Method for regulating transcription of plant drought-resistant gene by Ca2+-CAMTA-ERF109 transcription cascade module

Abstract

The invention relates to a Ca < 2 + >-CAMTA-ERF109 transcription cascade module for regulating and controlling transcription of a plant drought-resistant gene. Specifically, the invention provides an application of a CAMTA-ERF109 signal channel in regulation and control of abiotic stress tolerance of plants. The invention explains a new signal channel participating in plant osmotic stress for the first time: a CAMTA-ERF109 signal channel; wherein the CAMTA and the ERF109 are both positive regulatory factors for regulating the osmotic stress tolerance of the plants. The invention has important theoretical significance for genetic improvement of plant traits.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and relates to the application of the ERF109 gene in improving plant tolerance to osmotic stress, specifically involving Ca 2+ The CAMTA-ERF109 transcriptional cascade module regulates the transcription of plant drought resistance genes. Background Technology

[0002] Plants exhibit remarkable plasticity in many growth and developmental processes, enabling them to better adapt to their surroundings. Once plant cells sense changes in their external or internal environment, they produce substances such as ROS, NO, and Ca. 2+ Ions and other second messengers transduce and activate downstream signaling pathways. In plants, Ca... 2+ Signal decoding is mainly achieved through Ca 2+ Ion-dependent protein kinase-mediated regulation of protein phosphorylation and CaM-mediated transcriptional regulation. When CaM7 and Ca... 2+ After ion binding, CaM can directly bind to the promoters of downstream genes, mediating the transcription process. Besides the direct interaction between CaM and DNA regulating transcription, CMLs can also participate in transcriptional regulation in plant cells by binding to Calmodulin-binding transcription activator (CAMTA). CAMTA is a CaM-binding transcription activator found in eukaryotes. 2+ Dependency Ca 2+ Six members of the CAMTA transcription factor family, which binds to CAMTA proteins, were identified in Arabidopsis thaliana. These CAMTA transcription factor proteins contain two conserved domains: a C-terminal Ca2+ domain and a C-terminal Ca2+ domain. 2+ The domain that binds to the cytokinin protein mediates its interaction with Ca. 2+ The interaction between CAMTA and its proteins; the N-terminal CG-1 domain mediates the binding of DNA cis-elements (CAMTA binding sites), including ABREs and CGCG cis-elements. CAMTA transcription can be rapidly induced by abiotic stresses such as cold and salt, as well as hormones such as ABA and JA, indicating that CAMTA transcription factors play an important role in multiple signaling pathways in plant responses to environmental stress.

[0003] As early as 1984, researchers discovered that within minutes of applying growth factor stimulation to 3T3 cells, the transcription of a class of c-fos genes was rapidly induced. Subsequent studies detected these rapidly induced genes in different cell types. Because these genes are often rapidly induced within minutes of stimulation, they are also known as Immediate Early Response Genes (IEGs). IEGs in mammals mediate transcriptional responses to growth factors, neuronal stimuli, and immune stimuli, primarily encoding transcription factors of the Fos, EGR, and NR4A families. Studies have found that IEG proteins can be rapidly activated by stimulation in all mammals, promoting the transcription of some cell-specific late-response genes (LRGs), which are crucial for the response to initial stimuli. Defective expression of IEGs can lead to cancer, immunodeficiency, and neurological diseases. It is speculated that the rapid induction of IEGs is closely related to the signaling mechanisms by which cells sense and transduce stimuli, possibly serving as the first signal for cellular perception and transduction of stimuli. Furthermore, the rapid transcriptional induction of IEGs is related to its transcriptional regulatory pathway. Studies have found that in neural synaptic cells, stimulation-induced Ca2+... 2+ Ionic signals can regulate the transcription of IEGs, and transcription can still be detected after treatment with the protein synthesis inhibitor CHX, indicating that the transcription of IEGs can be independent of de novo protein synthesis. This may explain why the transcription of IEGs can be so rapid. Summary of the Invention

[0004] To address the aforementioned problems, the present invention first provides a method for regulating plant tolerance to abiotic stress, comprising the steps of: regulating the expression or activity of CAMTA and / or ERF109 proteins in plants; wherein the CAMTA and / or ERF109 proteins include their homologs.

[0005] Preferably, the CAMTA includes one or more of CAMTA1, CAMTA2 and CAMTA3.

[0006] Preferably, the step further includes regulating the expression or activity of the ERF113 protein in the plant.

[0007] In one or more embodiments, the regulation of the expression or activity of CAMTA and / or ERF109 proteins in plants includes:

[0008] (a) Upregulation of CAMTA and / or ERF109 protein expression or activity leads to upregulation of response genes induced by abiotic stress in plants, thereby enhancing plant tolerance to abiotic stress; or

[0009] (b) Downregulation of the expression or activity of CAMTA and / or ERF109 proteins leads to downregulation of response genes induced by abiotic stress in plants, thereby reducing plant tolerance to abiotic stress.

[0010] In one or more embodiments, the regulation of the expression or activity of the ERF113 protein in plants includes: upregulating the expression or activity of the ERF113 protein, thereby upregulating response genes induced by abiotic stress in plants and enhancing the plant's tolerance to abiotic stress; or downregulating the expression or activity of the ERF113 protein, thereby downregulating response genes induced by abiotic stress in plants and weakening the plant's tolerance to abiotic stress.

[0011] In one or more embodiments, the abiotic stress-induced response genes include early response genes and late response genes.

[0012] In one or more embodiments, the abiotic stress-induced response genes include, but are not limited to, one or more of CML37, ERF11, COR15A, P5CS1, RD20, RD29A, ABF2, WRKY15, WRKY33, WRKY30, ERF4, ERF15, and MYB51.

[0013] In one or more embodiments, the upregulation of CAMTA and / or ERF109 protein expression or activity comprises: (1) transferring the gene encoding CAMTA and / or ERF109 protein or an expression construct or vector containing the gene into a plant; (2) performing gain-of-function mutations on CAMTA and / or ERF109 protein; (3) promoting CAMTA and / or ERF109 protein expression by expressing an enhancing promoter or a tissue-specific promoter; or (4) promoting CAMTA and / or ERF109 protein expression by an enhancer.

[0014] In one or more embodiments, the downregulation of CAMTA and / or ERF109 expression or activity comprises: knocking out or silencing the gene encoding CAMTA and / or ERF109 protein in a plant, or inhibiting the activity of CAMTA and / or ERF109 protein.

[0015] In one or more embodiments, the inhibition of CAMTA and / or ERF109 protein activity includes: transferring an inhibitor of CAMTA and / or ERF109 gene transcription, protein expression, or protein activity into a plant; preferably, it includes: silencing CAMTA and / or ERF109 protein with an interfering molecule that specifically interferes with the expression of the gene encoding CAMTA and / or ERF109 protein, gene editing using a CRISPR system to knock out the gene encoding CAMTA and / or ERF109 protein, knocking out the gene encoding CAMTA and / or ERF109 protein by homologous recombination, or performing a loss-of-function mutation on CAMTA and / or ERF109 protein in a plant containing CAMTA and / or ERF109 protein.

[0016] In one or more embodiments, the amino acid sequence of CAMTA is selected from one or more of the following: (a) a polypeptide having the sequence shown in any one or more of SEQ ID NO: 1, 3 or 5; (b) a polypeptide derived from (a) having the polypeptide function of (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in any one or more of SEQ ID NO: 1, 3 or 5; or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the polypeptide function of (a).

[0017] In one or more embodiments, the polypeptide of CAMTA is encoded by SEQ ID NO: 1, 3 or 5 or their degenerate sequences.

[0018] In one or more embodiments, the amino acid sequence of the ERF109 protein is selected from one or more of the following: (a) a polypeptide having the sequence shown in SEQ ID NO: 8; (b) a polypeptide derived from (a) having the polypeptide function of (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in SEQ ID NO: 8; or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the polypeptide function of (a).

[0019] In one or more embodiments, the polypeptide of the ERF109 protein is encoded by SEQ ID NO: 8 or its degenerate sequence.

[0020] In one or more embodiments, the amino acid sequence of the ERF113 protein is selected from one or more of the following: (a) a polypeptide having the sequence shown in SEQ ID NO: 9; (b) a polypeptide derived from (a) having the polypeptide function of (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in SEQ ID NO: 9; or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the polypeptide function of (a).

[0021] In one or more embodiments, the polypeptide of the ERF113 protein is encoded by SEQ ID NO: 10 or its degenerate sequence.

[0022] A second aspect of this invention provides the application of the CAMTA-ERF109 signaling pathway, a pathway protein therein or its gene, or the coding sequence of said protein, wherein the application is selected from:

[0023] As a molecular marker for identifying plant tolerance to abiotic stress; or

[0024] Molecular markers used for targeted screening of plants with tolerance to abiotic stress; or

[0025] Used to screen for substances that improve plant tolerance to abiotic stresses.

[0026] The third aspect of this invention provides a method for targeted selection of plants, comprising the steps of: identifying the expression or activity of a CAMTA-ERF109 signaling pathway protein in a test plant, said protein including CAMTA and / or ERF109, said protein optionally also including ERF113, wherein if said protein has one or more characteristics selected from the group consisting of:

[0027] The CAMTA expression or activity of the tested plants was increased.

[0028] The ERF109 expression or activity of the tested plants was increased, and

[0029] The ERF113 expression or activity was increased in the tested plants.

[0030] A fourth aspect of the present invention provides a method for screening substances that improve plant tolerance to abiotic stresses, comprising:

[0031] (1) Add the candidate material to a system expressing CAMTA-ERF109 signaling pathway protein, wherein the protein includes CAMTA and / or ERF109, and optionally also includes ERF113;

[0032] (2) Detect the system and observe the expression or activity of CAMTA-ERF109 signaling pathway proteins:

[0033] If CAMTA protein expression or activity is increased, then this candidate substance is a substance that can improve the tolerance of plants to abiotic stress.

[0034] If the expression or activity of ERF109 protein is increased, then this candidate substance is a substance that can improve the tolerance of plants to abiotic stress.

[0035] In one or more embodiments, the plant is selected from one or more of the following families: Poaceae, Brassicaceae, Bromeliaceae, Orchidaceae, Arecaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, and Laminariaceae.

[0036] In one or more embodiments, the abiotic stress is selected from one or more of osmotic stress, drought stress, or salt stress. Attached Figure Description

[0037] Figure 1 Transcriptome analysis of Arabidopsis thaliana induced by osmotic stress. A: A simplified schematic diagram of transcriptome sequencing of wild-type Arabidopsis samples subjected to simulated osmotic stress. B: After sequencing, mannitol-simulated osmotic stimulation and MS control samples were compared and analyzed to screen for differentially upregulated or downregulated genes. The screening criteria were expression level log2FC greater than or equal to 2 and p-value less than 0.05. C: Heatmap analysis of differentially upregulated genes compared to the control group at different time points under osmotic stress. Mannitol represents osmotic induction, and MS represents the 1 / 2 MS control group.

[0038] Figure 2 ERF109 is an osmotically induced IEG in Arabidopsis thaliana. A: Time-series analysis of gene expression of differentially expressed genes upregulated by osmosis at 0, 5, 10, 30, 90, 180, and 360 minutes. ERF109, CML37, ABF2, and COR15A represent four gene clusters with similar expression patterns. B: Relative expression levels of differentially expressed genes screened by transcriptome sequencing at different time points under osmotic stress. Genes such as ERF109 (left in B) represent osmotically induced immediate early response genes (IEGs), and genes such as COR15A (right in B) represent osmotically induced late response genes (LRGs).

[0039] Figure 3ERF109 is rapidly transcribed by osmosis and does not rely on de novo protein synthesis. A: LUC fluorescence signal in transgenic seedlings containing the ERF109 promoter-driven LUC reporter gene under osmotic stress. 1 / 2 MS liquid medium served as a control. B: Effect of protein synthesis inhibitors on osmotically induced early-response genes (left B) and late-response genes (right B). Samples before osmotic stimulation served as controls. Values ​​are mean ± SD (n = 3 independent experiments). In B, *p<0.05, **p<0.01, ***p<0.001, Student's t-test.

[0040] Figure 4 For Ca 2+ Signal-mediated transcription of early response genes in permeation. Col-0 wild-type Arabidopsis seedlings grown on normal culture medium for 8 days were treated with Ca, with or without (control) Ca. 2+ Two hours after pretreatment with the ion inhibitor (BAPTA-AM), mannitol was applied to simulate osmotic stress. The relative expression levels of genes responding to early osmosis were measured. Values ​​are mean ± SD (n = 3 independent experiments).

[0041] Figure 5 A: Venn diagram of differentially upregulated genes and CAMTA1 and CAMTA3 target genes induced by osmotic stress. Man-up DEGs refer to genes differentially upregulated by osmotic stress in transcriptome sequencing. B: Distribution of 508 target genes of CAMTA1 and CAMTA3 at different osmotic response time points.

[0042] Figure 6 To investigate CAMTA regulation of osmotic stress-induced transcription of the IEG gene ERF109. A: Distribution of CAMTA binding sites within the 2500 bp upstream ATG promoter region of the Arabidopsis thaliana ERF109 gene. Green sequences represent CAMTA binding element motifs, and red sequences represent dotted spikes of the binding elements. B: EMSA experiment demonstrating in vitro binding of CAMTA1-GST protein to ERF109 promoter region binding elements. C: Experimental design of a dual-luciferase reporter system for CAMTA1, CAMTA2, and ERF109 promoters. D: Statistical analysis of the dual-luciferase activity ratio of CAMTA1 and CAMTA2 binding to the ERF109 promoter. LUC represents the CAMTA-induced ERF109 transcription level, and REN represents the 35S promoter-driven transcription level as a control. LUC / REN represents the transcriptional activation activity of CAMTA on ERF109.

[0043] Figure 7The loss of function of CAMTA inhibited the response of Arabidopsis thaliana to osmotic stress. A: Relative expression levels of osmotic-inducing genes in Col-0 wild-type and camta1 / 2, camta1 / 3, and camta2 / 3 mutants. Values ​​are mean ± SD (n = 3 independent experiments). B: Growth of camta mutants under osmotic stress. Wild-type and mutant seedlings were transferred to MS medium containing 75 mmol mannitol after approximately 4 days and continued to grow for approximately 10 days, with 1 / 2 MS serving as a control. C: Fresh weight of aerial leaves from section B above. Values ​​are mean ± SD (n = 12 seedlings). In C, *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.

[0044] Figure 8 This study analyzes promoter elements of osmotically induced IEGs. A: Expression heatmap of 40 transcription factors in osmotically induced IEGs at different time points after osmotic stimulation. B: Conserved GCCG motifs bound by the ERF transcription factor family. C: Analysis of promoter cis-elements of 40 osmotically induced rapid-response transcription factors. Promoter sequences of 40 transcription factors were retrieved and analyzed using online databases. The ERF binding motif refers to the element recognized and bound by the ERF transcription factor family.

[0045] Figure 9 ERF109 regulates the expression of some transcription factors in IEGs. A: Distribution of ERF109 binding sites within the 2500bp upstream ATG promoter region of the IEG gene WRKY15, induced by osmosis. Green sequences represent ERF109 binding element motifs, and red sequences represent dotted spikes of the binding elements. B: EMSA experiment demonstrating in vitro binding of ERF109-GST protein to the WRKY15 promoter region binding elements. C: Experimental design of a dual-luciferase reporter system for ERF109 and the promoters of IEG genes WRKY15 and WRKY33. D: Statistical analysis of the dual-luciferase activity ratio of ERF109 binding to the promoters of IEG transcription factors WRKY15 and WRKY33. LUC represents the induced transcriptional level of WRKY15 and WRKY33, and REN represents the transcriptional level driven by the 35S promoter, serving as a control. LUC / REN represents the transcriptional activation activity of ERF109 on WRKY15 and WRKY33.

[0046] Figure 10The erf109 / 113 mutant is sensitive to osmotic stress. A: Relative expression of IEGs in Col-0 wild-type and erf109 / 113 mutants under mannitol-simulated osmotic stress. B: Relative expression of LRGs in Col-0 wild-type and erf109 / 113 mutants under mannitol-simulated osmotic stress. The values ​​in A and B are mean ± SD (n = 3). C: Growth of Col-0 wild-type and erf109 / 113 mutants on mannitol-simulated osmotic stress medium.

[0047] Figure 11 The fresh weight statistics of wild-type Arabidopsis thaliana Col-0 overexpressing ERF109 under mannitol-simulated osmotic stress are presented. A: B: Fresh weight statistics of aboveground leaves in A above. *p<0.05, **p<0.01, ***p<0.001, Student's t-test.

[0048] Figure 12 For Ca 2+ -CAMTA-ERF109 transcriptional cascade module regulates osmotic stress-induced transcriptome reprogramming.

[0049] Figure 13 This is an infographic of the pCAMBIA1300 overexpression vector. Detailed Implementation

[0050] Studies have found that plants, when subjected to osmotic or drought stress, can induce intracellular calcium levels in their cytoplasm. 2+ Increased ion concentration activates Ca 2+ Signals that promote transcriptional responses in downstream drought-responsive genes. Although it has been recognized that Ca... 2+ Signaling plays a crucial role in osmotic stress responses, but the mechanisms by which plants regulate numerous downstream drought-responsive genes at the transcriptional level remain unclear. The inventors discovered a mimicking drought-induced Ca2+ signaling pathway. 2+ The signal can activate Ca 2+ The ion-dependent transcription factor CAMTA mediates the rapid transcription of Immediate Early Genes (IEGs) under drought stress, such as ERF109. ERF109 and other IEGs are genes that are rapidly induced and quickly decline in the early stages of drought stress (10 to 30 minutes). Their transcription is dependent on Ca2+. 2+ Ionic signals, independent of de novo protein synthesis. IEGs such as ERF109 can affect the transcription of late-response genes (LRGs) such as P5CS1 and COR15A. 2+The CAMTA-ERF109 transcriptional cascade module mediates transcriptome reprogramming in plant cell nuclei under drought conditions, enabling plants to gradually adapt to drought stress through the rapid induction of IEG genes to regulate LRG transcription. The loss of function of IEGs such as CAMTA and ERF109 affects plant responses to drought stress. Therefore, enhancing the expression of CAMTA or ERF109 through molecular breeding techniques can promote the expression of drought-resistant genes in plants under drought stress, leading to the development of drought-resistant and stress-tolerant new crop varieties.

[0051] the term

[0052] In this article, "abiotic stress" refers to negative stress responses caused by abiotic factors, including osmotic stress, salt stress, high temperature stress, or drought stress. "Osmotic stress" refers to changes in environmental factors that prevent plants from obtaining sufficient water. Common osmotic stress factors include drought, salt damage, and frost damage. In this article, mannitol or sorbitol are used as osmotic agents and dehydrating agents.

[0053] In this article, "salt tolerance" and "salt stress tolerance" can be used interchangeably.

[0054] As used herein, “plant” refers to a plant in which CAMTA or ERF109 or their homologs exist. Preferably, the term "plant" includes (but is not limited to): grasses such as rice (Oryza sativa), millet (Setaria italica), foxtail grass (Setaria viridis), Panicum hallii var. hallii, millet (Panicum miliaceum), Dichanthelium oligosanthes, maize (Zea mays), sorghum (Sorghum bicolor), dandelion (Eragrostis curvula), barley (Hordeum vulgare), brachypodium distachyon, and wheat (Triticum aestivum); cruciferous plants such as Arabidopsis thaliana; bromeliads such as pineapple (Ananas comosus); orchids such as dendrobium catenatum; and palms such as date palm (Phoenix palm). Plants such as *Dactylifera*, oil palm (*Elaeis guineensis*); water lily family plants, such as lotus (*Nelumbo nucifera*); poppy family plants, such as *Macleaya cordata*; myrtaceae family plants, such as *Syzygium oleosum*; Rubiaceae family plants, such as *Coffea canephora*; Solanaceae family plants, such as potato (*Solanum tuberosum*), tobacco (*Nicotiana tabacum*), beautiful tobacco (*Nicotiana asylvestris*), and Gapsinam chilense; Sterculiaceae family plants, such as cacao (*Theobroma cacao*); legume family plants, such as peanut (*Arachis hypogaea*); or phycophyceae family plants, such as phycocyanin (*Zostera marina*).

[0055] In this document, "reprogramming factor" refers to a bioactive polypeptide (or a nucleic acid, such as DNA or RNA, encoding it) or small molecule or mixture thereof, which acts on cells to alter transcription and, upon expression, reprograms somatic cells into different cell types, or pluripotent or multipotent. In some embodiments, the reprogramming factor may be non-integrating, i.e., provided by the somatic cell in a form that does not lead to the integration of exogenous DNA into the genome of the recipient cell.

[0056] In this article, "CAMTA-ERF109 signal path" refers to "CAMTA1 and CAMTA2", "CAMTA2 and CAMTA3", "CAMTA1 and CAMTA3" or "CAMTA1, CAMTA2 or CAMTA3". CAMTA1, CAMTA2 and CAMTA3 are highly homogeneous and have a certain degree of functional redundancy.

[0057] As used herein, “upregulation” includes: promotion, overexpression, enhancement, etc., which are statistically significant or marked upregulation, promotion, enhancement, or enhancement, such as upregulation, promotion, enhancement, or enhancement of 20%, 40%, 60%, 80%, 90%, or higher.

[0058] As used herein, “downregulation” includes: weakening, reducing, lowering, inhibiting; indicating significant downregulation, weakening, lowering, inhibiting, such as downregulation, weakening, lowering, inhibiting or downregulation by 20%, 40%, 60%, 80%, 90% or lower.

[0059] As used herein, "abiotic stress tolerance" refers to a plant's ability to tolerate abiotic stress. Typically, the tolerance of a test plant is assessed by comparing it with a control plant under the same abiotic stress environment. A test plant is generally considered to have better tolerance when it grows and develops better, has a higher survival rate, and survives longer under abiotic stress than the control plant. For example, salt tolerance is assessed by comparing test plants with control plants under the same high-salt environment.

[0060] As used herein, “high tolerance to abiotic stress” refers to a statistically significant increase in the tolerance of a plant (e.g., a modified plant) to abiotic stress compared to that of similar or identical plants, such as an increase in survival rate of 5%, 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0061] As used herein, “high expression or high activity” means that the expression or activity of a target gene / protein in a specific plant (e.g., a modified plant) is statistically significantly increased compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0062] As used herein, “low expression or low activity” means that the expression or activity of a target gene / protein in a particular plant (e.g., a modified plant) is statistically significantly reduced compared to the average expression or activity of the same type or plant species, such as by 10%, 20%, 40%, 60%, 80%, 90%, or less.

[0063] As used herein, "loss-of-function mutations" include those that cause a target protein to lose its function, such as through mutations, deletions, or insertions in key regions of its protein chain. In some approaches, gene editing involves the insertion, deletion, or mutation of bases in the gene encoding the target protein, causing premature termination of translation of the target protein.

[0064] As used herein, “gain-of-function mutations” include: enabling the normal expression of a target protein that was previously restricted or not expressed; and in some ways, reverting the expression of the target protein by reversing bases in the target protein coding gene (e.g., reverting to a sequence identical or degenerate to the wild type) through gene editing.

[0065] As used herein and as will be understood by those skilled in the art, selecting an appropriate “control plant” is a routine part of experimental design and may include a corresponding wild-type plant or a transgenic plant without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants may also be individuals from which the transgenic plant has been lost due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0066] CAMTA-ERF109 signaling pathway

[0067] In this invention, the term "(signaling) pathway" refers to a signaling system formed by the mutual censorship or interaction of a series of proteins or genes, which generally leads to the occurrence of certain cellular events. The CAMTA-ERF109 signaling pathway includes (but is not limited to): the CAMTA (calmodulin-binding transcription factor) gene (and / or its encoded protein), the ERF109 (ethylene responsive factor 109) gene (and / or its encoded protein), and optionally the ERF113 (ethylene responsive factor 113) gene (and / or its encoded protein).

[0068] When used as targets for artificial regulation or in the creation of screening systems, the proteins or encoding genes mentioned above can be naturally occurring, such as those purified and isolated from mammals; or they can be recombinantly prepared, for example, recombinant proteins can be produced using conventional gene recombination techniques. Furthermore, any variations that do not affect the biological activity of these proteins are acceptable, such as derivatives or variants whose function remains unchanged.

[0069] The variant forms of the signaling pathway proteins described above are also included in this invention, including (but 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 additions or deletions 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. Any protein with high homology to the described polypeptide and having the same function as the polypeptide is also included in this invention. Polypeptides derived from species other than Arabidopsis thaliana that have high homology to the polypeptide sequence or play the same or similar role in the same or similar regulatory pathways are also included in this invention.

[0070] Homologs of the signaling pathway genes / proteins described above are also included in this invention. It should be understood that while this invention preferably studies corresponding signaling pathway genes / proteins obtained from the specific species Arabidopsis thaliana, other polypeptides or genes obtained from other species that are homologous to the aforementioned signaling pathway genes / proteins (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0071] Plant Transformation

[0072] Based on the inventors' new discovery, the present invention provides a method for improving plants, the method comprising: regulating the expression or activity of CAMTA protein and / or ERF109 protein, thereby enhancing the plant's tolerance to abiotic stress.

[0073] On the one hand, the present invention provides a method for enhancing plant tolerance to abiotic stresses, including upregulating the expression or activity of CAMTA protein.

[0074] On the other hand, the present invention provides a method for enhancing plant tolerance to abiotic stresses, including upregulating the expression or activity of the ERF109 protein. Optionally, it also includes upregulating the expression or activity of the ERF113 protein.

[0075] It should be understood that, once the function of the CAMTA-ERF109 signaling pathway (optionally including upstream and downstream genes) is known, various methods well known to those skilled in the art can be used to regulate the CAMTA-ERF109 signaling pathway. For example, various methods well known to those skilled in the art can be used to upregulate or overexpress CAMTA.

[0076] In this invention, the upregulators of the CAMTA-ERF109 signaling pathway protein or its encoding gene 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 the signaling pathway protein, improve the stability of the signaling pathway protein, upregulate the expression of the signaling pathway gene, increase the effective duration of the signaling pathway protein, or increase the phosphorylation / activation level of each protein can be used in this invention as a substance useful for upregulating CAMTA and / or ERF109 or the signaling pathway. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0077] In this invention, the downregulator of the CAMTA-ERF109 signaling pathway protein or its encoding gene refers to any substance that can reduce the activity of the CAMTA-ERF109 signaling pathway protein, reduce the stability of the signaling pathway protein or its encoding gene, downregulate the expression of other signaling pathway proteins, reduce the effective duration of the signaling pathway protein, inhibit the transcription and translation of the signaling pathway gene, or reduce the phosphorylation / activation level of each protein. These substances can all be used in this invention as substances useful for downregulating CAMTA and / or ERF109. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the signaling pathway gene; or a gene editing reagent that specifically edits CAMTA and / or ERF, etc.

[0078] This invention also provides a method for downregulating CAMTA and / or ERF109 in the CAMTA-ERF109 signaling pathway in plants, including targeted mutation, gene editing, or gene recombination to achieve downregulation. For ERF, as a more specific implementation, a CRISPR / Cas9 system is used for gene editing to knock out or downregulate the target gene. Suitable sgRNA target sites lead to higher gene editing efficiency; therefore, suitable target sites can be designed and identified before gene editing. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments.

[0079] As another embodiment of the present invention, a method for upregulating the expression of ERF109 in plants is provided, comprising: (1) transferring a vector that highly expresses the ERF109 gene into plant cells, tissues, organs, or seeds to obtain plant cells, tissues, organs, or seeds transformed with the ERF gene high-expression vector molecule; (2) regenerating plants from the plant cells, tissues, organs, or seeds transformed with the interfering molecule obtained in step (1). Preferably, the method further comprises: (3) selecting plant cells, tissues, or organs transformed with the vector; and (4) regenerating plants from the plant cells, tissues, or organs in step (3).

[0080] In this invention, it was discovered that ERF109 plays a positive regulatory role in the signaling pathway. Its promoter region interacts with CAMTA and is thus subject to transcriptional regulation by the latter. ERF109 can also regulate other early corresponding transcription factors, which in turn can regulate the transcription of downstream transcription factors or stress response genes, thereby enhancing the plant's perception and adaptation to osmotic stimuli.

[0081] Plant targeted screening or selective screening

[0082] Having learned about the CAMTA-ERF109 signaling pathway and the functions of its genes / proteins, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can target and regulate plant tolerance to abiotic stresses by modulating this mechanism.

[0083] Therefore, the present invention provides a method for targeted selection of plants, the method comprising: identifying the expression or activity of CAMTA-ERF109 signaling pathway proteins in test plants, the proteins including CAMTA and / or ERF109, the proteins optionally also including ERF113; if the proteins have one or more characteristics selected from the group consisting of: increased CAMTA expression or activity in the test plant, increased ERF109 expression or activity in the test plant, and increased ERF113 expression or activity in the test plant.

[0084] The present invention provides a method for screening substances that improve plant tolerance to abiotic stress, comprising: (1) adding candidate substances to a system expressing CAMTA-ERF109 signaling pathway proteins, wherein the proteins include CAMTA and / or ERF109, and optionally also include ERF113; (2) detecting the system and observing the expression or activity of CAMTA-ERF109 signaling pathway proteins therein: if CAMTA expression or activity is increased, then the candidate substance is a substance that improves plant tolerance to abiotic stress; if ERF109 expression or activity is increased, then the candidate substance is a substance that improves plant tolerance to abiotic stress.

[0085] 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.

[0086] Through large-scale screening, a class of potential substances that specifically act on the CAMTA-ERF109 signaling pathway or its pathway genes / proteins can be obtained, which have a regulatory effect on plant tolerance to abiotic stress.

[0087] The present invention has the following beneficial effects:

[0088] This invention uses molecular breeding technology to enhance the expression of CAMTA and / or ERF109, thereby promoting the expression of drought-resistant genes in plants under drought stress and cultivating new drought-resistant and stress-tolerant crop varieties.

[0089] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention. The methods and reagents used in the embodiments are conventional methods and reagents in the art, unless otherwise stated. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0090] Materials and Methods

[0091] All Arabidopsis materials used in this invention were of the Columbia wild type (Col-0). The three T-DNA insertion mutants, camta1 (SALK_008187C), camta2 (SALK_007027), and camta3 (SALK_001152C), were ordered from the Arabidopsis Mutant Library Center (NASC), and subsequently, three double mutant materials, camta1 / 2, camta1 / 3, and camta2 / 3, were obtained through hybridization. The homozygous erf109 (SALK_150614) mutant and the Col-0 background ERF109pro:LUC reporter strain were obtained from the Shanghai Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. In this paper, the double mutant erf109 / 113 was obtained by gene editing to knock out ERF113 based on the erf109 (SALK_150614) mutant. In this study, the ERF109-OE (ERF109 overexpression) material was obtained by overexpressing ERF109 in wild-type Arabidopsis thaliana Col-0.

[0092] 1. Transcriptome sequencing of Arabidopsis thaliana

[0093] 1) Sow Col-0 wild-type Arabidopsis thaliana seeds on 1 / 2 MS vertical medium and grow them in a light incubator for about 8 days;

[0094] 2) Prepare a 1 / 2 MS liquid culture medium solution containing 300 mmol mannitol to simulate osmotic stress, and use the 1 / 2 MS liquid culture medium as a control;

[0095] 3) Use a small spray bottle to evenly spray the two solutions mentioned above onto the seedlings in the petri dish, and continue to place them in the light incubator for growth;

[0096] 4) After spraying the treatment solution simulating osmotic stress, samples were taken at time points of 5, 10, 30, 90, 180, and 360 minutes. Seedlings before spraying the treatment solution served as the untreated control group.

[0097] 5) After sampling, the samples are flash-frozen in liquid nitrogen at -80℃ in preparation for RNA extraction and transcriptome sequencing.

[0098] 2. qPCR experiment

[0099] 1) First, according to the extracted RNA... The One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit (TransGene) is used for reverse transcription PCR, including reagents and procedures.

[0100] 2) Using the product of the above reverse transcription PCR as a template, set up the reaction system according to the system of the real-time PCR kit, with a volume of 10 μL;

[0101] 3) After mixing the above system samples, shake to mix evenly in a 96-well quantitative PCR plate, centrifuge, and seal with a quantitative PCR membrane;

[0102] 4) Place the above samples in a CFX96 real-time PCR instrument (Bio-Rad), with Arabidopsis thaliana Actin as an internal control, set up 3 replicates, and run according to the routine real-time PCR procedure;

[0103] 5) After the operation is complete, copy the data for quantitative analysis.

[0104] Example 1: Osmotic stress can induce a rapid response in the transcriptome.

[0105] To investigate the changes in transcriptional levels in plant cells during the early stages of osmotic stress, Arabidopsis seedlings were treated with mannitol to simulate osmotic stress, followed by transcriptome sequencing analysis. It is known that changes in transcriptional levels in plant and animal cells can be detected as early as approximately 10-30 minutes after stimulation. Therefore, in this embodiment, sampling was performed at 5 minutes after stress stimulation as the first time point, followed by sampling at time gradients of 10 minutes, 30 minutes, 90 minutes, 3 hours, and 6 hours. It is known that the expression of genes responding to osmotic stress primarily peaks and gradually declines after 3 hours of osmotic stress stimulation. Furthermore, because the sampling time is very short, starting at 5 minutes after stimulation, it is impossible to treat the seedlings by transferring them from normal culture dishes to simulated osmotic culture dishes. Hydroponic treatment or liquid immersion treatment would introduce too many other interfering factors. Therefore, this embodiment optimized the method of osmotic stress stimulation of seedlings. To minimize interference from unnecessary factors such as mechanical stimulation, a gentle spraying method was used to apply osmotic stress treatment. Seedlings grown on normal culture dishes for 8 days were evenly sprayed with a treatment solution containing 300 mM mannitol using a small spray bottle, ensuring the liquid was evenly sprayed over the entire surface of the seedlings. Samples were taken at predetermined time points (i.e., 5 minutes, 10 minutes, 30 minutes, 90 minutes, 3 hours, and 6 hours after stress stimulation). Seedlings sprayed with 1 / 2 MS liquid medium without mannitol served as a parallel control. Figure 1 A).

[0106] After obtaining read counts for transcripts from all treatment and control samples, differential expression analysis was performed on transcripts from the treatment and control groups at different time points using DESeq2 software. Genes with a fold change of more than 2 and a p-adjustment value less than 0.05 were screened. After screening, no genes (DEGs) showing significant upregulation or downregulation were identified in samples subjected to osmotic stress stimulation for 5 minutes. However, in samples subjected to osmotic stress stimulation for 10 minutes, 40 genes showed significant upregulation and 1 gene showed downregulation. Regarding the number of upregulated genes, 565 genes were upregulated at 30 minutes, 1399 at 90 minutes, and 1583 and 764 at 3 and 6 hours, respectively. Figure 1 (B). This result indicates that plant responses to osmotic stress at the transcriptional level can be detected as early as 10 minutes, and the number of differentially expressed genes gradually increases over time. For all screened differentially expressed genes, cluster analysis (Heatmap) was performed using the relative expression level value log2(ratio) of each gene, grouping genes with similar or identical expression patterns into clusters. According to the clustering results, in the left cluster, genes represented by green were rapidly expressed by osmosis within 5 to 10 minutes, after which their expression levels rapidly decreased; genes represented by purple and cyan showed high expression at 30 and 90 minutes, respectively, followed by a rapid decrease; and finally, most genes represented by yellow began to be expressed after 30 minutes and remained so for a certain period before gradually decreasing. Figure 1 C).

[0107] Example 2: ERF109 is an immediate early response gene induced by osmotic stress.

[0108] The preceding transcriptome analysis revealed that osmotic stress can induce transcriptome reprogramming in plants, and there may be some correlation between genes induced at different times. To investigate the relationship between genes that began to be induced at different times, this embodiment first performed time-series expression analysis on all differentially expressed genes and then clustered them. The clustering results showed that genes like ERF109 were induced from 5 minutes onwards, peaked at 10 minutes, and then rapidly declined. Genes represented by CML37 were induced to peak expression at 30 minutes and then began to decline. Genes such as ABF2 and COR15A, however, only began to show significant upregulation after 90 minutes. Figure 2 A). The results of qPCR experiments also showed that the expression of genes such as ERF109 and CML37 was highest around 30 minutes after osmotic stress induction, while the expression of marker genes such as COR15A and P5CS1, which are osmotic stress-induced, increased mainly after 90 minutes. Figure 2(B; primer sequences are shown in SEQ ID NO: 11-18). These results indicate that ERF109, CML37, etc. are early response genes induced by osmotic stress, while COR15A, etc. are late response genes induced by osmotic stress.

[0109] This embodiment also tested the induction of ERF109 by osmotic stress using the same ERF109pro:LUC system. The results showed that both 0.6M mannitol and sorbitol-mimicking osmotic stress stimulation activated the expression of the ERF109 promoter-driven LUC reporter gene. Figure 3 A).

[0110] Changes in the transcriptional levels of genes such as ERF109 were detected by pretreatment with the protein synthesis inhibitor cycloheximide (CHX) followed by osmotic stress. The results showed that the responses of genes such as ERF109 and CML37 to osmotic stress were not affected after CHX pretreatment, while the induction levels of genes such as COR15A were significantly decreased after CHX pretreatment compared to the untreated group. Figure 3 B).

[0111] Based on these results, genes that were rapidly induced by hyperosmotic stress 30 minutes before treatment, such as ERF109, are osmotic stress-induced IEGs in plant cells, while genes that were only induced after 90 minutes of osmotic treatment, such as COR15A, are osmotic stress-induced LRGs.

[0112] Example 3: Ca 2+ Signals are involved in regulating the rapid transcriptional response induced by osmotic stress.

[0113] This embodiment tested Ca under osmotic stress. 2+ Effects of the ion signaling inhibitor BAPTA-AM on IEG transcription. Results showed that the induction levels of genes such as CML37 and ERF109 decreased after the inhibitor was applied, indicating that Ca... 2+ The signal may be involved in the regulation of IEGs. Figure 4 The inventors hypothesize that the rapid response of IEGs such as ERF109 induced by osmotic stress may be related to Ca. 2+The regulation of ion-dependent CAMTA (Calmodulin-binding transcription activator) was investigated. By analyzing and comparing potential downstream target genes of CAMTA1 and CAMTA3 in published data with genes upregulated by osmotic stress in the aforementioned transcriptome sequencing data, it was found that among 2130 differentially regulated genes induced by osmotic stress, 256 were target genes of CAMTA1 and 294 were target genes of CAMTA3. Interestingly, more than half of the target genes of CAMTA1 and CAMTA3 were induced to express 30 minutes and 90 minutes before osmotic stress stimulation, respectively, and ERF109 was also one of the potential target genes of CAMTA. Figure 5 (AB). This point is consistent with the inventors' hypothesis that ERF109 and other IEGs that are rapidly activated under osmotic stress may be target genes of CAMTA.

[0114] To verify this hypothesis, cis-elements were analyzed in the promoter region sequence of ERF109. The results showed that the 2500 bp promoter region upstream of the ATG in the ERF109 gene contained multiple motifs for CAMTA transcription factor recognition and binding. Figure 6 A). Subsequently, based on the results of the gel migration (EMSA) assay, the prokaryotically induced CAMTA1-GST protein could bind to a Cy5-labeled DNA probe containing the p1 site sequence of the ERF109 promoter. The addition of cold probes without Cy5 labeling competitively inhibited their binding, while point-mutated probes did not, indicating that the binding of CAMTA1 protein to the ERF109 promoter is specific. Figure 6 (B). Next, we verified the binding of the two using a dual-luciferase reporter assay. Plasmids with CAMTA1 or CAMTA2 driven by the 35S promoter and LUC driven by the ERF109 promoter were transformed into Agrobacterium and co-transfected into tobacco leaves. The ratio of LUC to REN enzyme activity in tobacco leaves was measured. The results showed that co-expression of CAMTA1 or CAMTA2 and ERF109pro:LUC enhanced the LUC / REN specific activity. Figure 6 These results indicate that the CAMTA transcription factor can bind to the promoter of ERF109 and activate its transcription.

[0115] Since CAMTA transcription factors can regulate the transcription of IEGs such as ERF109, if osmotic-induced IEG transcription is suppressed, osmotic-induced transcriptome reprogramming may also be affected. This study examined the transcriptional levels of IEGs and LRGs under simulated osmotic stress treatment using combined mutants of CAMTA1, CAMTA2, and CAMTA3 (camta1 / 2, camta1 / 3, camta2 / 3). The results showed that, in CAMTA dual-mutant materials, the induction level of ERF109 was significantly lower than that of the wild type after 10 minutes of osmotic stress stimulation. Figure 7 (A). The expression of the late-response gene P5CS1, induced by osmotic stress 3 hours after treatment, was also significantly reduced in all three dual-mutation materials. This indicates that the immediate early-response gene expression induced by osmotic stress, such as ERF109, is affected by Ca. 2+ Regulation by the ion-dependent transcription factor CAMTA. This also indicates that transcriptional changes in IEGs can affect the transcription of late-response genes, thereby influencing transcriptional reprogramming in plant cell nuclei under osmotic stress. To investigate the association between osmotic stress-induced transcriptional reprogramming and plant tolerance to osmotic stress, this study examined the growth of camta1 / 2, camta1 / 3, and camta2 / 3 mutants under osmotic stress. Treatment of CAMTA double mutants and wild-type Arabidopsis with 75 mM mannitol to simulate osmotic stress showed that the growth of the aboveground leaves of the double mutant material was more significantly inhibited. Statistical results of the aboveground fresh weight of the mutants and wild-type under osmotic treatment also indicated that the growth of the mutants was more sensitive to osmotic stress. Figure 7 (BC). Based on the above results, it is indicated that Ca 2+ Signal-mediated cellular transcriptome reprogramming is biologically significant for plant adaptation to osmotic stress.

[0116] Example 4: ERF109 is a key factor in osmotic stress-induced transcriptional reprogramming.

[0117] Based on the above results, it was found that under osmotic stress, plant cells can sense Ca2+. 2+ Changes in ion concentration activate CAMTA, regulating the transcription of IEGs and the osmotic stress-induced transcriptional reprogramming process, thus affecting plant tolerance to osmotic stress. Although the aforementioned experiments show that CAMTA can mediate the rapid activation of IEGs under osmotic stress, how IEGs affect the transcriptional reprogramming process remains unclear. Based on the data from the previous transcriptome expression pattern clustering (… Figure 1(C) It was found that the induction time of different differentially expressed genes followed a certain pattern. Rapidly responding genes such as ERF109, mentioned earlier, could be induced and activated within a short time, but the duration was short. However, with the continuation of osmotic stress stimulation, a large number of differentially expressed genes had been induced and activated by 30 minutes. Analysis of these early-responding differentially expressed genes revealed that 40 transcription factors were significantly induced before 30 minutes. From the expression heatmap results ( Figure 8 Of the transcription factors, ERF109 showed the strongest induction at 10 minutes, except for the heat shock transcription factor HSFB2A, and then rapidly declined. Subsequently, transcription factors including WRKY15, WRKY33, and ERF8 began to be induced at 30 minutes. Figure 8 Analysis of the promoter region sequences of these 40 early response transcription factors revealed an enrichment of GCCG elements that can be recognized by ERF transcription factor family proteins. Figure 8 (B). Among them, the promoters of six transcription factors, including WRKY15, WRKY33, WRKY30, ERF4, ERF15, and MYB51, are predicted to contain motifs that ERF109 can bind to. Figure 8 (C) may be a potential downstream target gene of ERF109. Based on the above results, the inventors hypothesize that ERF109 can regulate the activation of other early response transcription factors such as WRKY15, which in turn can regulate other downstream genes, amplifying the permeation signal at the transcriptional level in a transcriptional cascade manner.

[0118] EMSA experiments confirmed that the ERF109-GST protein can bind to the GCCG motif in the P2 region of the WRKY15 promoter; however, point mutations weaken this binding. Figure 9 (AB). The results of the dual-luciferase reporter gene assay further demonstrate that ERF109 can bind to the promoters of WRKY15 and WRKY33 and activate their transcriptional expression. Figure 9 (CD). Based on these results, ERF109 is not only affected by Ca... 2+ Ion-dependent CAMTA regulation can also regulate the activation of other transcription factors in IEGs. Subsequently, the phenotype of the ERF109 loss-of-function mutant under osmotic stress was detected and validated. In previous studies, the inventors constructed dual mutants of ERF109 and its family protein ERF113, and used mannitol to simulate osmotic stress treatment to detect the expression of IEGs and LRGs. qPCR results showed that the induction levels of osmotic stress-induced rapid response genes such as WRKY15 and WRKY33 in the mutants were significantly lower than in the wild type. Figure 10The gene COR15A, a late-stage response gene induced by osmotic stress, was also affected, with its induction level significantly reduced in the mutant. Figure 10 B). Furthermore, similar to the CAMTA mutant, the double mutants of ERF109 and ERF113 showed significantly inhibited seedling growth under osmotic stress. Figure 10 Furthermore, by overexpressing ERF109 in wild-type Arabidopsis thaliana Col-0, it was found that its growth under mannitol-simulated osmotic stress was better than that of the control Col-0 and the mutant erf109 / 113. Fresh weight statistics showed that the fresh weight of the ERF109-overexpressing material under osmotic stress was greater than that of the wild-type and the mutant. Figure 11 This indicates that upregulation of ERF109 expression can improve the osmotic stress tolerance of Arabidopsis thaliana. These results suggest that the function of ERF109 is crucial for plant adaptation and tolerance to osmotic stress. It also indicates that Ca... 2+ The CAMTA-ERF109 transcriptional module mediates transcriptional reprogramming under Arabidopsis thaliana osmotic stimulation, with ERF109 being a key regulator in this process.

[0119] In summary, upon sensing osmotic stress, the transient calcium level inside plant cells... 2+ Ion signals can activate Ca 2+ The activity of the ion-dependent transcription factor CAMTA regulates the increased transcriptional levels of rapidly responding genes such as ERF109; and the rapidly induced upregulated transcription factors in IEGs such as ERF109 can further activate the transcription of downstream related transcription factors or stress-response genes such as P5CS1, thereby regulating the perception and adaptation of plant cells to osmotic stimuli through a transcriptional cascade amplification process. Figure 12 Ca 2 + The CAMTA-ERF109 transcriptional cascade module mediates transcriptome reprogramming in plant cells under osmotic stress, and the process of transcriptome reprogramming can influence plant adaptation to osmotic stress. Based on the results of this study, Ca... 2+ The CAMTA-ERF109 transcriptional cascade module can serve as a potential modification site for molecular breeding. By enhancing the transcriptional activity of this module, the overall stress response genes of plants under osmotic or drought stress can be improved, thereby breeding new crop varieties that are more resistant to stress.

[0120] Part of the sequence in this article

[0121] <1CAMTA1 AT5G09410

[0122]

[0123] <2CDS

[0124]

[0125] <3CAMTA2 AT5G64220

[0126]

[0127] <4CDS sequence

[0128]

[0129] <5CAMTA3 AT2G22300

[0130]

[0131] <6CDS sequence

[0132]

[0133] <7ERF109 AT4G34410

[0134] MHYPNNRTEFVGAPAPTRYQKEQLSPEQELSVIVSALQHVISGENETAPCQGFSSDSTVISAGMPRLDSDTCQVCRIEGCLGCNYFFAPNQRIEKNHQQEEEITSSSNRRRESSPVAKKAEGGGKIRKRKNKKNGYRGVRQRPWGKFAAEIRDPKRATRVWLGTFETAEDAARAYDRAAIGFRGPRAKLNFPFVDYTSSVSSPVAADDIGAKASASASVSATDSVEAEQWNGGGGDCNMEEWMNMMMMMDFGNGDSSDSGNTIADMFQ

[0135] <8CDS sequence

[0136] atgcattatcctaacaacagaaccgaattcgtcggagctccagccccaacccggtatcaaaaggagcagttgtcaccggagcaagagctttcagttattgtctctgctttgcaacacgtgatctcaggggaaaacgaaacggcgccgtgtcagggtttttccagtgacagcacagtgataagcgcgggaatgcctcggttggattcagacacttgtcaagtctgtaggatcgaaggatgtctcggctgtaactactttttcgcgccaaatcagagaattgaaaagaatcatcaacaagaagaagagattactagtagtagtaacagaagaagagagagctctcccgtggcgaagaaagcggaaggtggcgggaaaatcaggaagaggaagaacaagaagaatggttacagaggagttaggcaaagaccttggggaaaatttgcagctgagatcagagatcctaaaagagccacacgtgtttggcttggtactttcgaaaccgccgaagatgcggctcgagcttatgatcgagccgcgattggattccgtgggccaagggctaaactcaacttcccctttgtggattacacgtcttcagtttcatctcctgttgctgctgatgatataggagcaaaggcaagtgcaagcgccagtgtgagcgccacagattcagttgaagcagagcaatggaacggaggaggaggggattgcaatatggaggagtggatgaatatgatgatgatgatggattttgggaatggagattcttcagattcaggaaatacaattgctgatatgttccagtga

[0137] <9ERF113 AT5G13330

[0138] MVSALSRVIENPTDPPVKQELDKSDQHQPDQDQPRRRHYRGVRQRPWGKWAAEIRDPKKAARVWLGTFETAEEAALAYDRAALKFKGTKAKLNFPERVQGPTTTTTISHAPRGVSESMNSPPPRPGPPSTTTTSWPMTYNQDILQYAQLLTSNNEVDLSYYTSTLFSQPFSTPSSSSSSSQQTQQQQLQQQQQQREEEEKNYGYNYYNYPRE

[0139] <10CDS sequence

[0140] atggtctccgctctcagccgtgtcatagagaatccgacagacccgccggtcaaacaagagcttgataaatcggatcaacatcaaccagaccaagatcaaccaagaagaagacactatagaggcgtaaggcagagaccatggggtaaatgggcggcagaaatccgcgatccaaagaaagcagcccgtgtctggctcgggactttcgagacggcagaggaagctgctttagcctatgaccgagctgccctcaaattcaaaggcaccaaggctaaactgaacttccctgaacgggtccaaggccctactaccaccacaaccatttctcatgcaccaagaggagttagtgaatccatgaactcacctcctcctcgacctggtccaccttcaactactactacttcgtggccaatgacttataaccaggacatacttcaatacgctcagttgcttacgagtaacaatgaggttgatttatcatactacacgtcgactctcttcagtcaacctttttcaacgccttcttcatcttcttcttcctcccaacagacgcagcaacagcagctacaacaacaacaacagcagcgtgaagaagaagagaagaattatggttacaattattataactacccaagagaataa

[0141] <11 ERF109-qF

[0142] AGAGATCCTAAAAGAGCCACAC

[0143] <12 ERF109-qR

[0144] CAAAGGGGAAGTTGAGTTTAGC

[0145] <13 CML37-qF

[0146] GAAGGAGTTGAAAGAAGCGTTT

[0147] <14 CML37-qR

[0148] CTTACAAGCATCAACCGTACAC

[0149] <15 AtP5CS1-qF

[0150] AGCTTGATGACGTTATCGATCT

[0151] <16 AtP5CS1-qR

[0152] AGATTCCATCAGCATGACCTAG

[0153] <17 AtCOR15A-qF

[0154] CATTAGCAGATGGTGAGAAAGC

[0155] <18 AtCOR15A-qR

[0156] TCTCAGCTTCTTTACCCAATGT

Claims

1. A method for regulating plant tolerance to abiotic stress, comprising the steps of: regulating the expression or activity of CAMTA and / or ERF109 proteins in plants; wherein, The CAMTA and / or ERF109 proteins include their homologs; Preferably, the step further includes regulating the expression or activity of the ERF113 protein in the plant.

2. The method as described in claim 1, characterized in that, The regulation of the expression or activity of CAMTA and / or ERF109 proteins in plants includes: (a) Upregulation of CAMTA and / or ERF109 protein expression or activity leads to upregulation of response genes induced by abiotic stress in plants, thereby enhancing plant tolerance to abiotic stress; or (b) Downregulation of the expression or activity of CAMTA and / or ERF109 proteins leads to downregulation of response genes induced by abiotic stress in plants, thereby reducing plant tolerance to abiotic stress.

3. The method as described in claim 2, characterized in that, The regulation of ERF113 protein expression or activity in plants includes: upregulating ERF113 protein expression or activity, thereby upregulating response genes induced by abiotic stress in plants and enhancing plant tolerance to abiotic stress; or downregulating ERF113 protein expression or activity, thereby downregulating response genes induced by abiotic stress in plants and weakening plant tolerance to abiotic stress.

4. The method as described in claim 3, characterized in that, The response genes induced by abiotic stress include early response genes and late response genes; Preferably, the response gene induced by abiotic stress is selected from one or more of CML37, ERF11, COR15A, P5CS1, RD20, RD29A, ABF2, WRKY15, WRKY33, WRKY30, ERF4, ERF15 and MYB51.

5. The method as described in claim 2, characterized in that, The upregulation of CAMTA and / or ERF109 protein expression or activity includes: (1) transferring the gene encoding CAMTA and / or ERF109 protein or an expression construct or vector containing the gene into a plant; (2) performing gain-of-function mutations on CAMTA and / or ERF109 protein; (3) promoting CAMTA and / or ERF109 protein expression by expressing an enhancing promoter or a tissue-specific promoter; or (4) promoting CAMTA and / or ERF109 protein expression by an enhancer; and / or The downregulation of CAMTA and / or ERF109 protein expression or activity includes: knocking out or silencing the gene encoding CAMTA and / or ERF109 protein in plants, or inhibiting the activity of CAMTA and / or ERF109 protein.

6. The method as described in claim 5, characterized in that, The inhibition of CAMTA and / or ERF109 protein activity includes: transferring inhibitors of CAMTA and / or ERF109 gene transcription, protein expression, or protein activity into plants; preferably, it includes: silencing CAMTA and / or ERF109 with interfering molecules that specifically interfere with the expression of the gene encoding CAMTA and / or ERF109 protein, gene editing using a CRISPR system to knock out the gene encoding CAMTA and / or ERF109 protein, knocking out the gene encoding CAMTA and / or ERF109 protein by homologous recombination, or performing loss-of-function mutations on CAMTA and / or ERF109 protein in plants containing CAMTA and / or ERF109 protein.

7. The method according to any one of claims 1-6, characterized in that, The method has one or more of the following characteristics: The amino acid sequence of the CAMTA protein is selected from one or more of the following: (a) a polypeptide having the sequence shown in any one or more of SEQ ID NO: 1, 3 or 5; (b) a polypeptide derived from (a) having the polypeptide function of (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in any one or more of SEQ ID NO: 1, 3 or 5; or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the polypeptide function of (a); preferably, the polypeptide of CAMTA is encoded by a degenerate sequence of SEQ ID NO: 1, 3 or 5 or thereto; The amino acid sequence of the ERF109 protein is selected from one or more of the following: (a) a polypeptide having the sequence shown in AT4G34410; (b) a polypeptide derived from (a) having the function of the polypeptide shown in (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in (a); or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the function of the polypeptide shown in (a); preferably, the polypeptide of ERF109 is encoded by SEQ ID NO: 8 or its degenerate sequence; The amino acid sequence of the ERF113 protein is selected from one or more of the following: (a) a polypeptide having the sequence shown in AT5G13330; (b) a polypeptide derived from (a) having the function of the polypeptide of (a) formed by substitution, deletion or addition of one or more amino acid residues of the sequence shown in AT5G13330; or (c) a polypeptide derived from (a) having more than 90% homology to the polypeptide sequence of (a) and having the function of the polypeptide of (a); preferably, the polypeptide of ERF113 is encoded by SEQ ID NO: 10 or its degenerate sequence; The plants are selected from one or more of the following families: Poaceae, Brassicaceae, Bromeliaceae, Orchidaceae, Arecaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, and Laminariaceae; and The abiotic stress is selected from one or more of osmotic stress, drought stress, or salt stress.

8. The application of the CAMTA-ERF109 signaling pathway, its pathway proteins or their genes, or the coding sequence of said proteins, wherein said application is selected from: As a molecular marker for identifying plant tolerance to abiotic stress; or Molecular markers used for targeted screening of plants with tolerance to abiotic stress; or Used to screen for substances that improve plant tolerance to abiotic stress; Preferably, the plant is selected from one or more of the following families: Poaceae, Brassicaceae, Bromeliaceae, Orchidaceae, Arecaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, and Laminariaceae. Preferably, the abiotic stress is selected from one or more of osmotic stress, drought stress, or salt stress.

9. A method for targeted selection of plants, comprising the steps of: identifying the expression or activity of a CAMTA-ERF109 signaling pathway protein in a test plant, said protein comprising CAMTA and / or ERF109, said protein optionally further comprising ERF113; if said protein has one or more characteristics selected from the group consisting of, then the test plant is a plant with abiotic stress tolerance: The CAMTA expression or activity of the tested plants was increased. The ERF109 expression or activity of the tested plants was increased, and The ERF113 expression or activity of the tested plants was increased. Preferably, the plant is selected from one or more of the following families: Poaceae, Brassicaceae, Bromeliaceae, Orchidaceae, Arecaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, and Laminariaceae. Preferably, the abiotic stress is selected from one or more of osmotic stress, drought stress, or salt stress.

10. A method for screening substances that enhance plant tolerance to abiotic stress, comprising: (1) Add the candidate material to a system expressing CAMTA-ERF109 signaling pathway protein, wherein the protein includes CAMTA and / or ERF109, and optionally also includes ERF113; (2) Detect the system and observe the expression or activity of CAMTA-ERF109 signaling pathway proteins: If CAMTA protein expression or activity is increased, then this candidate substance is a substance that can improve the tolerance of plants to abiotic stress. If the expression or activity of ERF109 protein is increased, then this candidate substance is a substance that can improve the tolerance of plants to abiotic stress. Preferably, the plant is selected from one or more of the following families: Poaceae, Brassicaceae, Bromeliaceae, Orchidaceae, Arecaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, and Laminariaceae. Preferably, the abiotic stress is selected from one or more of osmotic stress, drought stress, or salt stress.

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