Function research of calcium ion channel and transporter in plant osmotic stress response

Abstract

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

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and botany, and more specifically, to a method and its application of a plant CCX protein or its mutant in regulating plant growth and development, hyperosmolar stress response, ion stress response, and ion concentration regulation. Background Technology

[0002] my country has a vast land area, but the soil conditions for cultivation are far from ideal. Drought and salt stress cause osmotic stress in plants, severely impacting crop yield and quality. Plants possess a complex signal network for sensing, transducing, and responding to salt and osmotic stress signals, among which Ca... 2+ Signaling is an indispensable component. When plants are subjected to environmental stress, intracellular calcium... 2+ A sudden increase in calcium concentration creates a space- and time-specific calcium signal. Cells recognize and decode this signal, subsequently influencing protein phosphorylation, transcriptional levels, and the activity of proteins such as ion channels. Early research on calcium signaling primarily focused on the decoding mechanisms of calcium signals. Recent studies have utilized calcium ion bioprobes to explore the effects of environmental stress on calcium metabolism. 2+ The study of signals has attracted much attention.

[0003] Numerous studies have shown that the expression of CCX family genes is regulated by abiotic stresses such as senescence, reactive oxygen species, salt stress, and drought stress, and mediates Cd in plants. 2+ and Ca 2+ CCX4 plays a crucial role in the translocation and homeostasis of plant components and may function as an important regulator in plant resistance to abiotic stress. CCX4 is involved in plant growth, osmotic stress response, and salt stress response, and phosphorylation modification may be a key regulatory mechanism.

[0004] However, its specific role in plants has not been thoroughly studied. Summary of the Invention

[0005] The first aspect of the present invention provides a polynucleotide, wherein the polynucleotide

[0006] (1) Insert a base into the coding sequence of the wild-type CCX4 protein as shown in SEQ ID NO:1.

[0007] (2)(1) complementary sequences.

[0008] In one or more embodiments, the base insertion position is before position 635 of the CCX4 gene sequence.

[0009] In one or more embodiments, the inserted base is preferably T.

[0010] A second aspect of the present invention provides a nucleic acid construct containing the polynucleotides described herein.

[0011] In one or more embodiments, the nucleic acid construct is a vector.

[0012] In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

[0013] The present invention also provides a host cell comprising the nucleic acid constructs described in the second aspect herein.

[0014] A third aspect of the present invention provides a method for regulating plant responses to hyperosmolar stress, salt stress, or ion stress, the method comprising:

[0015] (1) Upregulating the expression or activity of CCX4 protein in plants, thereby promoting plant growth and development and improving the plant's tolerance to hyperosmolar stress, salt stress, or ion stress; or

[0016] (2) Downregulate the expression or activity of CCX4 protein in plants, thereby increasing the sensitivity of plants to cations or increasing the cation concentration in plant cells.

[0017] In one or more embodiments, the upregulation of CCX4 protein expression in plants includes: transferring the coding sequence of CCX4 protein into plants to obtain transformed plants, and the downregulation of CCX4 protein expression or activity in plants includes: (a) specifically interfering with CCX4 gene transcription and / or expression, (b) downregulating CCX4 protein activity, or (c) expressing CCX4 protein with reduced activity in plants.

[0018] In one or more embodiments, (a) the specific interference is interference with the transcription of the CCX4 gene or the translation of its transcripts; (b) the downregulation of CCX4 protein expression in plants includes: transferring nucleic acids that specifically interfere with CCX4 gene transcription and / or expression into plants to obtain transformed plants; (c) the CCX4 protein with reduced activity includes CCX4 protein with a mutated phosphorylation site; preferably, the phosphorylation site includes one or more selected from the following: Tyr8, Ser47, Ser60, Thr63, Thr396 and Thr399; more preferably, Ser47 and Ser60; and even more preferably, the phosphorylation site mutation is selected from any one of alanine, cysteine, aspartic acid and glutamic acid.

[0019] 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.

[0020] In one or more embodiments, (a) includes gene editing or RNA interference of the CCX4 gene to reduce the transcription and / or expression of the gene, preferably, the gene editing includes gene editing performed by T-DNA insertion or gene editing using CRISPR technology, and (b) the nucleic acid that specifically interferes with the transcription and / or expression of the CCX4 gene is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (ii) nucleic acid constructs that can express or form (i).

[0021] In one or more embodiments, the downregulation of CCX4 protein activity described in (b) includes, in a plant: (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) of the CCX4 protein capable of downregulating CCX4 protein activity; (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i); (iii) mutating an amino acid at a phosphorylation site of the CCX4 protein; and (iv) downregulating the expression or activity of a phosphorylase capable of phosphorylating the CCX4 protein.

[0022] In one or more embodiments, the phosphorylation site includes one or more selected from: Tyr8, Ser47, Ser60, Thr63, Thr396, and Thr399; preferably Ser47 or Ser60. More preferably, the phosphorylation site is mutated to any one of alanine, cysteine, aspartic acid, or glutamic acid. In one or more embodiments, Ser47 is mutated to alanine, cysteine, aspartic acid, or glutamic acid. In one or more embodiments, Ser60 is mutated to alanine, cysteine, aspartic acid, or glutamic acid.

[0023] In one or more embodiments, the phosphorylase is OSMO2 and CPK; preferably, the CPK is CPK3, CPK6, or CPK11.

[0024] In one or more embodiments, the Gene ID of the OSMO2 is 824318.

[0025] In one or more embodiments, the Gene ID of the CPK3 is 828465.

[0026] In one or more embodiments, the Gene ID of the CPK6 is 816235.

[0027] In one or more embodiments, the CPK11 has a Gene ID of 840471.

[0028] In one or more embodiments, the CCX protein is the CCX4 protein. Preferably, the amino acid sequence of the CCX4 protein is shown in SEQ ID NO:3.

[0029] In one or more embodiments, the plant is a cruciferous plant, preferably a Arabidopsis thaliana plant, and more preferably, the plant is Arabidopsis thaliana.

[0030] In one or more embodiments, the plant cell cations are selected from sodium, potassium, calcium ions, etc.

[0031] The present invention also provides the use of substances that regulate the expression or activity of CCX4 protein in plants in regulating plant growth and development, plant tolerance to hyperosmolar stress, salt stress, or ion stress, regulating plant sensitivity to cations, or regulating the concentration of cations in plant cells.

[0032] In one or more embodiments, the substance is a promoter of CCX4 protein expression or activity, and the regulation is to upregulate CCX4 protein expression or activity in plants, thereby promoting plant growth and improving the plant's tolerance to hyperosmolar stress, salt stress, or ion stress; preferably, the promoter is the CCX4 protein or its coding sequence; or

[0033] In one or more embodiments, the substance is an inhibitor of CCX4 protein expression or activity, the regulation being to downregulate CCX4 protein expression or activity in plants, thereby increasing the plant’s sensitivity to cations or increasing the cation concentration in plant cells. Preferably, the inhibitor is selected from: (1) an inhibitor that specifically interferes with CCX4 gene transcription and / or expression, (2) an inhibitor that downregulates CCX4 protein activity, or (3) a downregulated CCX4 protein variant or its coding sequence.

[0034] In one or more embodiments, (1) the inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA or combinations thereof, and (ii) nucleic acid constructs that can express or form (i), and (2) the inhibitor is selected from the group consisting of (i) specific antibodies or ligands of the CCX4 protein, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i).

[0035] In one or more embodiments, (1) the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of the CCX4 gene.

[0036] The present invention also provides a use of the CCX4 gene as a molecular marker for identifying plant growth, the plant's tolerance to hyperosmolar stress, salt stress, or ion stress, the plant's sensitivity to cations, or the level of cation concentration in plant cells.

[0037] In one or more embodiments, the method includes the steps of comparing the expression or activity of the CCX4 gene in plants with that in wild-type plants, and if the expression or activity of the CCX4 gene is upregulated, the plants grow taller and are more tolerant to hyperosmolar stress, salt stress, or ion stress; if the expression or activity of the CCX4 gene is downregulated, the plants are more sensitive to cations or have higher intracellular cation concentrations.

[0038] The present invention also provides a method for reducing the high calcium sensitivity phenotype of yeast strain mutants, the method comprising overexpressing CCX protein or a mutant protein thereof in yeast strain mutants.

[0039] In one or more embodiments, the CCX protein is the CCX4 protein.

[0040] In one or more embodiments, the mutant protein includes a mutation at amino acid position 47 of CCX4 and / or a mutation at amino acid position 60.

[0041] In one or more embodiments, the 47th amino acid is mutated to A or E.

[0042] In one or more embodiments, the 47th amino acid mutation also includes a deletion mutation.

[0043] In one or more embodiments, the 60th amino acid is mutated to A.

[0044] In one or more embodiments, the mutant protein is selected from CCX4. S47A CCX4 Δ47 CCX4 S47E Or CCX4 S47 / 60A .

[0045] In one or more embodiments, the yeast strain mutant is preferably csg2.

[0046] In one or more embodiments, the high calcium sensitivity phenotype refers to a yeast strain with a calcium ion concentration >50 mmol / L. Attached Figure Description

[0047] Figure 1 Growth phenotypes of CCX family gene mutants under salt and hyperosmolar stress. Growth of wild-type (WT), ccx4-1, ccx1-1, ccx1-2, ccx2, ccx5-1, and ccx5-2 mutants on 1 / 2 MS medium and medium supplemented with 150 mM mannitol and 75 mM sodium chloride.

[0048] Figure 2: Schematic diagram of the ccx4 mutant gene and its growth defect phenotype. (a) Topological model of the CCX4 protein. The M1-M12 cylinders represent the transmembrane domains predicted by TMHMM 2.0; the dashed boxes contain the conserved α-1 and α-2 repeat domains; the green arrows indicate the two large cytoplasmic loops of about 100 amino acids each in CCX4; the red arrow indicates the position of the 47th serine residue. (b) Schematic diagram of the AtCCX4 gene. The black rectangles represent the CDS coding region of AtCCX4; the gray areas represent the 5'UTR and 3'UTR regions, and the yellow area represents a sequence after the 3'UTR; the red dashed line indicates the position of the 47th serine residue; the triangle indicates the T-DNA insertion site of the ccx4-1 mutant. The blue arrow indicates that the mutant ccx4-10 inserts a T at nucleotide position 636 and causes a frameshift, resulting in premature termination. F1 / R1 are a pair of primers for identifying T-DNA insertion of ccx4-1 (as shown in SEQ ID NO:4 and SEQ ID NO:5); F1 / R2 are primers for identifying CCX4 expression levels (as shown in SEQ ID NO:4 and SEQ ID NO:6). (c) Gene sequencing peak diagram of the ccx4-10 mutation site. (d) Growth defect phenotype of ccx4 mutant after 4 weeks of culture in a short-day light chamber.

[0049] Figure 3 The ccx4 mutant is more sensitive to hyperosmolar stress. (a) Growth phenotype of plants on media with different concentrations of mannitol. (b) Statistical graph of the experiment in Figure (a). The fresh weight of the aboveground parts of different mutants after 10 days of growth on different media (n≥9) was weighed, and the relative fresh weight was calculated. GraphPad Prism9 was used for plotting and significance analysis. (c) Changes in osmotic potential of plants under high salt and hyperosmolar stress. Plants grown on 1 / 2 MS vertical medium for 10 days were transferred to media with 100 mM NaCl and 300 mM mannitol for two days. The concentration of plant tissue sap was measured. The osmotic potential was then calculated using Excel according to the van der Hoff formula π = cRT. GraphPad Prism9 was used for plotting and significance analysis. *p<0.05, **p<0.01, ***p<0.001, Multiple t tests.

[0050] Figure 4AtCCX4 affects water loss and stomatal movement in plants. (a) Plants approximately 5 weeks old grown in a short-day incubator were weighed, and the fresh weight of the entire aboveground part at different detachment times and the dry weight after drying were calculated to determine the water loss rate. Graphs were created and significance analysis was performed using GraphPad Prism 9 software. Error bars are standard deviations (SD), n=3. (b) Detached leaves of plants grown in a short-day incubator for 4-5 weeks were immersed in stomatal opening buffer for 2 hours to open the stomata. After treatment with buffer containing 250 mM mannitol for 40 minutes, stomatal opening was observed and photographed under a microscope. (c) Statistical graph of (b). The length and width of stomata were measured using ImageJ software, the width / length ratio was calculated using Excel, and statistical graphs and difference analysis were performed using GraphPad Prism 9. Error bars are standard deviations (SD), n>100. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, Multiple t tests.

[0051] Figure 5 The ccx4 mutant is sensitive to high concentrations of sodium, potassium, and calcium salts. (a) Phenotypes of Col-0, ccx4-1, and ccx4-10 after 10 days of growth on 1 / 2 MS medium supplemented with 100 mM sodium chloride, 50 mM potassium chloride, and 50 mM calcium chloride. (b) Fresh weight statistics (n≥3, 2-way ANOVA analysis, p<0.05). (c) Relative fresh weight statistics.

[0052] Figure 6 Abiotic stress-induced calcium signaling was enhanced in ccx4-1. Based on Aequorin calcium ion indicator, seedlings aged 6-7 days were treated with 0.8 M mannitol (a), 0.3 M NaCl (c), and 10 mM H2O2 (e), and fluorescence values ​​were recorded in real time. Calcium ion concentration was calculated and plotted as a curve (mean ± SEM) using GraphPad Prism 9. (b), (d), and (f) are violin plots of the peak calcium ion concentration induced by 0.8 M mannitol treatment (n≥18), 0.3 M NaCl treatment (n≥7), and 10 mM H2O2 treatment (n≥12), respectively. Statistical plots and significance analysis were performed using GraphPad Prism 9 software. ****p<0.0001, Multiple t tests.

[0053] Figure 7 Prokaryotically purified OSMO2 and CPK3 can phosphorylate CCX4. Using a method based on... 32The in vitro phosphorylation assay using P-labeled ATP was used to detect the phosphorylation of CCX41-HIS (1-24 aa), CCX42-HIS (35-130 aa), CCX43-HIS (155-208 aa), CCX44-HIS (294-442 aa), and CCX45-HIS (521-549 aa) protein fragments and the point spike protein fragment of CCX42-HIS by OSMO2-GST, as well as the phosphorylation of MBP-CCX42 protein fragments and its point spike protein by GST-CPK3 / 6 / 11. Autoradiography (top image) showed the autophosphorylation signal of the kinase and the phosphorylation signal of the substrate. Coomassie brilliant blue staining (bottom image) was used for color development of protein samples.

[0054] Figure 8 Phosphorylation of CCX4 affects plant growth. (a) Semi-quantitative PCR was used to identify the transcriptional level of the CCX4 gene in seedlings of Col-0, ccx4-1, WT-MYC#1, WT-3'UTR, and S47A-MYC#2 / 3 / 6 / 8 / 10 days.

[0055] Primers F1 / R2 and F1 / MYC-R (primer MYC-R is shown in SEQ ID NO:7) both cross the T-DNA insertion site of ccx4-1, and the PCR product sizes are 129bp and 266bp, respectively. MYC-R is the reverse primer on the gene sequence of the MYC protein tag. (b) Real-time PCR was used to detect the transcription level of the CCX4 gene in seedlings of Col0, ccx4-1, WT-MYC#1, WT-3'UTR, and S47A-MYC#2 / 3 / 6 / 8 / 10 days old. The primers were C4-qF / C4-qR, and ACTIN2 was the internal reference gene for semi-quantitative PCR and quantitative PCR experiments (primer C4-qF sequence is shown in SEQ ID NO:8, C4-qR sequence is shown in SEQ ID NO:9, ACTIN2-F sequence is shown in SEQ ID NO:10, and ACTIN2-R sequence is shown in SEQ ID NO:11). (c) Phenotypes of Col-0, ccx4-1, WT-MYC#1, WT-3'UTR and S47A-MYC#2 / 3 / 6 / 8 after 6 weeks of growth in a short-day incubator.

[0056] Figure 9Phosphorylation of CCX4 affects plant responses to osmotic stress. (a) Phenotypic results of Col-0, ccx4-1, and transgenic supplemented materials WT-MYC#1 and S47A-MYC#2 / 8 after 10 days of growth on 1 / 2 MS media supplemented with 75 mM, 100 mM, and 140 mM mannitol. (b) Statistical graph of aboveground fresh weight (n≥14, 2-way ANOVA analysis, p<0.05). (c) Statistical graph of relative aboveground fresh weight. (d) Statistical graph of water loss rate of the entire rosette leaf after detachment from Col-0, ccx4-1, and transgenic supplemented materials WT-MYC#1 and S47A-MYC#2 / 8 (n≥3).

[0057] Figure 10 Phosphorylation of CCX4 plays a crucial role in plant tolerance to ion stress. (a) Growth phenotypes of wild-type, mutant, and transgenic complement materials on 1 / 2 MS medium and medium supplemented with 100 mM sodium chloride, 50 mM potassium chloride, and 50 mM calcium chloride. (b) Statistical graph of fresh weight. (c) Statistical graph of relative fresh weight (n≥3, 2-way ANOVA analysis, p<0.05).

[0058] Figure 11 CCX4 possesses calcium ion transport capabilities in yeast csg2. CCX4 can compensate for the sensitivity of the csg2 yeast strain to high concentrations of calcium ions. (a) Empty vector (pYES2) and CAX1, sCAX1, CCX4, CCX4Δ47, CCX4 S47A CCX4 S47E and CCX4 S4 / 607A Transformed into yeast strain csg2. Transformants were transferred to SC-Ura medium containing galactose as the carbon source and 100 mM or 200 mM CaCl2 and grown at 30°C for 3-4 days. Gene expression was regulated by the galactose-inducible promoter GAL1. (b) p416 empty vector, full-length CCX4, CCX4... S47A CCX4 S47E and CCX4 S47D Transformed into CSG2 yeast competent cells, positive transformants were screened on SC-Ura selective medium with glucose as the carbon source. The transformants were then transferred to YPDA yeast medium and YPDA medium supplemented with 50 mM, 75 mM, or 100 mM CaCl2, and grown in an incubator at 30°C for 3-4 days. Detailed Implementation

[0059] The inventors discovered the role of CCX4 in plant growth, osmotic stress response, and salt stress response, and that phosphorylation modification may be an important regulatory mechanism.

[0060] A first aspect of the present invention provides a polynucleotide, wherein (1) a base is inserted into the coding sequence of the wild-type CCX4 protein as shown in SEQ ID NO:1, and (2) a complementary sequence to (1). The polynucleotide may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0061] As those skilled in the art will understand, DNA is typically a double-stranded structure with complementary sense and antisense strands. The sense strand, also known as the sense line, is the strand in the DNA double helix that carries genetic information. It cannot be transcribed and contains the same nucleotide sequence as the mRNA encoding the functional protein, differing only in that the T in DNA is replaced by U in mRNA. The antisense strand, also known as the template strand, is the DNA strand used by RNA polymerase during transcription. It acts as a template for transcription; RNA polymerase moves along the template strand and transcribes it into mRNA, which is then translated into a polypeptide.

[0062] In a narrow sense, a "polynucleotide sequence encoding..." or "the coding sequence of..." refers to a sequence located on the antisense strand of DNA that directly guides transcription, or a sequence located on mRNA that directly guides translation. Polynucleotides can contain coding sequences (or coding regions) and non-coding sequences (or non-coding regions, such as introns). Coding sequences can be continuous or discontinuous, and discontinuous coding sequence segments can be separated by non-coding sequences. The complete coding region obtained by sequentially connecting the coding sequence segments is the coding sequence of the polypeptide.

[0063] As those skilled in the art will understand, due to the degeneracy of the genetic code, a vast number of nucleic acids can be produced, all of which encode the antibodies or antigen-binding fragments of the present invention. Therefore, given the identification of specific amino acid sequences, those skilled in the art can produce any number of different nucleic acids by simply modifying the sequence of one or more codons without altering the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides that hybridize with the aforementioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, hybridizable polynucleotide-encoded peptides have the same biological functions and activities as mature peptides.

[0064] This invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes. "Polynucleotide encoding a polypeptide" can include a polynucleotide encoding the polypeptide, or it can include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0065] Exemplarily, the CCX4 gene mutation of the present invention includes (a) the insertion of one base into the CCX4 gene sequence compared to the sequence shown in SEQ ID NO:1. Preferably, the base insertion position is before position 635 of the CCX4 gene sequence. More preferably, the inserted base is T.

[0066] The full-length nucleotide sequence or fragments of the CCX4 gene of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available DNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. In this document, the primer sequences are shown in SEQ ID NO: 12 and 13. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. Usually, it is cloned into a vector, then transformed into cells, and then the relevant sequence is isolated from the proliferated host cells using conventional methods.

[0067] Furthermore, the relevant sequences can be synthesized artificially, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0068] This invention also provides a nucleic acid construct comprising the polynucleotides described herein. In one or more embodiments, the nucleic acid construct is a vector, preferably an expression vector or a recombinant vector. As a preferred embodiment, the promoter downstream of the nucleic acid construct contains a multiple cloning site or at least one restriction enzyme site. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the nucleic acid construct comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If desired, the nucleic acid construct may further include elements selected from the group consisting of: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; an resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator.

[0069] The methods used to prepare nucleic acid constructs are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0070] Those skilled in the art can use well-known methods to construct expression vectors containing the genes described in this invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When constructing recombinant expression vectors using the genes of this invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0071] Nucleic acid constructs including the polynucleotides of this invention can be used to transform suitable host cells to enable the host to express proteins. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Streptomyces*, or *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells, preferably cruciferous plant cells, more preferably *Arabidopsis thaliana* cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), it can be treated with CaCl2 or electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.) can be used. Transformed plants can also be transformed using methods such as *Agrobacterium* transformation or gene gun transformation, for example, leaf disc transformation, embryo transformation, flower bud soaking, etc. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants. When the polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0072] Those skilled in the art know how to select appropriate vectors, promoters, enhancers, and host cells.

[0073] The peptides described herein may be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0074] Transforming a host with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Transformed plants can be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and rice embryo transformation. Transformed plant tissues or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits.

[0075] This invention provides a method for regulating plant responses to hyperosmolar stress, salt stress, or ion stress. The method includes: (1) upregulating the expression or activity of CCX4 protein in plants to promote plant growth and development and improve the plant's tolerance to hyperosmolar stress, salt stress, or ion stress; or (2) downregulating the expression or activity of CCX4 protein in plants to improve the plant's sensitivity to cations or increase the cation concentration in plant cells. Preferably, the upregulation of CCX4 protein expression in plants includes: transferring the coding sequence of CCX4 protein into plants to obtain transformed plants. The downregulation of CCX4 protein expression or activity in plants includes: (a) specifically interfering with CCX4 gene transcription and / or expression; (b) downregulating CCX4 protein activity; or (c) expressing active CCX4 protein in plants. More preferably, the specific interference described in (a) is interference with the transcription of the CCX4 gene or the translation of its transcript; the downregulation of CCX4 protein expression in plants described in (b) includes: transferring nucleic acids that specifically interfere with the transcription and / or expression of the CCX4 gene into plants to obtain transformed plants; and the CCX4 protein with reduced activity described in (c) includes CCX4 protein with a mutated phosphorylation site. Preferably, the phosphorylation site includes one or more selected from the following: Tyr8, Ser47, Ser60, Thr63, Thr396, and Thr399; more preferably, Ser47 and Ser60; and even more preferably, the phosphorylation site mutation is selected from any one of alanine, cysteine, aspartic acid, and glutamic acid.

[0076] In one embodiment, 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.

[0077] In one embodiment, (a) includes gene editing or RNA interference of the CCX4 gene to reduce the transcription and / or expression of the gene, preferably, the gene editing includes gene editing performed by T-DNA insertion or gene editing using CRISPR technology, and (b) the nucleic acid that specifically interferes with the transcription and / or expression of the CCX4 gene is selected from the group consisting of: (i) antisense nucleic acids, microRNA, siRNA, RNAi, dsRNA, sgRNA or combinations thereof, and (ii) nucleic acid constructs that can express or form (i), preferably, the downregulation of CCX4 protein activity in (b) includes in plants: (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) that can downregulate CCX4 protein activity. (ii) introducing the nucleic acid sequence encoding (i) and / or a nucleic acid construct capable of expressing (i), (iii) mutating the amino acid of the phosphorylation site of the CCX4 protein, and (iv) downregulating the expression or activity of the phosphorylase capable of phosphorylating the CCX4 protein. In this application, the phosphorylation site includes one or more selected from the following: Tyr8, Ser47, Ser60, Thr63, Thr396, and Thr399; preferably Ser47 and Ser60, more preferably, the phosphorylation site is mutated to any one selected from alanine, cysteine, aspartic acid, and glutamic acid. Exemplarily, the phosphorylase is OSMO2 and CPK; more preferably, the CPK is CPK3, CPK6, or CPK11.

[0078] In this document, "CCX protein" refers to a polypeptide with CCX activity, including but not limited to variants of the polypeptide. "Variations" include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, 1-20, 1-10, 1-8, or 1-5), and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. In the art, amino acids of similar properties often refer to amino acid families with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of the polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of the genetically encoded amino acids. The amino acid sequence of the polypeptide encoded by the CCX4 gene in this paper is shown in SEQ ID NO:3.

[0079] The variant forms of polypeptides include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants.

[0080] As used herein, the term "receptor-like kinase" refers to a class of single-transmembrane proteins located on the cell membrane, comprising an extracellular receptor domain for sensing external signals, a transmembrane domain, and an intracellular kinase domain. The receptor-like kinase OSMO2 described herein phosphorylates the CCX protein. Preferably, the Gene ID of OSMO2 is 824318.

[0081] "Calcium-dependent protein kinase" (CPK) is a plant-specific protein kinase that possesses both calcium signal sensing and direct decoding functions, amplifying calcium signals through phosphorylation of downstream target proteins. In this paper, the Gene ID of CPK3 is 828465. The Gene ID of CPK6 is 816235. The Gene ID of CPK11 is 840471.

[0082] Exemplarily, the phosphorylation sites include the following of the CCX4 protein: tyrosine at position 8, serine at position 47, serine at position 60, threonine at position 63, threonine at position 396, and threonine at position 399. Preferably, the phosphorylation sites are selected from one or two of the following sites: serine at position 47 and serine at position 60. The amino acid sequence of the CCX4 protein is shown in SEQ ID NO:3.

[0083] This invention also provides the use of substances that regulate the expression or activity of CCX4 protein in plants in regulating plant growth and development, plant tolerance to hyperosmolar stress, salt stress, or ion stress, regulating plant sensitivity to cations, or regulating cation concentration in plant cells. The substance is a promoter of CCX4 protein expression or activity, and the regulation is to upregulate the expression or activity of CCX4 protein in plants, thereby promoting plant growth and improving plant tolerance to hyperosmolar stress, salt stress, or ion stress. More preferably, the promoter is the CCX4 protein or its coding sequence; or

[0084] The substance is an inhibitor of CCX4 protein expression or activity, and the regulation is to downregulate CCX4 protein expression or activity in plants, thereby increasing the plant’s sensitivity to cations or increasing the cation concentration in plant cells. More preferably, the inhibitor is selected from: (1) an inhibitor that specifically interferes with CCX4 gene transcription and / or expression, (2) an inhibitor that downregulates CCX4 protein activity, or (3) a CCX4 protein variant or its coding sequence that downregulates activity.

[0085] In one embodiment, (1) the inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNA, siRNA, shRNA, dsRNA, sgRNA or combinations thereof, and (ii) nucleic acid constructs that can express or form (i), and (2) the inhibitor is selected from the group consisting of (i) specific antibodies or ligands of the CCX4 protein, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i), preferably, (1) the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of the CCX4 gene.

[0086] The present invention also provides a method for reducing the high calcium sensitivity phenotype of a yeast strain mutant, the method comprising overexpressing a CCX protein or a mutant protein thereof in the yeast strain mutant, wherein, exemplary, the CCX protein is a CCX4 protein, the amino acid sequence of the CCX4 protein is shown in SEQ ID NO:3, and the mutant protein comprises a mutation at amino acid position 47 and / or a mutation at amino acid position 60 of the CCX4 protein.

[0087] In one embodiment, (1) the 47th amino acid is mutated to A or E, or the 47th amino acid mutation is a deletion mutation, or (2) the 47th amino acid is mutated to A or E, and the 60th amino acid is mutated to A, wherein the mutant protein is selected from CCX4. S47A CCX4 Δ47 CCX4 S47E Or CCX4 S47 / 60A The yeast strain mutant is csg2, wherein the high calcium sensitivity phenotype refers to a calcium ion concentration in the yeast strain >50 mmol / L.

[0088] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. The invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0089] Example

[0090] Example 1: Response of AtCCX family gene mutants to hyperosmolar and salt stress

[0091] To investigate the function of CCX protein in plants to resist drought and salt stress, the inventors ordered mutants of the AtCCX1-5 gene. Through identification, homozygous mutants of ccx1-1, ccx1-2, ccx2, ccx4-1, ccx5-1, and ccx5-2 were obtained. Mutants of AtCCX3 were not obtained. Mutant information: The Arabidopsis plant material used in the project was in Columbia ecotype (Col-0) background.

[0092] T-DNA insertion mutants were ordered from NASC (The Nottingham Arabidopsis Stock Centre) and ABRC (Arabidopsis Biological Resource Centre). The following are T-DNA insertion mutants: ccx4-1 (SALK_113447C), ccx1-1 (SALK_083111C), ccx1-2 (SALK_035514C), ccx2 (SALK_201874C), ccx5-1 (SALK_120074), ccx5-2 (SALK_073669C), nced3 / 5 (nced5-2, GK_328D05); nced3-2 (GK_129B08).

[0093] The following is information about genetically modified materials:

[0094] Transgenic materials are obtained through the traditional Agrobacterium-mediated flower dipping method.

[0095] Arabidopsis seeds were disinfected with 75% ethanol for two minutes, the waste liquid was aspirated, and then disinfected with 5% sodium hypochlorite solution for 10 minutes, followed by rinsing with sterile water five times. After disinfection, the seeds were sputtered onto 1 / 2 MS solid medium and vernalized in a 4°C cold storage for two days in the dark before being transferred to a PERCIVAL constant temperature light box (11 hours of light exposure at 23°C).

[0096] The growth conditions of Arabidopsis thaliana materials used in the aboveground dehydration experiment were as follows: After absorbing water, Arabidopsis thaliana seeds were scattered in the soil and vernalized in a cold storage for two days. Then, they were transferred to a short-day light box (8 hours of light, 23°C, 43% humidity) to germinate. About 10 days later, the Arabidopsis thaliana seedlings were transplanted into the soil of flower pots, with 6 seedlings planted in each pot. They continued to grow in the short-day light box and were used for experiments after three to four weeks.

[0097] In the experiment, it was found that mutants of the AtCCX1, AtCCX2, and AtCCX5 genes did not exhibit significantly abnormal growth phenotypes compared to the wild type on culture media supplemented with mannitol and sodium chloride. Figure 1(a) The AtCCX2 mutant ccx2 (SALK_201874C) showed slightly shorter root length on 150 mM mannitol medium, but did not exhibit the salt and hypertonic sensitivity phenotype previously reported. This may be due to inconsistencies in the insertion position of the mutant T-DNA fragment used in the experiments and inconsistencies in the experimental conditions. Recent reports, in their supplementary data, indicate that ccx3-1 (Col-0) also did not show abnormal responses to high salt and hypertonic stress. In the experiments of this invention, only the AtCCX4 mutant ccx4-1 showed a sensitive phenotype to salt and hypertonic stress; on medium supplemented with 150 mM mannitol and 75 mM sodium chloride, ccx4-1 exhibited more severe growth defects than the wild type. Figure 1 (b). To this end, the inventors conducted functional studies on AtCCX4 in plant abiotic stress.

[0098] Example 2: AtCCX4 participates in plant response to hyperosmolar stress

[0099] 2.1 The AtCCX4 mutant exhibits defects in its response to high osmotic stress.

[0100] The AtCCX4 gene contains only one exon and is transcribed into a 644-amino acid membrane protein. Using the SignalP 5.0 signal peptide prediction tool, no signal peptide for the CCX4 protein has been identified. Based on the transmembrane domain prediction of the online biological software tool TMHMM 2.0, CCX4 may have 11-12 transmembrane domains, namely M1-M12 transmembrane domains. M6 has a lower expected value and may not constitute a transmembrane helix, therefore it is represented by a dashed line. The expected amount of transmembrane helices in the first 60 amino acids of the CCX4 protein is very high, and there is a certain probability that it will serve as a signal peptide for membrane protein targeting the endoplasmic reticulum. Between M1 and M2, and between M5 and M6, there are two large cytoplasmic loops of approximately 100 amino acids each, which may serve as key functional regions for intracellular regulation. Figure 2 a).

[0101] To verify the function of AtCCX4, the mutant ccx4-10 was constructed using CRISPR gene editing technology. The mutation types and locations of the ccx4-1 and ccx4-10 mutants are detailed below. Figure 2 b. ccx4-1 is a mutant with a C-terminal insertion in T-DNA, located between M10 and M11. The mutation type of ccx4-10 involves the insertion of a T base at the 635th base of the gene sequence, causing premature frameshift termination of the codon. Its gene sequencing peak diagram is shown below. Figure 2 c. Growth defects were found in ccx4-1 and ccx4-10 during the experiment, see [reference needed]. Figure 2 , d.

[0102] Compared to the wild type, the two AtCCX4 mutants exhibited more severe growth defects under hyperosmolar stress. On media supplemented with 75 mM, 100 mM, and 140 mM mannitol, the aerial parts of ccx4-1 and ccx4-10 were smaller than those of the wild type. Figure 3 (a, b). Since the ccx4 mutant itself grows smaller than the wild type, the inventors weighed the fresh weight of the aboveground parts and calculated the ratio of the fresh weight of the aboveground parts on hypertonic stress medium to that on 1 / 2 MS medium, i.e., the relative fresh weight of the plant on different media, to characterize the growth inhibition of the aboveground parts of the plant in media supplemented with different concentrations of mannitol. Among them, the aboveground growth inhibition of ccx4-1 and ccx4-10 showed a significant difference at 75 mM mannitol (a, b). Figure 3 c).

[0103] Osmotic regulation is an important form of plant response to osmotic stress. After treating 10-day-old Arabidopsis seedlings with high-salt and high-osmotic media for two days, the osmotic pressure of the seedling tissue sap increased two to three times. The ccx4-1 and ccx4-10 mutants showed higher osmotic pressures after treatment than the wild type, with salt treatment showing a significant difference, while high-osmotic treatment did not show a significant difference. This may be related to the intensity and duration of the high-osmotic treatment. Figure 3 ,d).

[0104] In addition to studying the growth phenotype and osmotic potential changes of the CCX4 mutants under hyperosmotic treatment, the water loss rate and stomatal closure induced by hyperosmotic treatment were also tested. In the aboveground water loss experiment, both the CCX4-1 and CCX4-10 mutants exhibited a faster water loss phenotype than the wild type. Hyperosmotic treatment can induce stomatal closure in Arabidopsis thaliana; however, in CCX4-1 and CCX4-10, the hyperosmotic-induced stomatal closure was defective. Compared with the significant reduction in stomatal aperture in the wild type, the reduction in stomatal aperture in the two CCX4 mutants was not significant. Figure 4 This indicates that after CCX4 function is lost, stomata are unable to close in response to hypertonic stress in order to reduce water loss.

[0105] Example 3: Ion disorder in AtCCX4 mutants

[0106] CCX4 is a cation / Ca 2+ Exchange pumps, involved in plant cell Na+ exchange + K + Ca 2+The absorption and redistribution of isocations. Previous reports indicated that the CCX4 mutant aod6(Bu-5) showed significantly increased calcium accumulation in the aerial parts compared to the wild type. The ccx1-1 / ccx4 mutant was sensitive to calcium starvation in the culture medium; however, single mutants of ccx4-1 and ccx4-10 did not show more pronounced growth defects in response to calcium starvation. The CCX4 mutant aod6(Bu-5) exhibited a sensitive phenotype to the addition of the calcium chelator EGTA and high concentrations of calcium ions in the culture medium, suggesting that CCX4 is closely related to calcium homeostasis in plants. Loss of CCX4 function may lead to calcium dysregulation in plants, potentially affecting calcium nutrition and signaling.

[0107] 3.1 Mutations in AtCCX4 lead to plant sensitivity to high concentrations of sodium, potassium, and calcium ions.

[0108] Consistent with previous experimental results, ccx4-1 and ccx4-10 are highly sensitive to high concentrations of NaCl. Furthermore, both were found to be highly sensitive to high concentrations of KCl and CaCl2. Unlike the severe inhibition of wild-type growth by high concentrations of sodium salts, the same concentrations of potassium and calcium salts did not inhibit the growth of wild-type Arabidopsis thaliana as severely as sodium salts. However, with the loss of AtCCX4 function, the plant exhibited more severe growth defects in response to high concentrations of sodium, potassium, and calcium salts than the wild-type, indicating that AtCCX4 plays a crucial role in the regulation of ion homeostasis within plants; the specific regulatory mechanisms require further investigation.

[0109] 3.2 Enhanced calcium signaling induced by abiotic stress in the ccx4-1 mutant

[0110] Previously, it was discovered that the AtCCX4 knockout mutant is highly sensitive to high concentrations of calcium ions, and the calcium ion concentration in the mutant accumulates and increases in the aboveground tissues of plants, suggesting that AtCCX4 may mediate the excretion of calcium ions from plant cells. Since the ccx4 mutant exhibits abnormal accumulation and sensitivity to calcium ions, whether its calcium signaling is impaired is also a matter of great concern. To address this, the inventors introduced calcium ion biological probes, jellyfish fluorescent protein (AEQ), GCaMP3, and YC3.6, into the ccx4-1 mutant via hybridization, obtaining materials with both homologous and background.

[0111] Currently, the abiotic stress-induced calcium signaling in the ccx4-1 mutant was investigated using the jellyfish fluorescent protein (AEQ) probe. It was found that intracytoplasmic calcium signals induced by 0.8 M mannitol, 10 mM hydrogen peroxide, and 300 mM sodium chloride were all higher than those induced by the wild type. Figure 6(a, c, e). The peak value of calcium signal for each plant was statistically analyzed. Similarly, in the wild type, the peak values ​​of the increase in intracytoplasmic calcium ion concentration induced by hyperosmolar stress, oxidative stress, and high salt stress were all lower than those of ccx4-1. This indicates that AtCCX4 affects plant growth not only at the ion nutrition level but also at the calcium signaling level, influencing the plant's signal response to external stress. Our laboratory will continue to further observe calcium signaling in mutants using ccx4-1 / GCaMP3 and ccx4-1YC3.6 plant materials.

[0112] Example 4: OSMO2 and CPK can phosphorylate CCX4 in vitro.

[0113] AtCCX4 participates in the regulation and coordination of intracellular cations in plant cells, playing a crucial role in plant growth and responses to hyperosmolar and high-salt stress. So how is AtCCX4 regulated? Phosphorylation is one of the known mechanisms for regulating ion transporters. CCX4 consists of 644 amino acids and is predicted to be a 12-transmembrane protein. After several attempts to express and purify the full-length soluble CCX4 protein, truncated versions of the non-transmembrane regions of CCX4 were designed: CCX41 (1-24 aa), CCX42 (35-130 aa), CCX43 (155-208 aa), CCX44 (294-442 aa), and CCX45 (521-549 aa). These were then fused with MBP and HIS protein tags for expression. Numerous studies have shown that protein kinases such as RAF kinase, OST1 kinase, receptor-like kinase OSMO2, and CPK kinase play important roles in plant responses to salt and osmotic stress. Using a mixture of five protein fragments of CCX4 fused with MBP as substrates, kinase screening experiments were conducted using in vitro phosphorylation assays. It was found that receptor-like kinase OSMO2 and kinases CPK3 / 6 / 11 could phosphorylate CCX4 in vitro. Mass spectrometry analysis of the phosphorylated protein products revealed several potential phosphorylation sites: Tyr8, Ser47, Ser60, Thr63, Thr396, and Thr399. These amino acids were mutated to alanine, and the proteins were purified for in vitro phosphorylation testing. In in vitro phosphorylation experiments, it was found that receptor-like kinase OSMO2 and kinases CPK3 / 6 / 11 could phosphorylate the MBP-CCX42 (35-130 aa) protein segment. Figure 7(b) The Ser47 mutation to Ala severely affected the phosphorylation of the MBP-CCX42 (35-130 aa) protein fragment. However, the CCX4 protein fragment fused with the MBP tag showed obvious heterogeneous bands. To further optimize the experimental results, the CCX4 protein fragment was fused with the HIS protein tag and expressed, and in vitro phosphorylation experiments were performed again. The results showed that OSMO2 can indeed only phosphorylate the second protein fragment of CCX4, CCX42 (35-130 aa), with serine at position 47 being the key phosphorylation site. In addition, the mutation at Ser60 (mutation to Ala) can also affect the phosphorylation of CCX42 (35-130 aa). Figure 7 (a) and (b) Recombining serine at position 47 with alanine and cysteine ​​to mimic the unphosphorylated state of serine, or recombining serine at position 47 with aspartic acid and glutamic acid to mimic the phosphorylated state of serine, significantly reduced the phosphorylation of CCX42 (35-130aa). The remaining phosphorylation may be related to site Ser60. The protein with double mutations of S47 and S60 will be purified for verification in the future.

[0114] 4.1 The Ser47 phosphorylation site plays a crucial role in plant growth.

[0115] In the aforementioned experiments, in vitro phosphorylation of CCX4 by receptor-like kinases OSMO2 and calcium-dependent protein kinases CPK3 / 6 / 11 was discovered, and two key phosphorylation sites, Ser47 and Ser60, were identified. To verify whether CCX4 phosphorylation plays a role in plants, transgenic materials with complemented ccx4-1 mutants driven by the CCX4 self-promoter were constructed: wild-type CCX4 fused with the MYC protein tag, WT-MYC; wild-type CCX4 with a 3'UTR, WT-3'UTR; and Ser47 point-protrusion CCX4 fused with the MYC protein tag, S47A-MYC and S47E-MYC. In subsequent identification of transgenic materials, it was found that the protein expression level of CCX4 could not be identified by Western blot using antibodies against the MYC protein tag. After confirming the background of the ccx4-1 mutant and successfully inserting the gene fragment into the mutant genome using PCR, wild-type WT-MYC#1 and WT-3'UTR, as well as four Ser47 mutant complement materials S47A-MYC#2 / 3 / 6 / 8, were identified using semi-quantitative PCR and quantitative real-time PCR. The quantitative real-time and semi-quantitative real-time PCR results for the transgenic materials are as follows: Figure 5 (a, b). Similar to the transgenic complement material with the 3'UTR region, CCX4 with the MYC tag was found to compensate for the dwarf phenotype of ccx4-1 in soil. However, the mutation of serine at position 47 to alanine failed to compensate for the growth defect of ccx4-1. Figure 8This indicates that phosphorylation of CCX4 is very important for the normal growth of plants.

[0116] 4.2 The effect of Ser47 phosphorylation site on plant response to hyperosmolar stress

[0117] Through in vitro phosphorylation experiments, the inventors realized that Ser47 is a key phosphorylation site, while the later-discovered Ser60 may also be another influential phosphorylation site. Since the full-length CCX4 protein is difficult to express, the inventors used a truncated protein expression method to purify the truncated form for in vitro phosphorylation experiments. Furthermore, because the full-length CCX4 gene sequence cannot be constructed into vectors containing other larger proteins for fusion expression, including n / cLUC, n / cYFP, AD, and BD, results of interactions between CCX4 and OSMO2 and CPK proteins have not yet been obtained. In other words, this phosphorylation may not actually occur and have regulatory significance in plants. To investigate whether CCX4 protein phosphorylation has a biological function, wild-type and S47A mutant complementation materials were used to examine whether the Ser47 mutation affects the function of CCX4 in plants under hyperosmolar stress. Experimental results showed that the wild-type CCX4 WT-MYC#1 could compensate for the growth defect of the ccx4-1 mutant in hyperosmolar stress medium, while S47A-MYC#2 / 8 could not compensate for the growth defect phenotype. Similarly, the water loss rate of S47A-MYC#2 / 8 was close to that of ccx4-1, while the water loss rate of WT-MYC#1 decreased after being compensated for in ccx4-1, and was even slightly lower than that of the wild type. Figure 9 These results indicate that the Ser47 phosphorylation site also plays an indispensable role in the plant's response to high osmotic stress.

[0118] 4.3 The Ser47 phosphorylation site plays a key role in the response to ion stress.

[0119] Phosphorylation of CCX4 affects plant growth and response to hyperosmolar stress. To investigate the effect of phosphorylation of serine at position 47 of AtCCX4 on plant salt stress, Arabidopsis seedlings were grown for 10 days on 1 / 2 MS medium supplemented with 100 mM NaCl, 50 mM KCl, and 50 mM CaCl2. Similar to the ccx4-1 mutant, the three parallel lines of S47A-MYC reintroduced ccx4-1 were highly sensitive to high concentrations of sodium, potassium, and calcium salts. However, the WT-MYC#1 and WT-3'UTR transgenic materials were able to compensate for the sensitivity of ccx4-1 to high concentrations of sodium, potassium, and calcium salts. Figure 10 ).

[0120] Example 5: Ion transport characteristics of AtCCX4

[0121] AtCCX4 is a potential cation / calcium ion exchange pump responsible for the regulation of cations in plant cells. In yeast strains AXT3 and wx1, it has the function of transporting Na+. + and K + However, the transport direction of ions remains unclear. Jay et al. pointed out in their article that AtCCX3 and AtCCX4 do not possess the ability to transport Ca... 2+ To explore the calcium ion transport properties of AtCCX4, the inventors, referring to previous research, expressed CCX4 and its mutant proteins in calcium-sensitive yeast strains K616, K667, and csg2 with different characteristics, and observed the calcium ion transport capacity of CCX4 and the effect of phosphorylation on the calcium ion transport capacity of CCX4.

[0122] 5.1 Ion transport characteristics of AtCCX4 in csg2 yeast strain

[0123] Csg2 is a calcium ion channel located in the endoplasmic reticulum (ER) that regulates ER calcium homeostasis in the yeast *Saccharomyces cerevisiae*. Csg2 is a highly calcium-sensitive yeast mutant (>30 mmol / L), exhibiting increased intracellular calcium accumulation. To test whether AtCCX4 can inhibit calcium accumulation in the Csg2 mutant... 2+ Sensitivity was assessed in csg2 yeast for the expression of CCX4 and its mutants. All transformants of csg2 were found to grow normally on SC-Ura culture dishes with galactose as the carbon source. However, after the addition of 100 and 200 mM CaCl2, pYES2-CCX4 and pYES2-CCX4 mutants showed abnormal growth. S47A pYESS-CCX4Δ47 can compensate for the high calcium sensitivity phenotype of csg2 to some extent, however pYES2-CCX4 S47E pYES2-CCX4 S47 / 60A pYES2-CAX1 and pYES2-sCAX1 cannot ( Figure 11 (a) This indicates that AtCCX4 possesses ion transport activity in yeast csg2, and Ser47 and Ser60 may play a key role in the ion transport function of AtCCX4. Unlike H on the vacuolar membrane... + / Ca 2+The function of the antitransporter CAX1 in yeast K616 and K667 was investigated, but CAX1 failed to eliminate the toxicity of high concentrations of calcium ions to yeast csg2. Using the p416 vector, the complementation of the high-calc-sensitive phenotype of csg2 by AtCCX4 was also observed; all three site mutations expressing CCX4 and Ser47 could compensate for the growth defects of csg2 in YPDA media supplemented with 50 mM, 75 mM, and 150 mM CaCl2. In summary, the inventors have demonstrated that AtCCX4 possesses the ability to transport calcium ions, but the effects of its phosphorylation sites Ser47 and Ser60 on calcium ion transport activity still require further detailed experimental verification. Additionally, to test whether the driving force of AtCCX4's calcium ion transport depends on Na+... + and K + Based on the concentration gradient, the inventors plan to add different concentrations of Na to this experiment. + and K + Observe whether there are any changes in the transport capacity of calcium ions.

[0124] Sequence of this article:

[0125] The full-length nucleotide sequence (CDS) of SEQ ID NO:1-CCX4:

[0126]

[0127]

[0128] The nucleotide sequence of the SEQ ID NO:2-ccx4-10 after mutation:

[0129]

[0130] The full-length amino acid sequence of SEQ ID NO:3-CCX4:

[0131]

[0132] The nucleotide sequence of the SEQ ID NO:14-CCX4-1 mutation:

[0133]

[0134]

Claims

1. A polynucleotide, said polynucleotide (1) Insert a base into the coding sequence of the wild-type CCX4 protein as shown in SEQ ID NO:1, or The complementary sequences of (2)(1), Preferably, the base insertion position is before position 635 of the CCX4 gene sequence. More preferably, the inserted base is T.

2. A method for regulating plant responses to hyperosmolar stress, salt stress, or ion stress, the method comprising: (1) Upregulating the expression or activity of CCX4 protein in plants, thereby promoting plant growth and development and improving the plant's tolerance to hyperosmolar stress, salt stress, or ion stress; or (2) Downregulating the expression or activity of CCX4 protein in plants, thereby increasing the plant's sensitivity to cations or increasing the cation concentration in plant cells. Preferably, The upregulation of CCX4 protein expression in plants includes: transferring the coding sequence of the CCX4 protein into plants to obtain transformed plants. The downregulation of CCX4 protein expression or activity in plants includes: (a) specifically interfering with CCX4 gene transcription and / or expression, (b) downregulating CCX4 protein activity, or (c) expressing CCX4 protein with reduced activity in plants. More preferably, The specific interference described in (a) refers to interference with the transcription of the CCX4 gene or the translation of its transcripts. (b) describes downregulating the expression of the CCX4 protein in plants by: transferring nucleic acids that specifically interfere with the transcription and / or expression of the CCX4 gene into plants to obtain transformed plants. (c) The CCX4 protein with reduced activity includes CCX4 protein with a mutated phosphorylation site; preferably, the phosphorylation site includes one or more selected from the following: Tyr8, Ser47, Ser60, Thr63, Thr396 and Thr399; more preferably, Ser47 and Ser60; and even more preferably, the phosphorylation site mutation is selected from any one of alanine, cysteine, aspartic acid and glutamic acid.

3. The method as described in claim 2, characterized in that, 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.

4. The method as described in claim 2, characterized in that, (a) Includes gene editing or RNA interference of the CCX4 gene to reduce the transcription and / or expression of the gene, preferably, the gene editing includes gene editing via T-DNA insertion or gene editing using CRISPR technology. The nucleic acids that specifically interfere with the transcription and / or expression of the CCX4 gene as described in (b) are selected from the group consisting of (i) antisense nucleic acids, microRNAs, siRNAs, RNAi, dsRNAs, sgRNAs or combinations thereof, and (ii) nucleic acid constructs that can express or form (i).

5. The method as described in claim 2, characterized in that, The downregulation of CCX4 protein activity described in (b) includes, in plants: (i) expressing a specific antibody or ligand (e.g., an inhibitory antibody) that can downregulate CCX4 protein activity; (ii) introducing a nucleic acid sequence encoding (i) and / or a nucleic acid construct that can express (i); (iii) mutating an amino acid at a phosphorylation site of the CCX4 protein; and (iv) downregulating the expression or activity of a phosphorylase that phosphorylates the CCX4 protein. Preferably, the phosphorylation site includes one or more selected from: Tyr8, Ser47, Ser60, Thr63, Thr396, and Thr399; more preferably, Ser47 and Ser60; more preferably, the phosphorylation site is mutated to any one selected from alanine, cysteine, aspartic acid, and glutamic acid. Preferably, the phosphorylase is OSMO2 and CPK; more preferably, the CPK is CPK3, CPK6 or CPK11.

6. The method as described in claim 2, characterized in that, It also has one or more of the following characteristics: The plant is a cruciferous plant, preferably a Arabidopsis thaliana plant, and more preferably, Arabidopsis thaliana. The plant cell cations are selected from sodium, potassium, calcium ions, etc.

7. The uses of substances that regulate the expression or activity of CCX4 protein in plants in regulating plant growth and development, plant tolerance to hyperosmolar stress, salt stress, or ionic stress, regulating plant sensitivity to cations, or regulating cation concentration in plant cells. Preferably, the substance is a promoter of CCX4 protein expression or activity, and the regulation is to upregulate CCX4 protein expression or activity in plants, thereby promoting plant growth and improving the plant's tolerance to hyperosmolar stress, salt stress, or ion stress; more preferably, the promoter is CCX4 protein or its coding sequence; or Preferably, the substance is an inhibitor of CCX4 protein expression or activity, and the regulation is to downregulate CCX4 protein expression or activity in plants, thereby increasing the plant's sensitivity to cations or increasing the cation concentration in plant cells. More preferably, the inhibitor is selected from: (1) an inhibitor that specifically interferes with CCX4 gene transcription and / or expression, (2) an inhibitor that downregulates CCX4 protein activity, or (3) a CCX4 protein variant or its coding sequence that downregulates activity.

8. The use as described in claim 7, characterized in that, (1) The inhibitor is selected from the group consisting of (i) antisense nucleic acids, microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, or combinations thereof, and (ii) nucleic acid constructs that can express or form (i). (2) The inhibitor is selected from the group consisting of (i) specific antibodies or ligands of the CCX4 protein, and (ii) nucleic acid sequences encoding (i) and / or nucleic acid constructs capable of expressing (i). Preferably, (1) the inhibitor is a nucleic acid that specifically interferes with the transcription and / or expression of the CCX4 gene.

9. The use of a CCX4 gene as a molecular marker for identifying plant growth, tolerance to hyperosmolar stress, salt stress, or ionic stress, plant sensitivity to cations, or the concentration of cations within plant cells. Preferably, the method includes the following steps: comparing the expression or activity of the CCX4 gene in plants with that in wild-type plants; if the expression or activity of the CCX4 gene is upregulated, the plants grow taller and are more tolerant to hyperosmolar stress, salt stress, or ion stress; if the expression or activity of the CCX4 gene is downregulated, the plants are more sensitive to cations or have higher intracellular cation concentrations.

10. A method for attenuating the high calcium sensitivity phenotype of a yeast strain mutant, said method comprising overexpressing CCX protein or a mutant protein thereof in a yeast strain mutant. Preferably, the CCX protein is the CCX4 protein, and the amino acid sequence of the CCX4 protein is shown in SEQ ID NO:

3. More preferably, the mutant protein includes a mutation at amino acid position 47 and / or amino acid position 60 of the CCX4 protein.

11. The method as described in claim 10, characterized in that, (1) The 47th amino acid mutation is A or E, or the 47th amino acid mutation is a deletion mutation, or (2) The 47th amino acid is mutated to A or E, and the 60th amino acid is mutated to A. Preferably, the mutant protein is selected from CCX4. S47A CCX4 Δ47 CCX4 S47E Or CCX4 S47 / 60A , Preferably, the yeast strain mutant is csg2. Preferably, the high calcium-sensitive phenotype refers to a yeast strain with a calcium ion concentration >50 mmol / L.

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