Methods for modulating plant resistance to clubroot and related sag101 proteins and biological materials

Abstract

This application discloses a method for regulating clubroot resistance in plants, along with the related SAG101 protein and biological materials, belonging to the field of genetic engineering technology. The technical problem this application aims to solve is: how to regulate clubroot resistance in plants. To address this problem, this application provides a method for regulating clubroot resistance in plants, comprising regulating the expression level of the gene encoding the SAG101 protein in the recipient plant and / or regulating the content of the SAG101 protein in the recipient plant to regulate the clubroot resistance of the recipient plant; the SAG101 protein may be a protein with the amino acid sequence SEQ ID NO:2. Studying the function of SAG101 and applying it to the disease resistance mechanism and breeding of cruciferous plants is of great significance for the study of the mechanism of clubroot and / or clubroot resistance in cruciferous plants and for resistance breeding.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically involving methods for regulating plant clubroot resistance and related SAG101 protein and biomaterials. Background Technology

[0002] Arabidopsis thaliana is a widely used model organism in plant science research. Many genes associated with leaf senescence have been identified in Arabidopsis, known as senescence-associated genes (SAGs). SAG101 is one of them. In Arabidopsis, SAG101 is expressed in the early stages of leaf senescence, suggesting that it plays a regulatory role in the process. Leaf senescence is a complex biological process involving the interaction of multiple genes and signaling pathways. SAG101 may be involved in the following aspects: 1. Gene expression regulation: SAG101 may act as a transcription factor or interact with other transcription factors to regulate the expression of other senescence-related genes. 2. Cellular degradation: During senescence, intracellular macromolecules and organelles are degraded and recycled. SAG101 may be involved in regulating this process. 3. Signal transduction: SAG101 may be involved in the transduction of responses to internal and external signals (such as light signals, hormone signals, etc.), thereby influencing the leaf senescence process. No studies have been reported on the function of SAG101 in regulating plant resistance to clubroot bacteria.

[0003] Clubroot is a highly destructive root disease of Brassica crops, often referred to as the "cancer of cruciferous vegetables." It is widespread globally, occurring in almost every country and region that cultivates cruciferous crops. In China, clubroot is also rapidly expanding, affecting an annual area of ​​3.2 to 4 million hectares, accounting for more than one-third of the total cruciferous crop planting area, resulting in yield losses of 10% to 15% of total production, severely impacting the economic benefits for vegetable farmers. With the continuous expansion of cruciferous crop cultivation and the intensification of global climate change, the control of clubroot faces even greater challenges. Future research needs to further strengthen basic research on clubroot, including the biological characteristics of the pathogen, its pathogenic mechanisms, and the discovery and utilization of resistance genes. Summary of the Invention

[0004] The technical problem this application aims to solve is: how to regulate clubroot resistance in plants. Specifically, the technical problem this application aims to solve is: how to regulate clubroot resistance in cruciferous plants.

[0005] To address the aforementioned technical problems, this application provides a method for regulating clubroot resistance in plants. The method may include regulating the clubroot resistance of recipient plants by regulating the expression level of the gene encoding SAG101 protein in the recipient plant and / or regulating the content of the SAG101 protein in the recipient plant.

[0006] The SAG101 protein is any of the following proteins:

[0007] A1) A protein with the amino acid sequence SEQ ID NO:2;

[0008] A2) Proteins obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in A1) that have more than 80% similarity to the amino acid sequence shown in a1) and are associated with plant clubroot resistance.

[0009] A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] In this application, the SAG101 protein may be derived from Arabidopsis thaliana.

[0011] In this application, SEQ ID NO:2 consists of 537 amino acid residues.

[0012] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0014] Further, the connection described in A3) may be a peptide bond formed by dehydration condensation between the N-terminus of the tag and the C-terminus of the protein described in A1) or A2). Alternatively, the connection described in A3) may be a peptide bond formed by dehydration condensation between the C-terminus of the tag and the N-terminus of the protein described in A1) or A2).

[0015] Further, the SAG101 protein described in A1) may be a protein with the amino acid sequence SEQ ID NO:2.

[0016] Furthermore, the method includes reducing the clubroot resistance of the recipient plant by reducing the expression level of the gene encoding the SAG101 protein in the recipient plant and / or reducing the content of the SAG101 protein in the recipient plant, wherein the recipient plant contains the gene encoding the SAG101 protein.

[0017] This application also provides a method for obtaining a target plant with reduced clubroot resistance, the method comprising obtaining a target plant with reduced clubroot resistance by reducing the expression level of the gene encoding the SAG101 protein in the recipient plant and / or reducing the content of the SAG101 protein in the recipient plant, wherein the recipient plant contains the gene encoding the SAG101 protein.

[0018] Furthermore, the method can reduce the expression level of the gene encoding the SAG101 protein in the recipient plant and / or reduce the content of the SAG101 protein in the recipient plant by means of the manner described in B1) below.

[0019] B1) Reduce the expression level of the SAG101 protein encoding gene and / or the content of the SAG101 protein in the recipient plant by knocking out the SAG101 protein encoding gene in the recipient plant using a genome editing system.

[0020] Furthermore, the genome editing system is a CRISPR / Cas system.

[0021] Furthermore, the CRISPR / Cas system can express gRNA encoding the gene that targets the SAG101 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein.

[0022] Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0023] Furthermore, the method can achieve the purpose of reducing the expression level of the gene encoding the SAG101 protein in the recipient plant and / or reducing the content of the SAG101 protein in the recipient plant through the manner described in B2) below.

[0024] B2) Perform any of the following mutations on the genome sequence of the recipient plant:

[0025] B2-1) The deoxynucleotides at positions 1122-1566 of SEQ ID NO:3 of the SAG101 gene are deleted, thereby causing the SAG101 gene to be knocked out.

[0026] B2-2) The deoxynucleotides at positions 1526-1568 of SEQ ID NO:3 of the SAG101 gene are deleted, resulting in SAG101 gene knockout.

[0027] This application also provides a composition for genome editing, said composition being a genome editing system for knocking out the gene encoding the SAG101 protein in a recipient plant.

[0028] Furthermore, the genome editing system may be a CRISPR / Cas system;

[0029] Furthermore, the CRISPR / Cas system may include gRNA targeting the gene encoding the SAG101 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein;

[0030] Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5.

[0031] In some embodiments of this application, the genome editing system is introduced into the recipient plant in the form of a recombinant plasmid via Agrobacterium-mediated transformation.

[0032] This application also provides biological materials related to the SAG101 protein, which may be any of the following:

[0033] C1) Nucleic acid molecules that inhibit or reduce the expression of the SAG101 protein-encoding gene;

[0034] C2) Expression cassettes and / or constructs containing the nucleic acid molecules described in C1);

[0035] C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette and / or construct described in C2);

[0036] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), recombinant microorganisms containing the expression cassette and / or construct described in C2), or recombinant microorganisms containing the recombinant vector described in C3);

[0037] C5) A transgenic plant cell line containing the nucleic acid molecule described in C1), a transgenic plant cell line containing the expression cassette and / or construct described in C2), or a transgenic plant cell line containing the recombinant vector described in C3;

[0038] C6) Transgenic plant tissue containing the nucleic acid molecules described in C1), transgenic plant tissue containing the expression cassette and / or construct described in C2), or transgenic plant tissue containing the recombinant vector described in C3);

[0039] C7) Transgenic plant organs containing the nucleic acid molecules described in C1), transgenic plant organs containing the expression cassette and / or construct described in C2), or transgenic plant organs containing the recombinant vector described in C3);

[0040] C8), a nucleic acid molecule encoding the SAG101 protein;

[0041] C9) expression cassettes and / or constructs containing the nucleic acid molecules described in C8), recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues and / or transgenic plant organs.

[0042] Furthermore, the nucleic acid molecule described in C1) is either RNA encoding a gene that targets the SAG101 protein or DNA encoding the RNA.

[0043] Furthermore, the nucleotide sequence of the target site of the nucleic acid molecule described in C1) may be the first 20th positions of SEQ ID NO:4 and / or the first 20th positions of SEQ ID NO:5.

[0044] Furthermore, the nucleic acid molecule described in C8) can be any one of the following DNA molecules described in g1)-g3):

[0045] g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1;

[0046] g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3;

[0047] g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates plant clubroot resistance.

[0048] In some embodiments of this application, the expression cassette described in C2) may be an expression cassette for transcribing the sgRNA of the gene encoding the SAG101 protein described above.

[0049] The recombinant vector described in C3) can be pHEE401E-SAG101. The pHEE401E-SAG101 vector can transcribe sgRNA targeting the SAG101 gene shown in SEQ ID NO:3 and encode the effector protein of the CRISPR / Cas9 system: Cas9 protein.

[0050] Furthermore, the expression cassette described in C9) refers to a DNA sequence encoding the SAG101 protein that is capable of expressing the SAG101 protein in a host cell. This DNA sequence may include not only a promoter that initiates transcription of the SAG101 protein encoding gene, but also a terminator and / or enhancer sequence that terminates transcription of the SAG101 protein encoding gene.

[0051] Furthermore, the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi.

[0052] Furthermore, the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0053] Furthermore, the transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0054] Furthermore, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0055] This application also provides the use of the above-described compositions and / or biological materials in any of the following:

[0056] D1) Application in regulating plant clubroot resistance;

[0057] D2) Application in the preparation of products that regulate plant clubroot resistance;

[0058] D3) Application in regulating plant clubroot resistance;

[0059] D4) Application in the preparation of products that regulate plant clubroot resistance;

[0060] D5) Applications in plant breeding or plant-assisted breeding;

[0061] D6) Application in the preparation of plant breeding or plant-assisted breeding products.

[0062] In this application, the indicators for plant breeding may include clubroot resistance.

[0063] In this application, the purpose of plant breeding may include cultivating plants with altered resistance to clubroot disease. Specifically, the purpose of plant breeding includes cultivating plants with reduced resistance to clubroot disease and / or cultivating plants with increased resistance to clubroot disease.

[0064] The plants with reduced clubroot resistance obtained in this application can be used for research on the clubroot resistance mechanism of plants.

[0065] The plants with enhanced clubroot resistance obtained in this application can be used for clubroot resistance breeding.

[0066] Furthermore, in this application, the plant is a dicotyledonous plant.

[0067] In this application, the dicotyledonous plant may be selected from plants of the Brassicaceae family.

[0068] In this application, the cruciferous plants may be selected from, but are not limited to, the Brassica genus: Chinese cabbage, purple cabbage (red rapeseed, Guangdong cabbage), cabbage (cabbage, lotus white), kohlrabi (cabbage variety), cauliflower (cauliflower) (cabbage variety), broccoli (cabbage variety), mustard greens (pickled mustard greens, pickled mustard greens, turnip mustard greens (root mustard greens), yellow mustard (seed powder)), snow cabbage (mustard green variety), bok choy (small bok choy), Shanghai bok choy (small bok choy variety), European rapeseed (rapeseed);

[0069] and / or Radish genus: red-skinned radish, white-skinned radish, green-skinned radish, etc.;

[0070] and / or Capsella: Capsella bursa-pastoris;

[0071] And / or horseradish: horseradish (horse radish, wasabi);

[0072] And / or Arabidopsis: Arabidopsis.

[0073] In this application, the regulation may be to increase, promote, or adjust.

[0074] In this application, the regulation may also be a reduction, suppression, or downregulation.

[0075] In this application, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

[0076] In this application, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0077] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0078] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0079] The beneficial technical effects achieved by this application are as follows:

[0080] This application discloses for the first time the application of the SAG101 protein and its encoding gene in regulating clubroot resistance and / or clubroot resistance in cruciferous plants. Studying the function of SAG101 and applying it in cruciferous breeding is of great significance for understanding the mechanisms of clubroot resistance and / or clubroot resistance in cruciferous plants, disease control, and resistance breeding.

[0081] First, although we obtained susceptible material, this is the first confirmation of the SAG101 protein's involvement in the clubroot infection cycle, and this susceptible material can be utilized as a potential breeding resource. The discovery and enrichment of breeding resources will greatly accelerate the breeding process of clubroot-resistant cruciferous plants and strengthen disease control. Furthermore, resistance to clubroot can be achieved by overexpressing the SAG101 protein in cruciferous plants through transgenics or other means.

[0082] Secondly, disease-resistant mutants of Arabidopsis or tobacco are often used for high-throughput drug screening, such as testing the effects of novel bactericides or elicitors.

[0083] Furthermore, the diversity of organisms determines that "negative-effect" biomaterials prepared using bioengineering techniques have significant utilization value, and not all are meaningful only if they have "positive effects." Knocking out the SAG101 gene made Arabidopsis thaliana more susceptible to clubroot infection, yielding susceptible materials with a "negative effect." The clubroot resistance response in plants is a species-cascade response involving many pathways. Our susceptible materials can be used to study which genes involved in the clubroot infection cycle after SAG101 protein gene silencing make cruciferous plants more susceptible to clubroot infection. Therefore, confirming the involvement of the SAG101 protein in the clubroot resistance cycle provides new ideas and methods for revealing and applying genes related to the SAG101 protein involved in clubroot resistance research. The susceptible materials can be used as germplasm materials with the SAG101 protein gene lacking function. Attached Figure Description

[0084] Figure 1 Sequencing mutation site diagrams for the SAG101 gene knockout mutants sag101-1 and sag101-2. sag101-1 involves the deletion of deoxyribonucleotides from positions 1122 to 1566 of the SAG101 gene (SEQ ID NO:3). sag101-2 involves the deletion of deoxyribonucleotides from positions 1526 to 1568 of the SAG101 gene (SEQ ID NO:3).

[0085] Figure 2 This study analyzed the resistance of transgenic Arabidopsis thaliana plants to clubroot disease. Figure A shows wild-type Arabidopsis thaliana Est-1, SAG101 gene knockout mutants (sag101-1 and sag101-2) inoculated with *Plasmodiophora stylosa* collected from Dayi County, Sichuan Province. Symptoms were observed and photographed 21 days after inoculation. Whole plant images were taken using a Canon EOS 6D Mark II SLR camera (white bar indicates 1 cm); root images were taken using a stereomicroscope (red bar indicates 1 mm). Figures B and C show the disease index and incidence rate of wild-type Arabidopsis thaliana Est-1, SAG101 gene knockout mutants (sag101-1 and sag101-2) 21 days after inoculation with *Plasmodiophora stylosa*. The experiment was repeated in triplicate, with at least 12 Arabidopsis thaliana plants inoculated each time. Different letters in the figures indicate statistically significant differences (P < 0.05, mean ± SE, one-way ANOVA, Tukey's post-test). Detailed Implementation

[0086] Terminology in this application:

[0087] Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0088] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0089] For ease of understanding of this disclosure, several terms and abbreviations used herein are defined as follows:

[0090] In this application, "identity" refers to the similarity of amino acid sequences or nucleotide sequences. The similarity of amino acid sequences (or nucleotide 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, by 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 a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0091] The aforementioned 80% or higher consistency can be 80%, 85%, 90%, or 95% or higher.

[0092] The aforementioned 80% or higher level of consistency can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 85% or higher level of consistency can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 90% or higher level of consistency can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The aforementioned 95% or higher level of consistency can be at least 95%, 96%, 97%, 98%, or 99%.

[0093] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0094] As used in this article, "plant" includes explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0095] The term "cruciferous plants" as used in this article may be selected from, but is not limited to, any of the following:

[0096] Brassica genus: Chinese cabbage, purple cabbage (red rapeseed, Cantonese cabbage), cabbage (cabbage, lotus root), kohlrabi (a variety of cabbage), cauliflower, broccoli (a variety of cabbage), mustard greens (pickled mustard greens, pickled mustard greens, turnips (root mustard greens), yellow mustard (seed powder)), pickled mustard greens (mustard green variety), bok choy (small bok choy), Shanghai bok choy (small bok choy variety), European rapeseed (rapeseed);

[0097] and / or Radish genus: red-skinned radish, white-skinned radish, green-skinned radish, etc.;

[0098] and / or Capsella genus:: Capsella bursa-pastoris;

[0099] And / or horseradish: horseradish (horse radish, wasabi);

[0100] And / or Arabidopsis: Arabidopsis.

[0101] As used herein, "plant part" can refer to any organ or intact tissue of a plant, such as meristem, bud organ / structure (e.g., leaf, stem, or node), root, flower or floral organ / structure (e.g., flower, bract, sepal, petal, stamen, carpel, anther, and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., mature ovary), propagule, or other plant tissue (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof. The plant parts disclosed herein can be viable, non-viable, renewable, and / or non-renewable. "Propagule" can include any plant part that can grow into a whole plant.

[0102] Plant cells are biological cells of plants, derived from plants or derived from cultures obtained by culturing cells taken from plants. As used herein, “transgenic plant cell” means any plant cell transformed with a stably integrated recombinant DNA molecule, construct, expression cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0103] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0104] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0105] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0106] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0107] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.

[0108] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0109] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0110] In this application, "editing" or "genome editing" means using targeted genome editing technology to produce a targeted mutation, deletion, inversion, or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, or at least 10,000 nucleotides of endogenous plant genome nucleic acid sequence.

[0111] In this application, “editing” or “genome editing” also covers the use of targeted genome editing technology to target or integrate at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides into the endogenous genome of a plant.

[0112] In this application, a “target site” for genome editing refers to a location within a plant genome of a polynucleotide sequence that is targeted and cleaved by a site-specific nuclease, thereby introducing a double-strand break (or single-strand nick) into the nucleic acid backbone and / or its complementary DNA strand. The site-specific nuclease may bind to the target site, for example, via a non-coding guide RNA (e.g., but not limited to CRISPR RNA (crRNA) or single-strand guide RNA (sgRNA)). The non-coding guide RNA provided herein may be complementary to the target site (e.g., complementary to the strand of a double-stranded nucleic acid molecule or the chromosome of the target site). A “target site” also refers to a location within the plant genome of a polynucleotide sequence that is bound and cleaved by another site-specific nuclease, which may not be guided by a non-coding RNA molecule, such as a broad-spectrum nuclease, zinc finger nuclease (ZFN), or transcription activator-like effector nuclease (TALEN), to introduce a double-strand break (or single-strand nick) into the polynucleotide sequence and / or its complementary DNA strand.

[0113] In this application, the term "guide RNA" or "gRNA" is a short RNA sequence comprising (1) a structural or scaffold RNA sequence required to bind or interact with RNA-guided nucleases and / or other RNA molecules (e.g., tracrRNA), and (2) an RNA sequence that is identical or complementary to the target sequence or target site (referred to herein as the "guide sequence"). A "single-stranded guide RNA" (or "sgRNA") is an RNA molecule comprising tracrRNA and crRNA covalently linked by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or target sequence ("guide sequence") that is identical or complementary to a target site within the plant genome, for example, at or near the GA oxidase gene. An interphase sequence adjacent motif (PAM) may be present immediately adjacent to the 5' end of a genomic target site sequence complementary to the target sequence of the guide RNA and upstream of it in the genome, i.e., downstream (3') of the sense (+) strand immediately adjacent to the genomic target site (relative to the target sequence of the guide RNA), as is known in the art. The genomic PAM sequence (relative to the target sequence of the guide RNA) on the sense (+) strand adjacent to the target site may contain 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') of the target sequence of the guide RNA) is typically not complementary to the genomic PAM sequence. The guide RNA can usually be a non-coding RNA molecule that does not encode a protein.

[0114] In this application, "RNA-directed nuclease" refers to RNA-directed DNA endonucleases associated with the CRISPR system. Unrestricted examples of RNA-directed nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs or modified forms thereof. In some embodiments, the RNA-directed nuclease is Cas9. In some embodiments, the RNA-directed nuclease includes N-terminal and C-terminal nuclear localization sequences (NLS).

[0115] In some embodiments of this application, the composition for genome editing may be co-delivered with a DNA molecule containing a selection or screening marker gene.

[0116] Furthermore, the Cas9 protein described in this application is not limited to a specific protein, as long as it can be used in conjunction with the sgRNA described in this application. Furthermore, the Cas9 protein described herein is selected from Streptococcus pyogenes Cas9 (spCas9, subtype II-A), spCas9HF (high fidelity), nicked Cas9 (nCas9), Staphylococcus aureus Cas9 (saCas9, subtype II-A), Neisseria meningitidis Cas9 (NmCas9, subtype II-C), Francisella novicida Cas9 (FnCas9, subtype II-B), Streptococcus thermophilus Cas9 (St1Cas9, St3Cas9), Campylobacter jejuni Cas9 (CjCas9), and Treponema sp. Cas9, as well as other orthologs of Cas9 from other organisms, but not limited to these. The Cas9 protein may also include high-fidelity Cas9 mutants (such as SpCas9-HF1, eSpCas9-1.1, and TrueCut). TM HiFiCas9 protein, etc.

[0117] SAG101 encodes flavin monooxygenase, which participates in regulating plant disease resistance. This application uses Arabidopsis thaliana, a model plant in the Brassicaceae family, as material to study the contribution of the SAG101 gene to clubroot resistance. The results show that knocking out the SAG101 gene in the resistant material Est-1 significantly reduces the plant's resistance to clubroot fungus. This indicates that the SAG101 gene positively regulates plant clubroot resistance, and the SAG101 protein and its encoding gene can provide new gene resources for clubroot resistance breeding in Brassicaceae crops.

[0118] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are 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 present application in any way.

[0119] Unless otherwise specified, the experimental methods used in the following examples 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 examples are commercially available.

[0120] The pCBC-DT1T2 and pHEE401E vectors used in the following examples are disclosed in the literature “Ge, Z. et al. LLG2 / 3 areco-receptors in BUPS / ANX-RALF signaling to regulate Arabidopsis pollen tubeintegrity. Current Biology 29, 3256-3265.e5 (2019).”. These materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The aforementioned biological materials are only for repeating the experiments of this application and may not be used for other purposes.

[0121] The Agrobacterium GV3101 used in the following examples is owned by the applicant and is described in the literature "Liang, X. et al. Ligand-triggered de-repression of Arabidopsis heterotrimeric G proteins coupled to immune receptor kinases. Cell Research 28, 529-543 (2019)". It is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this application and may not be used for other purposes.

[0122] The Plasmodiophora brassicae fieldisolate Dayi (PbDy) and Arabidopsis thaliana Est-1 from Dayi County, Sichuan Province, used in the following examples are owned by the applicant and described in the literature "Wang W. et al. Wei Tsing, apericycle-expressed ion channel, safeguards the stele to confer clubroot resistance. Cell 186, 2656-2671 (2023)". They are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. These biological materials are only used to replicate the relevant experiments of this application and may not be used for other purposes.

[0123] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0124] The following examples used GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA combined with Tukey's test was used. Different letters indicate that there is a significant difference between the two groups of data (P < 0.05), and the same letters indicate that there is no significant difference between the two groups of data (P > 0.05).

[0125] Example 1: Analysis of resistance to clubroot bacteria in SAG101 knockout Arabidopsis thaliana

[0126] The genomic sequence of the SAG101 gene in Arabidopsis thaliana is a DNA molecule with the nucleotide sequence of SEQ ID NO:3, and its coding sequence (CDS) is a DNA molecule with the nucleotide sequence of SEQ ID NO:1, encoding the SAG101 protein with the amino acid sequence of SEQ ID NO:2.

[0127] I. Obtaining SAG101 knockout Arabidopsis

[0128] 1. Construction of SAG101 knockout vector

[0129] 1) Knockout target design

[0130] The target sites for knocking out the SAG101 gene were designed using the website http: / / skl.scau.edu.cn / . The target site sequences are as follows:

[0131] Target site 1: 5'-GCGGTCACAGAGTTGCTAAACGG-3' (SEQ ID NO:4), where positions 1 to 20 are the target site sequence and positions 21 to 23 are the PAM sequence;

[0132] Target site 2: 5'-TAATATTCAGCAATATCGAGTGG-3' (SEQ ID NO: 5), wherein positions 1 to 20 are the target site sequence and positions 21 to 23 are the PAM sequence; the target site sequence is inversely complementary to positions 1564 to 1583 of SEQ ID NO: 3.

[0133] 2) Primer design

[0134] Primers were designed based on the target site sequence. The primer sequences (5' to 3') are as follows (the target site sequence is in uppercase):

[0135] Target1-BsF: atatatggtctcgattgGCGGTCACAGAGTTGCTAAAgtt;

[0136] Target1-F0:tgGCGGTCACAGAGTTGCTAAAgttttagagctagaaatagc;

[0137] Target2-R0:aacCTCGATATTGCTGAATATTAcaatctcttagtcgactctac;

[0138] Target2-BsR:attattggtctcgaaacCTCGATATTGCTGAATATTAc.

[0139] 3) PCR amplification

[0140] Four-primer PCR amplification was performed using the pCBC-DT1T2 vector as a template. A 50 μL reaction mixture was prepared with the following components: 25 μL 2×KOD Buffer, 10 μL 2 mM dNTPs, 1 μL KOD DNA Polymerase, 1.5 μL pCBC-DT1T2, 1.5 μL Target1-BsF, 1.5 μL Target2-BsR, 1.5 μL Target1-F0, 1.5 μL Target2-R0, and 6.5 μL ddH2O. The reaction conditions were: pre-denaturation at 94℃ for 2 min, denaturation at 98℃ for 2 min, annealing at 55℃ for 30 s, extension at 68℃ for 30 s / kbp, final extension at 68℃ for 10 min, and storage at 4℃ for 2 min. PCR products were recovered by agarose gel electrophoresis.

[0141] 4) Enzyme digestion and ligation

[0142] The PCR fragment was ligated into the pHEE401E vector using a cut-and-ligate method to obtain the recombinant vector pHEE401E-SAG101, i.e., the SAG101 knockout vector. A 15 μL reaction mixture was prepared: 2 μL of the PCR product from step 3, 2 μL of the pHEE401E vector, 1.5 μL of 10×T4 DNA ligase buffer, 1.5 μL of 10×BSA, 1 μL of Bsa I, 1 μL of T4 DNA ligase, and 6 μL of ddH2O. The reaction conditions were: 37℃ for 5 h, 50℃ for 5 min, 80℃ for 10 min, and storage at 4℃ for 2 min.

[0143] 5) Reaction product conversion and coating

[0144] Add 15 μL of the reaction product to 100 μL of *E. coli* DH 5α competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, incubate on ice for 2 min, add 500 μL of LB medium, and incubate at 37°C in a shaker for 1 h. Spread the bacterial culture evenly on LB agar plates containing 50 mg / L kanamycin. Invert the plates and incubate overnight at 37°C.

[0145] 6) Cloning identification

[0146] Following colony PCR, PCR-positive single colonies were selected and sent to a sequencing company for routine Sanger sequencing. Sequencing results showed that the recombinant vector pHEE401E-SAG101 was obtained by replacing the fragment between the BsaI restriction sites of the pHEE401E vector with the DNA molecule shown in positions 463 to 1060 of SEQ ID NO:6. The recombinant vector pHEE401E-SAG101 contains the DNA molecule shown in SEQ ID NO:6, meaning it contains two sgRNA expression cassettes and one Cas9 protein expression cassette located within the pHEE401E vector. It can express sgRNA1 targeting positions 1-20 of SEQ ID NO:4, sgRNA2 targeting positions 1-20 of SEQ ID NO:5, and the CRISPR / Cas9 effector protein Cas9. After being introduced into the receptor, the two transcribed guide RNAs (sgRNA1 and sgRNA2) can target the target sequence near the PAM of the receptor genome, namely the SAG101 gene, through base complementarity pairing. The Cas9 protein causes a double-strand break in the DNA at the target site of the SAG101 gene. Through the organism's own DNA damage repair response mechanism, gene mutation occurs in the cleaved region during the repair process, thereby knocking out the SAG101 gene.

[0147] 2. Obtaining transgenic Arabidopsis thaliana

[0148] 1) Agrobacterium-mediated transformation

[0149] The pHEE401E-SAG101 plasmid was introduced into Agrobacterium GV3101 strain via electroporation. The specific steps are as follows: 1 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent cells, transferred to an electroporation cuvette, and electroporated at 2500V / 6ms. Then, 500 μL of LB medium was added, and the mixture was incubated at 28℃ in a shaker for 1 h. The bacterial suspension was then evenly spread onto LB agar plates containing 50 mg / L kanamycin and 50 mg / L gentamicin. The plates were inverted and incubated at 28℃ for 48 h to screen for positive colonies.

[0150] 2) Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0151] Agrobacterium GV3101 carrying the pHEE401E-SAG101 plasmid was streaked onto LB agar plates containing 50 mg / L kanamycin and 50 mg / L gentamicin, and incubated at 28°C for 48 h. Single colonies were picked and transferred to 2 mL of LB liquid medium containing 50 mg / L kanamycin and 50 mg / L gentamicin, and incubated overnight at 28°C in a shaker for 12 h.

[0152] Inoculate 2 mL of bacterial culture into 300 mL of LB liquid medium containing the corresponding antibiotic and incubate in a shaker at 28 °C for 12 h.

[0153] Collect bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, resuspend in 5% sucrose aqueous solution, and adjust OD. 600 Add 0.017% Silwet L-77 solution to a concentration between 0.8 and 1.0, and mix thoroughly.

[0154] Est-1 Arabidopsis thaliana plants with good growth were selected after about 8 weeks of growth in a long-day plant culture room (temperature 23℃, relative humidity 60%, light cycle 16h light / 8h darkness). Mature pods were cut off, and the inflorescences were soaked in bacterial solution for 5 minutes for transformation. After 24 hours of cultivation in the dark, they were cultured under normal light.

[0155] After the seeds mature, they are surface-sterilized with 30% 84 disinfectant for 10 minutes, sterilized by washing ddH2O 5 times, and spread evenly on 1 / 2 MS plates containing 25 mg / L hygromycin and 50 mg / L carbenicillin. They are vernalized at 4℃ for 48 hours. The plates are then transferred to a plant culture chamber (temperature 22℃, relative humidity 60%, photoperiod 16h light / 8h dark). After 10-12 days, healthy seedlings are selected and transplanted into a short-day plant culture room (temperature 23℃, relative humidity 60%, photoperiod 10h light / 14h dark).

[0156] 3) Obtaining SAG101 knockout Arabidopsis thaliana

[0157] Transgenic T0 generation Arabidopsis thaliana was continuously self-pollinated to obtain T2 generation transgenic lines. T2 generation transgenic Arabidopsis thaliana plants grown for approximately 4 weeks in a short-day plant culture chamber (temperature 23℃, relative humidity 60%, photoperiod 10h light and 14h dark) were selected. One to two leaves were taken from each plant, and genomic DNA was extracted using the CTAB method. The target sequence was amplified using primers SAG101-crispr-F and SAG101-crispr-R. The PCR products were sent to a sequencing company for routine Sanger sequencing, identifying two different mutation forms of SAG101 knockout Arabidopsis thaliana mutants. Figure 1 The primers were named sag101-1 and sag101-2, respectively. The primer sequences are as follows:

[0158] SAG101-crispr-F:5'-GAAGCTTGTGGGTGACCTCTG-3';

[0159] SAG101-crispr-R:5'-CACATCGTCTCTCATCCCCAC-3'.

[0160] SAG101 knockout Arabidopsis thaliana sag101-1 is an Arabidopsis mutant with a homozygous mutation in the SAG101 gene (the same mutation occurred on both chromosomes). The difference between the genome sequence of wild-type Arabidopsis thaliana Est-1 and the wild-type Arabidopsis thaliana Est-1 is the deletion of deoxynucleotides at positions 1122-1566 of the SAG101 gene in SEQ ID NO:3, resulting in the knockout of the SAG101 gene.

[0161] SAG101 knockout Arabidopsis thaliana sag101-2 is an Arabidopsis mutant with a homozygous mutation in the SAG101 gene (the same mutation occurred on both chromosomes). The difference between the genome sequence of wild-type Arabidopsis thaliana Est-1 and the wild-type Arabidopsis thaliana Est-1 is the deletion of deoxynucleotides at positions 1526-1568 of the SAG101 gene in SEQ ID NO:3, resulting in the knockout of the SAG101 gene.

[0162] II. Analysis of resistance to *Plasmodiophora stylosa* in SAG101 knockout *Arabidopsis thaliana*

[0163] 1. Inoculation with *Cladosporium brassicae*

[0164] Twenty-four wild-type Arabidopsis thaliana Est-1 and 24 SAG101 knockout mutant lines (sag101-1 and sag101-2) were taken and planted in holes, four plants per hole, in a short-day plant culture chamber (temperature 23℃, relative humidity 60%, photoperiod 10h light and 14h dark). After the plants emerged and grew for 20 days, a root drenching experiment was conducted to investigate the infection of *Platycodon brassicae*. The experiment was repeated three times, with each inoculation consisting of at least 12 wild-type Arabidopsis thaliana Est-1, sag101-1, and sag101-2 mutant lines. The specific steps are as follows:

[0165] 1) Chop the root tissue of Chinese cabbage containing *Cladosporium brassicae* from Dayi County, Sichuan Province, add an appropriate amount of sterilized ddH2O, and grind it with a tissue homogenizer.

[0166] 2) Filter the plant fragments through 8 layers of gauze and transfer the filtrate into a clean Erlenmeyer flask.

[0167] 3) Measure the spore concentration of *Plasmodiophora brassicae* in the bacterial culture using a hemocytometer.

[0168] 4) Dilute the concentration of *Plantochoria brassicae* to 1×10⁻⁶. 71 mL of diluted bacterial solution was pipetted onto the roots of Arabidopsis thaliana using a pipette.

[0169] 2. Disease index and incidence rate statistics

[0170] Twenty-one days after Arabidopsis thaliana root inoculation with *Plasmodiophora brassicae*, the incidence rate and disease index were statistically analyzed using the *Plasmodiophora brassicae* grading system. The statistical criteria for the *Plasmodiophora brassicae* grading system in Arabidopsis thaliana are as follows:

[0171] Grade 0, no symptoms, intact root system;

[0172] Grade 1: Small nodules on lateral roots, which pose negligible damage to the main root;

[0173] Grade 2, fewer lateral roots, small lumps on the main root;

[0174] Grade 3, with large lumps on the lateral roots and main root;

[0175] Grade 4, the main root is completely swollen and rotten.

[0176] The formulas for calculating the incidence rate and disease index are as follows:

[0177] Incidence rate = (Number of diseased plants / Total number of inoculated plants) × 100%

[0178] Disease index = (2(Disease level × Number of diseased plants at each level)) / (Total number of inoculated plants × Highest disease level 4)) × 100

[0179] Symptoms, incidence, and disease index of plants 21 days after inoculation with clubroot fungus are as follows: Figure 2 As shown in AC, the results indicate that wild-type Arabidopsis thaliana Est-1, after inoculation with clubroot fungus, exhibited resistance, meaning root development was generally normal, with only a few plants showing mild symptoms. However, against the resistant material Est-1 background, the SAG101 gene knockout mutants sag101-1 and sag101-2 showed susceptibility, characterized by swollen taproots and a reduced number of lateral roots. These results demonstrate that the SAG101 gene positively regulates Arabidopsis resistance to clubroot fungus, providing a new gene resource for clubroot resistance breeding in plants.

[0180] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented 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 are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method of modulating the resistance of a plant to clubroot, characterized in that, The method includes regulating clubroot resistance in recipient plants by regulating the expression level of the gene encoding SAG101 protein in the recipient plant and / or regulating the content of the SAG101 protein in the recipient plant. The SAG101 protein is any of the following proteins: A1) A protein with the amino acid sequence SEQ ID NO:2; A2) Proteins obtained by substituting, deleting and / or adding amino acid residues of the amino acid sequence shown in A1) that have more than 80% similarity to the amino acid sequence shown in a1) and are associated with plant clubroot resistance. A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. The method of claim 1, wherein, The method includes reducing clubroot resistance in recipient plants by decreasing the expression level of the gene encoding the SAG101 protein and / or reducing the content of the SAG101 protein in the recipient plants, wherein the recipient plants contain the gene encoding the SAG101 protein.

3. A method for obtaining a plant of interest with reduced resistance to clubroot, characterized in that, The method includes obtaining a target plant with reduced clubroot resistance by reducing the expression level of the gene encoding the SAG101 protein of claim 1 and / or reducing the content of the SAG101 protein in the recipient plant, wherein the recipient plant contains the gene encoding the SAG101 protein.

4. The method according to claim 2 or 3, characterized in that, The purpose of reducing the expression level of the gene encoding the SAG101 protein in the recipient plant and / or reducing the content of the SAG101 protein in the recipient plant is achieved through the method described in B1) or B2) below. B1) By knocking out the gene encoding the SAG101 protein in the recipient plant using a genome editing system, the expression level of the gene encoding the SAG101 protein and / or the content of the SAG101 protein in the recipient plant are reduced; Furthermore, the genome editing system is a CRISPR / Cas system; Furthermore, the CRISPR / Cas system includes a gRNA that targets the gene encoding the SAG101 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein; Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:5; B2) Perform any of the following mutations on the genome sequence of the recipient plant: B2-1) The deoxyribonucleotides at positions 42-71 of SEQ ID NO:3 of the SAG101 gene were deleted, and an adenine deoxyribonucleotide was inserted between positions 367 and 368 of SEQ ID NO:

3. B2-2) The deoxyribonucleotides at positions 45-58 of SEQ ID NO:3 of the SAG101 gene were deleted, and an adenine deoxyribonucleotide was inserted between positions 44 and 45 and between positions 367 and 368 of SEQ ID NO:

3.

5. A composition for genome editing, characterized by, The composition is a genome editing system for knocking out the gene encoding the SAG101 protein in a recipient plant; Furthermore, the genome editing system is a CRISPR / Cas system; Furthermore, the CRISPR / Cas system includes a gRNA that targets the gene encoding the SAG101 protein and the effector protein of the CRISPR / Cas system: the Cas9 protein; Furthermore, the target nucleotide sequence of the gRNA is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:

5.

6. A biomaterial relating to the SAG101 protein of claim 1, wherein the biomaterial is any of the following: C1) Nucleic acid molecules that inhibit or reduce the expression of the SAG101 protein-encoding gene; C2) Expression cassettes and / or constructs containing the nucleic acid molecules described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or a recombinant vector containing the expression cassette and / or construct described in C2); C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), recombinant microorganisms containing the expression cassette and / or construct described in C2), or recombinant microorganisms containing the recombinant vector described in C3); C5) A transgenic plant cell line containing the nucleic acid molecule described in C1), a transgenic plant cell line containing the expression cassette and / or construct described in C2), or a transgenic plant cell line containing the recombinant vector described in C3; C6) Transgenic plant tissue containing the nucleic acid molecules described in C1), transgenic plant tissue containing the expression cassette and / or construct described in C2), or transgenic plant tissue containing the recombinant vector described in C3); C7) Transgenic plant organs containing the nucleic acid molecules described in C1), transgenic plant organs containing the expression cassette and / or construct described in C2), or transgenic plant organs containing the recombinant vector described in C3); C8), a nucleic acid molecule encoding the SAG101 protein; C9) expression cassettes and / or constructs containing the nucleic acid molecules described in C8), recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues and / or transgenic plant organs.

7. The biomaterial of claim 6, wherein, C1) The nucleic acid molecule is either RNA encoding a gene that targets the SAG101 protein or DNA encoding the RNA; C8) The nucleic acid molecule is any one of the following (g1)-g3): g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:1; g2) The nucleotide sequence of the coding strand is the DNA molecule of SEQ ID NO:3; g3) is a DNA molecule that has more than 80% similarity to the DNA molecule described in g1) or g2) and regulates plant clubroot resistance.

8. The biomaterial of claim 7, wherein, The nucleotide sequence of the target site of the nucleic acid molecule described in C1) is positions 1-20 of SEQ ID NO:4 and / or positions 1-20 of SEQ ID NO:

5.

9. Use, characterized in that, The application is the use of the composition of claim 5 and / or the biomaterial of any one of claims 6 to 9 in any of the following: D1) Application in regulating plant clubroot resistance; D2) Application in the preparation of products that regulate plant clubroot resistance; D3) Application in regulating plant clubroot resistance; D4) Application in the preparation of products that regulate plant clubroot resistance; D5) Applications in plant breeding or plant-assisted breeding; D6) Application in the preparation of plant breeding or plant-assisted breeding products.

10. Use according to claim 9, characterized in that, The plant in question is a dicotyledonous plant.

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