Barley zinc-finger protein transcription factor hvdo f17 and application thereof in regulating salt tolerance of barley

Abstract

The application provides a barley zinc finger protein transcription factor HvDOF17 and application thereof in regulating salt tolerance of barley. The nucleotide sequence of the barley zinc finger protein transcription factor HvDOF17 is shown as SEQ ID No:1, and the full-length nucleotide sequence of the transcript cDNA is shown as SEQ ID No:2. The application first clones and analyzes the zinc finger protein gene HvDOF17 responding to salt stress in barley, and discloses the nucleotide sequence, amino acid sequence and promoter sequence of the gene, which has important significance for clarifying the molecular mechanism of salt tolerance regulation of barley and cultivation of salt-tolerant materials. In addition, the application first proves that the HvDOF17 gene is involved in salt tolerance of barley through a transgenic method, and after knocking out the HvDOF17 gene, the salt tolerance of barley can be significantly enhanced, and the adverse effects of salt stress can be effectively alleviated, so that the normal growth of the plant can be maintained.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to barley zinc finger protein transcription factor HvDOF17 and its application in regulating barley salt tolerance. Background Technology

[0002] Soil salinization is one of the major abiotic stresses threatening global crop production and food security, and hindering sustainable agricultural development. Soil salinity will have a long-term negative impact on crop growth and food security, and cultivating salt-tolerant crops to adapt to saline-alkali land has become a consensus for the effective utilization and transformation of such land. Salt stress causes osmotic stress, ion imbalance, and oxidative stress in plants, and ion balance is a prerequisite for avoiding or mitigating osmotic stress and oxidative damage. Ion balance, dominated by calcium signaling and transcriptional regulation, is a key mechanism for regulating plant salt tolerance, involving complex gene and molecular regulation. Therefore, exploring crop salt-tolerant germplasm and gene resources, and elucidating the physiological and molecular mechanisms of crop salt-tolerant genes, is of great significance for cultivating or creating new salt-tolerant crop materials adapted to saline-alkali land.

[0003] The most significant harm of salt stress to plants is the osmotic stress and ion imbalance caused by high concentrations of salt ions, and the secondary oxidative stress resulting from the combination of these two factors. Generally, excessive salt accumulation in the soil alters its physicochemical properties, leading to physiological drought in plant cells, ion imbalance, and the accumulation of reactive oxygen species (ROS). When plants are exposed to prolonged high-concentration salt stress environments, they experience severe growth inhibition, developmental changes, metabolic disorders, premature aging, and sterility. Specifically, osmotic stress is primarily caused by excessively high concentrations of soluble salts in the soil, reducing the plant's absorption and utilization of water, resulting in water deficit in plant cells. Secondly, ion stress is caused by the accumulation of excessive salt ions (mainly sodium ions, Na+) within plant cells. + ), inhibiting the absorption of potassium ions (K) by plant cells. + The absorption and transport of mineral nutrients such as oxygen and ions affect metabolic pathways such as photosynthesis, stomatal regulation, and nutrient synthesis in plants, resulting in specific ion toxicity. Furthermore, secondary oxidative stress is caused by both osmotic and ion stress. When excessive toxic substances accumulate in plant cells, large amounts of free reactive oxygen species (ROS) are produced, further affecting various physiological and biochemical processes within the plant and causing multiple metabolic disorders.

[0004] In summary, given that salt stress causes physiological damage to plants, including osmotic, ionic, and oxidative stress, studying and elucidating the plant response mechanisms to salt stress is fundamental to improving the salt tolerance potential of crops.

[0005] Barley (Hordeum vulgare L.) is the world's fourth largest cereal crop and the most salt-tolerant grass. It also boasts wide adaptability and abundant wild resources, making it an ideal model plant for studying the salt tolerance mechanisms of cereal crops. However, there are currently no reports on barley transcription factors (DOF).

[0006] Therefore, discovering superior salt-tolerant genes, such as members of the DOF family, in barley can enrich the theory of plant salt tolerance and provide theoretical and technical support for salt-tolerant breeding of barley and cereal crops. Summary of the Invention

[0007] The purpose of this invention is to provide a barley zinc finger protein transcription factor HvDOF17 and its application in regulating barley salt tolerance. This gene participates in the salt stress response of barley, providing a basis for the preparation of barley salt-tolerant germplasm resources.

[0008] The specific technical solution is as follows:

[0009] This invention provides a barley zinc finger protein transcription factor HvDOF17, the nucleotide sequence of which is shown in SEQ ID No:1. This invention is the first to clone a barley zinc finger protein transcription factor gene, named HvDOF17, and its full-length nucleotide sequence of the transcript cDNA is shown in SEQ ID No:2.

[0010] SEQ ID No. 1:

[0011] ATGATCTTCCCTCCTGCCTTCCTAGATTCATCAAGCTGCTGGAACACCAACCACAACCA

[0012] GCTTCAGGTATGCATGCATATATCCTTGTGTTCAATTAGTCGTTCTCTCAAGATTTTGTTC

[0013] ACACAAGAAAGAAAGAGGGAGAATATGTTCTAGCCAAGCTAGGGTTTGCTGATGGCAG

[0014] ATATATATCCCCTCTGCTGATTGCTGCACTATGTATCTTTGAATATGCTCCATATATACATCT

[0015] TTGCTGATGCTTAATTCCTGACCACTTAATTTGCAGCTGCAGCAAATCGGCAGTAACAC

[0016] TCATATCACCGCTACTCCTTCACCTGCTGGTCATGGAGATGGAGGAGGCAGCAACAACA

[0017] ACAACCATGGTCAGCAGGAAGGATTAATGGCCACGGCCGGGGCCGGAGGAGGTGGTG

[0018] GTGATGGTGGAGGCGGCGGCGGTGGGGATGGTGATAGCGCCGGCGGTGGGAACAACA

[0019] AGCCGATGTCGATGTCAGAGCGGGCACGGCTGGCACGGGTGCCGCAGCCGGAGCCGG

[0020] GGCTCAACTGCCCGCGCTGCGACTCCACCAACACCAAGTTCTGCTACTTCAACAACTA

[0021] CTCCCTCACCCAGCCCCGCCACTTCTGCCGCGCCTGCCGCCGCTACTGGACCCGCGGC

[0022] GGCGCGCTCCGCAACGTCCCCGTCGGTGGCGGGTACCGCCGCCACGCCAAGCGCAGC

[0023] GCCAAGCCCAAGGCGGGGTCGGCTGGATCCGGAACCACCACGGCAGGGACGTCGTCC

[0024] GCGACGTCGACGACGCCCAGCACCACTGCTTGCACCACCGGCACTGCCACTGCGCCGC

[0025] CCGCTCTGCAGTACTCCATGTTCGGCAGCGCGCCGCCGCACGGCAGCCGGTTCGCCGA

[0026] CACCTTCGACCCCGCGAGCCTCGGCCTCAGCTTCCCCGCCAGGCTGCTCTTCCCCGAC

[0027] AGCGGCGCCTACGGGGCCGACGGTGGCCCGCAGCAGCAGCACCACCACCAGGGGAAC

[0028] GGGAACGGCATGGAGCAGTGGGCGGCTGCGCAGATGCAGAGCTTCCCGTTCCTGCAC

[0029] GCCATGGATCACCAGATGTCCGGGAATCAGCCGCCGGCTTCGGCAATGCCGACCACAA

[0030] TGGCGGCGATGCAGGGCATGTTCCACCTAGGGCTACAGAGCGGGGGTGGCGGCAATGG

[0031] CGACGATGGAGGAAATCACCAGTTCCATCACCTGCCGGCCAAGAGGGACTACCAGCAG

[0032] CAGCAGCAGCAGCAGCAGCATGAGTACCCAAGCAGCAGGGGCATGTACGGGGACGTG

[0033] GTCAATGGCAATGGCGGCGGCTTCAATTTCTATTCCAGCACTAGCAATGCAGCTGGTAATTAG.

[0034] SEQ ID No.2:

[0035] ATGATCTTCCCTCCTGCCTTCCTAGATTCATCAAGCTGCTGGAACACCAACCACAACCA

[0036] GCTTCAGCTGCAGCAAATCGGCAGTAACACTCATATCACCGCTACTCCTTCACCTGCTG

[0037] GTCATGGAGATGGAGGAGGCAGCAACAACAACAACCATGGTCAGCAGGAAGGATTAA

[0038] TGGCCACGGCCGGGGCCGGAGGAGGTGGTGGTGATGGTGGAGGCGGCGGCGGTGGGG

[0039] ATGGTGATAGCGCCGGCGGTGGGAACAACAAGCCGATGTCGATGTCAGAGCGGGCACG

[0040] GCTGGCACGGGTGCCGCAGCCGGAGCCGGGGCTCAACTGCCCGCGCTGCGACTCCAC

[0041] CAACACCAAGTTCTGCTACTTCAACAACTACTCCCTCACCCAGCCCCGCCACTTCTGCC

[0042] GCGCCTGCCGCCGCTACTGGACCCGCGGCGGCGCGCTCCGCAACGTCCCCGTCGGTGG

[0043] CGGGTACCGCCGCCACGCCAAGCGCAGCGCCAAGCCCAAGGCGGGGTCGGCTGGATC

[0044] CGGAACCACCACGGCAGGGACGTCGTCCGCGACGTCGACGACGCCCAGCACCACTGC

[0045] TTGCACCACCGGCACTGCCACTGCGCCGCCCGCTCTGCAGTACTCCATGTTCGGCAGC

[0046] GCGCCGCCGCACGGCAGCCGGTTCGCCGACACCTTCGACCCCGCGAGCCTCGGCCTCA

[0047] GCTTCCCCGCCAGGCTGCTCTTCCCCGACAGCGGCGCCTACGGGGCCGACGGTGGCCC

[0048] GCAGCAGCAGCACCACCACCAGGGGAACGGGAACGGCATGGAGCAGTGGGCGGCTG

[0049] CGCAGATGCAGAGCTTCCCGTTCCTGCACGCCATGGATCACCAGATGTCCGGGAATCA

[0050] GCCGCCGGCTTCGGCAATGCCGACCACAATGGCGGCGATGCAGGGCATGTTCCACCTA

[0051] GGGCTACAGAGCGGGGGTGGCGGCAATGGCGACGATGGAGGAAATCACCAGTTCCATC

[0052] ACCTGCCGGCCAAGAGGGACTACCAGCAGCAGCAGCAGCAGCAGCAGCAGCATGAGTACC

[0053] CAAGCAGCAGGGGCATGTACGGGGACGTGGTCAATGGCAATGGCGGCGGCTTCAATTTCTATTCCAGCACTAGCAATGCAGCTGGTAATTAG.

[0054] The present invention also provides barley zinc finger protein, HvDOF17 protein encoded by barley zinc finger protein transcription factor HvDOF17, the amino acid sequence of which is shown in SEQ ID No:3.

[0055] SEQ ID No. 3:

[0056] MIFPPAFLDSSSCWNTNHNQLQLQQIGSNTHITATPSPAGHGDGGGSNNNNHGQQEGLMAT

[0057] AGAGGGGGDGGGGGGGDGDSAGGGNNKPMSMSERARLARVPQPEPGLNCPRCDSTNTK

[0058] FCYFNNYSLTQPRHFCRACRRYWTRGGALRNVPVGGGYRRHAKRSAKPKAGSGTTT

[0059] AGTSSATSTTPSTTACTTGTATAPPALQYSMFGSAPPHGSRFADTFDPASLGLSFPARLLFPD

[0060] SGAYGADGGPQQQHHHQGNGNGMEQWAAAQMQSFPFLHAMDHQMSGNQPPASAMPTT

[0061] MAAMQGMFHLGLQSGGGGNGDDGGNHQFHHLPAKRDYQQQQQQQQHEYPSSRGMYGDVVNGNGGGFNFYSSTSNAAGN.

[0062] This invention also provides an HvDOF17 gene promoter, the nucleotide sequence of which is shown in SEQ ID No:4. This promoter is 2000 bp in length and is used to guide the gene to be expressed in large quantities in plant cells to meet the needs of rapid synthesis of calmodulin-binding transcription factors and response to various stimuli or stresses.

[0063] SEQ ID No.4:

[0064] TACCTAATAGTATAAATGTCTTTAAATTGTTTATACTTCTTATTATATTTGTAATACTATGCA

[0065] TACCATTATGTTGAATAATAAATATTAATACGAACAACATATATCTTTGACATCATGTTGA

[0066] GTTATAATGAAGTAAAAGTTCAATATAAATAGGTCCCCTTATCTAGGCACACAAGGCC

[0067] AATTTATATTGATGCATTACCTCGGAAGTTGAATGGATAATAAATAAGTAATTTTATTTTC

[0068] ACATTAATAATATTTGGAAATTTGTTGGATAACATCACATGAGGTGCGAAGGAACAAAC

[0069] TATTTTTCCCAATCCGATGGTCTGGATTCCTGATATTTTACGACTCTTTTTCCAAAAGTTA

[0070] TGATTTATTTTGTATATGGCATATTAATAATTTCAAAATGGAATAGAATGAAACAAAATTA

[0071] CGAATTCATAAACTTGAAGCATTTTAAATCGTTTTTCAAAAATGCAGAATTGTTTGAA

[0072] TTAATGTTTAAAAATATTTTTAAGATAATAATCATAAATATTCATGATTTAGTTATGAGTTT

[0073] TTTACAATTTACAAGTACATACTTGTGATTTATTATCTCTTGTTATGTTTGCTTCTTATTTTT

[0074] TTCTTATTGTGTTCTTTGTGCCGATTGGGTTTGTGTGCAAAACCATTGACGCGTAATGTGT

[0075] CATATCACTTTCACTATCTCAACGGTCAAATAACCAACCTTTATATATTTGATTCCTTAGG

[0076] GCTTTTAAATGTTTGTTGAACTTGGGATTTCAAAATAGAGTGAGTGAAACAAAATTACC

[0077] TAAATAGTGATAGCACCATTATACTGTATGGGTGGGTCTTGAAAATTATGCGACTATAAAT

[0078] CAACTATTAACAAGCACCCCTAGAAACCATCCTTTCCTCCTTCCCACGAAAAGACTGCA

[0079] AGGCGATTAGATTTCCTCCTCTTTCGGTCGATGTCGTGATGGTCTACCCGTCTCTGATTG

[0080] CCTTTGGAACATGGAGGTGCGAAGGACACCCTCTCCCCCAGTGCGCTAGCATGAGGGC

[0081] TCCTGCTCTCATTCCTTTTAGTTTCGACGTCTTGGTTGGAGATGTATGGCAACGGCCATG

[0082] TCCTCAGTACGTATAGTTCTTTCACATTCGATCCTTGTTCTGATGGTGCGTCTAGCGTCG

[0083] TCGGAGAGCGTCTGGAGGCATGTCTCCGTCACATATCATGGGATTGACCCGTGCCAATC

[0084] TTCAATGGATATGTTTGGATCCGATCTTCATTCGTGTGTTCAGAGGTTTGATCCTTCCATT

[0085] CTACGACTCTCTTCATCTATGATGGTTTAGTTACAACAAGTTTGTTTGACTCTAGTGAGG

[0086] GAGGGGCGATAACGGCCGCGCGGCTCCGACCTGCTGCAGTGTCTGTATAGTAGTCCTTA

[0087] AGTGGTCTACAAAACCTGGCTGTAGTTTTTATTACTGTCAAGGTTGATTATGAACAGATC

[0088] ATCCCGAAAAGACACCCATATAACAAGCTTGCTAACTAGTACTTCTATATTGACTATTCT

[0089] TTATTGCGACTGTAGCTAGCAACACTGCATGCATCAGCAAGGTAGCATGGGTAGCCGTC

[0090] TCAGCACCGTCAGCACTCACCATGGCAAAAAGCAAAAGTGTGCAAAAAAAGGAAAAC

[0091] ATCAACAAGATGATGCCCTCTCCCTCCTCCAAATCGCCACAAGCTAGAGCGAGAGAGA

[0092] CCCTTCTCTCTCATCTCTCATCTCTTGGTGCTGCTGCTACCTGGCTCTCTCTCTCTCCTTT

[0093] TTCTCTCTCCTCTTGGGTAGCTCTCGCTCCCTCTCAAAGCAGTCAAGAGCTAGTCCCTC

[0094] CCTGTCTCTCTAGCTTCCATTCCATTCCTTTCCTTGGTACTAGTACTCTGATTCCCTTTGA

[0095] TTTCCCCAGCTGCCAAGTCTCTTCCTCCCACTATCTCTTCTCTCCAACCTCCAGCCCAGC

[0096] CGCCCAAACACCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCCCAACAACTCTCTGCAAGTCTAGATCGCCGGCC.

[0097] The present invention also provides a knockout recombinant vector, which is a CRISPR / Cas9 vector for editing barley zinc finger protein transcription factor HvDOF17. The original expression vector is barley Cas9, and the nucleotide sequence shown in SEQ ID No:11 or SEQ ID No:12 is inserted into the BasI restriction region.

[0098] Furthermore, the barley variety is the barley cultivar Golden Promise.

[0099] The present invention also provides a genetically engineered bacterium containing a CRISPR / Cas9 vector with HvDOF17 gene edited; the nucleotide sequence of the HvDOF17 gene is shown in SEQ ID No:1, and the full-length nucleotide sequence of its transcript cDNA is shown in SEQ ID No:2.

[0100] Furthermore, the host cell of the genetically engineered bacteria is Escherichia coli or Agrobacterium.

[0101] This invention also provides the application of the barley zinc finger protein transcription factor HvDOF17 in regulating plant growth.

[0102] This invention also provides the application of the aforementioned barley zinc finger protein transcription factor HvDOF17 in regulating barley salt tolerance. This gene is involved in the regulation of barley salt tolerance, and inhibiting the expression of this gene can improve the salt tolerance of barley.

[0103] Furthermore, the indicators reflecting the salt tolerance of barley are: the dry weight of the aboveground and underground parts of the barley plant, and the Na+ content of the aboveground and underground parts of the barley plant. + Content and / or K + content.

[0104] Furthermore, the regulatory pathway is as follows: by knocking out the HvDOF17 gene, the salt tolerance of barley is improved.

[0105] Furthermore, the knockout method is as follows:

[0106] (1) Based on the HvDOF17 genome sequence, target sequence sgRNA was designed and a CRISPR / Cas9 vector for barley HvDOF17 gene editing was constructed.

[0107] (2) The vector was transferred into Agrobacterium competent cells to obtain Agrobacterium containing the HvDOF17 gene editing CRISPR / Cas9 vector;

[0108] (3) Agrobacterium containing the HvDOF17 gene-edited CRISPR / Cas9 vector was used to infect wild-type barley embryos, and seedlings were obtained again through tissue culture. Genetically stable homozygous HvDOF17 gene deletion mutants were screened.

[0109] This invention also provides the application of the aforementioned barley zinc finger protein, the aforementioned HvDOF17 gene promoter, the aforementioned knockout recombinant vector, and the aforementioned genetically engineered bacteria in regulating barley salt tolerance.

[0110] Furthermore, the nucleotide sequence of the sgRNA is shown in SEQ ID No:11 or SEQ ID No:12.

[0111] This invention cloned the genomic DNA sequence of the HvDOF17 gene from barley, as shown in SEQ ID No:1, and simultaneously cloned the full-length cDNA sequence of the HvDOF17 gene, as shown in SEQ ID No:2. The amino acid sequence encoded by this gene is shown in SEQ ID No:3. This invention also successfully knocked out the HvDOF17 gene in barley Golden Promise using CRISPR, and used this material to demonstrate that the negative regulatory role of the HvDOF17 gene under salt stress can effectively enhance the salt tolerance of barley.

[0112] Compared with the prior art, the present invention has the following beneficial effects:

[0113] 1. This invention is the first to clone and analyze the zinc finger protein gene HvDOF17 in barley in response to salt stress, and publishes the nucleotide sequence, amino acid sequence and promoter sequence of the gene, which is of great significance for elucidating the molecular mechanism of salt tolerance regulation in barley and the breeding of salt-tolerant materials.

[0114] 2. This invention is the first to demonstrate, through transgenic means, that the HvDOF17 gene is involved in barley salt tolerance. Knocking out the HvDOF17 gene can significantly enhance the salt tolerance of barley, effectively mitigate the adverse effects of salt stress, and thus maintain and promote the normal growth of the plant. Attached Figure Description

[0115] Figure 1 This is a schematic diagram of the structure of the HvDOF17 gene in Example 1; where the black box represents the exon and the white box represents the non-coding region.

[0116] Figure 2 This is a schematic diagram of the structure of the CRISPR knockout vector constructed in Example 2; wherein, the TaU6 promoter drives sgRNA; the maize Ubi promoter (Ubi pro.) drives the expression of Cas9 protein; and the 35S promoter drives the resistance gene hygromycin.

[0117] Figure 3 These are the results of the hydroponic experiment with the transgenic material in Example 2;

[0118] Wherein, shoot represents the above-ground part or seedling, and root represents the underground part or root. (a) Fresh weight measurement results of above-ground and underground parts, unit: g; (b) Seedling height and root length measurement results, unit: cm.

[0119] Figure 4 These are the results of the hydroponic experiment with the transgenic material in Example 2;

[0120] Where shoot represents the aboveground part and root represents the underground part, (a) Phenotypic differences of knockout transgenic plants after four weeks of salt treatment (200 mM NaCl), WT: wild type, dof17-1, dof17-2, dof17-3: CRISPR knockout transgenic materials; (b) Dry weight measurements of aboveground and underground parts, in g; (c) NaCl of aboveground and underground parts. + Concentration measurement results, unit: mg.g -1 DW; (d) K of the above-ground and underground parts + Content measurement results, unit: mg.plant -1 (e) Na in the aboveground and underground parts + / K + Calculation results. Detailed Implementation

[0121] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0122] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0123] The barley cultivar used in the following examples is Golden Promise.

[0124] KOD One high-fidelity enzyme was purchased from TOYOBO Ltd., Japan; Agrobacterium tumefaciens AGL1 was purchased from Shanghai Weidi Biotech Ltd.

[0125] Example 1: Cloning of the barley zinc finger protein transcription factor HvDOF17 gene and its promoter

[0126] 1. Treatment of plant materials

[0127] Barley Golden Promise seeds were sterilized with 2% H2O2 for 20 min, rinsed 5 times with ddH2O, and then germinated in a germination box with the ventral side facing up. They were then cultured in the dark for 3 days in a culture room (14h / 10h, 22℃ / 18℃), and supplemented with light when the aboveground parts showed a 2cm tip. Seven-day-old barley seedlings were flash-frozen in liquid nitrogen and stored at -70℃ for DNA and total RNA extraction.

[0128] 2. Extraction of barley genomic DNA

[0129] Genomic DNA was extracted from barley using the CTAB method.

[0130] 3. Extraction of total RNA from barley

[0131] Total RNA was extracted from barley using the Trizol method. Genomic DNA was then removed using DNase I enzyme (Takara, Japan).

[0132] 4. cDNA Synthesis

[0133] Using Takara The Perfect Real Time (DRR037A) RT reagent kit reverse transcribes the total RNA extracted in step 3 above into single-stranded cDNA.

[0134] 5. Cloning of the HvCAMTA4 gene and its promoter

[0135] Using the DNA from step 2 and the cDNA from step 4 as templates, primers were designed according to the ORF frame of the target gene, and PCR amplification was performed using KOD One high-fidelity enzyme:

[0136] PCR system (25 μL):

[0137]

[0138] PCR procedure:

[0139]

[0140]

[0141] Specific target bands were obtained, and after gel extraction and recovery, the fragments were ligated into the T vector (Vazyme), transformed into E. coli DH5α, and positive clones were obtained by PCR verification. The clones were then sent to the company for sequencing. Primer synthesis and sequencing were both performed by Hangzhou Shangya Biotechnology Co., Ltd.

[0142] The primer and sequence information involved includes:

[0143] Using DNA as a template, the full-length genome sequence amplified by DNA-F / R is 1342 bp, as shown in SEQ ID No. 1. Primer information is as follows:

[0144] DNA-F:5'-ATGATCTTCCCTCCTGCCTTCC-3';

[0145] DNA-R:5'-CTAATTACCAGCTGCATTGCTAGTG-3';

[0146] Using cDNA as a template and cDNA-F / R as primers, a full-length cDNA sequence of 1311 bp was amplified, as shown in SEQ ID No. 2. Primer information is as follows:

[0147] cDNA-F:5'-ATGATCTTCCCTCCTGCCTTCC-3',

[0148] cDNA-R:5'-CTAATTACCAGCTGCATTGCTAGTG-3';

[0149] Using DNA as a template, the promoter sequence amplified by Promoter-F / R is 2000 bp, as shown in SEQ ID No:4. The primer information is as follows:

[0150] Promoter-F:5'-TGGTGCTCCCGGTCATTGC-3',

[0151] Promoter-R:5'-ATGAGTGTTACTGCCGATTTGC-3';

[0152] The CDS nucleotide sequence of the HvDOF17 gene was translated into amino acid sequence 377aa using BioXM 2.6, as shown in SEQ ID No:3.

[0153] Analysis of the gene structure revealed that the gene contains two exons and one intron. The gene structure diagram is attached. Figure 1 .

[0154] Example 2: Verification of HvDOF17 gene function using CRISPR transgenic method

[0155] 1. Construction of pUB-Cas9-TaU6-sgRNA:HvDOF17 CRISPR knockout vector

[0156] To generate CRISPR / Cas9 plasmids, two single guide RNA sequences of HvCAMTA4 were designed and ligated into the pTaU6:Cas9 vector via the restriction enzyme site BsaI. The target information is as follows:

[0157] TaU6-DOF17-sgF:CTTGGAAGGATTAATTGCCACGGCCGG;

[0158] TaU6-DOF17-sgR:AAACGCCGTGGCAATTAATCCTTC;

[0159] After ligation with T4 ligase, the pTaU6-sgRNA-Cas9 knockout vector was finally obtained (see appendix). Figure 2 ).

[0160] 2. Obtaining transgenic barley plants

[0161] The expression vectors constructed in step 1 were introduced into Agrobacterium AGL1 cells using a heat shock method. Agrobacterium containing the CRISPR knockout vector was then sequentially transformed into barley Golden Promise embryos. After hygromycin selection, dozens of transgenic candidate plants were obtained. DNA was then extracted from both the transgenic candidate plants and wild-type barley Golden Promise. Transgenic positive plants were obtained through PCR verification. The verification primers were DOF17-SG-F / R, and the amplified fragment lengths were 570 bp. Primer information is as follows:

[0162] DOF17-SG-F: 5'-ATGATCTTCCCTCCTGCCT-3';

[0163] DOF17-SG-R: 5'-GCTGGGCGTCGTCG-3'.

[0164] After propagation, enough transgenic seeds are harvested for experiments.

[0165] 3. Comparison of salt tolerance in barley plants with HvDOF17 gene knockout

[0166] (1) Hydroponic experiment: Three barley HvDOF17 CRISPR knockout transgenic materials (dof17-1, dof17-2, dof17-3) and barley Golden Promise wild-type plants (WT) were selected as materials. Seedlings of the above materials after 14 days of germination were used as experimental materials. The same number of seedlings of each genotype were transferred to 15L culture boxes. Culture boxes with nutrient solution containing 0 and 200mM NaCl were set up as normal conditions and salt treatment conditions, respectively. The nutrient solution with the same NaCl concentration was changed every 3 days.

[0167] The culture chamber conditions were: light intensity of 250 μm. -2S-1, day / night temperature 22 / 18℃, day / night duration 14 / 10h. Four plants were taken from each knockout transgenic material (dof17-1, dof17-2, dof17-3) and wild-type plant material. After 3 weeks of salt treatment, the plants were washed three times with deionized water to remove surface salt. Excess moisture was drained, and the plants were then photographed as a whole. The fresh weight of the aboveground and underground parts was measured. Root length and seedling height data for barley are shown in Table 1. Figure 3 As shown in b, the fresh weight results of the aboveground and underground parts of barley are shown in Table 2 and Figure 3 As shown in a.

[0168] Table 1. Root length and seedling height of barley

[0169] Seedling height (cm) Root length (cm) WT 19.58±1.13 11.62±3.81 dof17-1 21.87±1.59 12.53±1.29 dof17-2 21.90±0.57 11.45±1.90 dof17-3 21.93±1.55 12.38±1.96

[0170] Table 2 Fresh weight of barley aboveground and underground parts

[0171] Fresh weight Aboveground parts (g / plant) underground part (g / plant) WT 0.280±0.041 0.144±0.019 dof17-1 0.506±0.059 0.416±0.090 dof17-2 0.532±0.090 0.289±0.033 dof17-3 0.395±0.088 0.198±0.055

[0172] After rinsing the aboveground and underground parts of barley with purified water, four plants were taken from each of the knockout transgenic materials (dof17-1, dof17-2, dof17-3) and wild-type plants. The aboveground and underground parts of the barley were dried in an oven at 65℃, and the dry weights of the aboveground and underground parts were measured. The results are shown in Table 3. The Na+ content of the aboveground and underground parts of the barley was determined by ICP-MS. + K + The content and results are shown in Tables 4, 5, and 6. Figure 4 As shown.

[0173] Table 3 Dry weight of barley aboveground and underground parts

[0174] dry weight Aboveground parts (g / plant) underground part (g / plant) WT 0.056±0.004 0.013±0.001 dof17-1 0.104±0.010 0.033±0.006 dof17-2 0.092±0.011 0.024±0.003 dof17-3 0.070±0.014 0.017±0.005

[0175] Table 4. Na content in barley aboveground and underground parts + content

[0176] Na conc Aboveground parts (mg / g DW) Underground portion (mg / g DW) WT 126.2650±32.7541 30.6812±2.8606 dof17-1 35.0207±7.4775 34.3634±2.9409 dof17-2 59.6125±10.745 38.2309±4.4563 dof17-3 72.5965±12.2214 24.3020±8.1578

[0177] Table 5. K of barley aboveground and underground parts + content

[0178] K conc Aboveground parts (mg / g DW) Underground portion (mg / g DW) WT 31.9262±7.1631 5.8837±1.6321 dof17-1 25.8247±7.1828 5.6268±2.4152 dof17-2 27.3655±3.7663 5.9224±4.6570 dof17-3 29.9971±3.4711 6.9801±3.4641

[0179] Table 6. Na from the aboveground and underground parts of barley + / K + relative content

[0180] Na / K above ground Underground WT 4.4027±0.3750 5.4074±1.0172 dof17-1 1.3867±0.2742 6.8747±2.3939 dof17-2 2.2297±0.5935 8.8726±4.0998 dof17-3 2.4115±0.1987 5.4197±4.1437

[0181] Hydroponic experiments showed that after four weeks of salt treatment (200 Mm NaCl), CRISPR knockout transgenic plants and wild-type WT plants exhibited significant phenotypic differences (see appendix). Figure 4 a) Knockout plants exhibited superior salt tolerance compared to wild-type plants, as evidenced by fewer yellowed old leaves in the aboveground parts of the knockout plants, slower new leaf growth in the aboveground parts of the wild-type plants, and greater root biomass in the knockout plants. Therefore, HvDOF17 can negatively regulate barley salt tolerance, and this negative regulatory effect may be due to HvDOF17's involvement in root potassium (K) regulation. + Accumulation and Na + Allocation in progress.

[0182] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims. All transcripts, genomes, and promoters of the HvDOF17 gene, the amino acid sequences encoding proteins, and biological materials containing any of the above-mentioned genes are within the scope of protection of the present invention.

Claims

1. A barley zinc finger protein transcription factor HvDOF17 application in regulating the salt tolerance of barley by knocking out the gene to improve the salt tolerance of barley, wherein the nucleotide sequence of the barley zinc finger protein transcription factor is shown as SEQ ID No: 1, and the full-length nucleotide sequence of the transcript cDNA is shown as SEQ ID No:

2. HvDOF17 application in regulating the salt tolerance of barley by knocking out the gene to improve the salt tolerance of barley, wherein the nucleotide sequence of the barley zinc finger protein transcription factor is shown as SEQ ID No: 1, and the full-length nucleotide sequence of the transcript cDNA is shown as SEQ ID No:

2. HvDOF17 application in regulating the salt tolerance of barley by knocking out the gene to improve the salt tolerance of barley, wherein the nucleotide sequence of the barley zinc finger protein transcription factor is shown as SEQ ID No: 1, and the full-length nucleotide sequence of the transcript cDNA is shown as SEQ ID No:

2.

2. Use according to claim 1, wherein The indices reflecting the salt tolerance of barley are: the dry weights of the aboveground and underground parts of the barley plant, and the Na + contents and / or K + contents of the aboveground and underground parts of the barley plant, respectively.

3. The use according to claim 1, wherein the compound is ###0002### The method of knocking out is: (1) According to HvDOF17 genomic sequences, target sequences sgRNA were designed, and CRISPR / Cas9 vectors for barley HvDOF17 gene editing were constructed; (2) transferring the vector into Agrobacterium competent cells to obtain Agrobacterium containing HvDOF17 Agrobacterium of the gene editing CRISPR / Cas9 vector; (3) using a bacterial infection with Agrobacterium containing a CRISPR / Cas9 vector HvDOF17 Wild-type barley young embryos were infected with Agrobacterium containing a CRISPR / Cas9 vector, and young seedlings were reobtained by tissue culture, and a genetically stable homozygous HvDOF17 gene deletion mutant was obtained by screening.

4. A barley zinc finger protein transcription factor HvDOF17 The nucleotide sequence of the barley zinc finger protein transcription factor is shown as SEQ ID No: 1, and the full-length nucleotide sequence of its transcript cDNA is shown as SEQ ID No:

2. HvDOF17 The application relates to the application of the barley zinc finger protein transcription factor in regulating the growth of barley. HvDOF17 The application relates to the application of the barley zinc finger protein transcription factor in regulating the growth of barley.

5. Application of a genetically engineered bacterium in regulating salt tolerance of barley, wherein the genetically engineered bacterium comprises HvDOF17 a CRISPR / Cas9 vector for gene editing; the HvDOF17 nucleotide sequence of the gene is shown as SEQ ID No: 1, and the full-length nucleotide sequence of the transcript cDNA is shown as SEQ ID No: 2, and the regulated pathway is: by knocking out HvDOF17 HvDOF17 the gene, the salt tolerance of the barley is improved.

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