1 and a coding gene and application thereof; the TaVOZ1 can regulate plant salt tolerance and be used for breeding salt tolerance varieties.

[0005] The first object of the present invention is to provide a plant salt tolerance related protein, wherein the protein comprises an amino acid sequence as shown in any one of the following: 1) an amino acid sequence as shown in SEQ ID No. 1;

[0006] 2) The amino acid sequence of a derivative protein related to plant salt tolerance obtained by replacing, deleting and / or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No. 1.

[0007] The second object of the present invention is to provide a gene encoding the above protein.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the gene includes a nucleotide sequence as shown in any one of the following: 1) a nucleotide sequence as shown in SEQ ID No. 2;

[0009] 2) the CDS sequence in the nucleotide sequence shown in SEQ ID No. 2;

[0010] 3) A DNA molecule that has at least 70% homology with the nucleotide sequence defined in 1) or 2) and encodes the protein of claim 1;

[0011] 4) A DNA molecule that hybridizes with the DNA sequence defined in 1), 2) or 3) under stringent conditions and encodes the above-mentioned protein.

[0012] The third object of the present invention is to provide a biomaterial for regulating the content or activity of the above-mentioned protein and / or the expression amount of the above-mentioned gene.

[0013] In a preferred embodiment of the present invention, the types of biological materials include vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses.

[0014] The fourth invention objective of the present invention is to provide the use of the above protein or the above gene in improving the salt tolerance of plants.

[0015] In a preferred embodiment of the present invention, the plant includes a monocotyledonous plant or a dicotyledonous plant.

[0016] A fifth object of the present invention is to provide the use of the above-mentioned biological material in regulating plant salt tolerance.

[0017] The sixth invention object of the present invention is to provide a method for improving plant salt tolerance, comprising increasing the content or activity of the above protein in the target plant, or increasing the expression level of the above gene in the target plant.

[0018] In a preferred embodiment of the present invention, the method for increasing the expression level of the gene in the target plant comprises transforming the target plant using a recombinant expression vector, wherein the recombinant expression vector uses a plant expression vector as a base vector and connects the gene to the base vector.

[0019] Beneficial effects: The present invention provides a protein TaVOZ1 derived from wheat (Triticum aestivum L.), wherein TaVOZ1 is a transcription factor protein, and TaVOZ1 acts as a transcription activator, which activates the expression of stress response genes by binding to 5'-CTTCTT-3' or its reverse complementary sequence 5'-AAGAAG-3' in the promoter of downstream target genes to improve plant salt tolerance. In an embodiment of the present invention, the gene encoded by the transcription factor protein TaVOZ1 is introduced into wheat, and the salt tolerance of the obtained transgenic plants is enhanced; and after silencing the TaVOZ1 gene by RNAi, the salt tolerance of the obtained transgenic plants is weakened. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression pattern analysis diagram of TaVOZ1 gene in wheat leaves (A) and roots (B);

[0021] Figure 2 The results of the analysis of TaVOZ1 gene expression level (A) and protein level (B) in TaVOZ1 overexpression (OE) and silenced (RNAi) strains;

[0022] Figure 3 This is the result of identifying the salt tolerance phenotype of TaVOZ1 OE and RNAi strains;

[0023] Figure 4 Biomass analysis in leaves of TaVOZ1 OE and RNAi lines under normal growth (A) or 150 mM NaCl treatment (B).

[0024] Figure 5 Figure 2 NaCl levels in leaves of TaVOZ1 OE and RNAi strains under normal growth (A) or 150 mm NaCl treatment (B). + Content analysis result diagram;

[0025] Figure 6 Figure 1 shows the root system of TaVOZ1 OE and RNAi lines grown under 150 mm NaCl or hydroponic conditions for 20 days;

[0026] Figure 7 Comparison of root lengths of TaVOZ1 OE and RNAi strains grown under normal growth (A) and 150 mM NaCl conditions (B) for 20 days;

[0027] Figure 8 This is the result of alignment of TaVOZ1 homologous protein sequences;

[0028] Fig. 9 This is the phylogenetic analysis diagram of TaVOZ1 homologous genes;

[0029] Fig.10 This is the result of subcellular localization of TaVOZ1 in wheat protoplasts;

[0030] Fig.11 This is a diagram of the yeast transcription activity assay results;

[0031] Fig.12 This is a graph showing the results of dual luciferase assay to measure the transcriptional activity of TaVOZ1;

[0032] Fig.13 The binding motif results of TaVOZ1 were analyzed by DNA affinity purification sequencing (DAP-seq);

[0033] Fig.14Results of screening candidate target genes (A) and GO enrichment analysis (B and C) for the combination of transcriptome sequencing (RNA-seq) and DAP-seq. DETAILED DESCRIPTION

[0034] The present invention provides a plant salt tolerance related protein, the protein comprising an amino acid sequence shown in any one of the following: 1) an amino acid sequence shown in SEQ ID No. 1;

[0035] 2) The amino acid sequence of a derivative protein related to plant salt tolerance obtained by replacing, deleting and / or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No. 1.

[0036] The protein of the present invention is a transcription factor TaVOZ1 protein, which is derived from wheat and has an amino acid sequence as shown in SEQ ID No. 1:

[0037] ; Mutations can also be made based on the sequence shown in SEQ ID No. 1. For example, derivative proteins that are still related to plant salt tolerance after substitution, deletion and / or addition of one or several amino acid residues are all within the scope of protection of the present invention.

[0038] The present invention does not specifically limit the preparation method of the protein. The protein can be directly synthesized by artificial synthesis, or obtained by synthesizing the encoding gene and then expressing it. Of course, the N-terminus and / or C-terminus of the protein can also be chemically modified, such as the various tags shown in Table 1 for purification.

[0039] Table 1 Tags that can be linked to the proteins of the present invention (part)

[0040] Label Residue sequence SEQ ID No. Poly-Arg 5-6 (usually 5) RRRRR 3 Poly-His 2-10 (usually 6) HHHHHH 4 FLAG 8 DYKDDDDK 5 Strep-tagII 8 WSHQ 6 c-myc 10 EQKLISEEDL 7 HA 9 YPYDVPDYA 8

[0041] The present invention provides a gene encoding the above protein.

[0042] The gene of the present invention includes both genomic sequences and CDS sequences, sequences homologous to the genomic sequences or CDS sequences, and all DNA sequences that hybridize with all the above sequences under stringent conditions and encode the above amino acid sequences.

[0043] In a specific embodiment of the present invention, the genome sequence is shown in SEQ ID No. 2:

[0044]

[0045] At the same time, the gene of the present invention can also have more than 70% homology with the genome sequence or CDS sequence and encode the DNA molecule of the above-mentioned protein, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology.

[0046] In the present invention, DNA molecules that hybridize with the above-mentioned genomic sequence, CDS sequence or homologous sequence under stringent conditions and encode the above-mentioned protein also fall within the scope of protection of the gene described in the present invention. It is worth noting that the stringent conditions referred to in the present invention may be hybridization under heating conditions using a mixed solution containing SDS, followed by rinsing or washing the membrane using a solution containing SSC, specifically, under any of the following conditions:

[0047] ①: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA; 50°C, rinse in a mixed solution of 2×SSC and 0.1% SDS;

[0048] ②: 50℃, hybridize in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 50℃, rinse in a mixed solution of 1×SSC and 0.1% SDS;

[0049] ③: 50℃, hybridize in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 50℃, rinse in a mixed solution of 0.5×SSC and 0.1% SDS;

[0050] ④: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 50°C, rinse in a mixed solution of 0.1×SSC and 0.1% SDS;

[0051] ⑤: 50°C, hybridization in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 65°C, rinse in a mixed solution of 0.1×SSC and 0.1% SDS;

[0052] ⑥: Hybridize at 65°C in a mixed solution of 6×SSC and 0.5% SDS; wash the membrane once with a mixed solution of 2×SSC and 0.1% SDS, and once with a mixed solution of 1×SSC and 0.1% SDS.

[0053] The present invention provides a biological material for regulating the content or activity of the above protein and / or the expression amount of the above gene.

[0054] The regulation and control of the present invention includes up-regulation, and when the regulation is up-regulated, the biological material comprising the above-mentioned gene and expressing the above-mentioned protein or the biological material improving the activity of the protein is included. The present invention does not have any special restrictions on the type of the biological material, and can be a vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus. In a preferred embodiment of the present invention, the vector can be a recombinant expression vector formed after connecting the gene on the basis of a plant expression vector, and the plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company), etc. The plant expression vector can also include the 3' end non-translated region of the foreign gene, i.e., include a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylation to the 3' end of the mRNA precursor. For example, the non-translated region transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as annatto synthase Nos gene) and plant genes (such as soybean storage protein gene) all have similar functions.

[0055] When using the gene to construct a recombinant plant expression vector, the present invention can add any enhanced promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin)), constitutive promoter or tissue-specific expression promoter (such as seed-specific expression promoter) before the transcription initiation nucleotide, and the promoter can be used alone or in combination with other plant promoters. 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 the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene. 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 be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to methatrexate, and the EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose.

[0056] Specifically, in one embodiment of the present invention, the wheat overexpression vector is based on LGY-OE3, and the gene is inserted between the AvrII and BamHI restriction sites of the LGY-OE3 vector to obtain the overexpression vector LGY-OE3-TaVOZ1.

[0057] The biomaterial of the present invention also includes a recombinant vector for down-regulating the gene expression, such as RNAi. In one embodiment, pC336 (Ubi: GWRNAi: Nos) is used as a basic vector, and a targeting sequence is inserted between the attR1 and attR2 sites of the pC336 (Ubi: GWRNAi: Nos). The targeting sequence in the embodiment selects 664 to 868 bp in the sequence shown in SEQ ID No. 2 to construct an RNAi vector pC336-TaVOZ1.

[0058] The biomaterial of the present invention also includes a transcription activator of the protein. The transcription activator may be the protein, or may be the transcription activator prepared by using the protein as a raw material.

[0059] The present invention also provides the use of the above protein or the above gene in improving the salt tolerance of plants.

[0060] The protein or gene of the present invention can be used to increase the content or activity of the protein, or to increase the expression level of the gene, so as to improve the salt tolerance of plants.

[0061] The present invention also provides application of the above biological material in regulating plant salt tolerance.

[0062] In the present invention, the various materials in the biological material that can upregulate the content or activity of the protein, or upregulate the gene expression amount, can improve the salt tolerance of the plant, and the plant can be a dicot or a monocot. For example, in one embodiment of the present invention, wheat (Triticum aestivum L.) was used as a representative of monocot plants for experiments, and it was found that the gene encoding the transcription factor protein TaVOZ1 was introduced into wheat, and the salt tolerance of the transgenic plants obtained was enhanced. In one embodiment of the present invention, the material in the biological material that downregulates the gene expression, such as silencing the TaVOZ1 gene by RNAi, the salt tolerance of the transgenic plants obtained was weakened, that is, the above-mentioned RNAi vector pC336-TaVOZ1 can reduce the salt tolerance of plants. Therefore, the biological material described in the present invention can be used to regulate the salt tolerance of plants.

[0063] The present invention finds that the gene TaVOZ1 acts as a transcription activator, and activates the expression of stress response genes to improve plant salt tolerance by binding to 5'-CTTCTT-3' or its reverse complement sequence 5'-AAGAAG-3' in the promoter of the downstream target gene.

[0064] The present invention also provides a method for improving plant salt tolerance, comprising increasing the content or activity of the above protein in the target plant, or increasing the expression level of the above gene in the target plant.

[0065] The present invention increases the content or activity of the above protein in the target plant, or increases the expression level of the nucleic acid molecule encoding the above protein in the target plant, all by introducing the nucleic acid molecule encoding the above protein into the target plant.

[0066] To further illustrate the present invention, a plant salt tolerance-related transcription factor TaVOZ1 and its encoding gene and application provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods; and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0068] The vector LGY-OE3 used in the embodiments of the present invention has been disclosed in the literature (Niaz M, Zhang L, Lv G, HuH, Yang X, Cheng Y, Zheng Y, Zhang B, Yan X, Htun A, Zhao L, Sun C, Zhang N, Ren Y, ChenF. Identification of TaGL1-B1 gene controlling grain length through regulation of jasmonic acid in common wheat. Plant Biotechnol J. 2023, 21 (5): 979-989.); the vector pC336 (Ubi: GWRNAi: Nos) is recorded in the literature: WangY, Liu C, Du Y, Cai K, Wang Y, Guo J, Bai X, Kang Z, Guo JA stripe rust fungal effector PstSIE1 targets TaSGT1 to facilitate pathogen infection. Plant J.2022,112(6):1413-1428., vector pJIT163 is recorded in the literature: Li S, Zhang Y, Liu Y, Zhang P, Wang X, Chen B, Ding L, Nie Y, Li F, Ma Z, Kang Z, Mao H.The E3 ligase TaGW2mediates transcription factor TaARR12 degradationto promote drought resistance in wheat. Plant Cell.2024,36(3):605-625., which is publicly available from Northwest Agriculture and Forestry University.

[0069] The Agrobacterium tumefaciens GV3101+pSoup strain used in the embodiment of the present invention is recorded in the literature: Scholthof HB, Alvarado VY, Vega-Arreguin JC, Ciomperlik J, Odokonyero D, Brosseau C, Jaubert M, Zamora A, Moffett P. Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana. Plant Physiol. 2011, 156 (3): 1548-55.; the wheat variety Chinese Spring is recorded in the literature: Mao H, Li S, Wang Z, Cheng X, Li F, Mei F, Chen N, Kang Z. Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings. Plant Biotechnol J. 2020, 18 (4): 1078-1092.; the wheat variety Fielder is recorded in the literature: Li S, Li T, Zhang P, Wang X, Feng W, Zhang Y, Chen B, Liu Y, Zhan G, Hao C, Zhang X, Kang Z, Mao H. The E3 ubiquitin ligase TaGW2 facilitates TaSnRK1γandTaVPS24 degradation to enhance stripe rust susceptibility in wheat. Plant Biotechnol J. 2024, doi:10.1111 / pbi.14536., publicly available from Northwest Agriculture and Forestry University.

[0070] Example 1. Obtaining the protein TaVOZ1 and its encoding gene

[0071] 1. Cloning of protein TaVOZ1 and its encoding gene

[0072] Seeds of the cultivated wheat variety Chinese Spring were germinated in a petri dish at room temperature for three days, and then the germinated seeds were transferred to nutrient soil or Hoagland's nutrient solution and cultured for two weeks. The whole plant was quickly frozen in liquid nitrogen and ground, and total RNA was extracted and reverse transcribed to obtain cDNA. PCR amplification was performed using the cDNA as a template, and the amplified product was subjected to agarose gel electrophoresis. A 1917 bp DNA fragment was separated and purified for sequencing. The results showed that the sequence of the DNA fragment was shown in positions 79-1664 of SEQ ID No.2.

[0073] Amplification primer sequences:

[0074] cDNA-F (SEQ ID No. 9): 5'-ATGACACTGTTACCGCACGA-3';

[0075] cDNA-R (SEQ ID No. 10)5'-TCAAGCCCCGTCGTTTGAGT-3'.

[0076] The sequence shown in SEQ ID No. 2 is the full-length cDNA sequence encoding the protein TaVOZ1 shown in SEQ ID No. 1 in the wheat cultivar Chinese Spring, wherein the 1st to 78th positions are the 5' non-coding region, the 79th to 1664th positions are the coding sequence, and the 1665th to 2227th positions are the 3' non-coding region. The gene encoding the protein TaVOZ1 is named as gene TaVOZ1.

[0077] 2. Analysis of the expression pattern of TaVOZ1 gene in wheat leaves and roots under 100 mM NaCl treatment

[0078] The seeds of the wheat cultivar Chinese Spring were germinated in a petri dish at room temperature for three days, and then the germinated seeds were transferred to Hoagland's nutrient solution for two weeks. Then 100mM NaCl was added to the Hoagland's nutrient solution of the two-leaf and one-heart stage seedlings, and the roots and leaves of the three-leaf stage seedlings were quickly frozen in liquid nitrogen for 0, 1, 3, 6, 12 and 24 hours. When treated with ABA, the roots of the three-leaf stage seedlings were immersed in 100μM ABA aqueous solution, and the roots and leaves of the three-leaf stage seedlings were quickly frozen in liquid nitrogen for 0, 0.5, 1, 3, 6, 12, 24, 48 and 72 hours. The above samples were ground, total RNA was extracted, reverse transcribed, and cDNA was obtained. The cDNA was used as a template for real-time fluorescence quantitative PCR (qRT-PCR) to analyze the expression pattern of the TaVOZ1 gene.

[0079] TaVOZ1-F (SEQ ID No. 11): 5'-GGATCCCGCCGTCCGAGA-3';

[0080] TaVOZ1-R (SEQ ID No. 12): 5'-ACTCCCGGACCCAGCGA-3'.

[0081] The results are as follows Figure 1 As shown in Figures A and B, the expression of TaVOZ1 gene was upregulated in leaves and roots under salt stress, indicating that TaVOZ1 gene may play a role in regulating salt tolerance in wheat.

[0082] Example 2: Analysis of the role of TaVOZ1 gene in regulating salt tolerance in wheat

[0083] Using the vector LGY-OE3 as the base vector, the sequence amplified in Example 1 was inserted between the AvrII and BamHI restriction sites of LGY-OE3 to construct the overexpression recombinant vector LGY-OE3-TaVOZ1;

[0084] Using vector pC336 (Ubi:GWRNAi:Nos) as the basic vector, a DNA fragment of 664-868 bp of the sequence described in SEQ ID No.2 was inserted between attR1 and attR2 sites to construct the TaVOZ1 RNAi recombinant vector pC336-TaVOZ1.

[0085] TaVOZ1 overexpression (OE) or RNAi knockdown transgenic lines were constructed in the wheat variety Fielder background. DNA was extracted from each generation of transgenic plants and PCR detection was performed using primers; RNA was extracted and reverse transcribed to obtain cDNA, and qRT-PCR analysis was performed using primers.

[0086] PCR-F (SEQ ID No. 13): 5'-ATGTGGATTTTTTTAGCCCTGCCTT-3';

[0087] PCR-R (SEQ ID No. 14): 5'-TAGCGAAAACCGAATAAAAAACACA-3';

[0088] qPCR-F (SEQ ID No. 15): 5'-GAGGAAGAAAAACGACACCAAATTA-3';

[0089] qPCR-R (SEQ ID No. 16): 5'-GTACTGGAGGCCGGAGAACAT-3'.

[0090] Three overexpression lines with higher expression levels (OE1, OE2 and OE3) and three RNAi lines with higher silencing efficiency (RI1, RI2 and RI3) were selected, and total plant protein was extracted for western bolt analysis. The results are as follows: Figure 2 As shown in A and B, the protein levels of TaVOZ1 in the three overexpression lines were significantly increased relative to WT Fielder, while the protein levels of TaVOZ1 in the three silenced lines were relatively decreased.

[0091] TaVOZ1 OE, RNAi and WT Fielder wheat seeds were germinated in a petri dish at room temperature for three days, and then the germinated seeds were transferred to a small box of 7 cm × 7 cm filled with nutrient soil, watered and cultured with 150 mM NaCl, 9 plants were planted in each box, and at least three boxes were planted for each strain and each treatment. Phenotypic analysis of wheat seedlings at the two-leaf and one-heart stage found that the increase or decrease in TaVOZ1 expression did not lead to obvious changes in plant morphology or structure ( Figure 3 In the high-salt soil condition treated with 150 mM NaCl, the growth of plants was gradually inhibited from TaVOZ1 OE to WT Fielder and then to TaVOZ1 RNAi strain ( Figure 3 The biomass of the leaves of each strain was measured and it was found that under high-salt soil conditions, the biomass of TaVOZ1OE plants was significantly higher than that of WT Fielder, while the biomass of TaVOZ1 RNAi strains was significantly lower than that of WT Fielder; under normal growth conditions, the biomass of the transgenic strains was not significantly different from that of the wild type ( Figure 4 (A and B) Leaves from each strain were taken for Na + The content of Na was detected in the TaVOZ1 RNAi plants under high-salt soil conditions. + concentration, while the Na + The content of Na in leaves of each strain was lower than that of wild type; under normal growth conditions, + There was no significant difference in the level ( Figure 5 A and B).

[0092] TaVOZ1 OE, RNAi and WT Fielder wheat seeds were germinated in a petri dish at room temperature for three days, and then the germinated seeds were transferred to black 96-well boxes filled with Hoagland's nutrient solution or Hoagland's nutrient solution containing 150mM NaCl for culture. Each box contained 8 plants of each OE, RNAi and WT strain, and each strain treatment group and control group had three boxes. After growing in 150mM NaCl solution for 20 days, the root length of TaVOZ1 RNAi seedlings was significantly shorter than that of WT Fielder, while the root length of TaVOZ1 OE seedlings was significantly longer than that of WT Fielder; in the control group (no salt treatment), there was no significant difference in root length between the wild type and transgenic strains ( Figure 6 The taproot lengths of TaVOZ1 OE, RNAi, and WT Fielder wheat grown in 150 mM NaCl solution and salt-free solution for 20 days were measured. Twenty seedlings were measured for each line, and the experiment was repeated three times independently. The measurement results were consistent with the phenotypes. There was no difference in root length between the untreated WT and transgenic lines ( Figure 7 In the A), under the condition of 150 mM NaCl solution treatment, the root length of TaVOZ1 RNAi seedlings was significantly shorter than that of WT Fielder, while that of TaVOZ1 OE seedlings was significantly longer than that of WT Fielder ( Figure 7 (B).

[0093] Example 3: TaVOZ1 is a key regulatory factor in regulating salt stress response

[0094] 1. Homology comparison

[0095] TaVOZ1 is a NAC transcription factor. Sequence alignment of TaVOZ1 homologous proteins in wheat subgenomes A, B, and D revealed that the protein sequences of TaVOZ1-A, TaVOZ1-B, and TaVOZ1-D have 90% to 94% similarity ( Figure 8 ).

[0096] The amino acid sequence of TaVOZ1 was used as the seed sequence in Ensembl Plants online website (http: / / plants.ensembl.org / index.html), and the amino acid sequences of all candidate VOZ1s homologous proteins in wheat, Arabidopsis, rice, corn, Brachypodium distachyon, and tomato were blasted and downloaded. All VOZ1s sequences were merged and repeated sequences and incomplete sequences were removed. The protein sequences were aligned using MEGA7.0 software. The default parameters were used for double alignment and multiple alignment. The phylogenetic tree was constructed using the Neighbor-Joining method. The results are shown in the figure. Fig. 9As shown, TaVOZ1 is most closely related to OsVOZ1 in rice and BRADI_2g50070 in Brachypodium distachyon.

[0097] 2. Protein localization experiment

[0098] Using Chinese Spring (CS) cDNA as template, PCR amplification was performed, and the cloned target gene was cloned and connected into the expression vector pJIT163, and transformed into wheat (Chinese Spring) protoplasts. The empty vector pJIT163 was used as a control, and the protoplasts were observed under a laser confocal microscope.

[0099] Amplification primer F (SEQ ID No. 17): 5′-CTTGCATGCCTGCAGGTCGACATGACACTGTTACCGCACGA-3′;

[0100] Amplification primer R (SEQ ID No. 18): 5'-GCCCTTGCTCACCATGGATCCAGCCCCGTCGTTTGAGTTCC-3'.

[0101] The results are as follows Fig.10 As shown, the green fluorescence in the protoplasts transformed with the empty vector pJIT163 was distributed throughout the cells, while the green fluorescence in the protoplasts transformed with the TaVOZ1-GFP fusion protein vector was mainly distributed in the nucleus, indicating that TaVOZ1 may be a nuclear localized protein.

[0102] 3. The transcriptional regulatory function of TaVOZ1 was evaluated using yeast transcriptional activity detection assay.

[0103] The transcriptional activation region of herpes simplex virus protein 16 (VP16, Gene ID: 24271473) was fused with the GAL4 DNA binding domain (BD, Gene ID: 855828) to construct the pGBKT7-VP16 vector. Subsequently, TaVOZ1 was fused with pGBKT7 and pGBKT7-VP16 to construct the pGBKT7-TaVOZ1 and pGBKT7-TaVOZ1-VP16 vectors, and the constructed vectors were sequenced to verify their correctness and transformed into Saccharomyces cerevisiae Y2HGold cells. The diluted yeast cells were inoculated on SD / -Trp, SD / -Trp-His and SD / -Trp-His-Ade media, respectively. pGBKT7 and pGBKT7-VP16 were used as negative and positive controls, respectively.

[0104] The results are as follows Fig.11As shown, similar to the pGBKT7-VP16 positive control, TaVOZ1 acts as a transcriptional activator. In addition, the pGreen II-0800-LUC vector was used as a reporter, and the above-constructed vector plasmids were used as effectors to transform Agrobacterium. TaActin1-BD was used as a negative control and VP16-BD was used as a positive control. TaVOZ1-BD and pGreenII-0800-LUC bacterial solutions were mixed in a ratio of 1:2 and injected into tobacco leaves. After culturing in an artificial climate chamber at 22°C for 48 hours, the activity of firefly luciferase was detected. The results are shown in Fig.12 As shown, TaVOZ1 fused to the GAL4 binding domain (BD) can induce significantly enhanced expression of LUC in tobacco leaves, further verifying its possibility as a transcriptional activator.

[0105] 4. Study on the mechanism of action of TaVOZ1 in alleviating salt stress

[0106] DNA affinity purification sequencing (DAP-seq) was used to identify potential binding sites and regulated target genes of TaVOZ1 in the wheat genome. This analysis found 6574 candidate binding sites in 4436 genes ( Fig.13 Further analysis revealed that the potential binding sites of TaVOZ1 were located in the distal intergenic region, promoter region, intron, exon, 5'-UTR, 3'-UTR and terminator region with probabilities of 33.6%, 29.1%, 16.3%, 8.9%, 2.7%, 3.4% and 5.9%, respectively ( Fig.13 Middle B). Based on the prediction of potential TaVOZ1 binding motifs based on MEME-ChIP, 5'-CTTCTT-3' or its reverse complement 5'-AAGAAG-3' was the most significantly enriched core recognition motif ( Fig.13 C). Combining DAP-seq and transcriptome sequencing (RNA-seq) analysis, the present invention determined that TaVOZ1 may bind to 2347 candidate target genes for transcriptional regulation, of which 1402 genes were significantly upregulated in the TaVOZ1 OE strain, while 945 genes were significantly downregulated ( Fig.14 GO enrichment analysis showed that the upregulated TaVOZ1 target genes were mainly enriched in stress response-related aspects, such as “response to salt stress”, “response to water deficiency”, “ion homeostasis”, “transcriptional regulation” and “response to abscisic acid” ( Fig.14 B), while the down-regulated TaVOZ1 target genes were enriched in the processes of “protein autophosphorylation”, “response to salicylic acid”, “defense response”, “salicylic acid catabolism”, and “auxin response” ( Fig.14These results suggest that TaVOZ1 improves plant salt tolerance by activating the expression of stress response genes.

[0107] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A plant salt tolerance-related protein, characterized in that: The protein includes an amino acid sequence as shown in any of the following: 1) an amino acid sequence as shown in SEQ ID No. 1; 2) The amino acid sequence of a derivative protein related to plant salt tolerance obtained by replacing, deleting and / or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.

1.

2. A gene encoding the protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene includes the nucleotide sequence shown in any one of the following: 1) the nucleotide sequence shown in SEQ ID No. 2; 2) the CDS sequence in the nucleotide sequence shown in SEQ ID No. 2; 3) A DNA molecule that has at least 70% homology with the nucleotide sequence defined in 1) or 2) and encodes the protein of claim 1; 4) A DNA molecule that hybridizes with the DNA sequence defined in 1), 2) or 3) under stringent conditions and encodes the protein of claim 1.

4. A biological material for regulating the content or activity of the protein according to claim 1 and / or the expression amount of the gene according to claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that: The types of biological materials include vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses.

6. Use of the protein according to claim 1 or the gene according to claim 2 or 3 in improving the salt tolerance of plants.

7. The use according to claim 6, characterized in that: The plants include monocots or dicots.

8. Use of the biological material according to claim 4 or 5 in regulating plant salt tolerance.

9. A method for improving plant salt tolerance, characterized in that: The method comprises increasing the content or activity of the protein of claim 1 in the target plant, or increasing the expression level of the gene of claim 2 or 3 in the target plant.

10. The method according to claim 9, characterized in that: The method for increasing the expression level of the gene in the target plant comprises transforming the target plant with a recombinant expression vector, wherein the recombinant expression vector takes a plant expression vector as a basic vector and connects the gene to the basic vector.

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