CPK phosphorylation activates snrk2 kinases to enhance drought stress tolerance

Abstract

The present application provides that CPK phosphorylation activates SnRK2 kinase to enhance drought stress tolerance. The present application provides a method for improving the drought resistance of a plant or a method for preparing a plant with increased drought resistance, comprising: up-regulating the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 in the plant.

Description

Technical Field

[0001] This invention belongs to the fields of botany and genetic engineering. More specifically, this invention relates to CPK phosphorylation activating SnRK2 kinase to enhance drought stress tolerance. Background Technology

[0002] To survive in conditions such as drought and high salinity, terrestrial plants have evolved complex osmotic sensing and signal transduction mechanisms to appropriately activate abiotic stress responses. Despite extensive research, most knowledge about osmotic regulation is limited to the inventors' understanding of the stress hormone abscisic acid (ABA). Hyperosmotic stress triggers ABA accumulation within hours. In Arabidopsis, ABA binds to the receptor PYL protein, subsequently interacting with A-branch 2C protein phosphatase (PP2C-A), thereby releasing SnRK2 kinase. In Arabidopsis and *Bryum spp.*, after SnRK2.2 / 2.3 / 2.6 is released, it is subsequently activated by persistently active B2-type RAF protein kinases (RAF) and stress-activated B3 RAF phosphorylation. Although ABA signaling is crucial in these stress responses, it remains unclear whether other important positive regulators, besides RAF, exist upstream of SnRK2 in the early stages of osmotic stress.

[0003] The core osmotic signaling elements RAF, PP2C, and SnRK2 are conserved in green algae. After plants colonized the earth, they evolved the ABA receptor PYL. ABA signaling utilizes more conserved osmotic signaling elements by hijacking PP2C. In angiosperms, SnRK2 is divided into three subfamilies. SnRK2.1 / 4 / 5 / 9 / 10 belong to subfamily I; they respond to osmotic stress but not to ABA and are activated by the B4 RAF, which is activated by osmotic stress. SnRK2.2 / 2.3 / 2., which are activated by ABA signaling, belong to subfamily III and are activated by the B2 and B3 RAFs. The B2, B3, and B4 RAFs work together in the activation of SnRK2 and subsequent osmotic regulation. The decaplex mutants snrk2.1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 lacking all SnRK2, and the heptplex mutants raf16 / 18 / 20 / 24 / 35 / 40 / 42 lacking all B4 RAF, are hypersensitive to osmotic stress. The triplet mutants snrk2.2 / 3 / 6 lacking all subfamily III SnRK2, and the nine-fold mutants raf3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 lacking nine B2 and B3 RAF, are insensitive to ABA. However, research in *Symplocos spp.* has shown that osmotic regulation also involves unknown ABA- and RAF-independent SnRK2 function, prompting the inventors to further explore early osmotic stress signals beyond the RAF-SnRK2 kinase cascade.

[0004] Osmotic stress triggers cytoplasmic calcium2+ A transient increase in levels and rapid activation of RAF and SnRK2 protein kinases. OSCA1 mediates Ca under osmotic stress. 2+ Signal generation and stomatal closure. The plasma membrane-localized phospholipid-binding protein BONZAI (BON) regulates calcium metabolism under osmotic stress. 2+ Signal and downstream osmotic stress response. Ca 2+ The signal is composed of different Ca 2+ This involves protein decoding, including calcium-dependent protein kinases (CPKs, also known as CDPKs). The deactivation of CPK enzyme activity depends on its EF-hand domain interacting with Ca2+. 2+ The combination of CPK and SnRK2. CPK is a key regulator of ABA and abiotic stress signaling, and it co-phosphorylates and modifies multiple downstream substrates to mediate stress responses. CPK3 / 6 / 21 / 23 promotes stomatal closure by activating SLAC1 and its homolog SLAH3. Interestingly, Ca 2+ Induced stomatal closure requires subfamily III SnRK2, suggesting a positive regulation of SnRK2 by CPK. However, it remains unclear which CPKs are involved in Ca2+ osmotic stress. 2+ The decoding of the signal, and whether CPK transmits the signal to SnRK2. Summary of the Invention

[0005] The purpose of this invention is to provide CPK phosphorylation to activate SnRK2 kinase to enhance drought stress tolerance.

[0006] In a first aspect, the present invention provides a method for improving the drought resistance of plants or a method for preparing plants with increased drought resistance, comprising: upregulating the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 in plants.

[0007] In one or more embodiments, upregulating the expression or activity of CPK3, CPK4, CPK6, CPK11, and / or CPK27 in plants includes: introducing the encoding genes of CPK3, CPK4, CPK6, CPK11, and / or CPK27, or expression constructs or vectors containing such encoding genes, into plants; performing gain-of-function mutations on CPK3, CPK4, CPK6, CPK11, and / or CPK27; promoting the expression of CPK3, CPK4, CPK6, CPK11, and / or CPK27 by expressing an enhancing promoter or a tissue-specific promoter; or promoting the expression of CPK3, CPK4, CPK6, CPK11, and / or CPK27 by an enhancer.

[0008] In one or more embodiments, the method for preparing plants with increased drought resistance further includes: hybridizing transgenic plants whose expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 has been regulated with plants that have not been introduced with peptides or their encoding genes of CPK3, CPK4, CPK6, CPK11 and / or CPK27 to obtain hybrid offspring.

[0009] A second aspect of the present invention provides the use of CPK3, CPK4, CPK6, CPK11 and / or CPK27, or upregulators thereof, for upregulating the drought resistance of plants or for preparing plants with increased drought resistance.

[0010] In one or more embodiments, the regulator is an upregulator of CPK3, CPK4, CPK6, CPK11 and / or CPK27, which increases the drought resistance of plants. The upregulator includes: exogenous genes encoding CPK3, CPK4, CPK6, CPK11 and / or CPK27 or expression constructs or vectors containing such genes; preferably, the expression construct includes an enhancing promoter, a tissue-specific promoter or an enhancer; or, a reagent for gain-of-function point mutation of CPK3, CPK4, CPK6, CPK11 and / or CPK27.

[0011] In one or more embodiments, the plant is a cruciferous plant, a grass, or a solanaceous plant.

[0012] In one or more embodiments, the cruciferous plants include: Arabidopsis thaliana, rapeseed, and Chinese cabbage; the gramineous plants include: rice, wheat, corn, barley, oats, and rye; and the solanaceous plants include: tobacco, tomato, and pepper.

[0013] In one or more embodiments, the plant is Arabidopsis thaliana.

[0014] In one or more embodiments, the amino acid sequence of the CPK3 protein is selected from the group consisting of:

[0015] (1A) A protein having the amino acid sequence shown in SEQ ID NO:1;

[0016] (1B) A protein derived from (1A) having the function described in (1A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1.

[0017] (1C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:1, and having the function described in (1A);

[0018] (1D) The active fragment of the protein with the amino acid sequence shown in SEQ ID NO:1; or,

[0019] (1E) A protein formed by adding a tag sequence or restriction site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:1, or by adding a signal peptide sequence to its N end.

[0020] In one or more embodiments, the amino acid sequence of the CPK4 protein is selected from the group consisting of:

[0021] (2A) A protein having the amino acid sequence shown in SEQ ID NO:2;

[0022] (2B) A protein derived from (2A) having the function described in (2A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2;

[0023] (2C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:2, and having the function described in (2A);

[0024] (2D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:2; or,

[0025] (2E) A protein formed by adding a tag sequence or restriction site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

[0026] In one or more embodiments, the amino acid sequence of the CPK6 protein is selected from the group consisting of:

[0027] (3A) A protein having the amino acid sequence shown in SEQ ID NO:3;

[0028] (3B) A protein derived from (3A) having the function described in (3A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:3;

[0029] (3C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:3, and having the function described in (3A);

[0030] (3D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:3; or,

[0031] (3E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:3, or by adding a signal peptide sequence to its N end.

[0032] In one or more embodiments, the amino acid sequence of the CPK11 protein is selected from the group consisting of:

[0033] (4A) A protein having the amino acid sequence shown in SEQ ID NO:4;

[0034] (4B) A protein derived from (4A) having the function described in (4A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:4;

[0035] (4C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:4, and having the function described in (4A);

[0036] (4D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:4; or,

[0037] (4E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:4, or by adding a signal peptide sequence to its N end.

[0038] In one or more embodiments, the amino acid sequence of the CPK27 protein is selected from the group consisting of:

[0039] (5A) A protein having the amino acid sequence shown in SEQ ID NO:5;

[0040] (5B) A protein derived from (5A) having the function described in (5A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:5.

[0041] (5C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:5, and having the function described in (5A);

[0042] (5D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:5; or,

[0043] (5E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:5, or by adding a signal peptide sequence to its N end.

[0044] In one or more embodiments, the improvement of plant drought resistance further promotes the optimization of traits in plants including: the ability of plants to grow in drought conditions, or the biomass of plants in drought conditions.

[0045] In a third aspect, the present invention provides a plant cell, tissue, or organ containing an upregulator of exogenous CPK3, CPK4, CPK6, CPK11, and / or CPK27; wherein the upregulator of CPK3, CPK4, CPK6, CPK11, and / or CPK27 comprises: an exogenous gene encoding CPK3, CPK4, CPK6, CPK11, and / or CPK27 or an expression construct or vector containing the gene encoding the gene; preferably, the expression construct comprises an enhancing promoter, a tissue-specific promoter, or an enhancer; or, a reagent for gain-of-function point mutation of CPK3, CPK4, CPK6, CPK11, and / or CPK27.

[0046] In a fourth aspect, the present invention provides the use of plant CPK3, CPK4, CPK6, CPK11 and / or CPK27 as molecular markers for identifying plant drought resistance or for targeted screening of molecular markers for plant drought resistance.

[0047] A fifth aspect of the present invention provides a method for selecting or identifying the drought resistance of plants, the method comprising: identifying the expression or sequence characteristics of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes in a test plant; if the test plant has high expression or high activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes, then it is a plant with high drought resistance; if the test plant has low expression or low activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes, then it is a plant with low drought resistance.

[0048] A sixth aspect of the present invention provides a method for screening substances (including potential substances) that regulate plant drought resistance, comprising:

[0049] (1) Add the candidate substance to a system expressing CPK3, CPK4, CPK6, CPK11 and / or CPK27;

[0050] (2) Detect the system and observe the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27. If the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 is increased, it indicates that the candidate substance can be used to increase the drought resistance of plants; if the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 is decreased, it indicates that the candidate substance can be used to reduce the drought resistance of plants.

[0051] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0052] Figure 1 Dehydration-induced activation of CPK3 / 4 / 6 / 11 / 27.

[0053] Based on protein immunoprecipitation and 32 The in vitro phosphorylation assay of P-labeled ATP was used to detect the kinase activity of CPK in plants before and 30 minutes after dehydration treatment. Endogenous plant CPK proteins were purified by protein immunoprecipitation from transgenic positive plants of 35Spro::CPK3-MYC, 35Spro::CPK4-MYC, 35Spro::CPK11-MYC, 35Spro::CPK27-MYC, and 35Spro::CPK32-MYC. Western blotting based on anti-MYC antibody was used to detect the abundance of CPK3, CPK4, CPK11, CPK27, and CPK32 proteins in plants before and 30 minutes after dehydration treatment. 32 The in vitro phosphorylation assay of P-labeled ATP represented the kinase activity of CPK by detecting the autophosphorylation capacity of CPK in plants before and 30 minutes after dehydration treatment. The same batch of CPK protein was used for both Western blotting and in vitro phosphorylation.

[0054] Figure 2 CPK phosphorylates serine at position 175 of SnRK2.6 and directly activates SnRK2.6.

[0055] (A) CPK3 / 4 / 6 / 11 phosphorylation modification of SnRK serine position 175. The phosphorylation modification ability of CPK3 / 4 / 6 / 11 on SnRK2.6 serine position 175 was detected using a protein immunoblotting assay based on an antibody based on SnRK2.6 serine phosphorylation. Results from both anti-pS175 antibody and anti-GST antibody immunoblotting experiments were obtained from the same PVDF membrane. (B) CPK3 / 4 / 6 activation of SnRK2.6. Dephosphorylated HIS-SnRK2.6 (de-HIS-SnRK2.6) recombinant protein was purified from the BL21 strain co-expressing HIS-SnRK2.6 and GST-ABI1. AKS1 fragment protein was used as a substrate for SnRK2.6. 32 The radioactive signal of P and the results of Coomassie brilliant blue staining indicate the loading amount and phosphorylation modification level of each protein.

[0056] Figure 3 CPK is involved in dehydration-induced activation of SnRK2.6.

[0057] (A) Dehydration-induced SnRK2.6 activation deficiency in the cpk3 / 4 / 6 / 11 / 27 mutant. Protein immunoblotting based on an antibody phosphorylating at SnRK2.6 serine position 175 and... 32 Intracolloid kinase activity assay of P: Phosphorylation modification level of serine at corresponding sites of SnRK2 in Col-0, cpk3 / 4 / 6 / 11 / 27 and kinase activity (middle figure) at dehydration treatment for 0 / 15 / 30 / 60 / 120 / 240 minutes. The results of protein immunoblotting experiments based on anti-pS175 antibody and anti-actin antibody were obtained from the same PVDF membrane. All experiments used the same batch of test samples. (B) Mannitol-induced hyperactivation of SnRK2.6 in CPK overexpression transgenic positive lines (35Spro::CPK3-MYC, 35Spro::CPK4-MYC, 35Spro::CPK11-MYC, 35Spro::CPK27-MYC, 35Spro::CPK32-MYC). Protein immunoblotting experiments based on phosphorylation antibody at SnRK2.6 serine position 175 and based on... 32 The intracolloidal kinase activity assay for P detected the phosphorylation level (top figure) and kinase activity (middle figure) of serine residues at corresponding sites of SnRK2 in Col-0 and CPK overexpression lines after treatment with 0.6M mannitol for 0 / 30 minutes. Western blot results based on anti-pS175 antibody and anti-Actin antibody were obtained from the same PVDF membrane. All experiments used the same batch of test samples.

[0058] Figure 4The osmotic stress phenotype defect of the .cpk3 / 4 / 6 / 11 / 27 quintuplet mutant.

[0059] (A and B) Five-day-old acclimatized WT (Col-0) and cpk3 / 4 / 6 / 11 / 27 mutant seedlings were transferred to 1 / 2 MS medium with or without 150 mM mannitol for further growth (A). Acclimatized seedlings were those that germinated and grew on 1 / 2 MS medium containing 140 mM mannitol. The statistical values ​​of seedling fresh weight (B) are mean ± SD (n = 30 seedlings). (C) Growth of WT and CPK overexpression lines (35Spro::CPK3-MYC, 35Spro::CPK4-MYC, 35Spro::CPK11-MYC, 35Spro::CPK27-MYC, 35Spro::CPK32-MYC) before drought (i.e., control), approximately 3 weeks after drought (Drought), and 3 days after rewatering (Rewatered). Detailed Implementation

[0060] Through in-depth research, the inventors discovered that calcium-dependent protein kinases CPK3, 4, 6, 11, and 27 are activated by dehydration treatment and directly activate SnRK2 kinase through phosphorylation modification. Dehydration-induced SnRK2 activation is significantly defective in the cpk3 / 4 / 6 / 11 / 27 quintuple mutant, and dehydration-induced SnRK2 activation is enhanced in CPK overexpression lines. Phenotypic analysis revealed that the drought tolerance of CPK overexpression lines was significantly improved. This discovery elucidates the role of calcium-dependent protein kinases (CPK3, 4, 6, 11, and 27). 2+ The study investigated the key roles of CPK3 / 4 / 6 / 11 / 27 protein kinases in dehydration-induced activation of SnRK2s and revealed the important role of CPK-mediated dehydration signaling response in drought tolerance in plants.

[0061] CPK and Plants

[0062] As used herein, "plant" includes dicotyledonous or monocotyledonous plants. The term "plant" includes: Brassicaceae, Poaceae, Solanaceae, Euphorbiaceae, etc. For example, it may include, but is not limited to: Arabidopsis thaliana, rice, wheat, corn, soybean, etc. In a preferred embodiment, the plant is Arabidopsis thaliana.

[0063] As used herein, the term "target gene" refers to a gene of interest in the plant genome that requires overexpression. In this invention, it refers to CPK genes, such as CPK3, CPK4, CPK6, CPK11, and CPK27.

[0064] The CPK3, CPK4, CPK6, CPK11, and CPK27 proteins described in this invention also include fragments, derivatives, homologs, and analogs of the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins. As used herein, the terms "fragment," "derivative," and "analyte" refer to proteins that substantially retain the same biological function or activity as the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins of this invention. The protein fragments, derivatives, or analogs of this invention may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing additional amino acid sequences into the protein sequence, etc. These fragments, derivatives, and analogs, as defined herein, are within the scope well known to those skilled in the art. As used herein, “homology” includes homologous polypeptides (proteins) or genes of CPK3, CPK4, CPK6, CPK11, and CPK27 in multiple species.

[0065] Any bioactive fragment of CPK3, CPK4, CPK6, CPK11, or CPK27 protein can be used in this invention. Here, "bioactive fragment of CPK3, CPK4, CPK6, CPK11, or CPK27 protein" means a protein that retains all or part of the function of the full-length CPK3, CPK4, CPK6, CPK11, or CPK27 protein. Typically, the bioactive fragment retains at least 50% of the activity of the full-length CPK3, CPK4, CPK6, CPK11, or CPK27 protein. Under more preferred conditions, the bioactive fragment can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length CPK3, CPK4, CPK6, CPK11, or CPK27 protein.

[0066] In this invention, the term "CPK3 protein" refers to the protein with the SEQ ID NO:1 sequence having CPK3 protein activity, the term "CPK4 protein" refers to the protein with the SEQ ID NO:2 sequence having CPK4 protein activity, the term "CPK6 protein" refers to the protein with the SEQ ID NO:3 sequence having CPK6 protein activity, the term "CPK11 protein" refers to the protein with the SEQ ID NO:4 sequence having CPK11 protein activity, and the term "CPK27 protein" refers to the protein with the SEQ ID NO:5 sequence having CPK27 protein activity.

[0067] The terms "CPK3 protein," "CPK4 protein," "CPK6 protein," "CPK11 protein," and "CPK27 protein" also include variations of the sequences SEQ ID NO: 1-5 that have the same function as the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, and 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. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The terms “CPK3 protein”, “CPK4 protein”, “CPK6 protein”, “CPK11 protein”, and “CPK27 protein” also include active fragments and active derivatives of CPK3, CPK4, CPK6, CPK11, and CPK27 proteins.

[0068] Polynucleotide sequences (coding sequences) encoding CPK3, CPK4, CPK6, CPK11, CPK27 proteins or their conserved variants can also be used in this invention. The coding region sequences also include degenerate variants thereof. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes CPK3, CPK4, CPK6, CPK11, CPK27 proteins or their conserved variants, but differs from their coding region sequences. The term "coding gene" can include polynucleotides encoding said proteins, or it can include polynucleotides with additional coding and / or non-coding sequences.

[0069] Variants of the aforementioned polynucleotides are also available, encoding proteins or fragments, analogs, and derivatives of proteins having the same amino acid sequence as those of the present 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 protein it encodes.

[0070] It should be understood that although the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins / genes of the present invention are preferably obtained from Arabidopsis thaliana, other proteins / genes obtained from other plants that are highly homologous to the Arabidopsis CPK3, CPK4, CPK6, CPK11, and CPK27 proteins / genes (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) 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.

[0071] The coding sequences of the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates. Alternatively, the relevant sequences can also be synthesized artificially.

[0072] Vectors containing the aforementioned coding sequences, and host cells genetically engineered using the aforementioned vectors or the CPK3, CPK4, CPK6, CPK11, and CPK27 protein-coding sequences, are also included in this invention. Methods well-known to those skilled in the art can be used to construct expression vectors containing CPK3, CPK4, CPK6, CPK11, and CPK27 protein-coding sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The aforementioned sequences can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Vectors containing the aforementioned appropriate coding sequences and appropriate promoters or control sequences can be used to transform appropriate host cells to enable them to express proteins.

[0073] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method or rice embryo transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0074] Application of CPK in improving plant drought resistance

[0075] In their research, the inventors discovered that overexpression of CPK3, CPK4, CPK6, CPK11, or CPK27 significantly improves the drought resistance of plants.

[0076] Based on the inventor's hairstyle, the present invention provides the use of the CPK3, CPK4, CPK6, CPK11, CPK27 proteins or their encoding genes for improving the drought resistance of plants.

[0077] As used in this article, the terms "drought resistance" and "drought tolerance" can be used interchangeably, including but not limited to the ability of plants to grow in arid environments and the biomass of plants in arid environments.

[0078] As used herein, "improving the drought resistance of plants" refers to a statistically significant increase in the drought resistance (e.g., biomass in arid environments) and other characteristics of plants improved by the technical solution of this invention compared to unmodified plants (e.g., wild-type plants).

[0079] As used herein, the terms “enhancement,” “improvement,” or “enhancement” are interchangeable and, in their application, should mean an increase in drought resistance of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, or 40%, compared to the control plant as defined herein.

[0080] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design and can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even the same variety as the plant being evaluated. Control plants can also be individuals of transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts.

[0081] The present invention also provides a method for improving the drought resistance of plants, the method comprising transferring the encoding genes of the CPK3, CPK4, CPK6, CPK11, and CPK27 proteins into plants, thereby improving the plants' ability to resist drought environments.

[0082] The present invention also provides a method for increasing drought resistance in plants, particularly plants that express low (including no) CPK3, CPK4, CPK6, CPK11 and / or CPK27, comprising: upregulating the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27.

[0083] It should be understood that, after understanding the functions of CPK3, CPK4, CPK6, CPK11, and / or CPK27, various methods well known to those skilled in the art can be used to regulate the expression or activity of CPK3, CPK4, CPK6, CPK11, and / or CPK27. For example, various methods well known to those skilled in the art can be used to overexpress CPK3, CPK4, CPK6, CPK11, and / or CPK27.

[0084] In this invention, the upregulators of CPK3, CPK4, CPK6, CPK11, and / or CPK27 proteins or their encoding genes include promoters, agonists, and activators. The terms "upregulation" and "promotion" include "upregulation" and "promotion" of protein activity or protein expression, and these are statistically significant. Any substance that can increase the activity of CPK3, CPK4, CPK6, CPK11, and / or CPK27, improve the stability of CPK3, CPK4, CPK6, CPK11, and / or CPK27 proteins, upregulate the expression of CPK3, CPK4, CPK6, CPK11, and / or CPK27 genes, or increase the effective duration of action of CPK3, CPK4, CPK6, CPK11, and / or CPK27 proteins can be used in this invention as substances useful for upregulating CPK3, CPK4, CPK6, CPK11, and / or CPK27 or signaling pathways. These can be compounds, small chemical molecules, or biomolecules. The biomolecules mentioned can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0085] This invention also provides a method for upregulating the expression of CPK3, CPK4, CPK6, CPK11, and / or CPK27 in plants. The method includes: transferring the coding genes of CPK3, CPK4, CPK6, CPK11, and / or CPK27, or expression constructs or vectors containing said coding genes, into plants. Alternatively, gain-of-function mutations can be performed on the coding genes of CPK3, CPK4, CPK6, CPK11, and / or CPK27; to promote the expression of the coding genes of CPK3, CPK4, CPK6, CPK11, and / or CPK27 by expressing an enhancing promoter or a tissue-specific promoter; or, to promote the expression of the coding genes of CPK3, CPK4, CPK6, CPK11, and / or CPK27 by an enhancer. It should be understood that other methods for upregulating the expression of CPK3, CPK4, CPK6, CPK11, and / or CPK27 in plants should also be included in this invention.

[0086] In one embodiment of the present invention, plants can be overexpressed with CPK3, CPK4, CPK6, CPK11, and CPK27 proteins by introducing expression constructs containing genes encoding CPK3, CPK4, CPK6, CPK11, and CPK27 proteins. Transformation can also be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, leaf disc transformation or rice embryo transformation. The transformed plant tissues or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits.

[0087] As a preferred embodiment of the present invention, the method for obtaining transgenic plants is as follows:

[0088] (1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the encoding genes of CPK3, CPK4, CPK6, CPK11, and CPK27 proteins;

[0089] (2) Contact plant tissues or organs with Agrobacterium in step (1) so that the protein-coding genes of CPK3, CPK4, CPK6, CPK11, and CPK27 are transferred into the plant and integrated into the chromosome of the plant cell;

[0090] (3) Select plant tissues or organs into which the protein-coding genes of CPK3, CPK4, CPK6, CPK11, and CPK27 have been transferred; and

[0091] (4) Regenerate the plant tissues or organs from step (3) into a plant.

[0092] Molecular markers

[0093] After learning about the functions of CPK3, CPK4, CPK6, CPK11, and CPK27, they can be used as molecular markers for targeted screening of plants.

[0094] After learning about the functions of CPK3, CPK4, CPK6, CPK11, and CPK27, this new discovery can also be used to screen for substances or potential substances that can regulate plant drought resistance by modulating this mechanism.

[0095] Therefore, this invention provides a method for targeted selection or identification of plants, the method comprising: identifying the expression or sequence characteristics of CPK3, CPK4, CPK6, CPK11, and / or CPK27 in a test plant; if the test plant highly expresses CPK3, CPK4, CPK6, CPK11, and / or CPK27 proteins or their genes, it is a plant with increased drought resistance; if the test plant low expresses or does not express CPK3, CPK4, CPK6, CPK11, and / or CPK27 proteins or their genes, it is a plant with decreased drought resistance. This method can be applied to early identification, such as for the identification of plant seed / root tissues.

[0096] The present invention provides a method for screening substances (potential substances) that enhance plant drought resistance, comprising: (1) adding candidate substances to a system expressing CPK3, CPK4, CPK6, CPK11 and / or CPK27; (2) detecting the system and observing the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 therein. If their expression or activity is increased, it indicates that the candidate substance is a substance that can be used to increase plant drought resistance.

[0097] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0098] A variety of conventional techniques can be used to identify gene transcription or expression in a system. These techniques include, but are not limited to, oligonucleotide hybridization (e.g., probes), polymerase chain reaction (PCR), and polyacrylamide gel electrophoresis. Detecting protein-protein interactions and their strength can be achieved using various techniques well-known to those skilled in the art, such as immunoprecipitation, GST precipitation, phage display, or yeast two-hybrid systems. Nuclear localization of proteins is also a well-known technique in the field.

[0099] In addition, the tobacco bimolecular fluorescence complementarity (BIFC) assay can also be used to analyze protein interactions. The principle is that fluorescent proteins (YFP, GFP, Luciferase, etc.) have many specific sites on their loop structures between the two β-sheets that allow for the insertion of exogenous proteins without affecting the fluorescent activity of the fluorescent protein. BiFC technology utilizes this characteristic of the fluorescent protein family, splitting the fluorescent protein into two non-fluorescent molecular fragments, which are then fused separately with target proteins for expression. If the two target proteins approach each other due to physical interactions, the two molecular fragments of the fluorescent protein spatially approach each other, reforming an active fluorescent group and emitting fluorescence.

[0100] Through large-scale screening, a class of potential substances that specifically act on signaling pathways involving CPK3, CPK4, CPK6, CPK11 and / or CPK27, or those involved therein, can be obtained, thus regulating the drought resistance of plants.

[0101] The present invention will be 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 in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0102] Materials and methods

[0103] 1. Plant materials and growing conditions

[0104] Both the wild-type and mutant strains used in this paper are Columbia ecotype (Col-0). cpk3 / 4 / 6 / 11 / 27 was obtained by CRISPR-Cas9 systemic mutation in the cpk3 / 4 / 6 / 11 background.

[0105] Transgenic plants such as 35Spro::CPK3-MYC / Col-0, 35Spro::CPK4-MYC / Col-0, 35Spro::CPK6-MYC / Col-0, 35Spro::CPK11-MYC / Col-0, and 35Spro::CPK27-MYC / Col-0 were obtained through Agrobacterium-mediated transformation.

[0106] Arabidopsis thaliana rooted in soil is generally sown on 1 / 2 MS solid medium. After being cold-treated at 4°C for 24 or 48 hours, it is placed vertically at 23°C under constant temperature and light (long-day or short-day) for about 10 days. After that, the seedlings are transplanted into Arabidopsis thaliana-specific soil and placed in a long-day or short-day greenhouse for growth, depending on the experimental purpose.

[0107] 2. Carrier Construction

[0108] The vectors used in these examples were modified based on pCAMBIA1300 or pCAMBIA3301, with bacterial resistance being kanamycin resistance and plant resistance being either hygromycin resistance or herbicide resistance. Vector construction employed homologous recombination, enzyme digestion and ligation, and transfer PCR. The constructed plasmids were transformed into *E. coli* DH5α using a 42°C heat shock transformation method for propagation. After plasmid extraction and sequencing confirmation, the plasmids were transformed into *Agrobacterium* GV3101 for infecting target plants.

[0109] 3. Total plant protein extraction – used for in vivo kinase activity detection

[0110] 1) Sow on 1 / 2 MS solid medium, keeping the number of sowing rows and the number of seeds per row consistent, and cold treat at 4℃ for 24 hours.

[0111] 2) Cultivate vertically in a long-day, constant-temperature, and light-controlled incubator for 7-9 days until the true leaves begin to emerge.

[0112] 3) Take an appropriate amount of material from the corresponding location, transfer it to 1 / 2 MS liquid culture medium, and pretreat it for 2 hours at room temperature and under light.

[0113] 4) Add the appropriate concentration of ABA or mannitol solution to the 1 / 2MS liquid medium prepared in the above experiment, and adjust the pH to the original pH value of the 1 / 2MS liquid medium.

[0114] 5) After briefly and gently removing the liquid from the material, quickly transfer it to 1 / 2 MS liquid culture medium containing ABA, mannitol, etc., and start timing.

[0115] 6) After processing, quickly remove the material, absorb the residual culture medium with a paper towel and transfer it to a domestic 1.5mL centrifuge tube, add 50μL of extraction solution and 2 steel balls, and quickly transfer it to liquid nitrogen for flash freezing.

[0116] 7) After the automatic grinder module is flash-frozen with liquid nitrogen, place the sample inside, set the crushing program to 60Hz, run for 25 seconds, with a 5-second interval as one cycle, and crush for 8 minutes.

[0117] 8) After grinding, quickly open the cap and pour out the steel ball, add the protein extraction solution, and place the centrifuge tube on ice to wait for it to thaw. During this time, you can vortex to promote full contact between the extraction solution and the sample.

[0118] 9) Centrifuge at 4℃ and 18000g for 40 minutes, then transfer the supernatant to a new centrifuge tube.

[0119] 10) Take 5 μL of supernatant and add 1 mL of Bradford solution. Vortex to mix. The mixture should be blue at this point. The intensity of the color indicates the total protein concentration.

[0120] 11) Pipette 600 μL of the mixed liquid into a disposable cuvette, quantify the total protein concentration using the Bradford method, and then prepare a solution of equal concentration for later use.

[0121] Extract formulation:

[0122]

[0123]

[0124] 4. Prokaryotic protein purification—taking Gst-tagged proteins as an example

[0125] 1) Transform the correctly sequenced plasmid into E. coli strain BL21 (similar to the method for transforming DH5α).

[0126] 2) Take out the bacterial plate that has been incubated upside down overnight in a 37℃ constant temperature dark incubator, pick 10 single colonies and mix them into 3mL of LB medium containing antibiotics.

[0127] 3) 37℃ constant temperature shaker, 220rpm overnight gentle shake.

[0128] 4) Inoculate the small-scale bacterial culture at a ratio of 1:1000 into 350ml of fresh LB liquid medium.

[0129] 5) Shake the bacterial culture at 37℃ constant temperature shaker and 220rpm until the OD600 value is about 1.0.

[0130] 6) Transfer the large-volume bacterial culture to an incubator at 18°C ​​and 100 rpm, and continue incubation for 30 minutes.

[0131] 7) Add 1M IPTG of the stock solution to the bacterial culture at a ratio of 1:1000, and continue to culture at low temperature and low speed for 14-18 hours.

[0132] 8) Collect the bacterial culture by centrifugation at 4℃ and 4000g for 20 minutes, and resuspend the bacterial culture in 30mL TBS buffer.

[0133] 9) Add 0.1M PMSF ethanol solution to the bacterial solution at a ratio of 1:1000. Use an ultrasonic instrument with 85% power, low temperature, and a cycle of 5 seconds on and 5 seconds off to ultrasonically break up the bacterial solution for 5 minutes until it becomes clear.

[0134] 10) Centrifuge at 4℃ and 20000g for 40 minutes, transfer the supernatant to a 50mL centrifuge tube, add agarose resin with the corresponding label, and spin at low speed for 3 hours.

[0135] 11) Centrifuge at 4℃ and 500g for 2 minutes, discard the supernatant, gently resuspend the precipitate with 10mL TBS buffer, and repeat the washing 3 times.

[0136] 12) Resuspend the precipitate with 1 mL TBS and transfer it to a new 1.5 mL centrifuge tube.

[0137] 13) Centrifuge at 4℃ and 500g for 2 minutes, discard the supernatant, add 120μL of Gst tag protein elution buffer, and elute at 4℃ and the lowest rotation speed for 10 minutes.

[0138] 14) Centrifuge at 4℃ and 500g for 2 minutes, collect the supernatant into a new 1.5mL centrifuge tube, and repeat steps 11 / 12 twice to obtain a total of 360μL of elution buffer containing the target protein.

[0139] 15) Add 1 mL of Bradford solution to the 15 mL centrifuge tube left over from step 14, mix well, estimate the concentration of the target protein based on the blue intensity, take an appropriate volume of the target protein, and quantify it by electrophoresis, Coomassie brilliant blue staining (5 minutes), destaining (15 minutes * 3), etc. The remaining protein is aliquoted, flash-frozen in liquid nitrogen and stored at -80 for later use.

[0140] The staining solution formula is: 50% methanol, 10% glacial acetic acid, 40% dd H2O and 0.1% R250, where all percentages are by volume.

[0141] Decolorizing solution formula: 50% methanol, 10% glacial acetic acid and 40% dd H2O, where all percentages are by volume.

[0142] 5. Immunoprecipitation (IP)

[0143] 1) Take a plate of seedlings (about 100 plants) that have germinated about 10 days after sprouting on 1 / 2 MS solid medium, wrap them in aluminum foil, label them and then freeze them quickly with liquid nitrogen.

[0144] 2) Prepare the protein extraction buffer as follows:

[0145]

[0146] dd H2O

[0147] 3) Pre-cool the mortar and grinding rod with liquid nitrogen, add liquid nitrogen to the mortar, put in the plant material, and grind it quickly into powder with the grinding rod.

[0148] 4) Add 400 μL of protein extraction buffer, grind quickly into powder, mix thoroughly with plant powder, and place on ice.

[0149] 5) When the powder shows signs of melting, continue grinding until it is completely liquefied, then let it stand for 10 minutes.

[0150] 6) Gently pipette the liquid mixture to mix it, transfer it to a 1.5 mL centrifuge tube, and centrifuge at 4°C and 18000 g for 40 minutes.

[0151] 7) Dilute the remaining 2× protein extraction buffer with double-distilled water to make 1× protein extraction buffer.

[0152] 8) Take an appropriate amount of immunomagnetic beads coupled with the corresponding tags (10 μL per sample), remove the original buffer on the magnetic rack, add 1× protein extraction buffer, and rotate at room temperature for 30 minutes to equilibrate.

[0153] 9) After equilibration, aspirate the original buffer solution from the magnetic rack and add 10 μL of 1× protein extraction buffer to each sample for later use.

[0154] 10) After centrifugation, transfer the supernatant to a new 1.5mL centrifuge tube, add 10μL of equilibrated immunomagnetic beads to each sample, and incubate at 4℃, low speed, and rotation for 4-6 hours.

[0155] 11) Place the magnetic rack on ice, place the 1.5mL centrifuge tube in the designated position, and after the magnetic beads are completely adsorbed, remove the liquid with a pipette.

[0156] 12) Add 1 mL of 1× protein extraction buffer, gently invert to mix the magnetic beads, wash at 4°C, low speed, and rotate, and repeat twice.

[0157] 13) Remove the old buffer solution on a magnetic rack, add 60 μL of 1× protein extraction buffer, mix well, dispense, freeze in liquid nitrogen and store at -80°C for later use.

[0158] 6. In-gel kinase activity assay

[0159] 1) Extract total plant protein according to step 2.14, quantify and prepare electrophoresis samples.

[0160] 2) Prepare a dedicated PAGE adhesive. The formula for the lower layer adhesive is as follows:

[0161]

[0162] 3) Prepare the top layer adhesive - the same as the formula for the top layer adhesive of ordinary SDS-PAGE adhesive.

[0163] 4) After pre-prepared samples are treated at 98℃ for 2 minutes, they are loaded onto the gel and electrophoresed at 140V for the upper gel and 170V for the lower gel until the 25kDa protein band migrates to the edge of the gel.

[0164] 5) Prepare 300 mL of SDS elution buffer. After electrophoresis, immerse the gel block in 100 mL of buffer and elute on a horizontal shaker at low speed for 20 minutes. Repeat twice.

[0165]

[0166] 6) Prepare 300 mL of protein refolding buffer, immerse the gel block in 100 mL of buffer, and refold at room temperature on a horizontal shaker at low speed for 1 hour.

[0167]

[0168] 7) Immerse the gel block in fresh refolding buffer and refold at 4°C on a horizontal shaker at low speed for 16-18 hours. This step can be done overnight.

[0169] 8) Immerse the gel block in new refolding buffer and refold at room temperature for 1 hour.

[0170] 9) Prepare 150 mL of kinase reaction buffer:

[0171]

[0172] Immerse the gel block in 100 mL of reaction buffer and react at room temperature and low speed for 30 minutes.

[0173] Immerse the gel block in 30 mL of reaction buffer, add 3 μL of [γ-32P]-ATP, and react at low speed for 5 minutes.

[0174] *From this step onwards, operations must be performed under protective measures.

[0175] 10) Add 4.5 μL of ATP and react at room temperature for 1-2 hours.

[0176] 11) Prepare 500 mL of isotope elution buffer:

[0177]

[0178] Immerse the gel block in 100 mL of elution buffer and elute at room temperature and low speed for 1 hour. Repeat this process 4 times, using a new container each time.

[0179] 12) Perform PAGE gel drying, tableting, and signal acquisition according to the in vitro phosphorylation experimental procedure.

[0180] 7. In vitro phosphorylation assay (In vitro Kinase Assay)

[0181] 1) Prepare 5× in vitro phosphorylation experimental buffer:

[0182]

[0183] 2) The in vitro phosphorylation experimental system was prepared on ice.

[0184] *Taking CPK and SnRK2s as examples, an appropriate amount of Ca needs to be added to the CPK reaction system. 2+ ;

[0185] *The reaction system must be prepared using explosion-proof centrifuge tubes specifically designed for isotopes.

[0186]

[0187] 3) Add 2 μCi [γ-32P]ATP to 30 μL of 12.5 μM ATP, mix well, and dispense 2 μL into each sample.

[0188] *From this step onwards, operations must be performed under protective measures.

[0189] 4) Install explosion-proof centrifuge tube fittings and react in a metal bath at 30°C for 2 hours.

[0190] 5) Select an appropriate concentration of SDS-PAGE gel and perform electrophoresis, staining, destaining, and photography according to the Western Blot method.

[0191] 6) After taking the photos, put the glue block back into the decolorizing solution and immerse it on a horizontal shaker at low speed for 5 minutes.

[0192] *This step prevents the adhesive from cracking during the drying process due to prolonged photography.

[0193] 7) Place the gel block flat on the petri dish, gently cover the gel block with filter paper, hold the gel block down and gently flip the petri dish to remove the gel block and filter paper together from the petri dish.

[0194] *Prevents cracking during the drying process caused by adhesive stretching during transfer.

[0195] 8) Lay the glue side up and the filter paper side down on the glue dryer, cover with a layer of plastic wrap, seal the glue dryer, start vacuuming, start heating, and dry for 1 hour.

[0196] Once the plastic wrap touches the adhesive, it should not be moved, otherwise it is very easy to cause the adhesive to crack during the drying process.

[0197] 9) Use the phosphor screen removal instrument to remove impurities from the phosphor screen with strong light for more than 30 minutes.

[0198] 10) After drying, wrap the filter paper with glue in plastic wrap, stick the glued side against the phosphor screen, and press it into tablets for the appropriate time.

[0199] 11) Scan the phosphor screen with the instrument, adjust the signal strength, and save the experimental results.

[0200] 8. Sequence

[0201] CPK3 Protein Sequence (SEQ ID NO:1)

[0202] MGHRHSKSKSSDPPPSSSSSSSGNVVHHVKPAGERRGSSGSGTVGSSGSGTGGSRSTTSTQQNGRILGRPMEEVRRTYEFGRELGRGQFGVTYLVTHKETKQQVACKSIPTRRLVHKDDIEDVRREVQIMHHLSGHRNIVDLKGAYEDRHSVNLIMELCEGGELFDRIISKGLYSERAAADLCRQMVMVVHSCHSMGVMHRDLKPENFLFLSKDENSPLKATDFGLSVFFKPGDKFKDLVGSAYYVAPEVLKRNYGPEADIWSAGVILYILLSGVPPFWGENETGIFDAILQGQLDFSADPWPALSDGAKDLVRKMLKYDPKDRLTAAEVLNHPWIREDGEASDKPLDNAVLSRMKQFRAMNKLKKMALKVIAENLSEEEIIGLKEMFKSLDTDNNGIVTLEELRTGLPKLGSKISEAEIRQLMEAADMDGDGSIDYLEFISATMHMNRIEREDHLYTAFQFFDNDNSGYITMEELELAMKKYNMGDDKSIKEIIAEVDTDRDGKINYEEFVAMMKKGNPELVPNRRRM

[0203] CPK4 Protein Sequence (SEQ ID NO:2)

[0204] MEKPNPRRPSNSVLPYETPRLRDHYLLGKKLGQGQFGTTYLCTEKSSSANYACKSIPKRKLVCREDYEDVWREIQIMHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGCFSEREAAKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPSDDAKLKATDFGLSVFYKPGQYLYDVVGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKIDFKSDPWPTISEGAKDLIYKMLDRSPKKRISAHEALCHPWIVDEHAAPDKPLDPAVLSRLKQFSQMNKIKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLMDAADIDNSGTIDYGEFLAATLHINKMEREENLVVAFSYFDKDGSGYITIDELQQACTEFGLCDTPLDDMIKEIDLDNDGKIDFSEFTAMMKKGDGVGRSRTMRNNLNFNIAEAFGVEDTSSTAKSDDSPKMEAADVDNSGTIDYSEFIAATIHLNKLEREEHLVSAFQYFDKDGSGYITIDELQQSCIEHGMTDVFLEDIIKEVDQDNDGRIDYEEFVAMMQKGNAGVGRRTMKNSLNISMRDV

[0205] CPK6 protein sequence (SEQ ID NO:3)

[0206] MGNSCRGSFKDKIYEGNHSRPEENSKSTTTTVSSVHSPTTDQDFSKQNTNPALVIPVKEPIMRRNVDNQSYYVLGHKTPNIRDLYTLSRKLGQGQFGTTYLCTDIATGVDYACKSISKRKLISKEDVEDVRREIQIMHHLAGHKNIVTIKGAYEDPLYVHIVMELCAGGELFDRIIHRGHYSERKAAELTKIIVGVVEACHSLGVMHRDLKPENFLLVNKDDDFSLKAIDFGLSVFFKPGQIFKDVVGSPYYVAPEVLLKHYGPEADVWTAGVILYILLSGVPPFWAETQQGIFDAVLKGYIDFDTDPWPVISDSAKDLIRKMLCSSPSERLTAHEVLRHPWICENGVAPDRALDPAVLSRLKQFSAMNKLKKMALKVIAESLSEEEIAGLRAMFEAMDTDNSGAITFDELKAGLRRYGSTLKDTEIRDLMEAADVDNSGTIDYSEFIAATIHLNKLEREEHLVSAFQYFDKDGSGYITIDELQQSCIEHGMTDVFLEDIIKEVDQDNDGRIDYEEFVAMMQKGNAGVGRRTMKNSLNISMRDV

[0207] CPK11 protein sequence (SEQ ID NO:4)

[0208] METKPNPRRPSNTVLPYQTPRLRDHYLLGKKLGQGQFGTTYLCTEKSTSANYACKSIPKRKLVCREDYEDVWREIQIMHHLSEHPNVVRIKGTYEDSVFVHIVMEVCEGGELFDRIVSKGHFSEREAVKLIKTILGVVEACHSLGVMHRDLKPENFLFDSPKDDAKLKATDFGLSVFYKPGQYLYDVVGSPYYVAPEVLKKCYGPEIDVWSAGVILYILLSGVPPFWAETESGIFRQILQGKLDFKSDPWPTISEAAKDLIYKMLERSPKKRISAHEALCHPWIVDEQAAPDKPLDPAVLSRLKQFSQMNKIKKMALRVIAERLSEEEIGGLKELFKMIDTDNSGTITFEELKAGLKRVGSELMESEIKSLMDAADIDNSGTIDYGEFLAATLHMNKMEREENLVAAFSYFDKDGSGYITIDELQSACTEFGLCDTPLDDMIKEIDLDNDGKIDFSEFTAMMRKGDGVGRSRTMMKNLNFNIADAFGVDGEKSDD

[0209] CPK27 protein sequence (SEQ ID NO:5)

[0210] MGCFSSKELQQSKRTILEKPLVDITKIYILGEELGRGNFGLTRKCVEKSTGKTFACKTILKTKLKDEECEEDVKREIRIMKQLSGEPNIVEFKNAYEDKDSVHIVMEYCGGGELYDKILAL YDVGKSYSEKEAAGIIRSIVNVVKNCHYMGVMHRDLKPENFLLTSNDDNATVKVIDFGCSVFIEEGKVYQDLAGSDYYIAPEVLQGNYGKEADIWSAGIILYILLCGKSPFVKEPEGQMFN EIKSLEIDYSEEPWPLRDSRAIHLVKRMLDRNPKERISAAEVLGHPWMKEGEASDKPIDGVVLSRLKRFRDANKFKKVVLKFIAANLSEEEIKGLKTLFTNITDKSGNITLEELKTGLTR LGSNLSKTEVEQLMEAADMDGNGTIDIDEFISATMHRYKLDDRDEHVYKAFQHFDKDNDGHITKEELEMAMKEDGAGDEGSIKQIIADADTDNDGKINFEEFRTMMRTESSLQPEGELLPIIN

[0211] Example 1: Dehydration-induced activation of CPK kinase activity

[0212] CPK kinase activity and free calcium in plant cytoplasm 2+ Concentration is directly related, while dehydration stimulation induces free calcium in plant cytoplasm. 2+ The concentration increased. Therefore, the inventors hypothesized that dehydration could directly activate the protein kinase activity of CPK. To verify this hypothesis, the inventors drove CPK gene expression with the 35S promoter in a Col-0 background, ultimately obtaining transgenic positive plants of 35Spro::CPK3-MYC, 35Spro::CPK4-MYC, 35Spro::CPK6-MYC, 35Spro::CPK11-MYC, 35Spro::CPK27-MYC, and 35Spro::CPK32-MYC. The inventors used material that had germinated and grown for 10 days on solid 1 / 2 MS medium, dehydrated it for 30 minutes, and extracted endogenous CPK from the plant using an immunoprecipitation system. The change in CPK protein abundance before and after treatment was detected by Western blotting as a quality control, and the change in CPK protein kinase activity before and after treatment was detected by in vitro phosphorylation. The experimental results showed that, with almost the same protein loading amount, the kinase activity of CPK in the plant was significantly enhanced after treatment. Figure 1Based on this, the inventors believe that dehydration stimulation can directly activate the kinase activity of CPK3, CPK4, CPK6, CPK11, and CPK27.

[0213] Example 2: CPK phosphorylation and activation of SnRK2

[0214] Dehydration stimulation can directly activate CPK. The inventors hypothesize that CPK can directly activate SnRK2 protein kinase activity as a protein kinase. To further investigate whether CPK can directly phosphorylate SnRK2 kinase, the inventors constructed CPK3, CPK4, CPK6, CPK11, and SnRK2.6 with pGEX-6P-1 as the backbone. K50N A prokaryotic protein expression vector for a non-kinase-active spike protein. GST-CPKs and GST-SnRK2.6 were obtained after induction of expression and purification. K50N Protein. Immunoblot results based on antibodies against serine at position S175 showed that CPK3, CPK4, CPK6, and CPK11 could phosphorylate and modify serine at position S175 of SnRK2.6. Figure 2 A).

[0215] To further verify whether CPK can directly activate the kinase activity of SnRK2.6 through phosphorylation modification, the inventors first purified dephosphorylated, kinase-free SnRK2.6 (de-His-SnRK2.6) from the BL21 strain co-transformed with SnRK2.6 and ABI1. The in vivo purified osmotic stress-activated CPK3 / 4 / 6 was co-incubated with de-His-SnRK2.6 for 1 hour, then separated on a magnetic rack. AKS1 protein (a substrate of SnRK2.6) was added to the remaining reaction solution, and the reaction continued for 2 hours. Experimental results showed that CPK could phosphorylate SnRK2.6 and activate SnRK2.6's phosphorylation of AKS1. Figure 2 B).

[0216] In summary, CPK can directly activate SnRK2 through phosphorylation modification.

[0217] Example 3: CPK-mediated dehydration-induced activation of SnRK2

[0218] Dehydration can activate the kinase activity of SnRK2 and also directly activate CPK. CPK directly phosphorylates and activates SnRK2. Therefore, the inventors wanted to know whether CPK mediates dehydration-induced SnRK2 activation in plants? What is the dehydration-induced SnRK2 activation status in the cpk3 / 4 / 6 / 11 / 27 mutant?

[0219] Through Western blotting experiments, the inventors discovered that dehydration enhances the phosphorylation of serine at position 175 of SnRK2.6; on the other hand, compared to the Col-0 wild type, the phosphorylation level of serine at position 175 of SnRK2.6 induced by dehydration was significantly decreased in the cpk3 / 4 / 6 / 11 / 27 mutants. Figure 3 A). Intracollagenary kinase activity assays based on radiolabeled ATP showed that dehydration-induced activation of SnRK2.6 was significantly defective in the cpk3 / 4 / 6 / 11 / 27 mutant. Figure 3 A).

[0220] The inventors further investigated the activation of SnRK2 induced by dehydration in the CPK overexpression lines. The results showed that the phosphorylation level of serine at position 175 of SnRK2.6 induced by mannitol and the SnRK2.6 kinase activity were significantly higher in the CPK overexpression lines than in the wild type.

[0221] Based on the above results, the inventors believe that CPK plays an important role in activating SnRK2 in the early stages of dehydration stress.

[0222] Example 4: CPK mediates the biological function of SnRK2

[0223] The inventors believe that CPK mediates the induced activation of SnRK2 by early dehydration stress independent of ABA. So how does the phenotypic output of dehydration stress manifest in CPK-related mutants and overexpression lines?

[0224] Seedlings acclimatized to 140 mM mannitol for 5 days were transferred to 1 / 2 MS medium containing 150 mM mannitol and continued to grow. The results showed that the cpk3 / 4 / 6 / 11 / 27 mutant exhibited a stress-hypersensitive phenotype compared to the wild-type plants. Figure 4 (A). The inventors further observed the drought resistance of the CPK overexpression lines. Experimental results showed that, without affecting the normal growth of the plants under non-stress conditions, the drought resistance of the CPK overexpression lines was significantly improved compared to the wild-type plants. Figure 4 B).

[0225] discuss

[0226] Plant drought tolerance requires rapid and accurate osmotic regulation. Osmotic stress is a physical stimulus that triggers a series of physicochemical changes occurring in the cell wall, plasma membrane, or within the cell, which are then perceived by the plant. 2+Signaling is one of the earliest chemical changes. The inventors' research shows that CPK3, CPK4, CPK6, CPK11, and CPK27 are rapidly activated under osmotic stress and modulate plant tolerance to osmotic stress by enhancing SnRK2 activation. Enhanced SnRK2 activation was achieved through overexpression of CPKs, which may contribute to enhanced stress responses without causing growth defects under non-stress conditions. Therefore, the inventors' findings not only provide a Ca2+ pathway for osmotic stress... 2+ The signal decoding mechanism also provides a strategy for enhancing early penetration stress response.

[0227] Although CPK3, 4, 6, 11, and 27 are the major CPKs in response to osmotic stress, how these CPKs are activated remains unclear. Activation of CPKs may require at least two processes: release of the CPKs from inhibition of their own inhibitory connective domains, followed by activation via autophosphorylation or phosphorylation by other early signaling elements. These five CPKs belong to subfamilies I and II of the CPK family and are activated in vitro via Ca2+. 2+ The mediated release of autoinhibition by cytoplasmic Ca 2+ Increased activation. The osca1 and bon1 mutants exhibited increased Ca under osmotic stress. 2+ The mutation showed increased defects, but further analysis is needed to determine whether CPK activation is defective in these mutants. Furthermore, hypotonic stress induced higher levels of cytoplasmic calcium compared to hypertonic stress. 2+ The increases were transient, but they triggered different downstream reactions, indicating that Ca... 2+ Elevated levels may be necessary for CPK activation, but not sufficient. Therefore, further investigation into early signals in response to osmotic stress and environment-dependent CPK phosphorylation may resolve the activation and signal specificity of CPK.

[0228] The inventors' research shows that under osmotic stress, transient Ca 2+ Elevated levels of this chemical signal enhance the activation of early kinases. Five members of the calciprotein kinase family, including CPK3, 4, 6, 11, and 27, are activated under osmotic stress, which further enhances SnRK2 activation and controls stress response and tolerance. This reveals that Ca... 2+ The key role of signal transduction and CPK-SnRK2 kinase cascade in early osmotic stress signal transduction and osmotic regulation in vascular plants.

[0229] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. Methods for improving plant drought resistance or methods for preparing plants with increased drought resistance, including: Upregulates the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 in plants.

2. The method as described in claim 1, characterized in that, Upregulation of the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 in plants includes: Introducing the encoding genes of CPK3, CPK4, CPK6, CPK11 and / or CPK27, or expression constructs or vectors containing such encoding genes, into plants; performing gain-of-function mutations on CPK3, CPK4, CPK6, CPK11 and / or CPK27; promoting the expression of CPK3, CPK4, CPK6, CPK11 and / or CPK27 by expressing an enhancing promoter or a tissue-specific promoter; or promoting the expression of CPK3, CPK4, CPK6, CPK11 and / or CPK27 by an enhancer.

3. The method as described in claim 1, characterized in that, The method for preparing plants with increased drought resistance further includes: hybridizing transgenic plants whose expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 has been regulated with plants that have not been introduced with peptides or their encoding genes of CPK3, CPK4, CPK6, CPK11 and / or CPK27 to obtain hybrid offspring.

4. Use of a CPK3, CPK4, CPK6, CPK11 and / or CPK27, or an upregulator thereof, for upregulating the drought resistance of plants or for preparing plants with increased drought resistance.

5. The use as described in claim 4, characterized in that, The regulator is an upregulator of CPK3, CPK4, CPK6, CPK11 and / or CPK27, which increases the drought resistance of plants. The upregulator includes: exogenous CPK3, CPK4, CPK6, CPK11 and / or CPK27 encoding genes or expression constructs or vectors containing such encoding genes; preferably, the expression construct includes an enhancing promoter, a tissue-specific promoter or an enhancer; or, a reagent for gain-of-function point mutation of CPK3, CPK4, CPK6, CPK11 and / or CPK27.

6. As described in any one of claims 1 to 5, characterized in that, The plants mentioned are cruciferous plants, grasses, or solanaceous plants; Preferably, the cruciferous plants include: Arabidopsis thaliana, rapeseed, and Chinese cabbage; the grasses include: rice, wheat, corn, barley, oats, and rye; and the solanaceous plants include: tobacco, tomato, and pepper. More preferably, the plant in question is Arabidopsis thaliana.

7. As described in any one of claims 1 to 6, characterized in that, The amino acid sequence of the CPK3 protein is selected from the following group: (1A) A protein having the amino acid sequence shown in SEQ ID NO:1; (1B) A protein derived from (1A) having the function described in (1A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

1. (1C) A protein having ≥80% homology with the amino acid sequence shown in SEQ ID NO:1, and having the function described in (1A); (1D) The active fragment of the protein with the amino acid sequence shown in SEQ ID NO:1; or, (1E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the amino acid sequence shown in SEQ ID NO:1, or by adding a signal peptide sequence to its N end; and / or, The amino acid sequence of the CPK4 protein is selected from the following group: (2A) A protein having the amino acid sequence shown in SEQ ID NO:2; (2B) A protein derived from (2A) having the function described in (2A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (2C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:2, and having the function described in (2A); (2D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:2; or, (2E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end; and / or, The amino acid sequence of the CPK6 protein is selected from the following group: (3A) A protein having the amino acid sequence shown in SEQ ID NO:3; (3B) A protein derived from (3A) having the function described in (3A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:3; (3C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:3, and having the function described in (3A); (3D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:3; or, (3E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the amino acid sequence shown in SEQ ID NO:3, or by adding a signal peptide sequence to its N end; and / or, The amino acid sequence of the CPK11 protein is selected from the following group: (4A) A protein having the amino acid sequence shown in SEQ ID NO:4; (4B) A protein derived from (4A) having the function described in (4A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:4; (4C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:4, and having the function described in (4A); (4D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:4; or, (4E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the amino acid sequence shown in SEQ ID NO:4, or by adding a signal peptide sequence to its N end; and / or, The amino acid sequence of the CPK27 protein is selected from the following group: (5A) A protein having the amino acid sequence shown in SEQ ID NO:5; (5B) A protein derived from (5A) having the function described in (5A) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

5. (5C) A protein having ≥80% homology between its amino acid sequence and the amino acid sequence shown in SEQ ID NO:5, and having the function described in (5A); (5D) The active fragment of the protein containing the amino acid sequence shown in SEQ ID NO:5; or, (5E) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the protein with the amino acid sequence shown in SEQ ID NO:5, or by adding a signal peptide sequence to its N end.

8. As described in any one of claims 1 to 7, characterized in that, The improvement of plant drought resistance further promotes the optimization of the following traits in plants: the ability of plants to grow in drought conditions, or the biomass of plants in drought conditions.

9. A plant cell, tissue, or organ containing an exogenous upregulator of CPK3, CPK4, CPK6, CPK11, and / or CPK27; wherein, The upregulators of CPK3, CPK4, CPK6, CPK11, and / or CPK27 include: exogenous genes encoding CPK3, CPK4, CPK6, CPK11, and / or CPK27, or expression constructs or vectors containing such genes; preferably, the expression construct includes an enhancing promoter, a tissue-specific promoter, or an enhancer; or, reagents for gain-of-function point mutations of CPK3, CPK4, CPK6, CPK11, and / or CPK27.

10. Use of a plant CPK3, CPK4, CPK6, CPK11 and / or CPK27 as molecular markers for identifying plant drought resistance or for targeted screening of plant drought resistance molecular markers.

11. A method for selecting or identifying the drought resistance of plants, characterized in that, The method includes: identifying the expression or sequence characteristics of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes in the test plant; if the test plant has high expression or high activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes, it is a plant with high drought resistance; if the test plant has low expression or low activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 proteins or their genes, it is a plant with low drought resistance.

12. A method for screening substances that regulate plant drought resistance, comprising: (1) Add the candidate substance to a system expressing CPK3, CPK4, CPK6, CPK11 and / or CPK27; (2) Detect the system and observe the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27. If the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 is increased, it indicates that the candidate substance can be used to increase the drought resistance of plants; if the expression or activity of CPK3, CPK4, CPK6, CPK11 and / or CPK27 is decreased, it indicates that the candidate substance can be used to reduce the drought resistance of plants.

Images