WO2021136255A1 - Hak基因在调控植物性状中的作用 - Google Patents

Hak基因在调控植物性状中的作用 Download PDF

Info

Publication number
WO2021136255A1
WO2021136255A1 PCT/CN2020/140748 CN2020140748W WO2021136255A1 WO 2021136255 A1 WO2021136255 A1 WO 2021136255A1 CN 2020140748 W CN2020140748 W CN 2020140748W WO 2021136255 A1 WO2021136255 A1 WO 2021136255A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
protein
hak
acid sequence
amino acid
Prior art date
Application number
PCT/CN2020/140748
Other languages
English (en)
French (fr)
Inventor
李彦莎
张金山
王飞
Original Assignee
山东舜丰生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东舜丰生物科技有限公司 filed Critical 山东舜丰生物科技有限公司
Priority to CN202080003873.1A priority Critical patent/CN113227383B/zh
Publication of WO2021136255A1 publication Critical patent/WO2021136255A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/06—Roots
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)

Definitions

  • the present invention relates to the field of biology, especially the field of plant biology, and specifically relates to the role of HAK genes in regulating plant traits.
  • KUP/HAK/KT family is the earliest discovered, most numerous, and most functional potassium transport family in the biological world, and it is widely present in many species. It mediates potassium absorption in plants under low potassium stress, and participates in physiological processes such as plant cell enlargement and root auxin transport. The expression of genes in this family is regulated by plant growth and development regulators and environmental stimuli, and participates in plants. The growth and development and adversity response mechanism.
  • KUP K+uptake permease
  • HAK high-affinity K+
  • KT K+transporter
  • KT is a transporter with potassium transport function found in Arabidopsis.
  • Salt stress is one of the main abiotic stresses in plants. It will cause changes in multiple metabolic pathways in plants, resulting in complex changes in physiological levels, mainly manifested in ion imbalance, water deficit, and oxidative toxicity, which in turn leads to a decline in plant photosynthesis , Increased energy consumption, accelerated senescence, restricted growth, decreased yield and quality.
  • One of the main ways for plants to improve their own salt stress resistance is to regulate the transport of potassium (K) and sodium (Na) in the body through xylem parenchyma. The tolerance to salt stress is reflected in the adaptation to the toxic effects of Na+ and the maintenance of its own K+ nutrition. Plants with a higher K+/Na+ ratio are more tolerant to salt stress.
  • the purpose of the present invention is to provide a method for enhancing the tolerance of plants to high-salt stress by regulating the HAK gene. It further provides a method for enhancing the salt resistance of plants by mediating the regulation of Na+.
  • Another object of the present invention is to provide a use of HAK gene and its mutants in enhancing the salt tolerance of plants. Overexpression of the gene or mutation to obtain a mutant protein with better activity can significantly enhance the salt tolerance of plants.
  • the use of a substance for improving plant traits or preparing a preparation or composition for regulating plant traits wherein the plant traits include one or more selected from the group consisting of Species traits: (i) root length, root branching and/or root weight; (ii) plant height; and (iii) salt tolerance;
  • the substance is selected from the following group: (a) HAK protein; (b) nucleic acid sequence encoding HAK protein; (c) HAK protein and promoters encoding nucleic acid sequence thereof; or a combination thereof.
  • the HAK protein includes wild-type HAK protein and HAK protein mutants.
  • the HAK protein has the function of mediating Na + transport in plants.
  • amino acid sequence of the wild-type HAK protein is shown in SEQ ID NO: 2.
  • sequence of the HAK protein mutant has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, less than 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% Sequence identity.
  • sequence derived from it is the amino acid sequence of the wild-type HAK protein.
  • the use also includes regulating the transport of Na + ions in plants, which is embodied in one or any of the following i-iv:
  • the plants include angiosperms and gymnosperms.
  • the gymnosperm is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cunninghamaceae, Cupressaceae, Tricuspidae Cunninghamaceae, Taxaceae, Ephedraceae, Maizeaceae, Monotypicaceae, Centipedeaceae, or a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, legumes, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, rice, soybean, tomato, corn, sorghum, quinoa, millet, potato, tobacco, wheat, sorghum, rape, spinach, lettuce, cabbage , Cucumber, chrysanthemum, water spinach, celery, lettuce or a combination thereof.
  • the HAK gene is derived from: gymnosperms or angiosperms.
  • the HAK gene is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the HAK gene is derived from herbaceous plants and woody plants.
  • the HAK gene is derived from a Solanaceae plant, a Gramineae plant, a legume plant, or a combination thereof.
  • the HAK gene is derived from Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the HAK gene is derived from a cruciferous plant, a gramineous plant, a leguminous plant, a Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or a combination thereof.
  • the HAK gene is derived from Arabidopsis, rice, soybean, tomato, corn, sorghum, quinoa, millet, potato, tobacco, wheat, sorghum, rape, spinach, lettuce, cabbage, cucumber, Chrysanthemum vulgare, water spinach, celery, lettuce or a combination thereof.
  • the nucleic acid sequence encoding the HAK protein is a nucleic acid sequence of a gene selected from the group consisting of SlHAK20, OsHAK4, OsHAK17, ZmHAK4, SbHAK9, SbHAK26, GrHAK8, GmHAK12, PtHAK21, MdHAK5, or a combination thereof .
  • the nucleic acid sequence encoding the HAK protein is selected from the group consisting of the SlHAK20 gene derived from tomato, and the OsHAK4 or OsHAK17 gene derived from rice.
  • amino acid sequence of the HAK protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is formed by substitution, deletion or addition of one or more (such as 1-10) amino acid residues, and has the described regulatory plant traits A functional polypeptide derived from (i); or
  • the amino acid sequence has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least the amino acid sequence shown in SEQ ID NO: 2, 4 or 6
  • nucleotide sequence encoding the HAK protein is selected from any one or a combination of the following groups:
  • the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5 has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least A nucleotide sequence with 90% or at least 95% sequence identity;
  • the HAK protein mutant is a SlHAK20 protein mutant.
  • the nucleic acid sequence encoding the HAK protein mutant is a SlHAK20 nucleic acid sequence mutant.
  • the mutant form of the protein includes one or more amino acid substitutions, deletions or additions.
  • amino acid sequence of the HAK protein mutant includes:
  • the amino acid fragments with unequal numbers and random sequences include 1-100 amino acids, preferably 1-50 amino acids, preferably 1-30 amino acids, preferably 5-25 amino acids, preferably 10-amino acids. 20 amino acids.
  • the deletion method is preferably continuous deletion of amino acids in the C segment.
  • the number of missing amino acids exceeds one-half of the number of amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • the HAK protein mutant has at least 10%, at least 20%, at least 30%, at least 40%, at least 50% compared with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. %, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, At least 98% or at least 99% sequence identity.
  • the mutein at least basically retains the biological function of the sequence from which it is derived.
  • the HAK protein mutant sequence is selected from the following group:
  • amino acid sequence shown in SEQ ID NO: 12 or 14 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the function of regulating plant traits A polypeptide derived from (i); or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 12 or 14 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), which can regulate Na+ in plants The functional polypeptide of transport.
  • nucleotide sequence encoding the HAK protein mutant is selected from the following group:
  • the 5'end and/or 3'end of the nucleotide sequence shown in SEQ ID NO: 11 or 13 is truncated or 1-60 (preferably 1-30, more preferably 1- 10) the nucleotide sequence of nucleotides;
  • the nucleic acid sequence encoding the mutant is obtained by natural mutagenesis or gene editing technology.
  • the gene editing technology includes TALEN, ZIN, and CRISPR.
  • the composition includes an agricultural composition.
  • the composition further includes an agronomically acceptable carrier.
  • the content of the substance in the composition is (a) HAK protein; (b) nucleic acid sequence encoding HAK protein; (c) HAK protein and promoters encoding nucleic acid sequence thereof; or
  • the combination is 0.0001-99wt%, preferably 0.1-90wt%, more preferably 1-80wt%, based on the total weight of the composition.
  • the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the promoter includes a small molecule compound that promotes the expression of the HAK gene or its encoded protein.
  • the promoter is selected from the following group: small molecule compounds, nucleic acid molecules, or a combination thereof.
  • a method for improving plant traits including the step of regulating the expression and/or activity of HAK protein or its encoding nucleic acid sequence in said plant, thereby improving plant traits.
  • the improved plant traits are one or more selected from the following group:
  • the improved traits also include: (iv) promoting the transport of Na+ ions in plants.
  • said regulating the expression and/or activity of HAK protein or its encoding nucleic acid in said plant includes:
  • the ratio of the activity E1 of the HAK protein mutant or its encoding nucleic acid sequence in the plant to the background activity E0 of the same wild-type HAK protein or its encoding nucleic acid sequence in the same type of plant (E1 /E0) ⁇ 1.5 times, ⁇ 2 times, preferably ⁇ 5 times, more preferably ⁇ 10 times.
  • the method includes the steps:
  • the HAK protein or its encoding nucleic acid, or its promoter, or the HAK protein mutant or its encoding nucleic acid is introduced into the plant or plant cell, thereby improving the properties of the plant.
  • the method includes the steps:
  • the method of introduction includes: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method, ultrasonic method and polyethylene glycol (PEG)-mediated method.
  • the deletion method is preferably continuous deletion of amino acids in the C segment.
  • the number of missing amino acids exceeds one-half of the number of amino acids in the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid fragments with unequal numbers and random sequences include 1-100 amino acids, preferably 1-50 amino acids, preferably 1-30 amino acids, preferably 5-25 amino acids, preferably 10-amino acids. 20 amino acids.
  • the amino acid sequence of the mutant protein is an amino acid sequence formed by consecutively deleting 473 amino acid residues at the C-terminus on the basis of the amino acid sequence shown in SEQ ID NO: 2.
  • 1-100 amino acids of random sequence preferably 1-50 amino acids, preferably 1-30 amino acids, preferably 5-25 amino acids, preferably 10-20 amino acids are added to the C-terminus of the aforementioned mutant protein. .
  • amino acid sequence of the mutant protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO: 12 or 14 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the function of regulating plant traits A polypeptide derived from (i); or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO: 12 or 14 is greater than or equal to 80% (preferably greater than or equal to 90%, more preferably greater than or equal to 95% or greater than or equal to 98%). At least substantially the same active polypeptide.
  • an isolated polynucleotide is provided, which encodes the mutein as described in the third aspect of the present invention.
  • nucleotide sequence of the polynucleotide is selected from the following group:
  • the 5'end and/or 3'end of the nucleotide sequence shown in SEQ ID NO: 11 or 13 is truncated or 1-60 (preferably 1-30, more preferably 1- 10) the nucleotide sequence of nucleotides;
  • a vector which contains the polynucleotide according to the fourth aspect of the present invention.
  • a host cell which contains the vector as described in the fifth aspect of the present invention or the polynucleotide as described in the fourth aspect of the present invention integrated into the genome.
  • the host cells include eukaryotic cells and prokaryotic cells.
  • a genetically engineered plant cell, tissue or organ which contains the HAK protein mutant or the nucleic acid sequence encoding the HAK protein in the third aspect of the present invention.
  • a method for preparing genetically engineered plant cells, plant tissues or organs as described in the seventh aspect of the present invention comprising the following method steps: regulating the HAK gene or its The expression level and/or activity of the encoded protein, thereby obtaining genetically engineered plant tissues or plant cells.
  • the method includes the steps:
  • the method includes the steps:
  • the method of introduction includes: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method, ultrasonic method and polyethylene glycol (PEG)-mediated method.
  • a method for preparing a transgenic plant or a gene-edited plant including the steps:
  • the plant cell, tissue or organ as described in the seventh aspect of the present invention is regenerated into a plant body, thereby obtaining a transgenic or gene-edited plant.
  • the host cell according to the sixth aspect of the present invention is cultured to express the fusion protein; and/or, the fusion protein is isolated.
  • a method for detecting salt tolerance in plants comprising detecting the expression activity and/or expression amount of HAK protein or its encoding nucleic acid or its mutant.
  • the HAK protein is selected from the group consisting of SlHAK20, OsHAK4, OsHAK17, ZmHAK4, SbHAK9, SbHAK26, GrHAK8, GmHAK12, PtHAK21, MdHAK5 or any combination thereof.
  • Figure 1 shows the transcription level of SlHAK20 in different tomato plants. Among them, SlEF1a was used as an internal reference.
  • FIG. 2 shows the protein expression levels of SlHAK20-YFP in different tomato plants.
  • Figure 3 shows the results of the salt tolerance test of genetically modified tomato plants.
  • a shows the growth state of the plant under 175mM NaCl salt stress
  • b shows the growth state of the plant two weeks after recovery.
  • Figure 4 shows the results of the survival rate analysis of transgenic tomato plants.
  • Figure 5 shows the Na+ content and K+ content of the roots of the mutants compared with the wild-type roots under salt stress conditions with time.
  • Figure 6 shows the Na+/K+ of the roots of the mutants compared with the wild-type roots under salt stress conditions with time.
  • Figure 7 shows the Na+ content and K+ content of the xylem of the mutants compared with the wild-type xylem under salt stress conditions with time.
  • Figure 8 shows the Na+ content and K+ content of the above-ground parts of the mutants compared with the wild-type under salt stress conditions with time.
  • Figure 9 shows the Na+/K+ of the aboveground part of the mutant compared with the wild type under salt stress conditions with time.
  • Figure 10 shows the changes in the root length and the number of root branches of the mutants compared to the wild type after salt treatment.
  • Figure 11 shows the change in plant height of the mutant compared to the wild type after salt treatment.
  • Figure 12 shows the Na+/K+ of the roots and aerial parts of the mutant type compared to the wild type under salt stress conditions.
  • a shows the Na+/K+ of the roots of the mutant type compared to the wild type
  • b shows the Na+/K+ of the aerial parts of the mutant type compared to the wild type.
  • SEQ ID NO: Sequence description 1 Source tomato wild-type SlHAK20 nucleic acid sequence 2 Amino acid sequence of wild-type SlHAK20 from tomato 3 Nucleic acid sequence of wild-type OsHAK4 from rice 4 Amino acid sequence of wild-type OsHAK4 from rice 5 Nucleic acid sequence of wild-type OsHAK17 from rice 6 Amino acid sequence of wild-type OsHAK17 from rice 7 SlHAK20-1 nucleic acid sequence of the present invention 8 SlHAK20-1 amino acid sequence of the present invention 9 SlHAK20-2 nucleic acid sequence of the present invention 10 SlHAK20-2 amino acid sequence of the present invention 11 SlHAK20-3 nucleic acid sequence of the present invention 12 SlHAK20-3 amino acid sequence of the present invention 13 SlHAK20-4 nucleic acid sequence of the present invention 14 SlHAK20-4 amino acid sequence of the present invention
  • HAK gene or its encoded protein or its promoter of a plant can be used to regulate plant traits, the traits including : Root length, root weight and root branching, plant height, salt tolerance.
  • a plant such as tomato
  • HAK protein transporter
  • HAK protein and its coding gene in regulating Na+ transport in plants.
  • HAK protein can regulate the loading and unloading of Na+ in plants to improve the salt tolerance of plants.
  • HAK protein can enhance the transport of Na+ to and unloading from the xylem in plants, and at the same time promote the transport of Na+ from roots to other tissues or the outside world.
  • the inventors also unexpectedly discovered that when the amino acid sequence of the HAK protein is deleted by the gene editing technology, the root length and root branching can be increased under salt stress, and the plant height of the plant can be increased, and the plant's Salt tolerance.
  • the improvement of the roots can enhance the lodging resistance of plants, promote the absorption of nutrients, and enhance the ability to adapt to the environment.
  • the increase in plant height can increase the biomass and/or yield of the plant.
  • HAK protein As used herein, the terms “HAK protein”, “HAK polypeptide”, and “HAK transporter” are used interchangeably and refer to a type of ion that exists on the membranes of different organelles, such as plasma membranes, vacuolar membranes, and thylakoid membranes. Transporter.
  • HAK protein mutant and mutant HAK protein can be used interchangeably and refer to a mutant obtained by mutation of the amino acid sequence of a wild-type HAK protein.
  • HAK gene of the present invention refers to DNA sequences, and all refer to plants derived from plants (such as tomato, wheat). Etc.) HAK gene or its variants.
  • the HAK gene of the present invention is the SlHAK20 gene, and its nucleotide sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2.
  • the present invention also includes at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% with the preferred gene sequence of the present invention (SEQ ID NO:1) , At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology nucleic acid
  • the nucleic acid can also effectively regulate the agronomic traits of plants (such as tomato).
  • “Homology” or “identity” refers to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine, so each molecule is the same at that position.
  • the "percent identity” between two sequences is a function of the number of positions compared by the number of matching positions shared by the two sequences x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the nucleotide sequence in SEQ ID NO:1 can be substituted, deleted or added one or more to generate a derivative sequence of SEQ ID NO:1. Due to the codon degeneracy, even if it is different from SEQ ID NO:1 has low homology and can basically encode the amino acid sequence shown in SEQ ID NO:2.
  • the nucleotide sequence in SEQ ID NO:1 has been substituted, deleted or added at least one nucleotide-derived sequence” means that it can be used under moderately stringent conditions, and more preferably under highly stringent conditions.
  • SEQ ID NO: The nucleotide sequence that hybridizes with the nucleotide sequence shown in 1.
  • variant forms include (but are not limited to): deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , Insertion and/or substitution, and adding several at the 5'and/or 3'end (usually within 60, preferably within 30, more preferably within 10, most preferably within 5 ) Nucleotide.
  • genes provided in the examples of the present invention are derived from tomato, they are derived from other similar plants (especially plants belonging to the same family or genus as tomato) and are related to the sequence of the present invention (preferably, the sequence is such as SEQ ID NO: 1) HAK gene sequence with certain homology (conservation) is also included in the scope of the present invention, as long as those skilled in the art can easily use the information provided in this application after reading this application The sequence was isolated from other plants.
  • the polynucleotide or nucleic acid sequence of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO:1 or a degenerate variant.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, It is more preferable that the hybridization occurs when more than 95%.
  • HAK gene of the present invention is preferably derived from tomato
  • other genes from other plants that have certain homology with the tomato HAK gene and have similar functions are also within the scope of the present invention.
  • Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.
  • the HAK gene of the present invention can also be the OsHAK4 gene derived from rice. Its nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence of its encoded protein is shown in SEQ ID NO: 4 is shown.
  • the HAK gene of the present invention can also be OsHAK17 derived from rice. Its nucleotide sequence is shown in SEQ ID NO: 5, and the amino acid sequence of its encoded protein is shown in SEQ ID. NO: shown in 6. It further includes ZmHAK4 derived from corn; SbHAK9 and SbHAK26 derived from sorghum; GrHAK8 derived from cotton; GmHAK12 derived from soybean.
  • the full-length sequence of HAK nucleotide of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. It is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • mutant protein of the present invention As used herein, the terms "mutant protein of the present invention”, “SlHAK20 mutein”, “SlHAK20 gene mutant encoding protein” and “polypeptide of the present invention” are used interchangeably, and all refer to the C-terminal of the protein encoded by the SlHAK20 gene of the present invention After deletion, the mutant protein formed.
  • a typical amino acid sequence of the SlHAK20 mutein of the present invention is shown in SEQ ID NO: 12.
  • SlHAK20 mutein of the present invention is shown in SEQ ID NO: 14.
  • HAK protein can improve the salt tolerance of plants by regulating the loading and unloading of Na+ in plants under salt stress conditions.
  • HAK protein can enhance the transport of Na+ to and unloading from the xylem in plants, and at the same time promote the transport of Na+ from roots to other tissues or the outside world.
  • the present invention relates to a SlHAK20 protein with improved plant traits and its mutant protein.
  • the amino acid sequence of the SlHAK20 protein is shown in SEQ ID NO: 2; the amino acid sequence of the SlHAK20 mutant protein As shown in SEQ ID NO: 12 or 14.
  • the polypeptide of the present invention can effectively regulate the traits of plants (such as rice).
  • the present invention also includes that the sequence shown in SEQ ID NO: 2, 12 or 14 of the present invention has at least 50% or more (at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%). , At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) homologous polypeptides or proteins with the same or similar functions .
  • the “same or similar function” mainly refers to: “improving the root length and root branching, plant height, salt tolerance and other traits of plants”.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptide of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plant, insect, and mammalian cells
  • the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes SlHAK20 mutein fragments and analogs having SlHAK20 mutein activity.
  • fragment and “analog” refer to polypeptides that substantially maintain the same biological function or activity as the native SlHAK20 protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention may be: (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the base may or may not be encoded by the genetic code; or (ii) a polypeptide with a substitution group in one or more amino acid residues; or (iii) the mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, polyethylene glycol) fused to form a polypeptide; or (iv) additional amino acid sequence is fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein).
  • these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • the polypeptide variant is the amino acid sequence shown in SEQ ID NO.: 2, 12 or 14, after several (usually 1-60, preferably 1-30, more preferably 1-20, preferably 1-10) derived sequence obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually within 20) at the C-terminus and/or N-terminus, preferably Within 10, more preferably within 5) amino acids.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
  • the present invention also includes analogs of the claimed protein.
  • the difference between these analogues and the sequence SEQ ID NO: 2, 12 or 14 of the present invention may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modifications include: chemically derived forms of proteins in vivo or in vitro, the modifications can maintain or enhance or partially inhibit the transport function of the protein; the modifications include chemical modifications of amino acid side chains, peptides
  • the chemical modification of the chain end group such as the chemical modification of the sulfhydryl group, the chemical modification of the amino group, the chemical modification of the carboxyl group, the chemical modification of the disulfide bond and other modifications; the chemical modification includes phosphorylation modification (such as phosphotyrosine, Phosphoserine, phosphothreonine), glycosylation modification (mediated by glycosylase, such as N-glycosylation, O-glycosylation), fatty acylation (such as acetylation, palmitoylation), etc. .
  • a loss-of-function transgenic plant includes one or more of the following characteristics: (i) Root length and root branching are reduced compared to the wild type; ii) The plant height is reduced compared to the wild type; and (iii) the salt tolerance is reduced compared to the wild type.
  • the loss-of-function transgenic plant of the present invention includes a HAK mutein whose amino acid sequence is shown in SEQ ID NO: 8, and its gene mutation sequence is shown in SEQ ID NO: 7.
  • the loss-of-function transgenic plant of the present invention includes a HAK mutein whose amino acid sequence is shown in SEQ ID NO: 10, and its gene mutation sequence is shown in SEQ ID NO: 9.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell containing the vector of the present invention or the mutein coding sequence of the present invention, and a method for producing the S1HAK20 mutein of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant mutein. Generally speaking, there are the following steps:
  • polynucleotide (or variant) of the present invention encoding the mutant protein of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
  • the present invention also provides a recombinant vector including the gene of the present invention.
  • the downstream of the promoter of the recombinant vector contains a multiple cloning site or at least one restriction site.
  • the target gene is ligated into a suitable multiple cloning site or restriction site, so that the target gene and the promoter are operably linked.
  • the recombinant vector includes (from 5'to 3'direction): a promoter, a target gene and a terminator.
  • the recombinant vector may also include elements selected from the following group: 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance and green fluorescent protein, etc.); enhancer; or operator.
  • the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the mutein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the gene, expression cassette or vector including the present invention can be used to transform an appropriate host cell so that the host expresses the protein.
  • the host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc.
  • the transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain transgenic plants.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the plant traits involved in the present invention include root length, root weight and root branching, plant height, salt tolerance, Na+ ion transport in the plant, yield or biomass.
  • the application of the present invention can significantly improve one or more of the above-mentioned traits in plants.
  • the application of the present invention may also improve other traits related to the above-mentioned traits, such as the increase in root length or root branching, and can improve the biological traits such as lodging resistance and drought resistance of plants.
  • Other traits related to the implementation of the present invention are all It is included in the protection scope of the present invention.
  • above ground part and “above ground part” can be used interchangeably, and refer to the part of the plant body that is exposed above the ground, such as the stems, leaves, flowers, and fruits of plants.
  • the plants obtained by applying the present invention have a survival rate increased by at least 1 times, preferably at least 2 times, preferably at least 3 times, and preferably at least 4 times under saline conditions.
  • the plants obtained by applying the present invention can increase plant height or biomass by at least 0.1 times, preferably at least 0.2 times, preferably at least 0.5 times, and preferably at least 1 times under saline conditions. .
  • the present invention discloses for the first time the regulation mechanism of HAK gene, especially SlHAK20 and its homologous genes, on Na+ ion transport. Overexpression of the HAK gene can enhance the salt resistance of plants.
  • the research results are of important scientific value and social significance for cultivating new salt-tolerant crop varieties, expanding crop planting areas, increasing crop yields, and improving farmers’ income.
  • the present invention also obtains hak mutants that are beneficial to phenotypic improvement.
  • hak mutants By regulating endogenous genes in plants, the regulation of lodging resistance, biomass, salt resistance and other traits can be achieved, which is useful for cultivating high-quality, high-yield, and stress-tolerant plants.
  • the new variety has important scientific value.
  • Example 1 The effect of overexpression of HAK20 (SEQ ID NO: 2) in tomato on plant salt resistance
  • the transgenic method was used to construct an overexpression vector of HAK20 to transfect tomato callus, and three groups of transgenic positive plants Hap1OE-1, Hap1OE-2, and Hap1OE-3 were obtained.
  • the upstream and downstream primers of the cloning primer with the stop codon removed in SlHAK20 CDS were synthesized with the 5'end CAAGAGACAGGATCCGAATTC and ACCTCCGACCGGTGCACTAGT linker sequences (derived from the target vector) together with the amplification primers.
  • CDS coding sequence
  • Agrobacterium-mediated callus infection uses Agrobacterium-mediated callus infection to achieve. Incubate the Agrobacterium carrying the target vector with the detoxified tomato cotyledons for 20 minutes for infection. After discarding the Agrobacterium, the tomato cotyledons are placed in a co-cultivation medium (component 4.3g/L MS salt(519), 0.2mg/L IAA , 2mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6) Cultivate in the dark for 2-3 days.
  • a co-cultivation medium component 4.3g/L MS salt(519), 0.2mg/L IAA , 2mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6
  • the co-cultured tomato cotyledons are transferred to a selection medium containing 6 mg/L Hygromycin and 300 mg/L Timentin antibiotics (the composition is the same as the co-cultivation medium) for 7-10 days each generation of selection and subculture until the tissue culture seedlings are differentiated. Cut the tissue culture seedlings and insert them into the rooting medium (components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8) for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • the rooting medium components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8 for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • qRT-PCR Real-time quantitative PCR
  • RNA extraction kit RNA extraction kit
  • reverse transcription with reverse transcription kit iScriptTM cDNA Synthesis Kit, BIO-RAD
  • upstream primer 5'-TGCATTACAGGTTCTGAAGC-3' and downstream primer 5'-TGACTTGCTACTATAGCAGCT-3' were used as amplification primers
  • the amplification level of SlHAK20 was detected with a qPCR fluorescent dye kit (AceQ qPCR SYBR Green Master Mix, Vazyme).
  • transcript of the housekeeping gene SlEF1a upstream and downstream primers are: 5'-GACAGGCGTTCAGGTAAGGA-3' and 5'-GGGTATTCAGCAAAGGTCTC-3', respectively.
  • the detection of protein level is achieved through Western Blot.
  • Hap1OE-1, Hap1OE-2, and Hap1OE-3 plants are higher than those of wild type (TS-670), and Hap1OE-1 plants are slightly higher than Hap1OE-2. Both are better than Hap1OE-3.
  • both transgenic and wild-type tomato plants wilt after being treated with 175mM NaCl.
  • the wilting state of the wild-type plant is significantly heavier than that of the wild-type plant, and the state of the Hap1OE-1 transgenic plant is slightly better than the other two groups. After rewatering, the transgenic plants can gradually return to their normal growth state, and the wild-type plants show no signs of reversion.
  • the survival rate of the transgenic tomato plants was significantly better than that of the wild type.
  • the survival rate of transgenic plants is positively correlated with the transcription level and protein expression level of the SlHAK20 gene.
  • SlHAK20 can significantly enhance the salt resistance of plants and improve the adaptability of plants to adversity.
  • the research results are helpful for cultivating more salt-tolerant new plant varieties, which have important scientific value and social significance for expanding the planting area, increasing the utilization rate of saline-alkali land, increasing the yield of agricultural products, and improving the income of farmers.
  • the gene editing method was used to knock out the slhak20 gene in the wild-type tomato TS-21 variety, and two mutants slhak20-1 and slhak20-2 were produced.
  • sgRNA1 Single guide RNA1
  • sgRNA2 5'CATGGATCGACAAACCGGA3' and sgRNA2 (5'TCAGATGCAGCTGTTACAG 3') near the ATG of the SlHAK20 genome.
  • the upstream and downstream primers are respectively added with GATTG and AAAC...C linkers and synthesized (Thermo Fisher Scientific).
  • For the final concentration of 10 ⁇ M take 10 ⁇ L of each and mix well.
  • the fragment was directly digested with the BsaI-HF (New England BioLabs) of the target vector pCAMBIA1300-Cas9, and the T4 link was carried out for 2 hours.
  • the correct recombinant binary vector was obtained by screening and using M13F and sgRNA downstream primers for colony PCR identification. After the PCR product was further confirmed by Sanger sequencing, the tomato was transformed by Agrobacterium-mediated callus soaking transformation method for subsequent experiments. .
  • Agrobacterium-mediated callus infection uses Agrobacterium-mediated callus infection to achieve. Incubate the Agrobacterium carrying the target vector with the detoxified tomato cotyledons for 20 minutes for infection. After discarding the Agrobacterium, the tomato cotyledons are placed in a co-cultivation medium (component 4.3g/L MS salt(519), 1mg/L IAA, 0.3mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6) Incubate in the dark for 2-3 days.
  • a co-cultivation medium component 4.3g/L MS salt(519), 1mg/L IAA, 0.3mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6
  • the co-cultured tomato cotyledons are transferred to a selection medium containing 6 mg/L Hygromycin and 300 mg/L Timentin antibiotics (the composition is the same as the co-cultivation medium) for 7-10 days each generation of selection and sub-generation, until the tissue culture seedlings are differentiated. Cut the tissue culture seedlings and insert them into the rooting medium (components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8) for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • the rooting medium components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8 for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • Determination of sodium and potassium ions in roots and aboveground parts cultivate 19-day-old tomato seedlings in 1/4MS liquid medium, and treat them with 1/4MS containing 50mM NaCl for 0, 1, 2, 7, 14 days or specified After time, take the aerial parts and roots separately (the roots need to be washed 3 times with deionized water and dried with absorbent paper). Dry in an oven at 75°C for more than 24 hours, take about 1mg (accurately weigh and record the weight) of the dry sample and add 1mL of concentrated nitric acid containing the internal standard indium (In) to a clean glass tube. After digestion at 115°C for 4 hours, add 9.2mL of deionized Dilute with water and mix well. The diluted sample was detected by ICP-MS (NexION 350D; PerkinElmer) and the sodium and potassium ion content was calculated.
  • Determination of sodium and potassium ions in the xylem stream cultivate 19-day-old tomato seedlings in 1/4MS liquid medium, treat them with 50mM NaCl and 1/4MS for 0, 1, 2 or a specified time, then use sharp The blade removes the cotyledons and above, and collects the remaining part of the xylem stream. Dilute 20 ⁇ L of xylem stream with 5% 1.78 mL of In-containing concentrated nitric acid and use ICP-MS (NexION 350D, PerkinElmer) to detect and calculate the sodium and potassium ion content.
  • the function of the SlHAK20 gene is closely related to the transport of Na+ ions in plants. It can regulate the accumulation of Na+ ions in the roots of plants, promote the transport of Na+ from roots to xylem, and unload Na+ from xylem to roots, thereby reducing root and aboveground parts of the plant.
  • the ratio of Na+/K+ enhances the salt resistance of plants.
  • Example 3 The effect of HAK20 mutation improvement on plant root development, plant height, and salt resistance
  • the gene editing method was used to generate mutants slhak20-3 and slhak20-4 of the slhak20 gene in tomato TS-21 variety.
  • the amino acid sequences of slhak20-3 and slhak20-4 are shown in SEQ ID NO: 12 and SEQ ID NO: 14, respectively.
  • the correct recombinant binary vector was obtained by screening and using M13F and sgRNA downstream primers for colony PCR identification. After the PCR product was further confirmed by Sanger sequencing, the tomato was transformed by Agrobacterium-mediated callus soaking transformation method for subsequent experiments. .
  • Agrobacterium-mediated callus infection uses Agrobacterium-mediated callus infection to achieve. Incubate the Agrobacterium carrying the target vector with the detoxified tomato cotyledons for 20 minutes for infection. After discarding the Agrobacterium, the tomato cotyledons are placed in a co-cultivation medium (component 4.3g/L MS salt(519), 1mg/L IAA, 0.3mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6) Incubate in the dark for 2-3 days.
  • a co-cultivation medium component 4.3g/L MS salt(519), 1mg/L IAA, 0.3mg/L ZT, 30g/L sucrose, 7.4g/L agar, pH 5.5-5.6
  • the co-cultured tomato cotyledons are transferred to a selection medium containing 6 mg/L Hygromycin and 300 mg/L Timentin antibiotics (the composition is the same as the co-cultivation medium) for 7-10 days each generation of selection and sub-generation, until the tissue culture seedlings are differentiated. Cut the tissue culture seedlings and insert them into the rooting medium (components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8) for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • the rooting medium components 4.3g/L MS salt(524), 20g/L sucrose, 300mg/L Timentin, 7.4g/L Agar, pH 5.7-5.8 for rooting, and wait for the roots to grow strong After that, the seedlings can be refined and transplanted.
  • Phenotype identification root development, plant height, sodium and potassium ion content
  • Determination of sodium and potassium ions in roots and aboveground parts cultivate 19-day-old tomato seedlings in 1/4MS liquid medium, and treat them with 1/4MS containing 50mM NaCl for 0, 1, 2, 7, 14 days or specified After time, take the aerial parts and roots separately (the roots need to be washed 3 times with deionized water and dried with absorbent paper). Dry in an oven at 75°C for more than 24 hours, take about 1mg (accurately weigh and record the weight) of the dry sample and add 1mL of concentrated nitric acid containing the internal standard indium (In) to a clean glass tube. After digestion at 115°C for 4 hours, add 9.2mL of deionized Dilute with water and mix well. The diluted sample was detected by ICP-MS (NexION 350D; PerkinElmer) and the sodium and potassium ion content was calculated.
  • SlHAK20 mutation can affect the development of plant roots
  • mutants slhak20-3 and slhak20-4 increased the root length and the number of root branches compared with the wild type.
  • mutants slhak20-3 and slhak20-4 increased their plant heights compared to the wild type.
  • the experimental results show that the large fragment deletion mutant of SlHAK20, under salt stress, the root length and plant height of the plant are significantly better than that of the wild type, which can increase plant biomass and even phenotypes such as yield; it can also reduce the Na+/ in plant tissues. K+ ratio, and enhance the salt resistance of plants.
  • amino acid sequences of slhak20-3 and slhak20-4 shows that the N-terminal positions 1-240 of these two mutants are completely identical; the amino acid sequence of slhak20-3 also includes the C-terminal HGLLPLLSLGYTVS sequence; the amino acid sequence of slhak20-4 also includes the C-terminal CMDSYHSSHWDIQYHKALSEHI sequence. This is because in the process of gene editing, the nucleic acid sequence encoding the amino acids after 1-240 has a frameshift mutation and terminated prematurely, causing the amino acid sequence after 1-240 to be inconsistent with the wild-type.
  • Both slhak20-3 and slhak20-4 can improve the salt resistance of plants.
  • the same amino acid region (N-terminal 1-240) lacks 473 amino acids at the C-terminal.
  • the SlHAK20 protein with a deletion of 473 amino acids at the C-terminus can be considered to play an important role in enhancing the salt resistance of plants.
  • the research results have application value for the improvement of traits.
  • the HAK gene of the plant itself By improving the HAK gene of the plant itself, the regulation of traits such as salt resistance can be realized, and it has important scientific value for the cultivation of new stress-tolerant plant varieties.

Abstract

一种物质的用途，用于改进植物的性状或制备调控植物形状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：(i)根长、根分枝和/或根重；(ii)株高；和(iii)耐盐性；其中，所述物质选自下组：(a)HAK蛋白；(b)编码HAK蛋白的核酸序列；(c)HAK蛋白及其编码核酸序列的促进剂；或其组合。该HAK蛋白或其突变蛋白以及相应的方法，可实现对抗倒伏、生物量、抗盐等性状的调控，对于培育优质、高产、耐逆的植物新品种具有重要的科学价值。

Description

HAK基因在调控植物性状中的作用 技术领域
本发明涉及生物学领域，尤其植物生物学领域，具体涉及HAK基因在调控植物性状中的作用。
背景技术
现有技术报道KUP/HAK/KT家族是生物界内最早发现、数目最多、功能最丰富的钾转运家族，广泛存在于多个物种。它介导植物在低钾胁迫中的钾吸收，又参与植物细胞增大、根部生长素的运输等生理过程该家族基因的表达受植物自身生长发育调节因子和环境刺激等因素的调控，参与植物的生长发育和逆境响应机制。KUP(K+uptake permease)首先在大肠杆菌中发现，负责编码大肠杆菌钾吸收透性酶；HAK(high-affinity K+)是在许旺氏酵母中鉴定得到的KUP同源基因。KT(K+transporter)是在拟南芥中发现的具有钾转运功能的转运体。
盐胁迫是植物主要的非生物胁迫之一，它会引发植物多条代谢通路的改变，因而产生复杂的生理水平变化，主要表现为离子失衡、水分亏缺及氧化毒害，进而导致植物光合作用下降、能耗增加、衰老加速、生长受限、产量和品质下降。植物提高自身盐胁迫抗性的主要方式之一是通过木质部薄壁组织调节体内钾(K)和钠(Na)的转运。对盐胁迫的耐受性体现在对Na+毒害作用的适应以及自身K+营养的维持，拥有较高K+/Na+比值的植物对盐胁迫的耐受力更强。
研究报道，KUP/HAK/KT家族基因也受高盐逆境胁迫的诱导，然而截止目前，HAK众多亚型中发挥抗盐性的基因及其机制并不是很清楚。
因此，本领域迫切需要解析相关基因的作用机制并开发出一种能够增强植物对高盐逆境的耐受性的方法。
发明内容
本发明的目的就是提供一种通过调控HAK基因，增强植物对高盐逆境的耐受性的方法。进一步的提供一种通过介导Na+的调控实现增强植物抗盐性的方法。
本发明的另一目的是提供一种HAK基因及其突变体在增强植物耐盐性中的用途，通过过表达该基因，或者突变获得活性更优的突变蛋白可以显著增强植物的耐盐性。
在本发明的第一方面，提供了一种物质的用途，用于改进植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：(i)根长、根分枝和/或根重；(ii)株高；和(iii)耐盐性；
其中，所述物质选自下组：(a)HAK蛋白；(b)编码HAK蛋白的核酸序列；(c)HAK蛋白及其编码核酸序列的促进剂；或其组合。
在另一优选例中，所述HAK蛋白包括野生型HAK蛋白和HAK蛋白突变体。
在另一优选例中，所述HAK蛋白具有介导植物中Na +转运的功能。
在另一优选例中，所述野生型HAK蛋白的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述的HAK蛋白突变体的序列与其所源自的序列相比具有至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性。
在另一优选例中，所述其所源自的序列为野生型HAK蛋白的氨基酸序列。
在另一优选例中，所述用途还包括调控植物体内Na +离子转运，体现在如下i-iv中的一种或任意几种：
i、调节植物根部Na+离子的积累；ii、促进中Na +向木质部的转运及从木质部卸载；iii、降低根部和地上部位的Na+/K+比。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、大豆、番茄、玉米、高粱、藜麦、谷子、马铃薯、烟草、小麦、高粱、油菜、菠菜、生菜、白菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜或其组合。
在另一优选例中，所述HAK基因来源于：裸子植物或被子植物。
在另一优选例中，所述HAK基因来源于单子叶植物或双子叶植物。
在另一优选例中，所述HAK基因来源于草本植物和木本植物。
在另一优选例中，所述HAK基因来源于茄科植物、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述HAK基因来源于猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述HAK基因来源于十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述HAK基因来源于拟南芥、水稻、大豆、番茄、玉米、高粱、藜麦、谷子、马铃薯、烟草、小麦、高粱、油菜、菠菜、生菜、白菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜或其组合。
在另一优选例中，所述的编码HAK蛋白的核酸序列为选自下组的基因的核酸序列：SlHAK20、OsHAK4、OsHAK17、ZmHAK4、SbHAK9、SbHAK26、GrHAK8、GmHAK12、PtHAK21、MdHAK5，或其组合。
在另一优选例中，所述的编码HAK蛋白的核酸序列选自下组：来源于番茄的SlHAK20基因、来源于水稻的OsHAK4或OsHAK17基因。
在另一优选例中，所述HAK蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO:2、4或6所示氨基酸序列的多肽；
(ii)将如SEQ ID NO:2、4或6所示的氨基酸序列经过一个或多个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述调控植物性状功能的由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO:2、4或6所示的氨基酸序列具有至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同一性的，并且具有介导植物中Na +转运的功能的多肽。
在另一优选例中，所述编码HAK蛋白的核苷酸序列选自下组任一种或其组合：
(a)编码如SEQ ID NO:2、4或6所示多肽的多核苷酸；
(b)如SEQ ID NO:1、3或5所示的核苷酸序列；或
(c)与SEQ ID NO:1、3或5所示的核苷酸序列相比具有一个或多个碱基的取代、缺失或添加(例如1-10个)的序列；
(d)与SEQ ID NO:1、3或5所示的核苷酸序列具有至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％序列同一性的核苷酸序列；
(e)在SEQ ID NO:1、3或5所示的核苷酸序列的5’端和/或3’端截短或添加1-60个(较佳地1-30个，更佳地1-10个)核苷酸的核苷酸序列；
(f)与(a)-(e)中任一所述的核苷酸序列互补的多核苷酸序列。
在另一优选例中，所述HAK蛋白突变体是SlHAK20蛋白突变体。
在另一优选例中，所述编码HAK蛋白突变体的核酸序列是SlHAK20核酸序列突变体。
在另一优选例中，所述的蛋白突变形式包括一个或多个氨基酸的取代、缺失或添加。
在另一优选例中，所述HAK蛋白突变体的氨基酸序列包括：
(i)在SEQ ID NO:2、4或6所示的氨基酸序列的基础上，在C端缺失10-600个(优选地100-500个，更优选地400-500个，更优选地450-480个，更优选地460-48个0，更优选地470-480个，更优选地472或473个)氨基酸残基，所形成的氨基酸序列；和
(ii)任选地，位于所述氨基酸所述序列的C端的通过细胞修复机制添加的数量不等、序列随机的氨基酸片段。
在另一优选例中，所述的数量不等、序列随机的氨基酸片段包括1-100个氨基酸、优选1-50个氨基酸，优选1-30个氨基酸，优选5-25个氨基酸，优选10-20个氨基酸。
在另一优选例中，所述的缺失的方式优选为在C段氨基酸的连续缺失。
在另一优选例中，所述的缺失的氨基酸数量超过SEQ ID NO:2、4或6所示的氨基酸序列中氨基酸数量的二分之一。
在另一优选例中，所述的HAK蛋白突变体与SEQ ID NO:2、4或6所示的氨基酸序列相比具有至少10％，至少20％，至少30％，至少40％，至少50％，至少60％，至少70％，至少80％，至少85％，至少90％，少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％序列同一性。
在另一优选例中，所述的突变蛋白至少基本保留其所源自的序列的生物学功能。
在另一优选例中，所述HAK蛋白突变体序列选自下组：
(i)具有SEQ ID NO:12或14所示氨基酸序列的多肽；
(ii)将如SEQ ID NO:12或14所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述调控植物性状功能的由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO:12或14所示的氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有调控植物中Na+转运的功能的多肽。
在另一优选例中，所述编码HAK蛋白突变体的核苷酸序列选自下组：
(a)编码如SEQ ID NO:12或14所示多肽的多核苷酸；
(b)如SEQ ID NO:11或13所示的核苷酸序列；或
(c)与SEQ ID NO:11或13所示的核苷酸序列相比具有一个或多个碱基的取代、缺失或添加(例如1-10个)的序列；
(d)与SEQ ID NO:11或13所示的核苷酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)的核苷酸序列；
(e)在SEQ ID NO:11或13所示的核苷酸序列的5’端和/或3’端截短或添加1-60个(较佳地1-30个，更佳地1-10个)核苷酸的核苷酸序列；
(f)与(a)-(e)中任一所述的核苷酸序列互补的多核苷酸序列。
在另一优选例中，所述的编码突变体的核酸序列是通过自然诱变或基因编辑技术获得。
在另一优选例中，所述基因编辑技术包括TALEN、ZIN、CRISPR。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述组合物进一步包括农学上可接受的载体。
在另一优选例中，所述组合物中，所述物质的含量即(a)HAK蛋白；(b)编码HAK蛋白的核酸序列；(c)HAK蛋白及其编码核酸序列的促进剂；或其组合，为0.0001-99wt％，较佳地0.1-90wt％，更佳地1-80wt％，以所述组合物的总重量计。
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述促进剂包括促进HAK基因或其编码蛋白表达的小分子化合物。
在另一优选例中，所述的促进剂选自下组：小分子化合物、核酸分子、或其组合。
在本发明的第二方面，提供了一种改良植物性状的方法，包括步骤：调节所述植物中HAK蛋白或其编码核酸序列的表达量和/或活性，从而改良植物的性状。
在另一优选例中，所述植物的性状改良选自下组一种或多种：
(i)增加根长和/或根分枝；
(ii)增加株高；和
(iii)增强耐盐性。
在另一优选例中，所述的性状改良还包括：(iv)促进植物体内Na+离子转运。
在另一优选例中，所述调节所述植物中HAK蛋白或其编码核酸的表达量和/或活性包括：
(a)增强所述植物中内源的HAK蛋白或其编码核酸序列的表达量和/或活性；
(b)导入外源的HAK蛋白或其编码核酸序列，或HAK蛋白突变体或其编码核酸序列，使所述植物具有外源的HAK蛋白或其编码核酸序列，或HAK蛋白突变体或其编码核酸序列的表达量和/或活性；或
(c)施用HAK蛋白及其编码核酸序列的促进剂。
在另一优选例中，所述植物中HAK蛋白突变体或其编码核酸序列的活性E1与同种类型的植物中的相同野生型HAK蛋白或其编码核酸序列的本底活性E0之比(E1/E0)≥1.5倍，≥2倍，较佳地≥5倍，更佳地≥10倍。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将HAK蛋白或其编码核酸、或其促进剂，或HAK蛋白突变体或其编码核酸导入所述植物或植物细胞，从而改良植物的性状。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)导入基因编辑的工具，使其与植物内核苷酸序列接触，突变获得HAK蛋白突变体的核苷酸序列,从而改良植物的性状。
在另一优选例中，所述的导入的方法包括：农杆菌转化法、基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法。
在本发明的第三方面，提供了一种突变蛋白，所述突变蛋白的氨基酸序列选自下组：
(i)在SEQ ID NO:2所示的氨基酸序列的基础上，在C端缺失10-600个(优选地100-500个，更优选地400-500个，更优选地450-480个，例如，472、473个)氨基酸残基，所形成的氨基酸序列；和
(ii)任选的，位于所述氨基酸所述序列的C端的通过细胞修复机制添加的数量不等、序列随机的氨基酸片段。
在另一优选例中，所述的缺失的方式优选为在C段氨基酸的连续缺失。
在另一优选例中，所述的缺失的氨基酸数量超过SEQ ID NO:2所示的氨基酸序列中氨基酸数量的二分之一。
在另一优选例中，所述的数量不等、序列随机的氨基酸片段包括1-100个氨基酸、优选1-50个氨基酸，优选1-30个氨基酸，优选5-25个氨基酸，优选10-20个氨基酸。
在一个实施方式中，所述突变蛋白的氨基酸序列为在SEQ ID NO:2所示的氨基酸序列的基础上，在C端连续缺失473个氨基酸残基所形成的氨基酸序列。任选的，在上述突变蛋白的C端还添加有序列随机的1-100个氨基酸、优选1-50个氨基酸，优选1-30个氨基酸，优选5-25个氨基酸，优选10-20个氨基酸。
在另一优选例中，所述突变蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO:12或14所示氨基酸序列的多肽；
(ii)将如SEQ ID NO:12或14所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或 添加而形成的，具有所述调控植物性状功能的由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO:12或14所示的氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有与(i)至少基本相同活性多肽。
在本发明的第四方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第三方面所述的突变蛋白。
在另一优选例中，所述多核苷酸的核苷酸序列选自下组：
(a)编码如SEQ ID NO:12或14所示多肽的多核苷酸；
(b)如SEQ ID NO:11或13所示的核苷酸序列；或
(c)与SEQ ID NO:11或13所示的核苷酸序列相比具有一个或多个碱基的取代、缺失或添加(例如1-10个)的序列；
(d)与SEQ ID NO:11或13所示的核苷酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)的核苷酸序列；
(e)在SEQ ID NO:11或13所示的核苷酸序列的5’端和/或3’端截短或添加1-60个(较佳地1-30个，更佳地1-10个)核苷酸的核苷酸序列；
(f)与(a)-(e)中任一所述的核苷酸序列互补的多核苷酸序列。
在本发明的第五方面，提供了一种载体，所述载体含有如本发明第四方面所述的多核苷酸。
在本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第五方面所述的载体或基因组中整合有如本发明第四方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括真核细胞、原核细胞。
在本发明的第七方面，提供了一种基因工程化的植物细胞、组织或器官，含有本发明第三方面所述的HAK蛋白突变体或其编码核酸序列。
在本发明的第八方面，提供了一种制备如本发明第七方面所述的基因工程化的植物细胞、植物组织或器官的方法，包括以下方法步骤：调节所述植物中HAK基因或其编码蛋白的表达量和/或活性，从而获得基因工程的植物组织或植物细胞。
在另一优选例中，所述方法包括步骤：
(1)提供植物细胞、植物组织或器官；和
(2)导入基因编辑的工具，使其与植物内核苷酸序列接触，突变获得本发明第三方面所述的编码HAK蛋白突变体的核苷酸序列。
在另一优选例中，所述方法包括步骤：
(1)提供植物细胞、植物组织或器官；和
(2)导入含有本发明第三方面所述的HAK蛋白突变体或其编码核酸序列的载体。
在另一优选例中，所述的导入的方法包括：农杆菌转化法、基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法。
在本发明的第九方面，提供了一种制备转基因植物或基因编辑植物的方法，包括步骤：
将如本发明第七方面所述的植物细胞、组织或器官再生为植物体，从而获得转基因或基因编辑植物。
在本发明的第十方面，提供了一种制备本发明第三方面所述突变蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第六方面所述的宿主细胞，从而表达融合蛋白；和/或，分离所述 融合蛋白。
在本发明的第十一方面，提供了一种检测植物耐盐性的方法，所述的方法包括检测HAK蛋白或其编码核酸或其突变体的表达活性和/或表达量。
在另一优选例中，所述的HAK蛋白选自下组：SlHAK20、OsHAK4、OsHAK17、ZmHAK4、SbHAK9、SbHAK26、GrHAK8、GmHAK12、PtHAK21、MdHAK5或其任一组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了SlHAK20在不同番茄植株中的转录水平。其中，以SlEF1a作为内参。
图2显示了SlHAK20-YFP在不同番茄植株中的蛋白表达水平。
图3显示了转基因番茄植株的耐盐性检测结果。
其中，a显示了175mMNaCl盐胁迫条件下植株生长状态；b显示了恢复两周后植株的生长状态。
图4显示了转基因番茄植株的存活率分析结果。
图5显示了盐胁迫条件下，随着时间延长突变体较野生型根部的Na+含量和K+含量。
图6显示了盐胁迫条件下，随着时间延长突变体较野生型根部的Na+/K+。
图7显示了盐胁迫条件下，随着时间延长突变体较野生型木质部的Na+含量和K+含量。
图8显示了盐胁迫条件下，随着时间延长突变体较野生型地上部位的Na+含量和K+含量。
图9显示了盐胁迫条件下，随着时间延长突变体较野生型地上部位的Na+/K+。
图10显示了经盐处理后，突变体较野生型的根长和根分枝数量的变化情况。
图11显示了经盐处理后，突变体较野生型的株高变化情况。
图12显示了盐胁迫条件下，突变型较野生型的根部和地上部分的Na+/K+。
其中，a显示了突变型较野生型的根部的Na+/K+；b显示了突变型较野生型的地上部分的Na+/K+。
本申请中的序列表如下：
SEQ ID NO: 序列描述
1 来源番茄的野生型SlHAK20核酸序列
2 来源番茄的野生型SlHAK20氨基酸序列
3 来源水稻的野生型OsHAK4核酸序列
4 来源水稻的野生型OsHAK4氨基酸序列
5 来源水稻的野生型OsHAK17核酸序列
6 来源水稻的野生型OsHAK17氨基酸序列
7 本发明的SlHAK20-1核酸序列
8 本发明的SlHAK20-1氨基酸序列
9 本发明的SlHAK20-2核酸序列
10 本发明的SlHAK20-2氨基酸序列
11 本发明的SlHAK20-3核酸序列
12 本发明的SlHAK20-3氨基酸序列
13 本发明的SlHAK20-4核酸序列
14 本发明的SlHAK20-4氨基酸序列
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次意外地发现了一种植物(如番茄)的HAK基因或其编码蛋白或其促进剂，可用于调控植物的性状，所述性状包括：根长、根重和根分枝、株高、耐盐性。研究表明，在番茄中过表达HAK蛋白(转运体)或其编码基因可增加植物的抗盐性。HAK蛋白及编码基因在调控植物Na+转运中的用途，盐胁迫条件下，HAK蛋白可通过调控植物内Na+的装载和卸载，以提高植物的耐盐性。具体地，HAK蛋白可增强植物中Na+向木质部的转运及从木质部卸载，同时促进Na+从根部向其他组织或外界转运。
此外，本发明人还意外地发现，通过基因编辑技术使得HAK蛋白的氨基酸序列发生C端缺失时，在盐胁迫下，可以增加根长和根分枝，增加植物的株高，并且增强植物的耐盐性。根部的改善可以增强植物的抗倒伏性，促进营养物质的吸收，增强环境适应能力。而株高的增加，可以增加植物的生物量和/或产量。
研究还发现，HAK基因启动子下游48bp处6个碱基的缺失及3093bp处碱基G到A的置换，会导致植物耐盐性的下降。
在此基础上，完成了本发明。
HAK蛋白及编码核酸序列
如本文所用，术语“HAK蛋白”、“HAK多肽”、“HAK转运体”可以互换使用，是指存在于不同细胞器膜上，如质膜、液泡膜和类囊体膜上的一类离子转运体。
如本文所用，术语“HAK蛋白突变体”、“突变型HAK蛋白”可以互换使用，是指由野生型HAK蛋白氨基酸序列突变所获得的突变体。
如本文所用，术语“本发明的HAK基因”、“HAK基因”、“编码HAK蛋白的核酸序列”在合适条件下可互换使用，均指DNA序列，均指来源于植物(如番茄、小麦等)的HAK基因或其变体。
在一优选实施方式中，本发明的HAK基因为SlHAK20基因，其核苷酸序列如SEQ ID NO:1所示，其编码的氨基酸序列如SEQ ID NO:2所示。
本发明还包括与本发明的优选基因序列(SEQ ID NO:1)具有至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列同源性的核酸，所述核酸也能有效地调控植物(如番茄)的农艺性状。
“同源性”或“同一性”是指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据，)那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目初一进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。通常，在将两个序列比对难以产生最大同一性时进行比较，这样的比对可以通过使用，例如计算机程序如Align程序(DNAstar,Inc.)(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.J.Mol.Biol.J.Mol.Biol.J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此的方法来实现。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。在本文中，所述基因的变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。
在本发明中，SEQ ID NO:1中的核苷酸序列可以经过取代、缺失或添加一个或多个，生成SEQ ID NO:1的衍生序列，由于密码子的简并性，即使与SEQ ID NO:1的同源性较低，也能基本编码出如 SEQ ID NO:2所示的氨基酸序列。另外，“在SEQ ID NO:1中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO:1所示的核苷酸序列杂交的核苷酸序列。这些变异形式包括(但并小限于)：若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、插入和/或取代，以及在5’和/或3’端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。
应理解，尽管本发明的实例中提供的基因来源于番茄，但是来源于其它类似的植物(尤其是与番茄属于同一科或属的植物)的、与本发明的序列(优选地，序列如SEQ ID NO:1所示)具有一定同源性(保守性)的HAK的基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该序列。
本发明所述的多核苷酸或核酸序列可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO:1所示的编码区序列相同或者是简并的变异体。
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酞胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
应理解，虽然本发明的HAK基因优选来自番茄，但是来自其它植物的与番茄HAK基因具有一定同源性且功能相近的其它基因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
例如，在本发明的另一个优选的实施方式中，本发明的HAK基因还可以是来源于水稻的OsHAK4基因，其核苷酸序列如SEQ ID NO:3所示，其编码蛋白的氨基酸序列如SEQ ID NO:4所示。而在本发明的另一个优选的实施方式中，本发明的HAK基因还可以是来源于水稻的OsHAK17，其核苷酸序列如SEQ ID NO:5所示，其编码蛋白的氨基酸序列如SEQ ID NO:6所示。进一步还包括来源于玉米的ZmHAK4；来源于高粱的SbHAK9、SbHAK26；来源于棉花的GrHAK8；来源于大豆的GmHAK12等。
本发明的HAK核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的DNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
如本文所用，术语“本发明突变蛋白”、“SlHAK20突变蛋白”、“SlHAK20基因突变体编码蛋白”、“本发明多肽”可互换使用，都是指本发明中SlHAK20基因编码蛋白的C端缺失后，所形成的突变蛋白。
在另一优选的实施方式中，本发明SlHAK20突变蛋白的一种典型的氨基酸序列如SEQ ID NO:12所示。
Figure PCTCN2020140748-appb-000001
在另一优选的实施方式中，本发明SlHAK20突变蛋白的一种典型的氨基酸序列如SEQ ID NO:14所示。
Figure PCTCN2020140748-appb-000002
研究表明，HAK蛋白在盐胁迫条件下，HAK蛋白可通过调控植物内Na+的装载和卸载，以提高植物的耐盐性。
具体地，HAK蛋白可增强植物中Na+向木质部的转运及从木质部卸载，同时促进Na+从根部向其他组织或外界转运。
本发明涉及一种改进植物性状的SlHAK20蛋白，以及其突变蛋白，在本发明的一个优选例中，所述SlHAK20蛋白的氨基酸序列如SEQ ID NO:2所示；所述SlHAK20突变蛋白的氨基酸序列如SEQ ID NO:12或14所示。本发明的多肽能够有效调控植物(如水稻)的性状。
本发明还包括与本发明的SEQ ID NO:2、12或14所示序列具有至少50％或以上(至少50％，至少60％，至少70％，至少80％，至少85％，至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％或至少99％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指：“改进植物的根长和根分枝、株高、耐盐性等性状”。
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有SlHAK20突变蛋白活性的SlHAK20突变蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然SlHAK20蛋白相同的生物学功能或活性的多肽。
本发明的多肽片段、衍生物或类似物可以是：(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的；或(ii)在一个或多个氨基酸残基中具有取代基团的多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明中，所述的多肽变体是如SEQ ID NO.:2、12或14所示的氨基酸序列，经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保 守性变异最好根据表1进行替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还包括所要求保护的蛋白的类似物。这些类似物与本发明序列SEQ ID NO:2、12或14的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式，所述的修饰能够保持或增强或部分抑制蛋白的转运功能；所述的修饰包括氨基酸侧链的化学修饰、肽链末端基团化学修饰，如巯基的化学修饰、氨基的化学修饰、羧基的化学修饰、二硫键的化学修饰及其他修饰；所述的化学修饰包括，磷酸化修饰(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)、糖基化修饰(由糖基化酶介导，如N-糖基化、O-糖基化)、脂酰化修饰(如乙酰化、棕榈酰化)等。
特别值得注意的是，在本发明中，还提供了一种功能缺失型转基因植物，所述功能缺失包括一种或多种以下特征：(i)根长和根分枝较野生型减少；(ii)株高较野生型减少；和(iii)耐盐性较野生型减弱。
在一优选实施方式中，本发明的功能缺失型转基因植物中包括氨基酸序列如SEQ ID NO:8所示的HAK突变蛋白，其基因突变序列如SEQ ID NO:7所示。
Figure PCTCN2020140748-appb-000003
在另一优选的实施方式中，本发明的功能缺失型转基因植物中包括氨基酸序列如SEQ ID NO:10所示的HAK突变蛋白，其基因突变序列如SEQ ID NO:9所示。
Figure PCTCN2020140748-appb-000004
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及含有本发明的载体或本发明突变蛋白编码序列的宿主细胞，以及经重组技术产生本发明所述SlHAK20突变蛋白的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明还提供了一种包括本发明的基因的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达本发明目的基因时，将目的基因连接入适合的多克隆位点或酶切位点内，从而将目的基因与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5’到3’方向)：启动子、目的基因和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3’多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。
在本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
包括本发明基因、表达盒或载体可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一 段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
植物性状
本发明中所涉及的植物性状包括根长、根重和根分枝、株高、耐盐性、植物体内Na+离子转运、产量或生物量。施用本发明可显著改善植物中上述一种或多种性状。进一步的，施用本发明还可能改善与上述性状相关的其他性状，比如根长或根分枝的增加，可提高植物的抗倒伏性、抗旱性等生物性状，与实施本发明关联的其他性状均包含在本发明的保护范围内。
本发明所述的“地上部分”、“地上部位”可互换使用，是指裸露于地表以上植物体部分，比如包括植物的茎、叶、花、果实。
通过施用本发明所获得的植株相比野生型植株，在盐性条件下，存活率提高至少1倍，较佳的至少2倍，较佳的至少3倍，较佳的至少4倍。
通过施用本发明所获得的植株相比野生型植株，在盐性条件下，株高或生物量增加约至少0.1倍，较佳至少0.2倍，较佳的至少0.5倍，较佳的至少1倍。
本发明的主要优点包括：
1)本发明首次揭示了HAK基因，尤其是SlHAK20及其同源基因，对Na+离子转运的调控机制。过表达HAK基因可增强植物的抗盐性，该研究成果为培育新型抗盐作物品种，扩大作物种植面积、提高作物产量、改善农民收入具有重要的科学价值和社会意义。
2)本发明还获得了有益于表型改善的hak突变体，通过调控植物内源性基因，可实现对抗倒伏、生物量、抗盐等性状的调控，对于培育优质、高产、耐逆的植物新品种具有重要的科学价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：番茄中HAK20(SEQ ID NO:2)过表达对植物抗盐性的影响
本实验里中采用转基因方法，构建HAK20的过表达载体转染番茄愈伤组织，获得三组转基因阳性植株Hap1OE-1、Hap1OE-2、Hap1OE-3。
1、载体构建
SlHAK20 CDS(coding sequence,CDS)去掉终止密码子的克隆引物上游和下游引物分别与5’端的CAAGAGACAGGATCCGAATTC和ACCTCCGACCGGTGCACTAGT的接头序列(来源于目的载体)一起合成扩增引物。利用PCR扩增，从野生品种TS-21总RNA反转获得的总cDNA产物中，克隆出对应的包含接头CDS片段。进一步，通过特异性位点重组，在重组酶的催化下与目的载体pCAMBIA1300-YFP的同源序列重组而使得SlHAK20 CDS穿梭进目的载体。
2、遗传转化
利用农杆菌介导的愈伤组织侵染来实现。将携带目的载体的农杆菌与脱毒的番茄子叶孵育20min进行侵染，弃掉农杆菌后，番茄子叶置于共培养培养基(成分4.3g/L MS salt(519),0.2mg/L IAA,2mg/L ZT,30g/L sucrose,7.4g/L agar,pH 5.5-5.6)暗培养2-3天。共培养的番茄子叶转移到含有6mg/L  Hygromycin和300mg/L Timentin抗生素的筛选培养基上(成分与共培养培养基相同)进行7-10天每代的筛选继代，直到分化出组培苗。将组培苗剪下插入生根培养基(成分4.3g/L MS salt(524),20g/L sucrose,300mg/L Timentin,7.4g/L Agar,pH 5.7-5.8)进行生根，待根系发育强壮后即可炼苗、移栽。
3、转化植株鉴定及培养
取生根后的组培苗叶片，提取总DNA后，通过PCR扩增YFP基因，并进行琼脂糖凝胶电泳，若有目的条带即为阳性苗，若无目的条带作为阴性对照，同阳性苗一起移栽至泥炭土中培养、扩繁种子用于后续试验。
4、分子检测
实时定量PCR(qRT-PCR)检测过表达株系Hap1OE-1，Hap1OE-2和Hap1OE-3中SlHAK20的转录本水平。通过RNA提取试剂盒提取过表达株系Hap1OE-1，Hap1OE-2和Hap1OE-3的总RNA后，用反转录试剂盒(iScriptTM cDNA Synthesis Kit,BIO-RAD)反转录获得cDNA，上游引物5’-TGCATTACAGGTTCTGAAGC-3’和下游引物5’-TGACTTGCTACTATAGCAGCT-3’作为扩增引物，用qPCR荧光染料试剂盒(AceQ qPCR SYBR Green Master Mix,Vazyme)对SlHAK20的扩增水平进行检测。最后，利用管家基因SlEF1a的转录本(上、下游引物分别是：5’-GACAGGCGTTCAGGTAAGGA-3’和5’-GGGTATTCAGCAAAGGTCTC-3’)作为内参计算SlHAK20转录本的相对水平。
蛋白水平的检测是通过Western Blot实现的。用1％SDS蛋白提取液提取过表达株系Hap1OE-1，Hap1OE-2和Hap1OE-3中总蛋白，各取20μg总蛋白跑SDS-PAGE胶，后将蛋白转膜至硝酸纤维素膜(0.45μm HATF,Nitrocellulose membrane,Merck Millipore)，5％的脱脂奶粉封闭1h，加1:2500稀释的抗体anti-GFP(Thermo Fisher)或1:3000稀释的抗体anti-ACTIN(Thermo Fisher)于5％脱脂奶粉于4℃孵育过夜，后将膜转移到1:5000稀释的二抗抗体IgG(Abmart)中孵育1h，用1×TBST(Tris pH8.0,NaCl,0.05％Tween 20)漂洗3次每次15min，将膜正面加显色液(Thermo Fisher)，孵育5min后，将膜置于洁净的保鲜膜中用成像系统ChemiDocTM XRS+with Image LabTM Software(BIO-RAD)显影。
5、抗盐性检测
将阳性苗和阴性苗种子用10％次氯酸钠消毒20min后，点终于1/4MS培养基，水平放置萌发后转移至1/4MS培养基竖直培养约10天。将幼苗移栽到土块或液体培养盒(1/4MS液体培养基)中，继续培养18天后在培养液中加入175mM NaCl或用其浇灌土块中培养的番茄。盐处理2-3周后，进行复水，即用不含NaCl的培养液或水培养、浇灌番茄，2周后统计不同转基因材料的存活率。
6、实验结果及分析
(1)过表达水平
如图1和图2所示，Hap1OE-1、Hap1OE-2、Hap1OE-3植株的转录水平和蛋白表达水平均高于野生型(TS-670)，Hap1OE-1植株略高于Hap1OE-2，两者均优于Hap1OE-3。
(2)转基因番茄植株抗盐性检测
如图3所示，转基因和野生型番茄植株经过175mM NaCl处理后均出现萎蔫，野生型植株萎蔫状态明显重于野生型植株，其中Hap1OE-1转基因植株状态略优于其他两组。经复水后，转基因植株可逐渐恢复正常生长状态，野生型植株无复转迹象。
(3)存活率分析
如图4所示，经盐处理后，转基因番茄植株的存活率明显优于野生型。转基因植株的存活率与SlHAK20基因的转录水平和蛋白表达水平呈正相关性。
7、实验结论
过表达SlHAK20可以显著增强植物的抗盐性，提高植物对逆境的适应性。
该研究成果有助于培育出更多抗盐的植物新品种，对于扩大植物种植面积、增加盐碱土地的利用率、提高农产品产量、改善农民收入具有重要的科学价值和社会意义。
实施例2：番茄中HAK20对Na+调控作用
本实验例中采用基因编辑方法敲除野生型番茄TS-21品种中slhak20基因，并产生两个突变体slhak20-1和slhak20-2。
1、CRISPR编辑工具的构建
于SlHAK20基因组ATG附近设计Single guide RNA1(sgRNA1)5’CATGGATCGACAAACCGGA3’和sgRNA2(5’TCAGATGCAGCTGTTACAG 3’)的上、下游引物分别加GATTG和AAAC...C接头并合成(Thermo Fisher Scientific)后，稀释为终浓度10μM，各取10μL混匀。在PCR仪(BioRAD)中退火形成双链后直接与目的载体pCAMBIA1300-Cas9的BsaⅠ-HF(New England BioLabs)酶切片段，进行T4链接2h，反应产物转化大肠杆菌DH5α感受态后，卡那霉素筛选获得正确的重组双元载体，并用M13F和sgRNA下游引物进行菌落PCR鉴定，PCR产物进行Sanger测序进一步确定后，通过农杆菌介导的愈伤组织浸花转化法转化番茄，用于后续实验。
2、遗传转化
利用农杆菌介导的愈伤组织侵染来实现。将携带目的载体的农杆菌与脱毒的番茄子叶孵育20min进行侵染，弃掉农杆菌后，番茄子叶置于共培养培养基(成分4.3g/L MS salt(519),1mg/L IAA,0.3mg/L ZT,30g/L sucrose,7.4g/L agar,pH 5.5-5.6)暗培养2-3天。共培养的番茄子叶转移到含有6mg/L Hygromycin和300mg/L Timentin抗生素的筛选培养基上(成分与共培养培养基相同)进行7-10天每代的筛选继代，直到分化出组培苗。将组培苗剪下插入生根培养基(成分4.3g/L MS salt(524),20g/L sucrose,300mg/L Timentin,7.4g/L Agar,pH 5.7-5.8)进行生根，待根系发育强壮后即可炼苗、移栽。
3、植株培养及突变体筛选
取生根后的组培苗叶片，提取总DNA后，通过sgRNA上游约300bp处的上游引物和其下游300bp处的下游引物进行PCR扩增获得目的条带后，对目的片段进行Sanger测序，测序结果与SlHAK20的参照基因组序列比对获得基因编辑结果，纯合阳性苗移栽至泥炭土中培养、扩繁种子用于后续试验。
4、钠、钾离子含量测定
根和地上部分钠、钾离子含量测定：在1/4MS液体培养基中，培养19天大的番茄幼苗，用含50mM NaCl的1/4MS处理0、1、2、7、14天或指定的时间后，分别取地上部分和根(根需要用去离子水洗3次，用吸水纸吸干)。75℃烘箱烘干24h以上，取1mg左右(精确称重并记录重量)干样品加入1mL含有内标铟(In)的浓硝酸于洁净玻璃管中，115℃消解4h后，加入9.2mL去离子水稀释混匀。稀释后的样品用ICP-MS(NexION 350D；PerkinElmer)检测并计算钠、钾离子含量。
木质部流中钠、钾离子含量测定：在1/4MS液体培养基中，培养19天大的番茄幼苗，用含50mM NaCl的1/4MS处理0、1、2或指定的时间后，用锋利的刀片去掉子叶及其以上部分，收集剩余部分的木质部流。取20μL木质部流加5％1.78mL含In浓硝酸稀释后用ICP-MS(NexION 350D,PerkinElmer)检测并计算钠、钾离子含量。
5、实验结果
如图5所示，盐胁迫条件下，随着时间延长，突变体slhak20-1、slhak20-2较野生型根部的Na+含量显著升高，K+含量相当。
如图6所示，盐胁迫条件下，随着时间延长，突变体slhak20-1、slhak20-2较野生型根部的Na+/K+逐渐升高。
如图7所示，盐胁迫条件下，突变体slhak20-1、slhak20-2较野生型木质部Na+含量显著降低，K+含量相当。
如图8所示，盐胁迫条件下，随着时间延长，突变体slhak20-1、slhak20-2较野生型地上部位的Na+含量显著升高，K+含量相当。
如图9所示，盐胁迫条件下，随着时间延长，突变体较野生型地上部分的Na+/K+逐渐升高。
6、实验结论
SlHAK20基因功能与植物体内Na+离子的转运密切相关，其可调节植株根部Na+离子的积累，促进Na+从根部向木质部的转运，及Na+从木质部向根系部位的卸载，以此降低根部和地上部位的 Na+/K+比，增强植株抗盐性。
实施例3：HAK20突变改善对植物根部发育、株高、抗盐性的影响
本实验例中采用基因编辑方法产生番茄TS-21品种中slhak20基因的突变体slhak20-3、slhak20-4。slhak20-3、slhak20-4的氨基酸序列分别如SEQ ID NO:12和SEQ ID NO:14所示。
1、CRISPR编辑工具的构建
于SlHAK20基因组ATG下游3093bp前设计Single guide RNA 5’GGTCTCCTATCATGGGTGCATGG 3’的上、下游引物分别加GATTG和AAAC...C接头并合成(Thermo Fisher Scientific)后，稀释为终浓度10μM，各取10μL混匀。在PCR仪(BioRAD)中退火形成双链后直接与目的载体pCAMBIA1300-Cas9的BsaⅠ-HF(New England BioLabs)酶切片段，进行T4链接2h，反应产物转化大肠杆菌DH5α感受态后，卡那霉素筛选获得正确的重组双元载体，并用M13F和sgRNA下游引物进行菌落PCR鉴定，PCR产物进行Sanger测序进一步确定后，通过农杆菌介导的愈伤组织浸花转化法转化番茄，用于后续实验。
2、遗传转化
利用农杆菌介导的愈伤组织侵染来实现。将携带目的载体的农杆菌与脱毒的番茄子叶孵育20min进行侵染，弃掉农杆菌后，番茄子叶置于共培养培养基(成分4.3g/L MS salt(519),1mg/L IAA,0.3mg/L ZT,30g/L sucrose,7.4g/L agar,pH 5.5-5.6)暗培养2-3天。共培养的番茄子叶转移到含有6mg/L Hygromycin和300mg/L Timentin抗生素的筛选培养基上(成分与共培养培养基相同)进行7-10天每代的筛选继代，直到分化出组培苗。将组培苗剪下插入生根培养基(成分4.3g/L MS salt(524),20g/L sucrose,300mg/L Timentin,7.4g/L Agar,pH 5.7-5.8)进行生根，待根系发育强壮后即可炼苗、移栽。
3、植株培养及突变体筛选
取生根后的组培苗叶片，提取总DNA后，通过sgRNA上游约300bp处的上游引物和其下游300bp处的下游引物进行PCR扩增获得目的条带后，对目的片段进行Sanger测序，测序结果与SlHAK20的参照基因组序列比对获得基因编辑结果，纯合阳性苗移栽至泥炭土中培养、扩繁种子用于后续试验。
4、表型的鉴定(根发育、株高、钠钾离子的含量)
将基因编辑纯合体和对应野生型的种子用10％次氯酸钠消毒20min后，点终于1/4MS培养基，水平放置萌发后转移至含200mM NaCl的1/4MS培养基竖直放置培养1周或1月后统计其鲜重等性状。对照组在不含NaCl的1/4MS培养基竖直放置培养10天。
或将萌发的幼苗转移到1/4MS培养基竖直培养约10天。将幼苗移栽到土块或液体培养盒(1/4MS液体培养基)中，继续培养18天后在培养液中加入200mM NaCl或用含200mM NaCl的水浇灌土块中培养的番茄。盐处理2-3周后，进行复水，即用不含NaCl的培养液或水培养、浇灌番茄共2周后统计株高等性状。对照组一直用不含NaCl的1/4MS培养基或水浇灌土块培养30天后统计株高。
5、钠、钾含量的测定
根和地上部分钠、钾离子含量测定：在1/4MS液体培养基中，培养19天大的番茄幼苗，用含50mM NaCl的1/4MS处理0、1、2、7、14天或指定的时间后，分别取地上部分和根(根需要用去离子水洗3次，用吸水纸吸干)。75℃烘箱烘干24h以上，取1mg左右(精确称重并记录重量)干样品加入1mL含有内标铟(In)的浓硝酸于洁净玻璃管中，115℃消解4h后，加入9.2mL去离子水稀释混匀。稀释后的样品用ICP-MS(NexION 350D；PerkinElmer)检测并计算钠、钾离子含量。
6、实验结果及分析
(1)SlHAK20突变可以影响植物根部的发育
如图10所示，经盐处理后，突变体slhak20-3、slhak20-4较野生型根长增加、根分枝数量增加。
(2)SlHAK20突变对植物株高、生物量的影响
如图11所示，经盐处理后，突变体slhak20-3、slhak20-4较野生型的株高增加。
(3)SlHAK20突变对植物钠离子和钾离子含量的影响
如图12a所示，盐胁迫条件下，突变型slhak20-3、slhak20-4较野生型根部，Na+/K+显著降低。
如图12b所示，盐胁迫条件下，突变型slhak20-3、slhak20-4较野生型地上部分，Na+/K+显著降低。
7、实验结论
实验结果表明，SlHAK20大片段缺失突变体，在盐胁迫下，植物的根长和株高明显优于野生型，能够提高植物的生物量，甚至产量等表型；还可以降低植物组织的Na+/K+比，而增强植物的抗盐性。
分析slhak20-3、slhak20-4的氨基酸序列(SEQ ID NO:12和SEQ ID NO:14)可知，这两个突变体N端1-240位完全相同；slhak20-3的氨基酸序列还包括C端的HGLLPLLSLGYTVS序列；slhak20-4的氨基酸序列还包括C端的CMDSYHSSHWDIQYHKALSEHI序列。这是由于在基因编辑过程中，编码1-240位氨基酸之后的核酸序列发生移码突变并提前终止，导致1-240位后面的氨基酸序列与野生型不一致。
slhak20-3和slhak20-4均可以提高植物的抗盐性，二者相同的氨基酸区域(N端1-240位)与野生型SlHAK20相比，缺失了C端的473个氨基酸。换句话说，C端缺失473个氨基酸的SlHAK20蛋白可以认为对增强植物抗盐性具有重要作用。
我们将slhak20-3、slhak20-4的氨基酸序列在水稻、玉米中过表达，其同样可以增强植株的抗盐性。
该研究成果对性状的改良具有应用价值，通过改善植物自身的HAK基因，可实现抗盐等性状的调控，对于培育耐逆的植物新品种具有重要的科学价值。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种物质的用途，其特征在于，用于改进植物的性状或制备调控植物的性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：(i)根长、根分枝和/或根重；(ii)株高；和(iii)耐盐性；
    其中，所述物质选自下组：(a)HAK蛋白；(b)编码HAK蛋白的核酸序列；(c)HAK蛋白及其编码核酸序列的促进剂；或其组合。
  2. 一种改良植物性状的方法，其特征在于，包括步骤：调节所述植物中HAK蛋白或其编码核酸序列的表达量和/或活性，从而改良植物的形状。
  3. 一种突变蛋白，其特征在于，所述突变蛋白的氨基酸序列选自下组：
    (i)在SEQ ID NO:2所示的氨基酸序列的基础上，在C端缺失10-600个(优选地100-500个，更优选地400-500个，更优选地450-480个，更优选472或473个)氨基酸残基，所形成的氨基酸序列；和
    (ii)任选的，位于(i)所述氨基酸序列的C端的添加的数量不等、序列随机的氨基酸片段。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求3所述的突变蛋白。
  5. 一种载体，其特征在于，所述载体含有如权利要求4所述的多核苷酸。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求5所述的载体或基因组中整合有如权利要求4所述的多核苷酸。
  7. 一种基因工程化的植物细胞、组织或器官，其特征在于，含有权利要求3所述的HAK蛋白突变体或其编码核酸序列。
  8. 一种制备如权利要求7所述的基因工程化的植物细胞、植物组织或器官的方法，其特征在于，包括以下方法步骤：调节所述植物中HAK基因或其编码蛋白的表达量和/或活性，从而获得基因工程的植物组织或植物细胞。
  9. 一种制备转基因植物或基因编辑植物的方法，其特征在于，包括步骤：
    将如权利要求7所述的植物细胞、组织或器官再生为植物体，从而获得转基因或基因编辑植物。
  10. 一种制备权利要求3所述突变蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养权利要求6所述的宿主细胞，从而表达融合蛋白；和/或，分离所述融合蛋白。
PCT/CN2020/140748 2019-12-30 2020-12-29 Hak基因在调控植物性状中的作用 WO2021136255A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080003873.1A CN113227383B (zh) 2019-12-30 2020-12-29 Hak基因在调控植物性状中的作用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911397423.0A CN113122566B (zh) 2019-12-30 2019-12-30 Hak基因在调控植物性状中的作用
CN201911397423.0 2019-12-30

Publications (1)

Publication Number Publication Date
WO2021136255A1 true WO2021136255A1 (zh) 2021-07-08

Family

ID=76686536

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/140748 WO2021136255A1 (zh) 2019-12-30 2020-12-29 Hak基因在调控植物性状中的作用

Country Status (2)

Country Link
CN (2) CN113122566B (zh)
WO (1) WO2021136255A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715609A (zh) * 2018-06-08 2018-10-30 中国农业大学 植物钾转运体蛋白GhHAK5及其编码基因和应用
CN110078807A (zh) * 2019-04-30 2019-08-02 北京市农林科学院 促进钾离子高效吸收的蛋白质及其编码基因

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1289523C (zh) * 2003-11-19 2006-12-13 中国科学院上海生命科学研究院 水稻钾、钠离子转运基因及其应用
WO2008025097A1 (en) * 2006-08-31 2008-03-06 Commonwealth Scientific And Industrial Research Organisation Salt tolerant plants
CN109369786B (zh) * 2018-11-12 2021-08-31 贵州省烟草科学研究院 一种烟草kup8蛋白及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108715609A (zh) * 2018-06-08 2018-10-30 中国农业大学 植物钾转运体蛋白GhHAK5及其编码基因和应用
CN110078807A (zh) * 2019-04-30 2019-08-02 北京市农林科学院 促进钾离子高效吸收的蛋白质及其编码基因

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEN GUANG, HU QINGDI, LUO LE, YANG TIANYUAN, ZHANG SONG, HU YIBING, YU LING, XU GUOHUA: "Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges : Rice potassium transporter OsHAK1", PLANT CELL AND ENVIRONMENT, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 38, no. 12, 1 December 2015 (2015-12-01), GB, pages 2747 - 2765, XP055825600, ISSN: 0140-7791, DOI: 10.1111/pce.12585 *
DATABASE NUCLEOTIDE 8 August 2018 (2018-08-08), ANONYMOUS: "PREDICTED: Solanum lycopersicum probable potassium transporter 17 (LOC101256474), transcript variant X1, mRNA", XP055825602, retrieved from NCBI Database accession no. XM_004236854 *
DATABASE PROTEIN 8 August 2018 (2018-08-08), ANONYMOUS: "probable potassium transporter 17 isoform X1 [Solanum lycopersicum]", XP055825605, retrieved from NCBI Database accession no. XP_004236902 *
HUANG PING: "Functional Identification of Chenopodium Glaucum NHX1 Gene and Functional Comparison between CgNHX1 and OsNHX1 C-terminal K+/Na+ Transport Regulatory Regions", CHINA MASTER’S THESES OF XINJIANG UNIVERSITY, 1 January 2007 (2007-01-01), XP055827947 *
LIU PEI: "Cloning and Functional Comparation of Various Vacuolar Na+/H+ Antiporter Genes from Different Species Under Salt-and Drought-stresses", SHANDONG AGRICULTURAL UNIVERSITY OF CHINA MASTER’S THESES, 1 January 2007 (2007-01-01), XP055827949 *
QIN LIJUN, SONG LA-LA, ZHAO DAN, ZHAO DE-GANG: "Overexpression of Nicotiana Tabacum High-affinity Potassium Ion Transporter Protein Gene(HAK1) Improves the Salt-tolerance in Tobacco (N.tabacum) Plants", JOURNAL OF AGRICULTURAL BIOTECHNOLOGY, vol. 23, no. 12, 1 January 2015 (2015-01-01), pages 1576 - 1587, XP055825797, DOI: 10.3969/j.issn.1674-7968.2015.12.006 *

Also Published As

Publication number Publication date
CN113122566A (zh) 2021-07-16
CN113122566B (zh) 2023-04-07
CN113227383B (zh) 2022-11-22
CN113227383A (zh) 2021-08-06

Similar Documents

Publication Publication Date Title
CN110256544B (zh) NsNHX1蛋白质及其相关生物材料在培育耐逆型杨树中的应用
WO2009021448A1 (en) A plant height regulatory gene and uses thereof
CN110452291A (zh) 铁皮石斛胚胎发育晚期丰富蛋白DoLEA43在促进植物愈伤组织形成中的应用
US20210198682A1 (en) Application of sdg40 gene or encoded protein thereof
WO2017185854A1 (zh) Spl基因及其在增强植物耐热性能中的应用
CN114958903A (zh) 一种增强大豆香味的方法
US20140373193A1 (en) Use of OsPP18 Gene in Controlling Rice Drought Resistance
CN107299103B (zh) 厚藤IpASR基因及其编码蛋白和应用
CN110218247B (zh) PwRBP1和PwNAC1两种蛋白互作协同提高植物耐逆性及其应用
US11492635B2 (en) Method for improving stress tolerance of plants
BRPI0809359A2 (pt) Desumidificador
WO2021047377A1 (zh) Tpst基因在调控植物性状中的应用
CN114231535B (zh) 木薯MeRSZ21b基因在提高植物抗干旱胁迫中的应用
WO2021136255A1 (zh) Hak基因在调控植物性状中的作用
CN111454963A (zh) 火龙果耐盐基因HuERF1基因及其应用
CN113481210B (zh) 棉花GhDof1.7基因在促进植物耐盐中的应用
WO2021155753A1 (zh) 抗除草剂基因、多肽及其在植物育种中的应用
US20120110697A1 (en) Method of constructing novel higher plant and method of promoting the growth of higher plant
CN101665803B (zh) 具有改变的生长特性的植物及其生产方法
US9297020B2 (en) Gene for increasing the production of plant biomass and method of use thereof
CN106544355B (zh) 调节植物花序形态和籽粒数目的基因及其应用
CN115044592B (zh) 一种调控玉米株型和瘤黑粉病抗性的基因ZmADT2及其编码蛋白和应用
CN114836469B (zh) OsRST2基因或其相关生物材料在调节植物耐盐性中的应用
CN114438103B (zh) 调控水稻对干旱和盐胁迫耐受性的转录因子OsNAC15基因及应用
CN114621978B (zh) 拟南芥糖基转移酶ugt84a1在促进植物叶片生长方面的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20910371

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20910371

Country of ref document: EP

Kind code of ref document: A1