A method for eliminating the synergistic damage of drought environment and plant fusarium diseases

Abstract

The application discloses a method for eliminating the synergistic harm of drought environment and fusarium disease of plants. TaMPK3 The application obtains a gene edited wheat by knocking out a gene in wheat. TaMPK3 Compared with a receptor control, TaMPK3 The gene edited wheat has weakened and / or eliminated the trend of aggravation of fusarium disease under drought conditions and / or the trend of aggravation of drought damage under fusarium disease stress. The TaMPK3 protein and the coding gene thereof can regulate the connection between fusarium disease and drought environment in plants, the connection between fusarium disease and drought environment can be weakened and / or eliminated by reducing the content and / or activity of the TaMPK3 protein in the target plants (such as knocking out the TaMPK3 gene), and then the fusarium disease resistance of the plants under drought conditions or the drought resistance of the plants under fusarium disease stress is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for eliminating the synergistic harm of drought environment and plant Fusarium diseases. Background Technology

[0002] Wheat stem rot is a serious and difficult-to-control soil-borne fungal disease, mainly caused by *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium moniliforme*, and *Fusarium xanthophyte*, leading to typical stem rot symptoms. In recent years, the frequency of outbreaks has been increasing in the Huang-Huai-Hai wheat region of China, Australia, and the Pacific Northwest of the United States, causing significant wheat yield losses. Statistics show that in Henan Province, my country, stem rot caused an average annual yield reduction of 36,000 tons from 2017 to 2021, with a peak of 44,000 tons. In 2020, the affected area in Shandong Province reached 800,000 hectares, with whitehead rates reaching 30%-50% in severely affected areas. The symptoms of stem rot are highly insidious, primarily infecting the base of the wheat stem, making it difficult to detect in the early stages. Once the fungus invades, the mycelium spreads extremely rapidly, and its damage can cover the entire growth period from seed to mature plant, significantly increasing the difficulty of integrated pest management. At present, the effective control of stem base rot is an urgent problem to be solved in my country's wheat production. Research on the pathogenic mechanism of stem base rot and the discovery of disease-resistant germplasm materials are imperative. However, the overall resistance level of wheat varieties in my country is currently low, and no varieties with high resistance or immunity to stem base rot have been found.

[0003] In the field control of wheat stem base rot, it has been gradually proven that environmental conditions have a significant impact on the severity of the disease, especially under drought conditions where the damage caused by stem base rot is more severe. However, wheat in my country is mainly distributed in arid and semi-arid regions, and the aggravating effect of drought on stem base rot is a major factor in its outbreak. Clarifying the intrinsic relationship between drought and stem base rot, and identifying the reasons for the increased disease severity under drought conditions, is of great value for research on resistance regulation of wheat stem base rot. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to eliminate the synergistic harm of drought environment and Fusarium diseases to plants.

[0005] To address the aforementioned technical problems, this invention first provides a novel use for substances that reduce the content and / or activity of TaMPK3 protein.

[0006] This invention provides the use of a substance that reduces the content and / or activity of TaMPK3 protein in any of the following A1)-A6):

[0007] A1) Improve the disease resistance of plants under drought conditions (reduce and / or eliminate the tendency of plant diseases to worsen under drought conditions). A2) Improve the drought resistance of plants under disease conditions (reduce and / or eliminate the tendency of drought damage to worsen under disease conditions). A3) Cultivate plants with improved disease resistance under drought conditions; A4) Cultivate plants with improved drought resistance under disease conditions; A5) Plant breeding or plant variety improvement; A6) Eliminate or reduce the synergistic harm of drought and disease to plants; The TaMPK3 protein is TaMPK3-4A protein and / or TaMPK3-4B protein and / or TaMPK3-4D protein; The TaMPK3-4A protein is any one of the following (B1)-B4): B1) The amino acid sequence is that of the protein shown in Sequence 1; B2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 1; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1. B4) Proteins that have 80% or more of the same amino acid sequence as shown in Sequence 1 and have the same function; The TaMPK3-4B protein is any one of the following C1)-C4): C1) The amino acid sequence is the protein shown in sequence 2; C2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; C3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2. C4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function; The TaMPK3-4D protein is any one of the following (D1)-D4): D1) The amino acid sequence is that of the protein shown in sequence 3; D2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3; D3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3. D4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 3 and has the same function.

[0008] In the proteins described in B2), C2), or D2), the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag includes, but is not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0009] In the protein described in B3), C3), or D3), the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues; substitution and / or deletion and / or addition of no more than 9 amino acid residues; substitution and / or deletion and / or addition of no more than 8 amino acid residues; substitution and / or deletion and / or addition of no more than 7 amino acid residues; substitution and / or deletion and / or addition of no more than 6 amino acid residues; substitution and / or deletion and / or addition of no more than 5 amino acid residues; substitution and / or deletion and / or addition of no more than 4 amino acid residues; substitution and / or deletion and / or addition of no more than 3 amino acid residues; substitution and / or deletion and / or addition of no more than 2 amino acid residues; or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0010] In the proteins described in B4), C4), or D4), the identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences that have 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology with the amino acid sequences shown in Sequence 1, Sequence 2, or Sequence 3 of the present invention.

[0011] Any of the proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] In the above applications, the substances that reduce the content and / or activity of TaMPK3 protein include substances that reduce the content and / or activity of TaMPK3-4A protein, and / or substances that reduce the content and / or activity of TaMPK3-4B protein, and / or substances that reduce the content and / or activity of TaMPK3-4D protein.

[0013] The substance that reduces the content of TaMPK3-4A protein may be a substance that inhibits the synthesis of TaMPK3-4A protein, promotes the degradation of TaMPK3-4A protein, inhibits or interferes with the expression of the TaMPK3-4A protein encoding gene, or knocks out the TaMPK3-4A protein encoding gene.

[0014] The substance that reduces the activity of the TaMPK3-4A protein can be a protein, polypeptide, or small molecule compound that inhibits the function of the TaMPK3-4A protein.

[0015] The substance that reduces the content of TaMPK3-4B protein may be a substance that inhibits the synthesis of TaMPK3-4B protein, promotes the degradation of TaMPK3-4B protein, inhibits or interferes with the expression of the TaMPK3-4B protein encoding gene, or knocks out the TaMPK3-4B protein encoding gene.

[0016] The substance that reduces the activity of the TaMPK3-4B protein can be a protein, polypeptide, or small molecule compound that inhibits the function of the TaMPK3-4B protein.

[0017] The substance that reduces the content of TaMPK3-4D protein may be a substance that inhibits the synthesis of TaMPK3-4D protein, promotes the degradation of TaMPK3-4D protein, inhibits or interferes with the expression of the TaMPK3-4D protein encoding gene, or knocks out the TaMPK3-4D protein encoding gene.

[0018] The substance that reduces the activity of the TaMPK3-4D protein can be a protein, polypeptide, or small molecule compound that inhibits the function of the TaMPK3-4D protein.

[0019] The TaMPK3-4A protein-coding gene is either the cDNA molecule shown in sequence 4 or the genomic DNA molecule shown in sequence 7.

[0020] The TaMPK3-4B protein-coding gene is either the cDNA molecule shown in sequence 5 or the genomic DNA molecule shown in sequence 8.

[0021] The TaMPK3-4D protein-coding gene is either the cDNA molecule shown in sequence 6 or the genomic DNA molecule shown in sequence 9.

[0022] Furthermore, the substance that reduces the content and / or activity of TaMPK3 protein is any one of the following (E1) to (E7): E1) Inhibit or interfere with the expression of the TaMPK3 protein-coding gene or knock out the nucleic acid molecule of the TaMPK3 protein-coding gene; E2) An expression cassette containing the nucleic acid molecules described in E1); E3) A recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2); E4) Recombinant microorganisms containing the nucleic acid molecules described in E1), or recombinant microorganisms containing the expression cassette described in E2), or recombinant microorganisms containing the recombinant vector described in E3); E5) A transgenic plant cell line containing the nucleic acid molecule described in E1), or a transgenic plant cell line containing the expression cassette described in E2), or a transgenic plant cell line containing the recombinant vector described in E3; E6) Transgenic plant tissue containing the nucleic acid molecule described in E2), or transgenic plant tissue containing the expression cassette described in E2), or transgenic plant tissue containing the recombinant vector described in E3; E7) A transgenic plant organ containing the nucleic acid molecule described in E1), or a transgenic plant organ containing the expression cassette described in E2), or a transgenic plant organ containing the recombinant vector described in E3).

[0023] In E1 above, the nucleic acid molecule that inhibits or interferes with the expression of the TaMPK3 protein-coding gene or knocks out the TaMPK3 protein-coding gene can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0024] In some embodiments, the nucleic acid molecule that knocks out the TaMPK3 protein-coding gene is any of the following: F1) The DNA molecule shown in sequence 12; DNA molecules that have 75% or more identity with the nucleotide sequences defined by F2 and F1, and have the same function.

[0025] In E2) above, the expression cassette may include a promoter, the nucleic acid molecule described in E1) above, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0026] In E3 above, the vector refers to a vector that can carry the nucleic acid molecule described in E1 above into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0027] The recombinant vector refers to a recombinant DNA molecule constructed by in vitro ligation of the nucleic acid molecule described in E1) above with the vector. Existing plant expression vectors can be used to construct recombinant vectors containing the nucleic acid molecule described in E1). These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor. Similar functions exist in the untranslated regions of 3'-terminal transcription of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the alkaloid synthase gene Nos) and plant genes (such as the soybean storage protein gene). When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signal and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector can be processed, such as by adding genes encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.) that can be expressed in plants, or antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes that confer resistance to glyphosate EPSPS Genes such as herbicide-resistant marker genes or mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0028] In some embodiments, the substance for knocking out the TaMPK3 protein-coding gene is a CRISPR / Cas9 gene-editing vector that knocks out the TaMPK3 protein-coding gene. The CRISPR / Cas9 gene-editing vector expresses sgRNA and Cas9 protein targeting the TaMPK3 protein-coding gene. Preferably, the target sequence of the sgRNA is shown in sequences 10 and 11.

[0029] In some specific embodiments, the CRISPR / Cas9 gene editing vector for knocking out the TaMPK3 protein-coding gene is the recombinant plasmid pBUE411-TaMPK3. The recombinant plasmid pBUE411-TaMPK3 is obtained by inserting the DNA molecule shown in sequence 12 into the BsaI restriction site of the vector pBUE411.

[0030] In E4 above, the microorganism can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The bacteria can be from the genus *Escherichia* (…). Escherichia sp. Erwinia ( ) Erwinia sp. ), Agrobacterium ( Agrobacterium sp. Flavobacterium ( Flavobacterium sp. ), Alcaligenes ( Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ), Bacillus spp. ( Bacillus sp. Examples of bacteria include, but are not limited to, Escherichia coli (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ) or Bacillus pumilus ( Bacillus pumilus The fungus may be a yeast, and the yeast may be from the genus *Saccharomyces* (such as *Saccharomyces cerevisiae*). Saccharomyces cerevisiae Kluyveromyces (such as Kluyveromyces lactis) Kluyveromyces lactis Pichia genus (such as Pichia pastoris) Pichia pastoris ), genus *Schizosaccharomyces* (such as *Schizosaccharomyces cerevisiae*) Schizosaccharomyces pombe ), Hansenula genus (such as polymorphic Hansenula) Hansenula polymorpha (and others, but not limited to these.) The fungi may also originate from the genus Fusarium (…). Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. Aspergillus ( ) Aspergillus sp. ), Cephalosporium ( Cephalosporium sp. Actinomycetes may be derived from Streptomyces (…), but are not limited to these. Streptomyces sp. Nocardia ( ) Nocardia sp. Micromonospora ( Micromonospora sp. ), genus *Neurospora* Streptosporangium sp. ), genus Actinomycetes (Actinoplanes sp. ), thermophilic actinomycetes ( Thermoactinomyces sp. (e.g., but not limited to these). The algae mentioned may come from the genus *Fucus* (…). Fucus sp. ), genus *Cyclocarya* ( Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpa ( Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus *Stellaria* ( Asteromonas sp. ), Golden-colored algae ( Boekelovia sp. The viruses mentioned may include, but are not limited to, rotavirus, herpesvirus, influenza virus, adenovirus, etc.

[0031] The recombinant microorganisms refer to those obtained by manipulating and modifying the genes of a target microorganism, resulting in a functional change. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into the target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0032] In E6 above, the transgenic plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0033] In E7 above, the transgenic plant organs can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0034] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described above may or may not include propagation material.

[0035] To address the aforementioned technical problems, the present invention also provides a method for improving the disease resistance of plants under drought conditions.

[0036] The method for improving plant disease resistance under drought conditions provided by the present invention includes the following steps: reducing the content and / or activity of TaMPK3 protein in the target plant to improve the plant's disease resistance under drought conditions.

[0037] To address the aforementioned technical problems, the present invention also provides a method for improving the drought resistance of plants under disease conditions.

[0038] The method for improving the drought resistance of plants under disease conditions provided by the present invention includes the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in the target plant, thereby improving the drought resistance of the plant under disease conditions.

[0039] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with enhanced disease resistance under drought conditions.

[0040] The method for cultivating transgenic plants with enhanced disease resistance under drought conditions provided by the present invention includes the following steps: reducing the content and / or activity of TaMPK3 protein in the target plant to obtain transgenic plants; the transgenic plants exhibit higher disease resistance under drought conditions than the target plants.

[0041] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with enhanced drought resistance under disease conditions.

[0042] The method for cultivating transgenic plants with improved drought resistance under disease conditions provided by the present invention includes the following steps: reducing the content and / or activity of TaMPK3 protein in the target plant to obtain transgenic plants; the transgenic plants exhibit higher drought resistance under disease conditions than the target plants.

[0043] To address the aforementioned technical problems, the present invention also provides a method for plant breeding or plant variety improvement.

[0044] The method for plant breeding or plant variety improvement provided by the present invention includes the following steps: using the transgenic plant prepared according to the above method as a parent for breeding.

[0045] To address the aforementioned technical problems, the present invention ultimately provides a method for eliminating or mitigating the synergistic harm of drought and disease to plants.

[0046] The method for eliminating or reducing the synergistic harm of drought and disease to plants provided by the present invention includes the following steps: reducing the content and / or activity of TaMPK3 protein in the target plant to eliminate or reduce the synergistic harm of drought and disease to plants.

[0047] In any of the methods described above, the method for reducing the content and / or activity of TaMPK3 protein in the target plant includes introducing a substance that knocks out the TaMPK3 protein encoding gene into the target plant.

[0048] Furthermore, the substance used to knock out the TaMPK3 protein-coding gene is a CRISPR / Cas9 gene-editing vector that knocks out the TaMPK3 protein-coding gene. The CRISPR / Cas9 gene-editing vector expresses targeted... TaMPK3 The gene's sgRNA and Cas9 protein.

[0049] Furthermore, the target sequence of the sgRNA is shown in sequences 10 and 11.

[0050] In some embodiments, the CRISPR / Cas9 gene editing vector for knocking out the TaMPK3 protein-coding gene is the recombinant plasmid pBUE411- TaMPK3 The recombinant plasmid pBUE411-TaMPK3 was obtained by inserting the DNA molecule shown in sequence 12 into the BsaI restriction site of the vector pBUE411.

[0051] The disease resistance mentioned above refers to Fusarium disease resistance, that is, resistance to diseases caused by pathogens of the genus Fusarium. These pathogens include, but are not limited to, Fusarium pseudogracilis, Fusarium gracilis, Fusarium xanthophylloides, Fusarium flocculationii, and Fusarium aromaticum.

[0052] In some implementations, the Fusarium disease resistance is stem base rot resistance.

[0053] In some specific implementation cases, the stem base rot resistance refers to stem base rot resistance caused by Fusarium pseudograss infection.

[0054] The above-mentioned disease conditions refer to Fusarium disease stress conditions, that is, disease stress conditions caused by Fusarium pathogens. The Fusarium pathogens include, but are not limited to, *Fusarium pseudogracilis*, *Fusarium gracilis*, *Fusarium xanthosporium*, *Fusarium laminarum*, and *Fusarium aromaticum*.

[0055] In some implementations, the condition described under Fusarium disease stress is the condition described under stem base rot stress.

[0056] In some specific implementation cases, the "stress conditions under stem base rot" refers to the "stress conditions under Fusarium graminearum infection".

[0057] Any of the diseases mentioned above are Fusarium diseases, that is, diseases caused by pathogens of the genus Fusarium. These pathogens include, but are not limited to, *Fusarium pseudogracilis*, *Fusarium gracilis*, *Fusarium xanthosporium*, *Fusarium laminarum*, and *Fusarium aromaticum*.

[0058] In some implementations, the Fusarium disease is stem base rot.

[0059] In some specific implementation cases, the stem base rot is caused by Fusarium pseudograss infection.

[0060] In some embodiments, the transgenic plant exhibits higher disease resistance under drought conditions than the target plant, as evidenced by the transgenic plant showing a lower disease severity index after drought stress under Fusarium graminearum infection stress.

[0061] In some embodiments, the transgenic plant exhibits higher drought resistance under disease conditions than the target plant, as demonstrated by any one of the following X1)-X2): X1) Under drought stress, the aboveground fresh weight of the transgenic plant after infection with Fusarium graminearum was higher than that of the target plant; X2) Under drought stress, the malondialdehyde content of the transgenic plant after infection with Fusarium oxysporum was lower than that of the target plant.

[0062] In some specific implementations, the method of Fusarium graminearum infection is to place diseased wheat grains infected with Fusarium graminearum into the soil near the base of the wheat stem. Specific steps can be found in the literature "A Method to Inoculate Millet Grain-Colonized Fusarium pseudograminearum The method described in "on Wheat to Obtain Reproducible Disease Symptoms".

[0063] In some specific embodiments, the *Fusarium graminearum* infection is achieved by directly dripping the spore solution of *Fusarium graminearum* onto the base of the wheat stem. Specific steps can be found in the method described in the literature "Wheat WRKY transcription factor TaWRKY24 confers drought and salt tolerance in transgenic plants". The concentration of the *Fusarium graminearum* spore solution is 1 × 10⁻⁶. 6 per mL.

[0064] In some specific implementations, the drought stress is described as not providing any additional water.

[0065] In some specific implementations, the *Fusarium pseudograss* is *Fusarium pseudograss* CS3096.

[0066] The above-mentioned methods of introduction include, but are not limited to: transfecting plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and then culturing the transfected plant cells or tissues into plants.

[0067] The aforementioned transgenic plants include not only first-generation transgenic plants but also their progeny. The gene can be propagated within the species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques.

[0068] The indicators for any of the above-mentioned plant breeding include disease resistance under drought conditions (such as resistance to stem base rot under drought conditions) and / or drought resistance under disease conditions (such as drought resistance under stem base rot stress conditions).

[0069] The purpose of any of the above-mentioned plant breeding includes breeding disease-resistant plant varieties under drought conditions (such as plant varieties resistant to stem base rot under drought conditions) or disease-resistant plant varieties under disease conditions (such as drought-resistant plant varieties under stem base rot stress conditions).

[0070] The plant mentioned above is any one of the following Z1)-Z4): Z1) Monocotyledons; Z2) Gramineae plants; Z3) Plants of the Triticum genus; Z4) Wheat (such as Fielder).

[0071] This invention utilizes wheat ( Triticum aestivum Key node genes in plant responses to stem base rot and drought in L.) TaMPK3 Knockout was performed, and the following was obtained. TaMPK3 Gene-edited wheat homozygous lines ( TaMPK3 -KO1、 TaMPK3 -KO4 and TaMPK3 -KO16). Experiments showed that under drought conditions, wheat stem rot in the recipient control Fielder was more severe, with an increased disease severity index; while under stem rot stress, drought damage in the recipient control Fielder was more severe (reduced aboveground fresh weight and increased malondialdehyde content); however, compared with the recipient control Fielder... TaMPK3 Gene-edited wheat showed a weakened and / or disappeared trend in aggravating wheat stem rot under drought conditions (the change in disease severity index weakened and / or disappeared), and a weakened and / or disappeared trend in aggravating drought damage under stem rot stress conditions (the trend of decreasing aboveground fresh weight weakened and / or disappeared, and the trend of increasing malondialdehyde content weakened and / or disappeared). These results indicate the presence of the TaMPK3 protein and its encoding gene in this invention. TaMPK3 It can regulate the link between stem rot disease and drought in plants by reducing the content and / or activity of TaMPK3 protein in the target plant (e.g., knocking it out). TaMPK3 Genes can significantly weaken and / or eliminate the link between stem base rot and drought conditions, thereby improving plant resistance to Fusarium diseases under drought conditions or drought resistance under Fusarium disease conditions. Attached Figure Description

[0072] Figure 1 for TaMPK3 A schematic diagram illustrating frameshift mutations and premature coding termination caused by genomic sequence variations in gene-edited wheat.

[0073] Figure 2 for TaMPK3 Gene editing status of gene-edited wheat and TaMPK3Gene expression analysis. Fielder represents the recipient control wild-type wheat; KO-1 represents... TaMPK3 -KO1 strain wheat; KO-4 indicates TaMPK3 -KO4 strain wheat; KO-16 indicates TaMPK3 -KO16 strain of wheat. The difference is significant (P<0.001).

[0074] Figure 3 for TaMPK3 Analysis of the impact of drought on stem base rot disease in gene-edited wheat and wild-type wheat. A shows the phenotypic pattern of stem base rot disease in wild-type wheat (Fielder) under normal conditions. B shows the phenotypic pattern of stem base rot disease in wild-type wheat (Fielder) after drought treatment. C shows the disease severity index analysis of stem base rot in wild-type wheat under normal conditions and after drought treatment. D shows... TaMPK3 Phenotypic diagram of stem base rot disease in gene-edited wheat under normal conditions. E represents... TaMPK3 Phenotypic representation of stem base rot disease in gene-edited wheat after drought treatment. F represents... TaMPK3 Analysis of stem base rot severity index in gene-edited wheat under normal conditions and drought treatment. Fielder represents the recipient control wild-type wheat; KO-1 represents... TaMPK3 -KO1 strain wheat; KO-4 indicates TaMPK3 -KO4 strain wheat; KO-16 indicates TaMPK3 -KO16 strain of wheat. The difference is significant (P<0.001).

[0075] Figure 4 for TaMPK3Analysis of the impact of stem base rot stress on the drought stress levels of gene-edited wheat and wild-type wheat. A shows the phenotypes of wild-type wheat Fielder wheat in the stem base rot stress treatment group (injected with *Fusarium graminearum* spores on day 25 of drought treatment) and the control group after 15 days of drought treatment. B shows the phenotypes of wild-type wheat Fielder wheat in the stem base rot stress treatment group (injected with *Fusarium graminearum* spores on day 25 of drought treatment) and the control group after 30 days of drought treatment. C shows the aboveground fresh weight analysis of wild-type wheat Fielder wheat in the stem base rot stress treatment group (injected with *Fusarium graminearum* spores on day 25 of drought treatment) and the control group after 30 days of drought treatment. D shows the malondialdehyde (MDA) content analysis of wild-type wheat Fielder wheat in the stem base rot stress treatment group (injected with *Fusarium graminearum* spores on day 25 of drought treatment) and the control group after 30 days of drought treatment. E represents the stem base rot stress treatment group (injected with Fusarium spores on day 25 of drought treatment) and the control group. TaMPK3 Phenotypic diagram of gene-edited wheat after 15 days of drought treatment. F represents the stem base rot stress treatment group (injected with Fusarium spores on day 25 of drought treatment) and the control group. TaMPK3 Phenotypic diagram of gene-edited wheat after 30 days of drought treatment. G represents the stem base rot stress treatment group (injected with Fusarium spores on day 25 of drought treatment) and the control group. TaMPK3 Analysis of aboveground fresh weight of gene-edited wheat after 30 days of drought treatment. H represents the stem base rot stress treatment group (injected with Fusarium spores on day 25 of drought treatment) and the control group. TaMPK3 Analysis of malondialdehyde (MDA) content in gene-edited wheat after 30 days of drought treatment. In this study, Fielder represents the recipient control wild-type wheat; F1, F2, F3, F4, F5, and F6 are the pot numbers of different Fielder plants; KO-1 indicates... TaMPK3 -KO1 strain wheat; KO-4 indicates TaMPK3 -KO4 strain wheat; KO-16 indicates TaMPK3 -KO16 strain of wheat. The difference is significant (P<0.001). Detailed Implementation

[0076] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0077] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0078] The wheat variety “Fielder” in the following examples is described in the literature “Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'”, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0079] The pEasyBlunt vector used in the following examples is a product of Beijing TransGen Biotech Co., Ltd.

[0080] The qPCR enzyme used in the following examples is a product of Beijing TransGen Biotech Co., Ltd.

[0081] The Agrobacterium tumefaciens EH105 used in the following examples is a product of Beijing Bairddi Biotechnology Co., Ltd.

[0082] The vector pBUE411 and plasmid pCBC-MT1T2 used in the following examples are described in the article “Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiple genome editing in plants. BMC Plant Biol. 2014 Nov 29;14:327.”, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0083] The wheat variety Jinhe 991 in the following examples was bred by the Institute of Biotechnology and Food Science of Hebei Academy of Agricultural and Forestry Sciences, with the approval number Ji Shen Mai 20210021.

[0084] Example 1: Obtaining the TaMPK3 protein and its encoding gene This invention isolates and clones a substance related to plant resistance to stem rot from the wheat variety "Jinhe 991". TaMPK3 Gene( TaMPK3-4A , TaMPK3-4B and TaMPK3-4D (Genes). The specific steps are as follows: 1. Wheat seedlings of the Jinhe 991 variety, which have grown for about 2 weeks under normal conditions, are quick-frozen with liquid nitrogen and stored at -80℃ for later use.

[0085] 2. Total RNA was extracted from wheat leaves using the Trizol method (TianGen), and the total RNA was reverse transcribed to obtain cDNA.

[0086] 3. Using the obtained cDNA as a template, primers were applied... TaMPK3 -F: 5'-ATGGACGGCGCTCCGGTGG-3' and TaMPK3 -R: 5'-GTATCGGAAGTTGGGGTTCAACTCCA-3' was used for PCR amplification to obtain the amplification product.

[0087] 4. After the amplification product was detected by agarose gel electrophoresis, a DNA fragment of approximately 1110 bp was separated and purified. Then, this PCR product was ligated into a cloning vector and sequenced.

[0088] Sequencing results show that three species exist in the wheat variety "Jinhe 991". TaMPK3 The coding region sequences are shown in sequences 4, 5, and 6, respectively. The gene shown in sequence 4 is named... TaMPK3-4A The gene, whose encoded amino acid sequence of the TaMPK3-4A protein is shown in Sequence 1, is named as shown in Sequence 5. TaMPK3-4B The gene, whose encoded amino acid sequence of the TaMPK3-4B protein is shown in Sequence 2, is named as shown in Sequence 6. TaMPK3-4D The gene encodes the TaMPK3-4D protein, whose amino acid sequence is shown in Sequence 3.

[0089] Example 2: The effect of TaMPK3 protein on the relationship between wheat stem base rot and drought environment I. Construction of CRISPR / Cas9 gene editing vectors 1. According to TaMPK3 Gene sequence was used to design sgRNA target sequences. The final designed sgRNA target sequences are as follows: Target 1 sequence: 5'-CGGCCTCAAGTACATCCACT CGG-3' (Sequence 10).

[0090] Target 2 sequence: 5'-CAGCCGCCGATCATGCCCATCGG-3' (Sequence 11).

[0091] 2. Using pCBC-MT1T2 plasmid as a template, PCR amplification was performed using the TaMPK3-crispr-F0 and TaMPK3-crispr-R0 primer pairs to obtain DNA fragment A; using DNA fragment A as a template, PCR amplification was performed using the TaMPK3-crispr-F and TaMPK3-crispr-R primer pairs to obtain DNA fragment B. The primer sequences are as follows: TaMPK3-crispr-F: 5'-AATAATGGTCTCAGGCGAGCCGCCGATCATGCCCAT-3'; TaMPK3-crispr-R: 5'-ATTATTGGTCTCTAAACAGTGGATGTACTTGAGGCC-3.

[0092] TaMPK3-crispr-F0: 5'-GAGCCGCCGATCATGCCCATGTTTTAGAGCTAGAAATAGC-3'; TaMPK3-crispr-R0: 5'-AGTGGATGTACTTGAGGCCCGCTTTCTTGGTGCC-3.

[0093] 3. The vector pBUE411 was linearized by restriction endonuclease BsaI to obtain the digested vector backbone.

[0094] 4. The PCR product recovered in step 2 (DNA fragment B) and the digested vector backbone from step 3 were ligated using restriction site ligation technology to obtain the ligation product. The ligation product was then amplified by PCR using TaMPK3-crispr-F2 and TaMPK3-crispr-R2 primers, and the amplified product was sequenced for verification. The correctly sequenced recombinant plasmid was named pBUE411-TaMPK3. The primer sequences are as follows: TaMPK3-crispr-F2: 5'-AGCCGCCGATCATGCCCAT-3'; TaMPK3-crispr-R2: 5'-AGTGGATGTACTTGAGGCC-3'.

[0095] Sequencing results showed that the recombinant plasmid pBUE411-TaMPK3 was obtained by inserting the DNA molecule shown in sequence 12 into the BsaI restriction site of the vector pBUE411. The recombinant plasmid pBUE411-TaMPK3 can express Cas9 protein and two target proteins. TaMPK3 sgRNA of the gene, 2 targets TaMPK3 The target sequences of the gene's sgRNA are shown in sequences 10 and 11, respectively.

[0096] two, TaMPK3 The acquisition of gene-edited wheat 1. The recombinant plasmid pBUE411-TaMPK3 was introduced into Agrobacterium EH105 to obtain Agrobacterium EH105 / pBUE411-TaMPK3 containing the recombinant plasmid pBUE411-TaMPK3.

[0097] 2. Agrobacterium EH105 / pBUE411-TaMPK3 containing recombinant plasmid pBUE411-TaMPK3 was inoculated into YEP liquid medium and cultured at 28°C and 3000 rpm for about 18 hours.

[0098] 3. Streak the bacterial culture obtained in step 2 onto YEP solid medium (containing 50 μg / L streptomycin and 50 μg / L kanamycin) and incubate at 28°C for about 2 days.

[0099] 4. Sterilize wheat Fielder seeds with chlorine for 8 hours, then sow them evenly in sterilized B5 medium and culture until the radicle forms.

[0100] 5. After completing step 4, remove the wheat cotyledons and the radicle below the cotyledon node, and then culture the wheat until callus grows. Infect the callus with Agrobacterium EH105 (EH105 / pBUE411-TaMPK3) containing the recombinant plasmid pBUE411-TaMPK3. After culturing, obtain T0 generation gene-edited wheat.

[0101] 6. DNA was extracted from T0 generation gene-edited wheat plants and amplified by PCR to detect the presence of DNA in various genomes. TaMPK3 The status of gene target site editing. TaMPK3 T0 generation gene-edited wheat, in which the gene target has been mutated, is called T0 generation. TaMPK3 Gene-edited wheat. Primer sequences are as follows: MPK8-crispr-AF: 5'-GAAGAACACTGCCAGGTAGTAAAGA-3'; MPK8-crispr-AR: 5'-GTCATCATGTCGCTCTCCGAT-3'; MPK8-crispr-BF: 5'-AAGGACTAAGGAGAAGAGAAGGGC-3'; MPK8-crispr-BR: 5'-CAGTAGCAGGTTGCTCGGTTTC-3'; MPK8-crispr-DF: 5'-CGGCAATTCAGGCGCTAAGCTGTTC-3'; MPK8-crispr-DR: 5'-GCACTGACACTGACCCCAACA-3'.

[0102] T0 generation TaMPK3 Gene-edited wheat was propagated, and three stable, normally growing transformants were selected from the third generation. TaMPK3 Gene-edited wheat homozygous lines were named as follows: TaMPK3 -KO1 strain, TaMPK3 -KO4 strain and TaMPK3 -KO16 strain.

[0103] Compared to the wild-type wheat Fielder genome sequence, TaMPK3 Gene-edited wheat homozygous lines TaMPK3 The differences in -KO1 are only as follows: an A-base insertion occurs in the gene sequence (sequence 7) encoding the TaMPK3-4A protein on wheat chromosome 4A, between bases 944 and 945; an A-base insertion occurs in the gene sequence (sequence 8) encoding the TaMPK3-4B protein on wheat chromosome 4B, between bases 987 and 988; and a C-base insertion occurs in the gene sequence (sequence 9) encoding the TaMPK3-4D protein on wheat chromosome 4D, between bases 932 and 933.

[0104] Compared to the wild-type wheat Fielder genome sequence, TaMPK3 Gene-edited wheat homozygous lines TaMPK3 The differences in -KO4 are only as follows: an A-base insertion occurs in the gene sequence (sequence 7) encoding the TaMPK3-4A protein on wheat chromosome 4A, between bases 944 and 945; an A-base insertion occurs in the gene sequence (sequence 8) encoding the TaMPK3-4B protein on wheat chromosome 4B, between bases 987 and 988; and an A-base insertion occurs in the gene sequence (sequence 9) encoding the TaMPK3-4D protein on wheat chromosome 4D, between bases 933 and 934.

[0105] Compared to the wild-type wheat Fielder genome sequence, TaMPK3 Gene-edited wheat homozygous lines TaMPK3The differences in -KO16 are only as follows: a base deletion occurs in the gene sequence (Sequence 7) encoding the TaMPK3-4A protein on wheat chromosome 4A, with the deleted base located at position 943 of Sequence 7; an A base insertion occurs in the gene sequence (Sequence 8) encoding the TaMPK3-4B protein on wheat chromosome 4B, with the A base inserted between positions 987 and 988; and an A base insertion occurs in the gene sequence (Sequence 9) encoding the TaMPK3-4D protein on wheat chromosome 4D, with the A base inserted between positions 933 and 934 of Sequence 9.

[0106] The A, B, and D genomes of the above gene-edited wheat homozygous lines TaMPK3 All gene sequences underwent frameshift mutations after editing, resulting in premature termination of stop codons and premature termination of protein translation. Figure 1 ).

[0107] three, TaMPK3 Detection of relative gene expression levels Pick TaMPK3 Gene-edited wheat homozygous lines TaMPK3 -KO1、 TaMPK3 -KO4 and TaMPK3 Total RNA was extracted from leaves of KO16 and reverse transcribed to obtain cDNA. qRT-PCR was then performed using the cDNA as a template. Actin Genes used as internal reference genes for detection TaMPK3 The relative expression level of genes.

[0108] Used for detection TaMPK3 The primer sequences for the gene are as follows: TaMPK3-A(RT)-Forward:5'-GCGATACGCACAGAAAAGAGCCA-3'; TaMPK3-A(RT)-Reverse: 5'-CGATTCTTGTAGGGGCGAGGAGG-3'; TaMPK3-B(RT)-Forward: 5'-CGTGTTGCCTCCTGTGATTGGG-3'; TaMPK3-B(RT)-Reverse:5'-GATCGGCGGCTGGTACTTTGC-3'; TaMPK3-D(RT)-Forward:5'-AACCCCAACTTCCGATACT-3'; TaMPK3-D(RT)-Reverse: 5'-AATCCATTTCTTCATAAACTAGAG-3'.

[0109] Used for detection Actin The primer sequences for the gene are as follows: Actin-F :5'-GGAATCCATGAGACCACCTAC-3'; Actin-R :5'-GACCCAGACAACTCGCAAC-3'.

[0110] The results are as follows Picture 2 As shown, the results indicate that compared to wild-type wheat Fielder (receptor control), TaMPK3 TaMPK3-4A in gene-edited wheat homozygous lines TaMPK3-4B and TaMPK3-4D Gene expression levels were all significantly downregulated (almost completely absent). This indicates... TaMPK3 Gene editing was successful, and it was able to directly influence gene-edited materials. TaMPK3 Gene expression levels.

[0111] Four, TaMPK3 Analysis of the Influence of Drought Environment on Stem Base Rot Disease in Gene-Edited Wheat and Wild-Type Wheat Test materials: TaMPK3 Gene-edited wheat homozygous lines ( TaMPK3 -KO1) and wild wheat (WT, Fielder).

[0112] Experimental methods: analysis TaMPK3 The severity of stem rot in gene-edited wheat and wild-type wheat was affected by drought. The experiment was divided into two groups: Normal group: Weigh out the same mass of soil and sow the same number of seeds. TaMPK3 Gene-edited wheat and recipient control were used. One-week-old wheat seedlings were inoculated with diseased wheat grains infected with Fusarium graminearum CS3096. After inoculation with diseased wheat grains, the seedlings were cultured under normal conditions (soil moisture content that maintains normal wheat growth). The disease severity index of the plants was investigated 30 days after stem base rot stress.

[0113] Drought treatment group: Weigh out the same mass of soil and sow the same number of seeds. TaMPK3 Gene-edited wheat and recipient control were used. One-week-old wheat seedlings were inoculated with diseased wheat grains infected with Fusarium graminearum CS3096. No water was added after inoculation to induce water shortage and drought stress in the plants. The disease severity index of the plants was investigated 30 days after stem rot stress.

[0114] Except for the drought environment, the normal group and the drought treatment group had the same experimental conditions. Three replicate experiments were set up, and phenotypic observations and statistics were performed on 10 plants of each test line in each replicate experiment.

[0115] The specific steps for inoculating millet grains infected with Fusarium graminearum CS3096 are described above. Refer to the reference "A Method to Inoculate Millet Grain-Colonized Millet Grain". Fusarium pseudograminearum The method described in "on Wheat to Obtain Reproducible Disease Symptoms".

[0116] The disease severity index mentioned above is calculated using the formula (ΣnX / 6N)×100, where X represents the severity level of crown rot, n represents the number of plants with a given score, and N is the total number of plants being evaluated. The severity level of crown rot is assessed using a 0-6 scale, including a new level inserted between levels 3 and 4 of the previous 0-5 scale system. This additional level specifically corresponds to the appearance of lesions on the third leaf sheath and severe necrosis on the second leaf sheath. For detailed calculation methods, please refer to the literature "A simple method for the assessment of crown rot disease severity in wheat seedlings inoculated with..." Fusarium pseudograminearum The method in "".

[0117] The results are as follows Picture 3 As shown, the results indicate that under drought conditions, the stem base rot disease in the recipient control Fielder was significantly more severe than under normal conditions, and the disease severity index was significantly increased ( Picture 3 C). And TaMPK3 Genetically edited wheat showed similar stem rot symptoms under drought conditions to those under normal conditions, with no significant change in the disease severity index. Picture 3 F). Explanation of knockout TaMPK3 The gene blocked the aggravating effect of drought on wheat stem base rot, eliminated the synergistic harm between drought and stem base rot, and improved wheat's resistance to stem base rot under drought conditions.

[0118] five, TaMPK3 Analysis of the Influence of Drought Stress on Stem Base Rot Stress on Gene-Edited Wheat and Wild-Type Wheat Test materials: TaMPK3 Gene-edited wheat homozygous lines ( TaMPK3 -KO1、 TaMPK3 -KO4、 TaMPK3 -KO16), wild wheat (WT, Fielder).

[0119] Experimental methods: analysis TaMPK3 The effects of stem rot stress on drought stress were investigated in gene-edited wheat and wild-type wheat. The experiment was divided into two groups: Stem base rot stress treatment group: Weigh out the same mass of soil and sow the same number of seeds in each pot. TaMPK3 Genetically edited wheat and a recipient control were subjected to drought treatment without any subsequent watering. After 25 days of growth under drought conditions, wheat seedlings were infected with *Fusarium graminearum* using the following method: *Fusarium graminearum* spore solution (containing 1 million spores per milliliter, injection volume 0.5 mL) was injected into the base of the wheat stem using a syringe. The fresh weight of the aboveground parts and malondialdehyde (MDA) content were measured 15 and 30 days after drought treatment.

[0120] Control group: Weigh out the same mass of soil and sow the same number of seeds. TaMPK3 Genetically edited wheat and recipient control were subjected to drought treatment without any additional water, and the fresh weight of the aboveground parts and malondialdehyde content were measured 15 and 30 days after the drought treatment.

[0121] The experimental conditions were the same for the stem base rot stress treatment group and the control group, except for the stem base rot stress treatment.

[0122] The results are as follows Picture 4 As shown, the results indicated that under stem base rot stress treatment, the drought stress damage to the recipient control Fielder was significantly more severe than under normal growth conditions, with a significant decrease in the fresh weight of the aboveground parts and a significant increase in the malondialdehyde content, which reflects cell damage. Picture 4 C and Picture 4 D). And TaMPK3 Genetically edited wheat showed similar drought stress damage under stem base rot treatment to that under normal growth conditions, with no significant changes in aboveground fresh weight and malondialdehyde (MDA) content. Picture 4 G and Picture 4 H). Explanation of knockout TaMPK3 The gene blocked the aggravating effect of stem base rot on wheat drought damage, eliminated the synergistic harm of drought environment and stem base rot, and improved the drought resistance of wheat under stem base rot stress.

[0123] In summary, inhibiting the TaMPK3 protein and its encoding gene... TaMPK3 The expression of TaMPK3 can regulate the relationship between stem rot and drought in plants by inhibiting the expression of TaMPK3 protein and its encoding gene. TaMPK3 Expressions (e.g., knockout) TaMPK3 (Genes) can block the aggravating effect of drought on plant stem base rot or the aggravating effect of stem base rot on drought damage to plants, thus eliminating the synergistic harm of drought and stem base rot to plants.

[0124] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of substances that reduce TaMPK3 protein content and / or activity in any of the following A1)-A6): A1) Improve the disease resistance of plants under drought conditions; A2) Improve the drought resistance of plants under disease conditions; A3) Cultivate plants with improved disease resistance under drought conditions; A4) Cultivate plants with improved drought resistance under disease conditions; A5) Plant breeding or plant variety improvement; A6) Eliminate or reduce the synergistic harm of drought and disease to plants; The TaMPK3 protein is TaMPK3-4A protein and / or TaMPK3-4B protein and / or TaMPK3-4D protein; The TaMPK3-4A protein is any one of the following (B1)-B4): B1) The amino acid sequence is that of the protein shown in Sequence 1; B2) A fusion protein with the same function obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 1; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 1. B4) Proteins that have 80% or more of the same amino acid sequence as shown in Sequence 1 and have the same function; The TaMPK3-4B protein is any one of the following C1)-C4): C1) The amino acid sequence is the protein shown in sequence 2; C2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; C3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2. C4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function; The TaMPK3-4D protein is any one of the following (D1)-D4): D1) The amino acid sequence is that of the protein shown in sequence 3; D2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 3; D3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 3. D4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 3 and has the same function.

2. The application according to claim 1, characterized in that: The substance that reduces the content and / or activity of TaMPK3 protein is any one of the following (E1) to (E7): E1) Inhibit or interfere with the expression of the TaMPK3 protein-coding gene or knock out the nucleic acid molecule of the TaMPK3 protein-coding gene; E2) An expression cassette containing the nucleic acid molecules described in E1); E3) A recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2); E4) Recombinant microorganisms containing the nucleic acid molecules described in E1), or recombinant microorganisms containing the expression cassette described in E2), or recombinant microorganisms containing the recombinant vector described in E3); E5) A transgenic plant cell line containing the nucleic acid molecule described in E1), or a transgenic plant cell line containing the expression cassette described in E2), or a transgenic plant cell line containing the recombinant vector described in E3; E6) Transgenic plant tissue containing the nucleic acid molecule described in E2), or transgenic plant tissue containing the expression cassette described in E2), or transgenic plant tissue containing the recombinant vector described in E3; E7) A transgenic plant organ containing the nucleic acid molecule described in E1), or a transgenic plant organ containing the expression cassette described in E2), or a transgenic plant organ containing the recombinant vector described in E3).

3. The application according to claim 2, characterized in that: E1) The nucleic acid molecule is any one of the following: F1) The DNA molecule shown in sequence 12; DNA molecules that have 75% or more identity with the nucleotide sequences defined by F2 and F1, and have the same function.

4. A method for improving the disease resistance of plants under drought conditions, comprising the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in the target plant, thereby improving the disease resistance of the plant under drought conditions.

5. A method for improving the drought resistance of plants under disease conditions, comprising the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in the target plant, thereby improving the drought resistance of the plant under disease conditions.

6. A method for cultivating transgenic plants with enhanced disease resistance under drought conditions, comprising the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in a target plant to obtain a transgenic plant; wherein the transgenic plant exhibits higher disease resistance under drought conditions than the target plant.

7. A method for cultivating plants with enhanced drought resistance under disease conditions, comprising the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in a target plant to obtain a transgenic plant; wherein the transgenic plant exhibits higher drought resistance under disease conditions than the target plant.

8. A method for plant breeding or plant variety improvement, comprising the following steps: using a transgenic plant prepared according to the method of claim 6 or 7 as a parent for breeding.

9. A method for eliminating or reducing the synergistic harm of drought and disease to plants, comprising the following steps: reducing the content and / or activity of the TaMPK3 protein as described in claim 1 in the target plant, thereby eliminating or reducing the synergistic harm of drought and disease to plants.

10. The application according to any one of claims 1-3 or the method according to any one of claims 4-9, characterized in that: The disease resistance mentioned refers to resistance to Fusarium diseases; Alternatively, the "disease conditions" refer to the conditions under Fusarium disease stress. Alternatively, the disease in question is a Fusarium disease.

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