Plant salt tolerance related transcription factor taVOZ1, and coding gene and application thereof

Abstract

The application provides a plant salt-tolerance related transcription factor TaVOZ1, a coding gene and application thereof, and belongs to the field of functional gene technology.The TaVOZ1 is related to plant salt tolerance, has an amino acid sequence shown as SEQ ID No.1, and the gene coding the protein has a nucleotide sequence shown as SEQ ID No.2.The application can regulate the content or activity of the protein or the expression amount of the gene to achieve the purpose of regulating plant salt tolerance, for example, when the gene is overexpressed, the salt tolerance of the plant is enhanced, and when the gene is knocked down, the salt tolerance is weakened.The TaVOZ1 can activate the expression of stress response genes by combining 5'-CTTCTT-3' or its reverse complementary sequence 5'-AAGAAG-3' in the promoter of a downstream target gene to improve the salt tolerance of plants.Therefore, the TaVOZ1 can be used to cultivate salt-tolerant wheat varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of functional gene technology, and particularly relates to a plant salt tolerance related transcription factor TaVOZ1, a coding gene thereof and application. BACKGROUND

[0002] Soil salinization is one of the most common abiotic stresses, which seriously affects global agricultural production. About 20% of the land in the world is affected by soil salinization, and the area of damaged arable land reaches 20 million hm 2 Wheat (Triticum aestivum L.) is the main food for 40% of the world's population and is the main source of carbohydrates and proteins for human beings.

[0003] Excessive cations (such as Na + ) or anions (such as Cl - ) in saline soil can cause the reduction of water and nutrient absorption of wheat, produce osmotic stress and ion stress, affect physiological and biochemical functions such as photosynthesis and protein synthesis of wheat, and thus reduce the yield and quality of wheat. Although wheat is the largest food crop in the world in terms of planting area, wheat is relatively sensitive to salt stress, and therefore, it is particularly important to improve the salt tolerance of wheat and breed salt-tolerant wheat varieties to ensure the food security and sustainable development of agriculture in China. However, crop salt tolerance is a complex quantitative trait controlled by multiple genes, and traditional breeding methods have been difficult to meet the demand for genetic improvement of crop salt tolerance, and it is urgent to mine new genetic resources through molecular breeding methods to improve the salt tolerance of wheat. Therefore, it is of great significance to mine salt tolerance genes in wheat for breeding salt-tolerant wheat varieties and improving the yield of wheat. SUMMARY

[0004] The application provides a plant salt tolerance related transcription factor TaVOZ1, a coding gene thereof and application, and the TaVOZ1 can regulate the salt tolerance of plants and be used for breeding salt-tolerant varieties.

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

[0006] 2) an amino acid sequence of a derived protein related to the salt tolerance of plants, which is obtained by substitution, deletion and / or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID No. 1.

[0007] The second application purpose of the application is to provide a gene encoding the above protein.

[0008] In a preferred mode of the present application, the nucleotide sequence of the gene comprises any one of the following nucleotide sequences: 1) the nucleotide sequence 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 having at least 70% homology with the nucleotide sequence defined in 1) or 2) and encoding the above-mentioned protein;

[0011] 4) a DNA molecule hybridizing with the DNA sequence defined in 1), 2) or 3) under stringent conditions and encoding the above-mentioned protein.

[0012] The third object of the present application is to provide a biological material 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 mode of the present application, the type of the biological material comprises a vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus.

[0014] The fourth object of the present application is to provide the above-mentioned protein or the above-mentioned gene for use in improving the salt tolerance of a plant.

[0015] In a preferred mode of the present application, the plant comprises a monocotyledonous plant or a dicotyledonous plant.

[0016] The fifth object of the present application is to provide the above-mentioned biological material for use in regulating the salt tolerance of a plant.

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

[0018] In a preferred mode of the present application, the method for increasing the expression amount of the gene in a target plant comprises transforming the target plant with a recombinant expression vector, wherein the recombinant expression vector is a plant expression vector-based vector and the gene is connected to the vector.

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

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

[0021] Figure 2 Figure is the analysis result diagram of TaVOZ1 gene expression level (A) and protein level (B) in TaVOZ1 overexpression (OE) and silencing (RNAi) lines;

[0022] Figure 3 Figure is the salt tolerance phenotype identification result diagram of TaVOZ1 OE and RNAi lines;

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

[0024] Figure 5 Figure is the Na content analysis result diagram in TaVOZ1 OE and RNAi lines in leaves under normal growth (A) or 150mM NaCl treatment (B); +

[0025] Figure 6 Figure is the root system diagram of TaVOZ1 OE and RNAi lines under 150mM NaCl or water culture conditions for 20 days;

[0026] Figure 7 Figure is the comparison result diagram of root length of TaVOZ1 OE and RNAi lines under normal growth (A) and 150mM NaCl conditions (B) for 20 days;

[0027] Figure 8 Figure is the sequence alignment result diagram of TaVOZ1 homologous protein;

[0028] Figure 9 Figure is the phylogenetic analysis diagram of TaVOZ1 homologous genes;

[0029] ​Figure 10 Figure for subcellular localization results of TaVOZ1 in wheat protoplasts;

[0030] Figure 11 Figure for yeast transcriptional activity assay results;

[0031] Figure 12 Figure for results of determining the transcriptional activity of TaVOZ1 by dual luciferase assay;

[0032] Figure 13 Figure for results of DNA affinity purification sequencing (DAP-seq) analysis of the binding motif of TaVOZ1;

[0033] Figure 14 Figure for results of screening candidate target genes by combining transcriptome sequencing (RNA-seq) and DAP-seq (A) and GO enrichment analysis (B and C). DETAILED DESCRIPTION

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

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

[0036] The protein according to the present application is a transcription factor TaVOZ1 protein, which is derived from wheat, and the amino acid sequence thereof can be as shown in SEQ ID No. 1:

[0037] MTLLPHDAFDFDESWAGFLPIYPARRPPTPASFLPRPSLFASRPSKVAAIPSEEEKRHQIIRASPSPLPWRKGWRERPPTPRRRSIGAAERTARASAMRKGPSRSGSARHQQFRARAKTRVDDLQDMFSGLQYARKEARSTDAVLLEAQLHQMLREWRAELSVPSPASSLQGNNNRDPPSETPRPPQLAAAEEEDDATSKLVEQKPRPSANQAHKHAQGDQDMKPEPREEAIADPVTVAQQPTSLGPGVITTPATAGFHDQMYYVNQELSVEDFLYDDDYKINLPGSNPEILNNLEGIGHQEYLQFNLPQELPPNAYLDMNNYGQNAGDGFLHMSDLLTTMSPAPASFLRPKCALWDCPRPAQGSESWQDYCSMYHAELAVKEEGPPGTMPVIRPRGIDLKDGPLFAALSAKIQGKHVGVPVCEGAATTKSPWNAPELFDLYIFEGESMREWLFFDKPRRAFDSGNRKQRSLPDYNGRGWHESRKQVMKDFGGLKRSYYMDPQPSSSYEWHLYEYEINDRDAFALYRLEFKSSDAKKSAKSKFTCSPLIEIQQQMVRLSADGPVENKRTARARTQDVSTNIYPVQNNTAQANAPDAYQAASQVDQMTFLNGSVVYGPHLPYGYSTEGGDFYWNSNDGA; or a derivative protein obtained by mutating the sequence shown in SEQ ID No. 1, such as a protein obtained by substituting, deleting and / or adding one or more amino acid residues while still being related to plant salt tolerance, all of which fall within the protection scope of the present application.

[0038] The present application does not have special limitations on the preparation method of the protein, which can be directly synthesized by artificial synthesis method, or obtained by synthesizing the coding gene and then expressing, and of course, chemical modification can be performed on the N terminal and / or C terminal of the protein, such as various tags shown in Table 1 for purification.

[0039] Table 1 Tags (part) that can be connected to the protein of the present application

[0040] Tag Residue Sequence SEQ ID No. Poly-Arg 5-6 (typically 5) RRRRR 3 Poly-His 2-10 (typically 6) HHHHHH 4 FLAG 8 DYKDDDDK 5 Strep-tag II 8 WSHPQFEK 6 c-myc 10 EQKLISEEDL 7 HA 9 YPYDVPDYA 8

[0041] The present application provides a gene encoding the above-mentioned protein.

[0042] The gene according to the present application comprises both the genomic sequence and the CDS sequence, homologous sequences of the genomic sequence or the CDS sequence, and all DNA sequences which hybridize to all the above sequences under stringent conditions and encode the above amino acid sequences.

[0043] In one specific embodiment of the present application, the genomic sequence is shown as SEQ ID No. 2:

[0044]

[0045] Meanwhile, the gene of the present application can also have more than 70% homology with the genomic sequence or the CDS sequence and encode 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 application, the DNA molecule hybridizing with the above-mentioned genomic sequence, CDS sequence or homologous sequence under stringent conditions and encoding the above-mentioned protein also belongs to the protection scope of the gene of the present application. It is worth mentioning that the stringent conditions of the present application can be hybridization under heating conditions using a mixed solution containing SDS, and then rinsing or washing the film using a solution containing SSC. Specifically, it can be performed under any of the following conditions:

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

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

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

[0050] ④: 50℃, hybridization in a mixed solution containing 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 50℃, rinsing in a mixed solution containing 0.1×SSC and 0.1% SDS;

[0051] ⑤: 50℃, hybridization in a mixed solution containing 7% SDS, 0.5M Na3PO4 and 1mM EDTA; 65℃, rinsing in a mixed solution containing 0.1×SSC and 0.1% SDS;

[0052] ⑥: 65℃, hybridization in a mixed solution containing 6×SSC and 0.5% SDS; rinsing the film once using a mixed solution containing 2×SSC and 0.1% SDS, and a mixed solution containing 1×SSC and 0.1% SDS.

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

[0054] The regulation includes up-regulation, at which the biological material containing the above-mentioned gene and expressing the above-mentioned protein or the biological material improving the activity of the protein is included. The type of the biological material is not specially limited in the present application, which can be a vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus. In a preferred mode of the present application, the vector can be a recombinant expression vector formed by connecting the gene to a plant expression vector, which includes binary Agrobacterium vector and vector available for plant microprojectile bombardment, such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pCAMBIA3301, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corporation) and the like. The plant expression vector can further contain the 3' untranslated region of the exogenous gene, i.e. the region containing the polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenosine to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end transcription of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the Nos gene of nopaline synthase) and plant gene (such as the soybean storage protein gene).

[0055] When the gene construct is used to construct a recombinant plant expression vector, any one of the enhanced promoters (such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize), constitutive promoters or tissue-specific expression promoters (such as seed-specific expression promoters) can be added before the transcription initiation nucleotide, and the promoters can be used alone or in combination with other plant promoters. When the gene construct of the present application is used to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of the start codon, but must be in 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 start codon is broad, and can be natural or synthetic. The translation initiation region can be 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 express enzymes or luminescent compounds that produce color changes in plants (GUS genes, luciferase genes, etc.), marker genes for antibiotics (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 agent-resistant marker genes (such as herbicide-resistant genes), and the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose.

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

[0057] The biological material of the present application also includes a recombinant vector that down-regulates the expression of the gene, such as RNAi. In one embodiment, the pC336 (Ubi:GWRNAi:Nos) is used as the base vector, and the target sequence is inserted between the attR1 and attR2 sites of the pC336 (Ubi:GWRNAi:Nos). In an embodiment, the target sequence is selected from 664-868 bp of the sequence shown in SEQ ID No. 2, and the RNAi vector pC336-TaVOZ1 is constructed.

[0058] The biological material of the present application also includes a transcription activator of the protein. The transcription activator can be the protein, or the transcription activator prepared from the protein as a raw material.

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

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

[0061] The application also provides the use of the above biological material in regulating the salt tolerance of plants.

[0062] In the application, the biological material can be used to up-regulate the content or activity of the protein, or to up-regulate the expression of the gene, so as to improve the salt tolerance of plants, which can be dicotyledonous plants or monocotyledonous plants. In an embodiment of the application, wheat (Triticum aestivum L.) is used as a representative of monocotyledonous plants for testing, and it is found that the salt tolerance of the transgenic plants is enhanced after the gene encoding the transcription factor protein TaVOZ1 is introduced into the wheat. In an embodiment of the application, the material for down-regulating the expression of the gene in the biological material is used, such as the transgenic plants with reduced salt tolerance obtained after the TaVOZ1 gene is silenced by RNAi, i.e., the RNAi vector pC336-TaVOZ1 can be used to reduce the salt tolerance of plants. Therefore, the biological material of the application can be used to regulate the salt tolerance of plants.

[0063] It is found in the application that the gene TaVOZ1 acts as a transcription activator, 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 the downstream target gene, so as to improve the salt tolerance of plants.

[0064] The application also provides a method for improving the salt tolerance of plants, which comprises increasing the content or activity of the above protein in target plants, or increasing the expression of the above gene in target plants.

[0065] In the application, the content or activity of the above protein in target plants is increased, or the expression of the nucleic acid molecule encoding the above protein in target plants is increased, which is achieved by introducing the nucleic acid molecule encoding the above protein into the target plants.

[0066] In order to further illustrate the application, the plant salt tolerance related transcription factor TaVOZ1, the encoding gene and the application thereof provided by the application are described in detail in combination with examples below, but they should not be understood as limiting the protection scope of the application.

[0067] The experimental methods used in the embodiments of the application are conventional methods unless otherwise specified, and the materials, reagents and the like used are commercially available unless otherwise specified.

[0068] The vector LGY-OE3 used in the embodiments of the present application has been disclosed in the literature (Niaz M, Zhang L, Lv G, Hu H, Yang X, Cheng Y, Zheng Y, Zhang B, Yan X, Htun A, Zhao L, Sun C, Zhang N, Ren Y, Chen F. 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: Wang Y, Liu C, Du Y, Cai K, Wang Y, Guo J, Bai X, Kang Z, Guo J. A stripe rust fungal effector PstSIE1 targets TaSGT1 to facilitate pathogen infection. Plant J. 2022, 112(6): 1413-1428. The 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 TaGW2 mediates transcription factor TaARR12 degradation to promote drought resistance in wheat. Plant Cell. 2024, 36(3): 605-625. and is available to the public from Northwest A&F University.

[0069] The Agrobacterium tumefaciens GV3101 + pSoup strain used in the embodiments of the present application is described 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 described 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 described 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 TaSnRK1γ and TaVPS24 degradation to enhance stripe rust susceptibility in wheat. Plant Biotechnol J. 2024, doi:10.1111 / pbi.14536., which is available to the public from Northwest A&F University.

[0070] Example 1, obtaining of the protein TaVOZ1 and its encoding gene

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

[0072] The seeds of the wheat cultivar Chinese Spring were taken, and the germinated seeds were transferred to Hoagland's nutrient solution for two weeks after being incubated in a culture dish at room temperature for three days. The whole plant was quickly frozen in liquid nitrogen, ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA. The cDNA was used as a template for PCR amplification. The amplified product was subjected to agarose gel electrophoresis, and the 1917 bp DNA fragment was separated and purified for sequencing. The results showed that the sequence of the DNA fragment is shown in SEQ ID No. 2 at positions 79-1664.

[0073] Amplification primer sequence:

[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 of the protein TaVOZ1 encoding sequence shown in SEQ ID No. 1 in the wheat cultivar Chinese Spring, wherein the first-78th is the 5' non-coding region, the 79th-1664th is the coding sequence, and the 1665th-2227th is the 3' non-coding region. The gene encoding the protein TaVOZ1 is named as the gene TaVOZ1.

[0077] 2. Expression pattern analysis of TaVOZ1 gene in wheat leaves and roots under 100 mM NaCl treatment

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

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

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

[0081] Results are shown in Table 1 Figure 1 As shown in A and B of Table 1, TaVOZ1 gene was up-regulated in leaf and root under salt stress, indicating that TaVOZ1 gene might play a function in regulating wheat salt tolerance.

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

[0083] Based on the vector LGY-OE3, the sequence amplified in Example 1 was inserted into the AvrII and BamHI enzyme cutting sites of LGY-OE3 to construct the overexpression recombinant vector LGY-OE3-TaVOZ1.

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

[0085] The transgenic lines of TaVOZ1 overexpression (OE) or RNAi knockdown were constructed in the background of wheat variety Fielder. The DNA of each generation of transgenic plants was extracted, and PCR detection was performed using primers; the 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 high expression levels (OE1, OE2, and OE3) and three RNAi lines with high silencing efficiency (RI1, RI2, and RI3) were selected, and total plant protein was extracted for Western blot analysis. The results are as follows: Figure 2 As shown in Figures A and B, the amount of TaVOZ1 protein in the three overexpression lines was significantly higher than that in the WT Fielder lines, while the amount of TaVOZ1 protein in the three silenced lines was relatively lower.

[0091] TaVOZ1 OE, RNAi, and WT Fielder wheat seeds were germinated in petri dishes at room temperature for three days. The germinated seeds were then transferred to 7cm × 7cm boxes filled with nutrient soil, watered, and cultured in 150mM NaCl. Nine seedlings were planted in each box, and at least three boxes were planted for each line and each treatment. Phenotypic analysis of wheat seedlings at the two-leaf stage revealed that increases or decreases in TaVOZ1 expression did not lead to significant changes in plant morphology or structure. Figure 3 (Top-middle side view), while under high-salt soil conditions treated with 150 mM NaCl, the growth of plants from TaVOZ1 OE to WT Fielder, and then to TaVOZ1 RNAi lines, was gradually inhibited. Figure 3 (Lower middle view). Biomass measurements of leaves from each line revealed that under high-salt soil conditions, the biomass of TaVOZ1OE plants was significantly higher than that of WT Fielder, while the biomass of the TaVOZ1 RNAi line was significantly lower than that of WT Fielder; however, under normal growth conditions, the biomass of the transgenic lines was not significantly different from that of the wild type. Figure 4 (A and B). Leaves from each strain were taken for Na24 analysis. + Content analysis revealed that TaVOZ1 RNAi plants accumulated higher levels of Na under high-salt soil conditions. + Concentration, while the Na of TaVOZ1 OE plants + The Na content was lower than that of the wild type; however, under normal growth conditions, the Na content in the leaves of each strain was higher. + No significant difference at the level ( Figure 5 (A and B in the middle).

[0092] TaVOZ1 OE, RNAi and WT Fielder seeds were germinated in Petri dishes at room temperature for three days, and then the germinated seeds were transferred to black 96-well boxes containing Hoagland's nutrient solution or Hoagland's nutrient solution containing 150 mM NaCl, with 8 plants of each OE, RNAi and WT line in each box, and three boxes for each line of treatment and control. After 20 days of growth in 150 mM NaCl solution, 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; while in the control group (no salt treatment), there was no significant difference in root length between wild type and transgenic lines Figure 6 The main root length of TaVOZ1 OE, RNAi and WT Fielder wheat grown in 150 mM NaCl solution and salt-free treatment solution was measured, 20 seedlings of each line were measured, and three independent repeated experiments were performed, the measurement results were consistent with the phenotype, there was no difference in root length between untreated WT and transgenic lines Figure 7 A), while under the condition of 150 mM NaCl solution treatment, the root length of TaVOZ1 RNAi seedlings was significantly shorter than that of WT Fielder, and TaVOZ1 OE was significantly longer than WT Fielder Figure 7 B).

[0093] Example 3, TaVOZ1 is a key regulatory factor for regulating salt stress response

[0094] 1. Homology alignment

[0095] TaVOZ1 is a NAC transcription factor, sequence alignment of A, B and D three subgenomic homologous proteins of TaVOZ1 in wheat was performed, and it was found that the protein sequences of TaVOZ1-A, TaVOZ1-B and TaVOZ1-D had 90%-94% similarity Figure 8 ).

[0096] At the Ensembl Plants online website (http: / / plants.ensembl.org / index.html), the amino acid sequence of TaVOZ1 was used as the seed sequence, and the amino acid sequences of all candidate VOZ1 homologous proteins in wheat, Arabidopsis, rice, corn, Brachypodium distachyon and tomato were downloaded by Blast respectively. All VOZ1 sequences were combined and repeated sequences and incomplete sequences were removed, protein sequences were aligned using MEGA7.0 software, default parameters were used for double alignment and multiple alignment, and a phylogenetic tree was constructed using the Neighbor-Joining method, and the results are as follows Figure 9As shown, TaVOZ1 is most closely related to OsVOZ1 in rice and BRADI_2g50070 in Brachypodium distachyon.

[0097] 2. Protein localization experiment

[0098] The cDNA of Chinese Spring (CS) was used as a template for PCR amplification, and the cloned target gene was cloned and ligated into the expression vector pJIT163, which was transformed into wheat (Chinese Spring) protoplasts. The transformed empty vector pJIT163 was used as a control, and the transformed 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 shown in Figure 10 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 is likely a nuclear localization protein.

[0102] 3. Evaluation of the transcriptional regulation function of TaVOZ1 using a yeast transcriptional activity detection test.

[0103] The transcriptional activation region of herpes simplex virus protein 16 (herpes simplex virus protein, 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, respectively, to construct the pGBKT7-TaVOZ1 and pGBKT7-TaVOZ1-VP16 vectors. The constructed vectors were sequenced to verify their correctness, and were transformed into Saccharomyces cerevisiae Y2HGold cells. The diluted yeast cells were inoculated on SD / -Trp, SD / -Trp-His, and SD / -Trp-His-Ade culture media, respectively. pGBKT7 and pGBKT7-VP16 were used as negative and positive controls, respectively.

[0104] The results are shown in Figure 11As shown, TaVOZ1 functions as a transcriptional activator similar to pGBKT7-VP16 positive control. In addition, pGreen II-0800-LUC vector was used as a reporter and the above constructed vector plasmid was used as effector to transform Agrobacterium. With TaActin1-BD as negative control and VP16-BD as positive control, TaVOZ1-BD was mixed with pGreen II-0800-LUC bacterial liquid at a ratio of 1:2 and injected into tobacco leaves. After 48h incubation in a 22℃ artificial climate chamber, the activity of Firefly luciferase was detected. As shown in Figure 12 , fusion of TaVOZ1 to GAL4 binding domain (BD) can induce significant enhancement of LUC expression in tobacco leaves, further verifying its possibility as a transcriptional activator.

[0105] 4. Mechanism of TaVOZ1 in alleviating salt stress

[0106] DNA affinity purification sequencing analysis (DAP-seq) was used to identify and analyze the potential binding sites and regulated target genes of TaVOZ1 in the wheat genome. The analysis found 6574 candidate binding sites in 4436 genes Figure 13 (A). Further analysis found 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 Figure 13 (B). Based on MEME-ChIP, the prediction of potential TaVOZ1 binding motifs found that 5'-CTTCTT-3' or its reverse complement sequence 5'-AAGAAG-3' was the most significantly enriched core recognition motif Figure 13 (C). Combined with DAP-seq and transcriptome sequencing (RNA-seq) analysis, the present application determined that TaVOZ1 can bind to 2347 candidate target genes for transcriptional regulation, of which 1402 genes were significantly up-regulated in the TaVOZ1 OE strain, and 945 genes were significantly down-regulated Figure 14 (A). GO enrichment analysis showed that the up-regulated TaVOZ1 target genes were mainly enriched in stress response-related aspects, such as "response to salt stress", "response to water deficit", "ion homeostasis", "transcriptional regulation" and "response to abscisic acid" Figure 14 (B), while the down-regulated TaVOZ1 target genes were enriched in "protein autophosphorylation", "response to salicylic acid", "defense response", "salicylic acid catabolism", and "auxin response" processes Figure 14In conclusion, these results suggest that TaVOZ1 improves plant salt tolerance by activating the expression of stress responsive genes.

[0107] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained by those skilled in the art without creativity on the basis of the above embodiments, and these embodiments also belong to the protection scope of the present application.

Claims

1. Use of a plant salt-related protein or a gene encoding said plant salt-related protein for increasing salt tolerance in wheat, characterized in that, The amino acid sequence of the plant salt tolerance related protein is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The nucleotide sequence of the gene is 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 method of increasing salt tolerance in wheat, characterized by, The method comprises increasing the content of the plant salt tolerance related protein in the target plant, or increasing the expression amount of the gene encoding the plant salt tolerance related protein in the target plant, and the amino acid sequence of the plant salt tolerance related protein is shown as SEQ ID No.

1.

4. The method of claim 3, wherein, The method for increasing the expression amount of the gene encoding the plant salt tolerance related protein in the target plant comprises transforming the target plant with a recombinant expression vector, and the recombinant expression vector is a plant expression vector-based vector, and a gene encoding the plant salt tolerance related protein is connected on the basic vector.

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