Barley zinc finger protein transcription factor HvDOF17 and application thereof in regulating salt tolerance of barley

Abstract

The invention provides a barley zinc finger protein transcription factor HvDOF17 and application of the barley zinc finger protein transcription factor HvDOF17 in regulation and control of barley salt tolerance. The nucleotide sequence of the barley zinc finger protein transcription factor HvDOF 17 is shown as SEQ ID No: 1, and the transcript cDNA full-length nucleotide sequence of the barley zinc finger protein transcription factor HvDOF 17 is shown as SEQ ID No: 2. The zinc finger protein gene HvDOF17 responding to salt stress in barley is cloned and analyzed for the first time, a nucleotide sequence, an amino acid sequence and a promoter sequence of the gene are disclosed, and the zinc finger protein gene HvDOF17 has important significance on clarification of a molecular mechanism of barley salt tolerance regulation and culture of salt-tolerant materials. Besides, the invention proves that the HvDOF17 gene participates in the salt tolerance of the barley through a transgenic means for the first time, and after the HvDOF17 gene is knocked out, the salt tolerance of the barley can be remarkably enhanced, and adverse effects caused by salt stress can be effectively relieved, so that normal growth of plants is maintained.

Description

Technical Field

[0001] The invention relates to the technical field of biological gene engineering, and in particular to a barley zinc finger protein transcription factor HvDOF17 and an application thereof in regulating barley salt tolerance. Background Art

[0002] Soil salinization is one of the main abiotic stresses that threatens global crop production and food security and restricts sustainable agricultural development. Soil salt damage will harm crop growth and food security in the long term. Breeding salt-tolerant crops to adapt to saline-alkali land has become a consensus for the effective utilization and transformation of saline-alkali land. Salt stress causes osmotic stress, ion imbalance, and oxidative stress to plants, and ion balance is a prerequisite for avoiding or alleviating osmotic stress and oxidative damage in plants. Ion balance, dominated by calcium signaling and transcriptional regulation, is a key mechanism for regulating plant salt tolerance, which involves complex genetic or molecular regulation. Therefore, exploring crop salt-tolerant germplasm and gene resources and revealing the physiological and molecular mechanisms of crop salt-tolerant genes are of great significance for breeding or creating new salt-tolerant crop materials to adapt to saline-alkali land.

[0003] The main harm of salt stress to plants is the osmotic stress and ion imbalance caused by high concentrations of salt ions, as well as the secondary oxidative stress caused by the superposition of the two. Usually, excessive salt accumulation in the soil will cause changes in the physical and chemical properties of the soil, resulting in physiological drought in plant cells, ion imbalance, and accumulation of reactive oxygen species (ROS). When plants are in a high-concentration salt stress environment for a long time, they will experience serious growth inhibition, developmental changes, metabolic disorders, premature aging and infertility. Specifically, osmotic stress is mainly caused by the excessive concentration of soluble salts in the soil, which reduces the absorption and utilization of water by plants, leading to water deficit in plant cells. Secondly, ionic stress is caused by the accumulation of excessive salt ions (mainly sodium ions Na + ), inhibiting plant cells from reacting with potassium ions (K + ) and other mineral nutrients, affecting the metabolic pathways of plant photosynthesis, stomatal regulation, and material synthesis, resulting in specific ion toxicity. Furthermore, secondary oxidative stress is caused by both osmotic and ionic stress. When excessive toxic substances accumulate in plant cells, a large amount of free ROS will be produced, further affecting various physiological and biochemical processes in the plant body and causing a variety of metabolic disorders.

[0004] In summary, considering that salt stress causes physiological damage to plants such as osmotic, ionic and oxidative stress, studying and clarifying the response mechanism of plants to salt stress is the research basis for improving the salt tolerance potential of crops.

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

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

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

[0008] The specific technical solutions are as follows:

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

[0010] SEQ ID No.1:

[0011] ATGATCTTCCCTCCTGCCTTCCTAGATTCATCAAGCTTGCTGGAACACCAACCACAACCA

[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 a barley zinc finger protein, an HvDOF17 protein encoded by the barley zinc finger protein transcription factor HvDOF17, and the amino acid sequence of the HvDOF17 protein 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] The present invention also provides a HvDOF17 gene promoter, the nucleotide sequence of which is shown in SEQ ID No: 4. The promoter is 2000 bp 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] CGAAATGTCATAAACTTGAAGCATTTTAAATCGTTTTTCAAAAATGCAGAATTGTTTGAA

[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, using barley Cas9 as the original expression vector, and inserting a nucleotide sequence as shown in SEQ ID No: 11 or SEQ ID No: 12 into the BasI restriction enzyme cutting region.

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

[0099] The present invention also provides a genetically engineered bacterium, which contains a CRISPR / Cas9 vector for editing the HvDOF17 gene; 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] The present invention also provides the application of the barley zinc finger protein transcription factor HvDOF17 in regulating plant growth.

[0102] The present invention also provides the use of the barley zinc finger protein transcription factor HvDOF17 in regulating barley salt tolerance. The gene is involved in the regulation of barley salt tolerance, and inhibiting the expression of the 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 and / or K + content.

[0104] Furthermore, the regulation approach is: improving the salt tolerance of barley by knocking out the HvDOF17 gene.

[0105] Furthermore, the knockout method is:

[0106] (1) Design target sequence sgRNA based on the HvDOF17 genome sequence and construct a CRISPR / Cas9 vector for barley HvDOF17 gene editing;

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

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

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

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

[0111] The present invention cloned the genomic DNA sequence of the gene from barley, as shown in SEQ ID No: 1, and cloned the full-length cDNA sequence of the HvDOF17 gene as shown in SEQ ID No: 2, and the amino acid sequence encoded by the gene is shown in SEQ ID No: 3. The present invention also successfully knocked out the HvDOF17 gene in the barley Golden Promise using the CRISPR method, and used the material to confirm that the negative regulation 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. The present invention cloned and analyzed for the first time the zinc finger protein gene HvDOF17 in barley that responds to salt stress, and published the nucleotide sequence, amino acid sequence and promoter sequence of the gene, which is of great significance for clarifying the molecular mechanism of salt tolerance regulation in barley and the cultivation of salt-tolerant materials.

[0114] 2. The present invention, for the first time, uses transgenic means to confirm that the HvDOF17 gene is involved in the salt tolerance of barley. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 It is a schematic diagram of the structure of the HvDOF17 gene in Example 1; wherein the black frame represents the exon, and the white frame represents the non-coding region.

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

[0117] Figure 3 This is the result of the hydroponic experiment of the genetically modified material in Example 2;

[0118] Wherein, shoot refers to the aboveground part or seedling, root refers to the underground part or root, (a) the fresh weight measurement results of the aboveground part and the underground part, unit: g; (b) the measurement results of the seedling height and the root length, unit: cm.

[0119] Figure 4 This is the result of the hydroponic experiment of the genetically modified material in Example 2;

[0120] Wherein, shoot refers to the aboveground part, and root refers to the underground part. (a) Phenotypic difference 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 measurement results of the aboveground and underground parts, unit: g; (c) NaCl concentration of the aboveground and underground parts + Concentration measurement results, unit: mg.g -1 DW; (d) K in aboveground and underground parts + Content measurement results, unit: mg.plant -1 ; (e) Na in aboveground and underground parts + / K + Calculate the result. DETAILED DESCRIPTION

[0121] The technical solution of the present invention is 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 form of modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0122] In the present invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

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

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

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

[0126] 1. Processing of plant materials

[0127] Golden Promise barley seeds were sterilized with 2% H2O2 for 20 minutes, rinsed with ddH2O 5 times, and germinated in a germination box with the ventral side facing up. They were cultured in the dark in a culture room (14h / 10h, 22℃ / 18℃) for 3 days, and supplemented with light when the aboveground part was 2cm white. The 7-day-old barley seedlings were taken, quickly frozen in liquid nitrogen, and stored at -70℃ for DNA and total RNA extraction.

[0128] 2. Extraction of barley genomic DNA

[0129] The CTAB method was used to extract barley genomic DNA.

[0130] 3. Extraction of total RNA from barley

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

[0132] 4. Synthesis of cDNA

[0133] Using Takara's The total RNA extracted in 3 above was reverse transcribed into single-stranded cDNA using the RT reagent Kit Perfect Real Time (DRR037A).

[0134] 5. Cloning of HvCAMTA4 gene and promoter

[0135] Using the DNA in step 2 and the cDNA in step 4 as templates, design primers according to the ORF frame of the target gene, and use KOD One high-fidelity enzyme to perform PCR amplification respectively:

[0136] PCR system (25 μL):

[0137]

[0138] PCR procedure:

[0139]

[0140]

[0141] The specific target band was obtained, and the fragment was connected to the T vector (Vazyme) after gel excision and recovery, and transformed into E. coli DH5α. The positive clone was verified by PCR and then sent to the company for sequencing. Primer synthesis and sequencing were completed by Hangzhou Shangya Biotechnology Co., Ltd.

[0142] Primers and sequence information involved, including:

[0143] Using DNA as a template, DNA-F / R amplified a full-length genomic sequence of 1342 bp, as shown in SEQ ID No. 1, and the primer information is:

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

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

[0146] Using cDNA as template and cDNA-F / R as primers, amplify the full-length cDNA sequence of 1311 bp, as shown in SEQ ID No. 2. The primer information is as follows:

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

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

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

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

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

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

[0153] Through the analysis of gene structure, it was found that the gene has two exons and one intron. The gene structure diagram is attached. Figure 1 .

[0154] Example 2 CRISPR transgenic method to verify the function of HvDOF17 gene

[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 was 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 above were introduced into Agrobacterium AGL1 by heat shock method. Agrobacterium containing CRISPR knockout vectors were transformed into barley Golden Promise embryos in sequence, and dozens of transgenic candidate plants were obtained after hygromycin screening. DNA of transgenic candidate plants and wild-type barley Golden Promise was extracted in sequence, and transgenic positive plants were obtained by PCR verification method. The verification primers were DOF17-SG-F / R, and the length of the amplified fragments was 570bp. The primer information was:

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

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

[0164] After multiplication, enough transgenic seeds are harvested for use in experiments.

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

[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 14 days after germination were used as experimental materials. The same number of seedlings of each genotype were transferred to a 15L culture box, and the culture boxes were set with nutrient solution containing 0 and 200 mM NaCl concentrations as normal conditions and salt treatment conditions, and the nutrient solution with the same NaCl concentration was replaced every 3 days.

[0167] The conditions in the culture room were: light intensity of 250 μm m -2s-1, day and night temperature was 22 / 18℃, day and night length was 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, they were washed three times with deionized water to remove the salt on the surface of the experimental materials. After the excess water was drained, the experimental materials were photographed as a whole, and the fresh weight of the aboveground and underground parts was measured. The root length and seedling height data of barley are shown in Table 1 and Figure 3 The fresh weight of the aboveground and underground parts of barley is 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 aboveground and underground parts of barley

[0171] Fresh weight Above ground (g / plant) Underground (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 the aboveground and underground parts of barley were rinsed with pure water, 4 plants were taken from each knockout transgenic material (dof17-1, dof17-2, dof17-3) and wild-type plant material, and the dry weights of the aboveground and underground parts of barley were measured in an oven at 65°C. The results are shown in Table 3. ICP-MS was used to measure the Na + , K + Content, the results are shown in Table 4, Table 5, Table 6 and Figure 4 shown.

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

[0174] Dry weight Above ground (g / plant) Underground (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 in the aerial and underground parts of barley + content

[0176] Na conc Aboveground part (mg / g DW) Underground part (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 in aerial and underground parts of barley + content

[0178] K Aboveground part (mg / g DW) Underground part (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 in the aerial 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] The results of the hydroponic experiment showed that after four weeks of salt treatment (200 mM NaCl), the CRISPR knockout transgenic plants and the wild type WT showed obvious phenotypic differences (see Appendix Figure 4 a). The knockout plants have better salt tolerance than the wild-type plants, and their growth performance is as follows: the knockout plants have fewer yellowing old leaves above ground than the wild-type plants, the new leaves of the wild-type plants grow more slowly above ground, and the knockout plants have more root biomass than the wild-type plants. Therefore, HvDOF17 can negatively regulate barley salt tolerance, and this negative regulation may be due to the involvement of HvDOF17 in root K + Accumulation and Na + Allocating.

[0182] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variants and modifications without exceeding the technical solution described in the claims. All nucleotide sequences of the transcript, genome and promoter of the HvDOF17 gene, the amino acid sequence of the encoded protein, and biological materials containing any of the above genes belong to the protection scope of the present invention.

Claims

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

2.

2. A barley zinc finger protein, characterized in that The amino acid sequence of the HvDOF17 protein encoded by the barley zinc finger protein transcription factor HvDOF17 is shown in SEQ ID No:

3.

3. HvDOF17 gene promoter, characterized in that The nucleotide sequence of the HvDOF17 gene promoter is shown in SEQ ID No:

4.

4. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contain a CRISPR / Cas9 vector for editing the HvDOF17 gene; 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.

5. Use of the barley zinc finger protein transcription factor HvDOF17 as claimed in claim 1 in regulating barley salt tolerance.

6. The use according to claim 5, characterized in that 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, respectively. + Content and / or K + content.

7. The use according to any one of claims 5 or 6, characterized in that The regulation approach is: improving the salt tolerance of barley by knocking out the HvDOF17 gene.

8. The use according to claim 7, characterized in that The knockout method is: (1) Design target sequence sgRNA based on the HvDOF17 genome sequence and construct a CRISPR / Cas9 vector for barley HvDOF17 gene editing; (2) transferring the vector into Agrobacterium competent cells to obtain Agrobacterium containing the HvDOF17 gene editing CRISPR / Cas9 vector; (3) Agrobacterium containing the HvDOF17 gene editing CRISPR / Cas9 vector was used to infect wild-type barley embryos, and seedlings were obtained through tissue culture. Genetically stable homozygous HvDOF17 gene deletion mutants were screened.

9. Use of the barley zinc finger protein transcription factor HvDOF17 as claimed in claim 1 in regulating plant growth.

10. Use of the barley zinc finger protein according to claim 2, the HvDOF17 gene promoter according to claim 3, or the genetically engineered bacteria according to claim 4 in regulating barley salt tolerance.

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