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CN-122012518-A - Functional study of calcium ion channel and transporter in plant osmotic stress response

CN122012518ACN 122012518 ACN122012518 ACN 122012518ACN-122012518-A

Abstract

The invention relates to a method for regulating plant growth and development, hypertonic stress response, ion stress response and ion concentration by CCX protein or a mutant thereof and application thereof.

Inventors

  • ZHAO YANG
  • HUANG HUILING
  • WANG HAO

Assignees

  • 中国科学院分子植物科学卓越创新中心

Dates

Publication Date
20260512
Application Date
20241112

Claims (11)

  1. 1. A polynucleotide, the polynucleotide (1) Inserting a base into the coding sequence of the wild type CCX4 protein shown as SEQ ID NO. 1, or (2) The complementary sequence of (1), Preferably, the base insertion position is before the 635 th position of the CCX4 gene sequence, More preferably, the insertion base is T.
  2. 2. A method of modulating a plant's response to hyperosmotic, salt, or ionic stress, the method comprising: (1) Up-regulating CCX4 protein expression or activity in plant to promote plant growth and development, raise tolerance of plant to high osmotic stress, salt stress or ion stress, or (2) Down-regulating CCX4 protein expression or activity in plants, thereby increasing the sensitivity of the plants to cations or increasing the cation concentration of plant cells, Preferably, the method comprises the steps of, The up-regulating of expression of CCX4 protein in plant includes transferring the coding sequence of CCX4 protein into plant to obtain transformed plant, The downregulation of expression or activity of a CCX4 protein in a plant includes (a) specifically interfering with CCX4 gene transcription and/or expression, (b) downregulating CCX4 protein activity, or (c) expressing a CCX4 protein with reduced activity in a plant, More preferably, the process is carried out, (A) Wherein the specific interference is interference with the transcription of the CCX4 gene or the translation of its transcript, (B) Wherein said down-regulating expression of CCX4 protein in a plant comprises transferring nucleic acid which specifically interferes with transcription and/or expression of the CCX4 gene into a plant to obtain a transformed plant, (C) The CCX4 protein with reduced activity comprises CCX4 protein with mutation of phosphorylation site, preferably, the phosphorylation site comprises one or more selected from Tyr8, ser47, ser60, thr63, thr396 and Thr399, more preferably Ser47 and Ser60, and even more preferably, the phosphorylation site is mutated into any one selected from alanine, cysteine, aspartic acid and glutamic acid.
  3. 3. The method of claim 2, wherein the nucleotide sequence encoding CCX4 protein in CCX4-10 is shown in SEQ ID No. 2 and/or the nucleotide sequence encoding CCX4 protein in CCX4-1 (salk_ 113447C) is shown in SEQ ID No. 14.
  4. 4. The method of claim 2, wherein, (A) Comprising subjecting the CCX4 gene to gene editing or RNA interference to reduce transcription and/or expression of the gene, preferably, the gene editing comprises gene editing by T-DNA insertion or gene editing by CRISPR technology, (B) Wherein said nucleic acid that specifically interferes with transcription and/or expression of the CCX4 gene is selected from the group consisting of (i) an antisense nucleic acid, microRNA, siRNA, RNAi, dsRNA, sgRNA, or a combination thereof, and (ii) a nucleic acid construct capable of expressing or forming (i).
  5. 5. The method of claim 2, wherein said downregulating CCX4 protein activity in (b) comprises (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) capable of downregulating CCX4 protein activity of CCX4 protein, (ii) introducing a nucleic acid sequence encoding (i) and/or a nucleic acid construct capable of expressing (i), (iii) mutating amino acids of the phosphorylation site of CCX4 protein, (iv) downregulating expression or activity of a phosphorylase capable of phosphorylating CCX4 protein, Preferably, the phosphorylation site comprises one or more selected from Tyr8, ser47, ser60, thr63, thr396 and Thr399, preferably Ser47, ser60, more preferably, the phosphorylation site is mutated to any one selected from alanine, cysteine, aspartic acid, glutamic acid, Preferably, the phosphorylase is OSMO and CPK, more preferably, the CPK is CPK3, CPK6 or CPK11.
  6. 6. The method of claim 2, further characterized by one or more features selected from the group consisting of: The plant is a crucifer plant, preferably the plant is an arabidopsis plant, more preferably the plant is arabidopsis thaliana; the plant cell cation is selected from sodium, potassium, calcium ion, etc.
  7. 7. The use of a substance that modulates CCX4 protein expression or activity in plants in modulating plant growth, plant tolerance to osmotic stress, salt stress, or ion stress, modulating plant sensitivity to cations, or modulating plant cell cation concentration, Preferably, the agent is an enhancer of CCX4 protein expression or activity, the modulation being an up-regulation of CCX4 protein expression or activity in a plant, thereby enhancing plant growth, enhancing tolerance of a plant to hyperosmotic, salt stress, or ionic stress, more preferably the enhancer is CCX4 protein or a coding sequence thereof, or Preferably, the agent is an inhibitor of CCX4 protein expression or activity, the modulation being down-regulation of CCX4 protein expression or activity in the plant, thereby increasing plant sensitivity to cations, or increasing plant cell cation concentration, more preferably the inhibitor is selected from (1) an inhibitor that specifically interferes with CCX4 gene transcription and/or expression, (2) an inhibitor that down-regulates CCX4 protein activity, or (3) an activity down-regulated CCX4 protein variant or coding sequence thereof.
  8. 8. The use according to claim 7, wherein, (1) The inhibitor is selected from the group consisting of (i) an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, sgRNA, or a combination thereof, and (ii) a nucleic acid construct capable of expressing or forming (i), (2) The inhibitor is selected from the group consisting of (i) an antibody or ligand specific for the CCX4 protein, and (ii) a nucleic acid sequence encoding (i) and/or a nucleic acid construct capable of expressing (i), Preferably, the inhibitor of (1) is a nucleic acid that specifically interferes with the transcription and/or expression of the CCX4 gene.
  9. 9. The application of CCX4 gene in identifying plant growth, tolerance of plant to high osmotic stress, salt stress or ion stress, sensitivity of plant to cation or cation concentration in plant cell, Preferably, the steps are performed to compare the expression or activity of the CCX4 gene in a plant with that of a wild type plant, and if the expression or activity of the CCX4 gene is up-regulated, the plant grows more, is more tolerant to hypertonic, salt-stress, or ion-stress, and if the expression or activity of the CCX4 gene is down-regulated, the plant is more sensitive to cations or the concentration of cations in the plant cell is higher.
  10. 10. A method of attenuating a high calcium-sensitive phenotype of a mutant yeast strain, said method comprising overexpressing a CCX protein or a mutant protein thereof in the mutant yeast strain, Preferably, the CCX protein is CCX4 protein, the amino acid sequence of the CCX4 protein is shown as SEQ ID NO. 3, More preferably, the mutant protein comprises an amino acid mutation at position 47, and/or an amino acid mutation at position 60 of the CCX4 protein.
  11. 11. The method of claim 10, wherein, (1) The 47 th amino acid mutation is A or E, or the 47 th amino acid mutation is deletion mutation, or (2) Said amino acid at position 47 is mutated to A or E, and said amino acid at position 60 is mutated to A, Preferably, the mutant protein is selected from CCX4 S47A 、CCX4 Δ47 、CCX4 S47E or CCX4 S47/60A , Preferably, the mutant of the yeast strain is csg2, Preferably, the high calcium sensitive phenotype refers to a calcium ion concentration >50mmol/L in the yeast strain.

Description

Functional study of calcium ion channel and transporter in plant osmotic stress response Technical Field The invention relates to the field of biotechnology and botanics, in particular to a method for regulating plant growth and development, hypertonic stress response, ion stress response and ion concentration of a plant CCX protein or a mutant thereof and application thereof. Background The soil area of China is large, but the cultivation and production conditions of soil are not optimistic. Drought and salt stress cause osmotic stress on plants, severely affecting crop yield and quality. Plants have complex signal networks for sensing, transduction and response of salt and osmotic stress signals, of which Ca 2+ signals are an integral component. After plants are subjected to environmental stress, the concentration of intracellular Ca 2+ is transiently increased to form a space-time specific calcium signal, and cells can influence protein phosphorylation, transcription level, activity of ion channels and other proteins by recognizing and decoding the calcium signal. Early calcium signaling studies have been focused mainly on the mechanism of calcium signal decoding, and in recent years, studies have been focused on the use of calcium ion biological probes to explore the environmental stress-mediated Ca 2+ signaling. A large number of researches show that CCX family gene expression is regulated and controlled by aging, active oxygen, salt stress, drought stress and other abiotic stress, and mediates transportation and steady maintenance of Cd 2+ and Ca 2+ in plants, and can play an important role in the adversity stress resistance of the plants. CCX4 plays a very important role in plant growth, osmotic stress and salt stress response, and phosphorylation modification may be an important regulatory way. However, their specific role in plants has not been studied intensively. Disclosure of Invention The first aspect of the present invention provides a polynucleotide, which polynucleotide (1) Inserting a base into the coding sequence of wild type CCX4 protein shown in SEQ ID NO.1, (2) The complement of (1). In one or more embodiments, the base insertion position is before position 635 of the CCX4 gene sequence. In one or more embodiments, the insertion base is preferably T. In a second aspect, the invention provides a nucleic acid construct comprising a polynucleotide as described herein. In one or more embodiments, the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector. The invention also provides a host cell comprising a nucleic acid construct as described in the second aspect herein. In a third aspect, the invention provides a method of modulating a plant's response to hyperosmotic, salt or ionic stress, the method comprising: (1) Up-regulating CCX4 protein expression or activity in plant to promote plant growth and development, raise tolerance of plant to high osmotic stress, salt stress or ion stress, or (2) Down-regulating CCX4 protein expression or activity in plants, thereby increasing plant sensitivity to cations, or increasing plant cell cation concentration. In one or more embodiments, the up-regulating expression of the CCX4 protein in the plant comprises transferring the coding sequence of the CCX4 protein into the plant to obtain a transformed plant, and the down-regulating expression or activity of the CCX4 protein in the plant comprises (a) specifically interfering with transcription and/or expression of the CCX4 gene, (b) down-regulating activity of the CCX4 protein, or (c) expressing the CCX4 protein with reduced activity in the plant. In one or more embodiments, the specific interference in (a) is interference of transcription of CCX4 gene or translation of its transcript, (b) the down-regulating expression of CCX4 protein in a plant comprises transferring nucleic acid specifically interfering with transcription and/or expression of CCX4 gene into a plant to obtain a transformed plant, (c) the ccX4 protein with reduced activity comprises a CCX4 protein with a mutation in a phosphorylation site, preferably the phosphorylation site comprises one or more selected from Tyr8, ser47, ser60, thr63, thr396 and Thr399, more preferably Ser47, ser60, further preferably the phosphorylation site is mutated to any one selected from alanine, cysteine, aspartic acid, glutamic acid. In one or more embodiments, the nucleotide sequence encoding the CCX4 protein in CCX4-10 is shown in SEQ ID NO. 2 and/or the nucleotide sequence encoding the CCX4 protein in CCX4-1 (SALK_ 113447C) is shown in SEQ ID NO. 14. In one or more embodiments, (a) comprises gene editing or RNA interference of the CCX4 gene to reduce transcription and/or expression of the gene, preferably the gene editing comprises gene editing by T-DNA insertion or gene editing by CRISPR techniques, (b) the nucleic acid that specifically interferes with CCX4 gene trans