Application of ta cyp51h37 protein and related biological materials in regulating resistance of plants to stem base rot

Abstract

This invention discloses the application of TaCYP51H37 protein and related biomaterials in regulating plant stem base rot resistance. This invention constructs a transgenic... TaCYP51H37 wheat and TaCYP51H37 Silent wheat, through identification of its resistance to stem base rot, was found to show that, compared with the recipient control, it was more susceptible to *Fusarium graminearum* infection. TaCYP51H37 The disease severity index of wheat was significantly reduced, while the aboveground length, aboveground fresh weight, and aboveground dry weight were all significantly increased. TaCYP51H37 The phenotype of silent wheat was the opposite, with a disease severity index significantly higher than that of the control plants, and significantly lower aboveground part length, aboveground fresh weight, and aboveground dry weight. This invention is the first to discover that the TaCYP51H37 protein positively regulates plant resistance to stem rot, which is of great significance and application value for improving plant disease resistance and cultivating new disease-resistant plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of TaCYP51H37 protein and related biomaterials in regulating plant stem base rot resistance. Background Technology

[0002] Wheat stem rot ( Fusarium Wheat stem rot (FCR) is a highly contagious soil-borne disease in arid and semi-arid regions, causing widespread lodging, yield reduction, and mortality in wheat production, seriously threatening grain production. The main pathogen causing wheat stem rot is *Fusarium* (*Fusarium* spp.). Fusarium sp. Fusarium pseudograss () F. pseudograminearum , Fpg Fusarium oxysporum can survive in soil for a long time, remaining active in crop residues for up to three years, causing the incidence of wheat stem rot to increase year by year. Furthermore, early-stage wheat stem rot is difficult to detect, and the mycelium spreads extremely rapidly, covering the entire growth period of wheat, significantly increasing the difficulty of integrated pest management. Wheat stem rot was first discovered and reported in Australia, and has since occurred in wheat-producing regions worldwide, including Asia, Africa, North America, South America, and Oceania. In recent years, the affected area of ​​wheat stem rot has gradually expanded in my country's major wheat-producing areas, causing severe yield reductions in provinces such as Henan and Shandong, becoming a major wheat disease and posing a serious threat to food security. The China Association for Science and Technology has listed scientific and effective control of wheat stem rot as one of my country's top ten industrial technology issues.

[0003] Wheat, as one of the world's most important food crops, is widely used as a staple food and industrial raw material. Currently, the damage caused by stem base rot in my country's main wheat-producing areas is gradually worsening. According to reports, the affected area of ​​wheat stem base rot in Henan Province has reached 4.974 million mu (approximately 328,667 hectares), with an average disease incidence rate of 13.9% across the province and a maximum rate of 70%. In Shandong Province, the affected area exceeds 12 million mu (approximately 800,000 hectares), spanning 123 counties (cities and districts) in 15 cities, with some poorly managed plots experiencing a whitehead rate of 30%-50%. This disease has caused a significant reduction in my country's wheat yield, and the incidence rate is increasing year by year. Furthermore, controlling stem base rot greatly increases the economic cost of cultivation, but it does not fundamentally solve the problem. Currently, large-scale screening of stem base rot-resistant germplasm has been conducted; however, only some moderately resistant germplasm resources have been discovered, and no varieties with highly effective resistance or immunity to stem base rot have been found. Breeding disease-resistant varieties is the most economical and effective way to control wheat stem rot. Therefore, it is urgent to accelerate the research on disease resistance mechanisms, explore major disease-resistant genes, and improve disease-resistant breeding strategies. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the resistance of plant stem base rot.

[0005] To address the aforementioned technical problems, the present invention first provides novel uses for proteins or substances that regulate the content and / or activity of said proteins.

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

[0007] A1) Regulate plant disease resistance; A2) Cultivating plants with altered disease resistance; A3) Plant breeding or plant variety improvement; A4) Prepare products that regulate plant disease resistance; A5) Prepare products from plants that have been modified to have disease resistance; A6) Prepare products for plant breeding or plant variety improvement; The protein is named TaCYP51H37 and is derived from wheat (…). Triticum aestivum L.), the TaCYP51H37 protein is any one of the following B1)-B4): B1) The amino acid sequence is that of the protein shown in SEQ ID No. 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 SEQ ID No. 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 SEQ ID No. 1; B4) is a protein that has 80% or more of the same amino acid sequence as the one shown in SEQ ID No. 1 and has the same function.

[0008] In the protein described in B2) above, 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) above, 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, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or 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 protein described in B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence 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 Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the identity of a pair of amino acid sequences, the identity value (%) can then be obtained. The identity includes amino acid sequences having 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology with the amino acid sequence shown in SEQ ID No. 1 of this invention.

[0011] The proteins described in B1), B2), B3), or B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] In the above applications, substances that regulate the content and / or activity of TaCYP51H37 protein include substances that increase the content and / or activity of TaCYP51H37 protein or substances that decrease the content and / or activity of TaCYP51H37 protein.

[0013] Furthermore, the substance that enhances the activity of TaCYP51H37 protein may be a protein, polypeptide, or small molecule compound that enhances or promotes the function of TaCYP51H37 protein.

[0014] The substance that increases the content of TaCYP51H37 protein may be a substance that promotes the synthesis of TaCYP51H37 protein or inhibits the degradation or overexpression of TaCYP51H37 protein.

[0015] The substance that reduces the activity of TaCYP51H37 protein may be a protein, polypeptide, or small molecule compound that inhibits the function of TaCYP51H37 protein.

[0016] The substance that reduces the content of TaCYP51H37 protein may be a substance that inhibits TaCYP51H37 protein synthesis, promotes TaCYP51H37 protein degradation, or inhibits the expression of the TaCYP51H37 protein-encoding gene, or a substance that knocks down (reduces) or eliminates the TaCYP51H37 protein-encoding gene.

[0017] Furthermore, the substance that overexpresses TaCYP51H37 protein is a vector that overexpresses the TaCYP51H37 protein encoding gene.

[0018] The substance that inhibits the expression of the TaCYP51H37 protein-coding gene is a VIGS vector containing a specific fragment of the TaCYP51H37 protein-coding gene.

[0019] In some embodiments, the vector overexpressing the TaCYP51H37 protein-coding gene is a recombinant plasmid pWMB110-TaCYP51H37. The recombinant plasmid pWMB110-TaCYP51H37 is obtained by inserting the DNA molecule shown in SEQ ID No. 2 into the Spe I restriction site of the vector pWMB110.

[0020] In some embodiments, the VIGS vector containing a specific fragment of the TaCYP51H37 protein-coding gene is the recombinant plasmid BSMV γ- TaCYP51H37 The recombinant plasmid BSMV γ- TaCYP51H37 The plasmid was obtained by inserting the DNA molecule shown in SEQ ID No. 3 into the NheⅠ restriction site of the vector BSMV-γ.

[0021] To address the aforementioned technical problems, this invention provides new uses for biomaterials related to the TaCYP51H37 protein.

[0022] This invention provides the use of biomaterials related to the TaCYP51H37 protein in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Cultivating plants with altered disease resistance; A3) Plant breeding or plant variety improvement; A4) Prepare products that regulate plant disease resistance; A5) Prepare products from plants that have been modified to have disease resistance; A6) Prepare products for plant breeding or plant variety improvement; The biomaterial is any one of the following E1) to E8): E1) The nucleic acid molecule encoding the TaCYP51H37 protein mentioned above; E2) Nucleic acid molecules that inhibit or interfere with the expression of the TaCYP51H37 protein-coding gene; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecule described in E1) or E2), or a recombinant vector containing the expression cassette described in E3); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2), or recombinant microorganisms containing the expression cassette described in E3), or recombinant microorganisms containing the recombinant vector described in E4).

[0023] E6) A transgenic plant cell line containing the nucleic acid molecule described in E1) or E2), or a transgenic plant cell line containing the expression cassette described in E3), or a transgenic plant cell line containing the recombinant vector described in E4); E7) Transgenic plant tissue containing the nucleic acid molecules described in E1) or E2), or transgenic plant tissue containing the expression cassette described in E3), or transgenic plant tissue containing the recombinant vector described in E4); E8) A transgenic plant organ containing the nucleic acid molecule described in E1) or E2), or a transgenic plant organ containing the expression cassette described in E3), or a transgenic plant organ containing the recombinant vector described in E4).

[0024] In E1 above, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA. The nucleic acid molecule can also be RNA, such as mRNA.

[0025] In this invention, the nucleic acid molecule is any one of the following: F1) The DNA molecule shown in SEQ ID No. 2; The nucleotide sequence defined by F2) has 75% or more identity with F1) and is a DNA molecule encoding the TaCYP51H37 protein.

[0026] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaCYP51H37 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity with the TaCYP51H37 nucleotide sequence isolated according to this invention, provided they encode the TaCYP51H37 protein and have the same function, are derived from and equivalent to the nucleotide sequence of this invention. This identity refers to sequence similarity to a natural nucleic acid sequence, including nucleotide sequences possessing 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0027] In E2 above, the nucleic acid molecule can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0028] In this invention, the nucleic acid molecule is any one of the following: G1) The DNA molecule shown in SEQ ID No. 3; DNA molecules that have 75% or more identity with the nucleotide sequences defined by G2 and G1 and regulate plant disease resistance.

[0029] The expression cassette described above may include a promoter, the nucleic acid molecule described in E1) or E2) 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.

[0030] The vector mentioned above refers to a vector capable of carrying the nucleic acid molecules described in E1) or E2) 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, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0031] The recombinant vector described above refers to a recombinant DNA molecule constructed by ligating the nucleic acid molecule described in E1) or E2) above with the vector in vitro. Recombinant vectors containing the nucleic acid molecule described in E1) or E2) above can be constructed using existing plant expression vectors. 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.

[0032] Any of the microorganisms mentioned above can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Among them, the bacteria may originate 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, 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.

[0033] The recombinant microorganisms mentioned above refer to those obtained by manipulating and modifying the genes of a target microorganism, thereby altering its function. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into a 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.

[0034] The transgenic plant tissues described above may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0035] The organs of any of the transgenic plants mentioned above may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0036] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described above may or may not include propagation material.

[0037] In any of the above applications, the regulation of plant disease resistance refers to either increasing or decreasing plant disease resistance. The regulation method is positive regulation, specifically manifested as follows: when the content and / or activity of TaCYP51H37 protein in the plant increases, the plant's disease resistance increases; when the content and / or activity of TaCYP51H37 protein in the plant decreases or is absent, the plant's disease resistance decreases.

[0038] In some implementation schemes, when plants TaCYP51H37 Increased gene expression levels enhance plant resistance to stem rot.

[0039] In some implementation schemes, when plants TaCYP51H37 When gene expression levels decrease, plant resistance to stem rot decreases.

[0040] To address the aforementioned technical problems, the present invention also provides a method for improving plant disease resistance.

[0041] The method for improving plant disease resistance provided by the present invention includes the following steps: increasing the content and / or activity of TaCYP51H37 protein in the target plant.

[0042] To address the aforementioned technical problems, this invention also provides a method for cultivating transgenic plants with enhanced disease resistance.

[0043] The method for cultivating transgenic plants with enhanced disease resistance provided by the present invention includes the following steps: increasing the content and / or activity of TaCYP51H37 protein in the target plant to obtain a transgenic plant; the transgenic plant has higher disease resistance than the target plant.

[0044] To address the aforementioned technical problems, the present invention also provides a method for plant breeding or plant variety improvement.

[0045] 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.

[0046] In the above method, the method for increasing the content and / or activity of TaCYP51H37 protein in the target plant is to overexpress the TaCYP51H37 protein in the plant.

[0047] Furthermore, the overexpression method involves introducing the gene encoding the TaCYP51H37 protein into the target plant.

[0048] Furthermore, the gene encoding the TaCYP51H37 protein is the DNA molecule shown in SEQ ID No. 2.

[0049] In some embodiments, the gene encoding the TaCYP51H37 protein is introduced into the target plant via the recombinant plasmid pWMB110-TaCYP51H37. The recombinant plasmid pWMB110-TaCYP51H37 is obtained by inserting the DNA molecule shown in SEQ ID No. 2 into the Spe I restriction site of the vector pWMB110.

[0050] To address the aforementioned technical problems, the present invention also provides a method for reducing plant disease resistance.

[0051] The method for reducing plant disease resistance provided by the present invention includes the following steps: reducing the content and / or activity of TaCYP51H37 protein in the target plant.

[0052] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with reduced disease resistance.

[0053] The method for cultivating transgenic plants with reduced disease resistance provided by the present invention includes the following steps: reducing the content and / or activity of TaCYP51H37 protein in the target plant to obtain a transgenic plant; the transgenic plant has lower disease resistance than the target plant.

[0054] Furthermore, the method for reducing the content and / or activity of TaCYP51H37 protein in the target plant is to introduce a substance that inhibits or interferes with the expression of the TaCYP51H37 protein encoding gene into the target plant.

[0055] Furthermore, the substance that inhibits or interferes with the expression of the TaCYP51H37 protein-coding gene is a substance that silences the TaCYP51H37 protein-coding gene.

[0056] Furthermore, the substance used to silence the TaCYP51H37 protein-coding gene is a VIGS vector containing a specific fragment of the TaCYP51H37 protein-coding gene.

[0057] In some embodiments, the VIGS vector containing a specific fragment of the TaCYP51H37 protein-coding gene is the recombinant plasmid BSMV γ- TaCYP51H37。 The recombinant plasmid BSMV γ- TaCYP51H37 The plasmid was obtained by inserting the DNA molecule shown in SEQ ID No. 3 into the NheⅠ restriction site of the vector BSMV-γ.

[0058] The disease resistance described above refers to stem base rot resistance. This stem base rot resistance includes, but is not limited to, resistance to stem base rot caused by infection with *Fusarium graminearum*, *Fusarium graminearum*, and other *Fusarium* species.

[0059] In some implementation cases, the stem base rot resistance refers to stem base rot resistance caused by Fusarium graminearum infection.

[0060] In some embodiments, the transgenic plant exhibits higher disease resistance than the target plant as manifested in any one of the following X1)-X5): X1) Under Fusarium oxysporum infection stress, the growth status of the transgenic plant is better than that of the target plant; X2) Under Fusarium oxysporum infection stress, the disease severity index of the transgenic plant is lower than that of the target plant; X3) Under Fusarium oxysporum infection stress, the aboveground part of the transgenic plant is longer than that of the target plant; X4) Under Fusarium oxysporum infection stress, the aboveground fresh weight of the transgenic plant was higher than that of the target plant; X5) Under Fusarium oxysporum infection stress, the aboveground dry weight of the transgenic plant was higher than that of the target plant.

[0061] In some embodiments, the disease resistance of the transgenic plant is lower than that of the target plant as manifested in any one of the following Y1)-Y5): Y1) Under Fusarium oxysporum infection stress, the growth status of the transgenic plant was worse than that of the target plant; Y2) Under Fusarium oxysporum infection stress, the disease severity index of the transgenic plant is higher than that of the target plant; Y3) Under Fusarium oxysporum infection stress, the aboveground part of the transgenic plant is shorter than that of the target plant; Y4) Under Fusarium oxysporum infection stress, the fresh weight of the aboveground parts of the transgenic plant is lower than that of the target plant; Y5) Under Fusarium oxysporum infection stress, the aboveground dry weight of the transgenic plant was lower than that of the target plant.

[0062] In some specific embodiments, the *Fusarium graminearum* infection is achieved by directly dripping a spore solution of *Fusarium graminearum* onto the base of the wheat stem, and the preferred concentration of the *Fusarium graminearum* spore solution is 1 × 10⁻⁶. 6 Cells / mL. For specific steps, please refer to the method in the literature "Wheat WRKY transcription factor TaWRKY24 confers drought and salttolerance in transgenic plants".

[0063] In some specific implementations, the *Fusarium graminearum* is *Fusarium graminearum* CS3096.

[0064] 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.

[0065] The transgenic plants mentioned above 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.

[0066] The indicators for plant breeding mentioned above include disease resistance (such as resistance to stem base rot).

[0067] The purpose of any of the above-mentioned plant breeding programs includes developing disease-resistant plant varieties (such as plant varieties resistant to stem rot).

[0068] 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).

[0069] This invention uses wheat ( Triticum aestivum L.) regulates plant disease resistance TaCYP51H37 The gene was introduced into the recipient control plant, wild-type wheat (Fielder), and transgenic wheat was obtained. TaCYP51H37 Wheat homozygous strains ( TaCYP51H37 -OE4、 TaCYP51H37 -OE8 and TaCYP51H37 -OE10). By converting TaCYP51H37 Identification of stem rot resistance in homozygous wheat lines revealed that, compared to the non-transgenic recipient control, overexpression of *Fusarium graminearum* was observed under *Fusarium graminearum* infection.TaCYP51H37 The disease severity index of transgenic wheat plants with the overexpressed gene was significantly lower than that of the recipient control, while the length, fresh weight, and dry weight of the aboveground parts were all significantly higher than those of the recipient control, indicating that the overexpression of the gene... TaCYP51H37 The gene can significantly enhance resistance to wheat stem rot. To further verify... TaCYP51H37 Genes positively regulate wheat disease resistance; this invention also constructs... TaCYP51H37 Silent wheat and its resistance to stem base rot were identified. The results showed that, compared with the recipient control, TaCYP51H37 The disease severity index of silent wheat was significantly increased, and the length, fresh weight, and dry weight of the aboveground parts were all significantly reduced. This invention is the first to discover that the TaCYP51H37 protein positively regulates plant resistance to stem rot, which is of great significance and application value for improving plant disease resistance and breeding new disease-resistant varieties. Attached Figure Description

[0070] Figure 1 For the transfer TaCYP51H37 wheat and wild-type wheat TaCYP51H37 Expression level analysis. Fielder represents the recipient control wild-type wheat; OE-4 represents... TaCYP51H37 -Wheat of OE4 strain; expressed by OE-8 TaCYP51H37 -OE8 wheat strain; OE-10 indicates TaCYP51H37 -OE10 strain of wheat. The difference is significant (P<0.05).

[0071] Figure 2 For the transfer TaCYP51H37 Identification of wheat resistance to stem rot during the seedling stage. 'a' represents the transition during the seedling stage. TaCYP51H37 Phenotypic photographs of wheat and the recipient control under normal conditions and stem rot stress treatment. b shows the transformation at the seedling stage. TaCYP51H37 Phenotypic photographs of wheat and the recipient control under stem rot stress treatment. c represents the transition at the seedling stage. TaCYP51H37 Statistical analysis of disease severity indices in wheat and the recipient control under stem rot stress treatment. d represents the transition during the seedling stage. TaCYP51H37 Aboveground part length of wheat and recipient control under normal and stem rot stress conditions, respectively. e represents the length of the transplanted wheat at the seedling stage. TaCYP51H37 The aboveground fresh weights of wheat and the recipient control under normal conditions and stem rot stress, respectively. f represents the conversion rate at the seedling stage. TaCYP51H37 Aboveground dry weights of wheat and the recipient control under normal conditions and stem base rot stress, respectively. (See figure.) The difference is statistically significant (P<0.05). Fielder represents the recipient control wild-type wheat; OE-4 represents... TaCYP51H37-Wheat of OE4 strain; expressed by OE-8 TaCYP51H37 -OE8 wheat strain; OE-10 indicates TaCYP51H37 -OE10 strain of wheat.

[0072] Figure 3 for TaCYP51H37 Identification of resistance to stem rot in silent wheat seedlings. (a) TaCYP51H37 Phenotypic photographs of silent wheat and control wheat under normal growth conditions. (b) TaCYP51H37 Phenotypic photographs of silent wheat and control wheat under stem base rot stress treatment. c represents... TaCYP51H37 Silent wheat and control wheat under normal conditions TaCYP51H37 Gene expression level. d represents... TaCYP51H37 Silent wheat and control wheat under stem rot stress treatment TaCYP51H37 Gene expression level. e represents... TaCYP51H37 Disease severity index of silent wheat and control wheat under stem base rot stress treatment. f represents... TaCYP51H37 Aboveground part length of silent wheat and control wheat under normal conditions and stem base rot stress. g is... TaCYP51H37 The aboveground fresh weight of silent wheat and control wheat under normal conditions and stem base rot stress. h is... TaCYP51H37 Aboveground dry weight of silent wheat and control wheat under normal conditions and stem base rot stress. (See figure.) The difference is significant (P<0.05). Detailed Implementation

[0073] 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.

[0074] 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.

[0075] The *Fusarium graminearum* CS3096 in the following examples is described in the literature “Comparative pathogenomicsreveals horizontally acquired novel virulence genes in fungi infecting cerealhosts”, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0076] 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.

[0077] The pEasyBlunt vector used in the following examples is a product of Beijing TransGen Biotech Co., Ltd.

[0078] The qPCR enzyme used in the following examples is a product of Beijing TransGen Biotech Co., Ltd.

[0079] The Agrobacterium tumefaciens EH105 used in the following examples is a product of Beijing Bairddi Biotechnology Co., Ltd.

[0080] The vector pWMB110 in the following examples is described in the literature "Liu Y, Yu TF, Li YT, Zheng L, LuZW, ZhouYB, ChengJ, ChenM, ZhangJP, SunGZ, CaoXY, Liu YW, Ma YZ and Xu ZS. (2022) Mitogen-activated protein kinase TaMPK3 suppresses ABA response by destabilizing TaPYL4 receptor in wheat". New Phytologist The information is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences, in ,236(1),pp.114-131.”

[0081] The virus-induced gene silencing vectors BSMVα, BSMVβ, and BSMVγ used in the following examples are described in the literature "QiH, Zhu X, Guo F, Lv L, Zhang Z. The Wall-Associated Receptor-Like KinaseTaWAK7D Is Required for Defense Responses to..." Rhizoctonia cerealis The article, available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences, is titled "Wheat. International Journal of Molecular Sciences, 22(11):5629."

[0082] 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.

[0083] Example 1: Obtaining the TaCYP51H37 protein and its encoding gene This invention isolates and clones a substance related to plant resistance to stem rot from the wheat variety "Fielder". TaCYP51H37 Genes. The specific steps are as follows: 1. Fielder wheat seedlings that have grown under normal conditions for about 2 weeks are flash-frozen with liquid nitrogen and stored at -80℃ for later use.

[0084] 2. Total RNA was extracted from wheat leaves using the Trizol method (TianGen), and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). ds cDNA was synthesized using the SMART method, and the PCR products were detected by 1.0% agarose gel electrophoresis.

[0085] 3. The DNA molecule shown in SEQ ID No. 2 was obtained by 5' RACE and 3' RACE methods, and the DNA molecule shown in SEQ ID No. 2 was named TaCYP51H37 Gene. The amino acid sequence of the TaCYP51H37 protein encoded by this gene is shown in SEQ ID No. 1.

[0086] 4. The DNA molecule shown in SEQ ID No. 2 was ligated into the pEasy-Blunt vector using the Peasyblunt kit to form the pEasyBlunt-TaCYP51H37 plasmid.

[0087] Example 2, Transfer TaCYP51H37 Preparation of wheat and analysis of its resistance to stem rot I. Construction of Recombinant Expression Vectors 1. Using the pEasyBlunt-TaCYP51H37 plasmid from Example 1 as a template, PCR amplification was performed using primer pairs consisting of TaCYP51H37-110F and TaCYP51H37-110R. The PCR amplification products were then recovered from the gel. The primer sequences are as follows (Note: the underlined parts are the recognition sequences of the restriction endonuclease Spe I): TaCYP51H37-110F: 5'-CTAGAGGATCC ACTAGT ATGGAGATGGCAAGTAGCGCC-3'; TaCYP51H37-110R: 5'-AGCTTGGCCG ACTAGT GCCTAGCAGCTGGCGTCTCTT-3.

[0088] 2. Digest the vector pWMB110 with the restriction endonuclease Spe I and recover the vector backbone.

[0089] 3. The PCR product recovered in step 1 and the vector backbone from step 2 are ligated using In-Fusion technology to obtain the ligation product. The ligation product is sequenced, and the recombinant plasmid with correct sequencing is named pWMB110-TaCYP51H37.

[0090] Sequencing results showed that the recombinant plasmid pWMB110-TaCYP51H37 was obtained by inserting the DNA molecule shown in SEQ ID No.2 into the Spe I restriction site of the vector pWMB110.

[0091] II. Obtaining Genetically Modified Wheat 1. The recombinant plasmid pWMB110-TaCYP51H37 was introduced into Agrobacterium EH105 to obtain Agrobacterium EH105 / pWMB110-TaCYP51H37 containing the recombinant plasmid pWMB110-TaCYP51H37.

[0092] 2. Agrobacterium EH105 / pWMB110-TaCYP51H37 containing recombinant plasmid pWMB110-TaCYP51H37 was inoculated into YEP liquid medium and cultured at 28°C and 3000 rpm for about 18 hours.

[0093] 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.

[0094] 4. Sterilize wheat (Fielder) seeds with chlorine for 8 hours, then sow them evenly in sterilized B5 medium and culture until the radicle forms.

[0095] 5. After completing step 4, remove the wheat cotyledons and the radicle below the cotyledon node, and then culture the wheat until callus tissue grows. Infect the callus tissue with Agrobacterium EH105 (EH105 / pWMB110-TaCYP51H37) containing the recombinant plasmid pWMB110-TaCYP51H37, which was cultured on the solid medium in step 3. After culturing, T0 generation transgenic wheat is obtained.

[0096] 6. DNA was extracted from transgenic wheat plants for PCR identification. The primer sequences for PCR identification were as follows: F: 5'-TTTAGCCCTGCCTTCATACG-3'; R: 5'-CCCATCTCATAAATAACGTCATGC-3'. The transgenic wheat with a size of 1723 bp obtained by PCR amplification was identified as T0 positive transgenic wheat. The T0 positive transgenic wheat was propagated, and three stable transformations with normal growth were screened in the third generation. TaCYP51H37 The homozygous wheat lines were named as follows: TaCYP51H37 -OE4 strain, TaCYP51H37 -OE8 strain and TaCYP51H37 -OE10 strain.

[0097] three, TaCYP51H37 Gene relative expression level detection Take T3 as the replacement TaCYP51H37 Wheat strains ( TaCYP51H37 -OE4、 TaCYP51H37 -OE8 and TaCYP51H37 Total RNA was extracted from leaves of type OE10 and reverse transcribed to obtain cDNA. qRT-PCR was performed using the cDNA as a template, with the Actin gene used as an internal control gene. [Detection / Survey] TaCYP51H37 The relative expression level of genes.

[0098] Used for detection TaCYP51H37 The primers for the gene are as follows: TaCYP51H37qPCR-F: 5'-TCCCTCGCTAGTACCTGGAC-3'; TaCYP51H37qPCR-R: 5'-TTGGTGGGTTCAACCGAAGT-3'.

[0099] The primers used to detect the Actin gene are as follows: Actin-F: 5'-GGAATCCATGAGACCACCTAC-3'; Actin-R: 5'-GACCCAGACAACTCGCAAC-3'.

[0100] The results are as follows Figure 1As shown, the results indicate that: TaCYP51H37 in wheat TaCYP51H37 The gene expression level was 20-50 times higher than that in wild-type wheat (recipient control), and the difference was statistically significant. This indicates that the exogenous gene... TaCYP51H37 The gene has not only been successfully integrated into the wheat genome, but it can also be used in transgenic wheat. TaCYP51H37 Normal transcriptional expression in wheat.

[0101] IV. Transfer TaCYP51H37 Identification of resistance to stem rot in wheat To verify overexpression TaCYP51H37 Regulation of resistance to wheat stem rot by genes, identification of transgenic genes TaCYP51H37 Resistance to stem rot in wheat and recipient control plants. The specific steps are as follows: Test material: The material obtained in step two TaCYP51H37 Wheat strains ( TaCYP51H37 -OE4、 TaCYP51H37 -OE8 and TaCYP51H37 -OE10) and wild wheat (WT, Fielder).

[0102] Experimental methods: The resistance of the tested materials to stem base rot was identified. The experiment was divided into the following two groups: Stem base rot stress treatment group: Weigh out the same mass of soil and sow the same number of seeds. TaCYP51H37 Wheat and recipient control. When the seedlings reached the 4-leaf stage, they were inoculated with wheat grains infected with Fusarium graminearum CS3096. After 7 days of normal growth, the disease severity index, aboveground part length, aboveground part fresh weight, and aboveground part dry weight were recorded.

[0103] Normal group (CK): Weigh out the same mass of soil and sow the same number of seeds. TaCYP51H37 Wheat and the recipient control. Disease severity index, aboveground length, aboveground fresh weight, and aboveground dry weight were measured after the same growth time as the treatment group at 22℃ (normal conditions).

[0104] The stem base rot stress treatment group and the normal group were identical in all experimental conditions except for the stem base rot inoculation. Three replicate experiments were set up, with 15 plants of each test line observed and their traits statistically analyzed in each replicate.

[0105] 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".

[0106] 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 "".

[0107] The results are as follows Figure 2 As shown, the results indicated that after infection with *Fusarium graminearum*, severe lesions appeared at the stem base of the recipient control wheat, and the disease severity index of the recipient control was significantly higher than that of the transgenic wheat. TaCYP51H37 Genetically modified wheat plants, TaCYP51H37 The aboveground part length of wheat infected with *Fusarium graminearum* was significantly longer than that of the recipient control, and both the fresh weight and dry weight of the aboveground parts were also significantly higher. These results indicate that overexpression... TaCYP51H37 Genes can significantly enhance a plant's resistance to stem base rot.

[0108] Example 3 TaCYP51H37 Preparation of Silent Wheat and Analysis of its Resistance to Stem Base Rot one, TaCYP51H37 Construction of silent carriers 1. Total RNA was extracted from wheat Fielder leaves and reverse transcribed to obtain cDNA.

[0109] 2. Website design using SGN VIGS tool TaCYP51H37 Optimal silence segment, TaCYP51H37 The optimal silencing fragment is shown in SEQ ID No. 3. Then, using the cDNA from step 1 as a template, in... TaCYP51H37 Specific primers were designed at both ends of the optimal silenced fragment to amplify the gene, and the PCR product was recovered. The amplification primers are as follows: BSMV- TaCYP51H37 -F:5'-GTCCAATCCATTTCTTTCAA-3'; BSMV- TaCYP51H37 -R:5'-TTACGTGACCTCACAGTTCCAG-3'.

[0110] 3. The amplified and recovered PCR products were ligated into the pEASY-Blunt vector (TransGen Biotech, Beijing) and transformed into *E. coli* Trans1-T1 competent cells (TransGen Biotech, CD501-02). The cells were then plated onto solid LB agar plates containing 50 μg / L kanamycin and incubated overnight at 37°C. Colonies of *E. coli* were subjected to culture PCR detection. Positive clones were sent to the company for sequencing. The correctly sequenced colonies were preserved, and the pEASY-Blunt vector was obtained. TaCYP51H37 Plasmid.

[0111] 4. Using the plasmid obtained in step 3 as a template, γ- TaCYP51H37 -F and γ- TaCYP51H37 PCR amplification was performed using primer pairs consisting of -R to obtain PCR amplification products, which were then recovered from the gel.

[0112] γ- TaCYP51H37 -F:5'-TTTTTTTTTTTTTTAGCTAGC GTCCAATCCATTTCTTTCAA-3'; γ- TaCYP51H37 -R:5'-GATTCTCTTCCGTTGCTAGC TTACGTGACCTCACAGTTCCAG-3'.

[0113] 5. Digest the vector BSMV-γ with the restriction endonuclease NheⅠ and recover the vector backbone.

[0114] 6. The PCR product from step 4 and the vector backbone from step 5 were ligated using Takara's In-Fusion technology to obtain the ligation product. The ligation product was sequenced, and the correctly sequenced recombinant plasmid was named BSMV γ- TaCYP51H37 .

[0115] Sequencing results showed that the recombinant plasmid BSMV γ- TaCYP51H37 The plasmid was obtained by inserting the DNA molecule shown in SEQ ID No. 3 into the NheⅠ restriction site of the vector BSMV-γ.

[0116] two, TaCYP51H37 The Acquisition of Silent Wheat 1. Linearization of VIGS vectors The vector BSMV-α was digested with the restriction endonuclease MluⅠ, and the vector backbone was recovered.

[0117] The vector BSMV-β was digested with the restriction endonuclease SpeⅠ, and the vector backbone was recovered.

[0118] The vector BSMV-γ was digested with the restriction endonuclease NheⅠ, and the vector backbone was recovered.

[0119] The vector BSMV γ- was digested with the restriction endonuclease MluⅠ. TaCYP51H37 , Recycle the carrier skeleton.

[0120] 2. After completing step 1, the recovered vectors were transcribed in vitro using the Promega RiboMAX™ Large Scale RNA Production Systems-T7 kit.

[0121] 3. Prepare FES buffer solution 5×GP buffer stock solution: 1.877g glycine, 2.613g dipotassium hydrogen phosphate, RNase-free ddH2O to a final volume of 50mL, sterilize in an autoclave for 20min and set aside.

[0122] Prepare FES buffer: 1×GP Buffer 100 mL, sodium pyrophosphate 5 g, bentonite 5 g, diatomaceous earth 5 g, RNase-free ddH2O to a final volume of 500 mL, sterilize in an autoclave for 20 min and set aside.

[0123] Composition of different treatment friction fluids: Products from pre-completed in vitro transcription, α, β, γ / γ- TaCYP51H37 2.5 μL of each solution were mixed thoroughly at a 1:1:1 (volume ratio), and diluted with an equal volume of RNase-free ddH2O. 5 μL of the diluted mixture was then pipetted and mixed with 90 μL of FES buffer to obtain the FES mixture. Four groups were set up for each experiment: a completely blank control group (WT), a viral blank control group (α+β+γ), and a gene silencing group (α+β+γ-). TaCYP51H37 ).

[0124] 4. After completing step 3, spray a small amount of RNase-free ddH2O onto the surface of the wheat leaves (Fielder) to be infected. Apply 8-10 μL of FES buffer to a clean glove and rub the second leaf of the seedling three times, controlling the pressure during rubbing. After rubbing, spray a small amount of RNase-free ddH2O from top to bottom to maintain humidity. Change to clean gloves after each treatment. After virus inoculation, incubate at 23±2℃ in the dark for 24 h, then adjust to a 16h / 8h light / dark cycle. Observe and record phenotypic changes regularly.

[0125] 5. After completing step 4, wait 10 days for the wheat to show signs of BSMV virus infection (yellow spots on leaves), and then determine the gene silencing efficiency of the wheat plants. Take wheat leaves infected with Fielder and BSMV viruses, extract total RNA, and reverse transcribe it to obtain cDNA. Use the cDNA template for qRT-PCR, using the Actin gene as an internal control gene, and detect... TaCYP51H37 The relative expression level of genes.

[0126] Used for detection TaCYP51H37 The primers for the gene are as follows: RT- TaCYP51H37 -F:5'-GATGGTTAGCTACAAGAGACG-3'; RT- TaCYP51H37 -R:5'-GCACACACTATGAGAAGACCA-3'.

[0127] The primers used to detect the Actin gene are as follows: RT-Actin-F: 5'-CCTCTCTGCGCCAATCGT-3'; RT-Actin-R:5'-TCAGCCGAGCGGGAAATTGT-3'.

[0128] The results are as follows Figure 3 As shown. The results indicate that compared with wheat plants infected with BSMV:γ (corresponding to the "virus blank control group (α+β+γ)" mentioned above), wheat plants infected with BSMV:γ- TaCYP51H37 Wheat plants (corresponding to the "gene silencing group (α+β+γ-)" mentioned earlier) TaCYP51H37 The transcript levels of ) were significantly reduced, indicating TaCYP51H37 The gene was successfully silenced.

[0129] three, TaCYP51H37 Identification of resistance to stem base rot in silent wheat To further verify TaCYP51H37 Gene resistance to wheat stem base rot, identification TaCYP51H37 Disease resistance of silent wheat during the seedling stage. The specific steps are as follows: Test materials: TaCYP51H37 Silent wheat plants (BSMV-) TaCYP51H37 Silent plant control wheat (BSMV:γ).

[0130] Experimental methods: The resistance of the tested materials to stem base rot was identified. The experiment was divided into two groups (each group was repeated three times): Stem base rot stress treatment group: infected with Fusarium graminearum spore liquid. TaCYP51H37Silent wheat plants and silent control wheat plants were observed at the base of their stems. After infection, lesions appeared one week after growth in a culture chamber under normal conditions. The plants were photographed and leaves were taken for qRT-PCR detection. The wheat disease severity index, aboveground part length, aboveground part fresh weight, and aboveground part dry weight were then recorded.

[0131] Normal group: The test materials were cultured under normal conditions in an incubator and photographed at the same time as the stem base rot stress treatment group. Leaves were also taken for qRT-PCR detection. Afterwards, the wheat disease grade index, aboveground part length, aboveground part fresh weight and aboveground part dry weight were counted.

[0132] The results are as follows Figure 3 As shown, the results indicate that after infection by *Fusarium graminearum*, TaCYP51H37 Silent wheat plants had a higher disease severity index than control wheat, but their expression level after Fusarium graminearum infection was significantly lower than that of control wheat. Furthermore, after Fusarium graminearum infection, TaCYP51H37 The aboveground length, aboveground fresh weight, and aboveground dry weight of the silent wheat were all significantly lower than those of the control wheat.

[0133] The above results indicate that the TaCYP51H37 protein and its encoding gene... TaCYP51H37 It can regulate plant resistance to stem rot by increasing the content and / or activity of TaCYP51H37 protein in the target plant (e.g., through overexpression). TaCYP51H37 (Gene) can significantly improve the resistance of the target plant to stem base rot.

[0134] 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 a protein or a substance that regulates the content and / or activity of said protein in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Cultivating plants with altered disease resistance; A3) Plant breeding or plant variety improvement; A4) Prepare products that regulate plant disease resistance; A5) Prepare products from plants that have been modified to have disease resistance; A6) Prepare products for plant breeding or plant variety improvement; The protein is any one of the following B1)-B4): B1) The amino acid sequence is that of the protein shown in SEQ ID No. 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 SEQ ID No. 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 SEQ ID No. 1; B4) is a protein that has 80% or more of the same amino acid sequence as the one shown in SEQ ID No. 1 and has the same function.

2. Use of the biomaterial related to the protein of claim 1 in any of the following A1)-A6): A1) Regulate plant disease resistance; A2) Cultivating plants with altered disease resistance; A3) Plant breeding or plant variety improvement; A4) Prepare products that regulate plant disease resistance; A5) Prepare products from plants that have been modified to have disease resistance; A6) Prepare products for plant breeding or plant variety improvement; The biomaterial is any one of the following E1) to E8): E1) A nucleic acid molecule encoding the protein described in claim 1; E2) Nucleic acid molecules that inhibit or interfere with the expression of the protein-coding gene as described in claim 1; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecule described in E1) or E2), or a recombinant vector containing the expression cassette described in E3); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2), or recombinant microorganisms containing the expression cassette described in E3), or recombinant microorganisms containing the recombinant vector described in E4); E6) A transgenic plant cell line containing the nucleic acid molecule described in E1) or E2), or a transgenic plant cell line containing the expression cassette described in E3), or a transgenic plant cell line containing the recombinant vector described in E4); E7) Transgenic plant tissue containing the nucleic acid molecules described in E1) or E2), or transgenic plant tissue containing the expression cassette described in E3), or transgenic plant tissue containing the recombinant vector described in E4); E8) A transgenic plant organ containing the nucleic acid molecule described in E1) or E2), or a transgenic plant organ containing the expression cassette described in E3), or a transgenic plant organ containing the recombinant vector described in E4).

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 SEQ ID No. 2; The nucleotide sequence defined by F2) has 75% or more identity with F1) and is a DNA molecule encoding the protein.

4. The application according to claim 2, characterized in that: E2) The nucleic acid molecule is any one of the following: G1) The DNA molecule shown in SEQ ID No. 3; DNA molecules that have 75% or more identity with the nucleotide sequences defined by G2 and G1 and regulate plant disease resistance.

5. A method for improving plant disease resistance, comprising the following steps: increasing the content and / or activity of the protein described in claim 1 in the target plant.

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

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

8. A method for reducing plant disease resistance, comprising the steps of: reducing the content and / or activity of the protein described in claim 1 in the target plant.

9. A method for cultivating transgenic plants with reduced disease resistance, comprising the following steps: reducing the content and / or activity of the protein described in claim 1 in a target plant to obtain a transgenic plant; wherein the transgenic plant has lower disease resistance than the target plant.

10. The application according to any one of claims 1-4 or the method according to any one of claims 5-9, characterized in that: The disease resistance mentioned refers to resistance to stem base rot.

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