Application of slwrky30 gene and its encoded protein in improving resistance of plants to root-knot nematode

Abstract

The application discloses an application of SlWRKY30 gene and its coded protein in improving resistance of plants to root-knot nematode disease. Specifically disclosed are a protein SlWRKY30 with an amino acid sequence of SEQ ID No. 1, a substance for regulating activity and / or content of the protein, and an application of a coded gene of the protein SlWRKY30 in regulating resistance of plants to root-knot nematode disease. The application introduces SlWRKY30 gene from tomato into a receptor tomato to obtain a tomato with overexpression of SlWRKY30 gene. Experiments prove that the root-knot index of the tomato with overexpression of SlWRKY30 gene is significantly reduced, indicating that overexpression of the SlWRKY30 gene can significantly improve the resistance of the tomato to southern root-knot nematode disease. The SlWRKY30 gene and its coded protein have a wide application prospect in tomato breeding and have a potential value for guaranteeing high and stable yield of the tomato.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the SlWRKY30 gene and its encoded protein in improving plant resistance to root-knot nematode disease. Background Technology

[0002] Tomato (Solanum lycopersicum) is an herbaceous plant belonging to the genus Solanum in the family Solanaceae. It is the most widely cultivated and consumed vegetable crop worldwide. In tomato production, pests and diseases frequently cause large-scale yield reductions. Currently, there are more than 40 common diseases affecting tomato production.

[0003] Root-knot nematodes (Meloidogyne spp.) are a major plant pathogen. Tomatoes are one of the vegetable crops most susceptible to root-knot nematodes. There are four common species of root-knot nematodes: the southern root-knot nematode, the Javan root-knot nematode, the peanut root-knot nematode, and the northern root-knot nematode. The southern root-knot nematode is the most damaging to tomatoes. When the southern root-knot nematode invades the tomato roots, it disrupts the normal transport of water and nutrients, causing wilting and yellowing of the leaves above ground. In severe cases, it can even cause the plant to wither and die, leading to reduced tomato yields and significant economic losses. In recent years, with the adjustment of the planting structure and the rapid expansion of protected cultivation areas for vegetables, coupled with severe continuous cropping throughout the year, root-knot nematode disease has become increasingly serious. At the same time, root-knot nematode disease exacerbates soil-borne diseases such as wilt and root rot, as well as some bacterial diseases, becoming a major obstacle to current tomato production. The occurrence and damage caused by root-knot nematode disease result in huge economic losses in agricultural production, making its control an urgent matter. Currently, there is still a lack of direct and effective methods, and indirect methods are mainly used, such as high-temperature soil disinfection. However, this method is labor-intensive and time-consuming, and it is only effective for soil depths of less than 30 cm, while root-knot nematodes have the ability to move into deeper soil layers. Chemical control can also be used, but it is costly and easily causes environmental pollution. Therefore, selecting nematode-resistant varieties is the best control measure for root-knot nematode disease. The key link is the discovery and identification of resistance-related genes. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the resistance of plants to root-knot nematode disease. The technical problem to be solved is not limited to the technical subject matter described herein; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, the present invention first provides the application of proteins or substances that regulate the activity and / or content of said proteins, wherein the application may be any of the following:

[0006] A1) The application of proteins or substances that regulate the activity and / or content of said proteins in regulating plant resistance to root-knot nematode disease;

[0007] A2) The use of proteins or substances that regulate the activity and / or content of said proteins in the preparation of products that regulate plant resistance to root-knot nematode disease;

[0008] A3) The application of proteins or substances that regulate the activity and / or content of said proteins in the cultivation of plants resistant to root-knot nematodes;

[0009] A4) The use of proteins or substances that regulate the activity and / or content of said proteins in the preparation of products for cultivating plants resistant to root-knot nematodes;

[0010] A5) The application of proteins or substances that regulate the activity and / or content of said proteins in plant breeding or plant germplasm resource improvement;

[0011] The protein is named SlWRKY30 and can be any of the following:

[0012] B1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0013] B2) A protein that has more than 80% identity with and has the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0014] B3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2).

[0015] In the above applications, the protein may be derived from tomato (Solanum lycopersicum).

[0016] To facilitate the purification or detection of proteins in B1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0017] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0018] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein SlWRKY30 of this invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the protein SlWRKY30 isolated in this invention, provided they encode and function as protein SlWRKY30, are derived from and equivalent to the nucleotide sequence of this invention.

[0019] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0020] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, 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 to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0021] In this document, the "identity of more than 80%" 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 "identity of more than 95%" can be at least 95%, 96%, 97%, 98%, or 99%.

[0022] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein SlWRKY30.

[0023] In the above text, the substance regulating gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (including regulation of modification, splicing, and / or processing of the transcript of the gene); 3) regulation of RNA transport of the gene (including regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (including regulation of the activity of the protein translated by the gene, such as regulation of protein precursor processing, protein transport, protein degradation, and / or protein folding).

[0024] The substance that regulates gene expression can be any of the biological materials described in D1)-D3) of this article.

[0025] Furthermore, the substance regulating gene expression may be a substance (including nucleic acid molecules or vectors) that increases or upregulates the expression of the gene encoding the protein SlWRKY30.

[0026] Furthermore, the substance regulating gene expression may also be a substance (including nucleic acid molecules or vectors) that reduces or downregulates the expression of the gene encoding the protein SlWRKY30.

[0027] The present invention also provides applications of biomaterials related to the protein SlWRKY30, wherein the applications may be any of the following:

[0028] C1) Application of biomaterials related to the protein SlWRKY30 in regulating plant resistance to root-knot nematode disease;

[0029] C2) Application of biomaterials related to the protein SlWRKY30 in the preparation of products that regulate plant resistance to root-knot nematode disease;

[0030] C3) Application of biomaterials related to the protein SlWRKY30 in the cultivation of plants resistant to root-knot nematode disease;

[0031] C4) Application of biomaterials related to the protein SlWRKY30 in the preparation of products for cultivating plants resistant to root-knot nematode disease;

[0032] C5) Application of biomaterials related to the protein SlWRKY30 in plant breeding or plant germplasm resource improvement;

[0033] The biomaterial may be any of the following:

[0034] D1) The nucleic acid molecule encoding the protein SlWRKY30;

[0035] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0036] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0037] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0038] D5) Recombinant host cells containing the nucleic acid molecules described in D1), or recombinant host cells containing the expression cassette described in D2), or recombinant host cells containing the recombinant vector described in D3);

[0039] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2);

[0040] D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

[0041] Furthermore, the expression cassette described in D2), the recombinant vector described in D3), the recombinant microorganism described in D4), the recombinant host cell described in D5), the transgenic plant tissue described in D6), and the transgenic plant organ described in D7 express the nucleic acid molecule described in D1.

[0042] In the above applications, the nucleic acid molecule described in D1) can be any of the following:

[0043] E1) The coding sequence is the DNA molecule at positions 268-1236 of SEQ ID No. 3;

[0044] E2) The nucleotide sequence is positions 268-1236 of SEQ ID No. 3 or the DNA molecule of SEQ ID No. 3;

[0045] The nucleotide sequence of E3 is the DNA molecule of SEQ ID No. 2.

[0046] The DNA molecule shown in positions 268-1236 of SEQ ID No. 3 may be the coding sequence (CDS) of the SlWRKY30 gene.

[0047] The DNA molecule shown at positions 268-1236 of SEQ ID No. 3 encodes the amino acid sequence of the protein SlWRKY30 of SEQ ID No. 1.

[0048] The DNA molecule shown in SEQ ID No. 2 may be the genomic nucleotide sequence of the SlWRKY30 gene.

[0049] The DNA molecule shown in SEQ ID No. 3 may be the cDNA sequence of the SlWRKY30 gene.

[0050] D1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in positions 268-1236 of SEQ ID No. 3.

[0051] D1) The nucleic acid molecules also include nucleic acid molecules that are more than 95% identical to the nucleotide sequences shown in SEQ ID No. 3, SEQ ID No. 3 or SEQ ID No. 2 at positions 268-1236 and originate from the same species.

[0052] The coding sequence (CDS) of the protein SlWRKY30 gene described in this invention can be any nucleotide sequence capable of encoding the protein SlWRKY30. Considering codon degeneracy and codon preferences among different species, those skilled in the art can use codons suitable for expression in specific species as needed.

[0053] The expression cassette described herein includes a promoter, a nucleic acid molecule encoding the protein SlWRKY30, and a terminator. The promoter may be a CaMV35S promoter, a NOS promoter, or an OCS promoter, and the terminator may be a NOS terminator or an OCSpolyA terminator.

[0054] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0055] The vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of this invention, the vector is a modified pCAMBIA1300 vector (Huang Huang, Wenchao Zhao, Hui Qiao, Chonghua Li, Lulu Sun, Rui Yang, Xuechun Ma, Jilin Ma, Susheng Song, and Shaohui Wang. SlWRKY45 interacts with jasmonate-ZIM domain proteins to negatively regulate defense against the root-knot nematode Meloidogyne incognita in tomato. 2022. Horticulture Research, 9:uhac197).

[0056] Recombinant expression vectors containing the coding sequence (CDS) of the SlWRKY30 gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for gene gun transformation. When constructing recombinant plant expression vectors using the SlWRKY30 gene coding sequence (CDS), any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, 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 sources of the translation control signals and start codons are broad, and they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0057] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0058] By introducing the encoding DNA of the SlWRKY30 gene provided in this invention into plant cells or recipient plants using any vector capable of guiding the expression of exogenous genes in plants, plants resistant to root-knot nematodes with higher resistance than the recipient plants can be obtained. The expression vector carrying the SlWRKY30 gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0059] The microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Among them, the bacteria may originate from genera such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, but are not limited to these. For example, the bacteria may be *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. The fungus may be a yeast, and the yeast may come from genera such as *Saccharomyces cerevisiae*, *Kluyveromyces* (e.g., *Kluyveromyces lactis*), *Pichia pastoris* (e.g., *Pichia pastoris*), *Schizosaccharomyces pombe* (e.g., *Schizosaccharomyces pombe*), and *Hansenula* (e.g., *Hansenula polymorpha*), but is not limited thereto. The fungus may also come from genera such as *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp., but is not limited thereto. The actinomycetes may originate from genera such as *Streptomycess*, *Nocardia*, *Micromonospora*, *Streptosporangium*, *Actinoplanes*, and *Thermoactinomyces*, but are not limited to these. The algae may originate from genera such as *Fucus*, *Achnanthes*, *Amphiprora*, *Amphora*, *Ankistrodesmus*, *Asteromonas*, and *Boekelovia*, but are not limited to these. The viruses may be rotavirus, herpesvirus, influenza virus, adenovirus, etc., but are not limited to these.In one or more embodiments of the present invention, the microorganism is Agrobacterium tumefaciens GV3101.

[0060] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" can be understood not only to refer to a specific recipient cell, but also to the offspring of such a cell, which may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations and / or alterations, but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryzasativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (e.g., grass carp, carp, rainbow trout or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc., but not limited to these.

[0061] The recombinant vectors described in this article refer to recombinant DNA molecules constructed by linking exogenous target genes with vectors in vitro. They can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0062] The recombinant microorganisms (or recombinant host cells) described herein refer to recombinant microorganisms (or recombinant host cells) whose functions have been altered by manipulating and modifying the genes of a target microorganism (or target host cell). Examples include recombinant microorganisms (or recombinant host cells) obtained by introducing exogenous target genes or recombinant vectors into a target microorganism (or target host cell), or recombinant microorganisms (or recombinant host cells) obtained by directly editing the endogenous genes of a target microorganism (or target host cell). The term "recombinant microorganism" (or recombinant host cell) can be understood to refer not only to a specific recombinant microorganism (or recombinant host cell), but also to its offspring. Due to natural, accidental, or intentional mutations and / or alterations, the offspring need not be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms (or recombinant host cells).

[0063] The recombinant vector described in D3 of this article can be the recombinant vector flag-SlWRKY30-OE.

[0064] The recombinant vector flag-SlWRKY30-OE is obtained by replacing the fragment (small fragment) between the Sal I and Spe I recognition sites of the modified pCAMBIA1300 vector with the DNA fragment whose nucleotide sequence is shown at positions 268-1236 of SEQ ID No. 3 in the sequence listing, while keeping the other nucleotide sequences of the modified pCAMBIA1300 vector unchanged. The recombinant vector flag-SlWRKY30-OE expresses the SlWRKY30 protein with the amino acid sequence shown in SEQ ID No. 1.

[0065] The recombinant microorganism mentioned in D4 of this article may be recombinant Agrobacterium GV3101 / flag-SlWRKY30-OE.

[0066] The recombinant Agrobacterium GV3101 / flag-SlWRKY30-OE is a recombinant microorganism obtained by introducing the recombinant vector flag-SlWRKY30-OE into Agrobacterium tumefaciens GV3101, which contains the DNA molecule shown in positions 268-1236 of SEQ ID No. 3.

[0067] The introduction can be achieved through recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, in-planta technology, freeze-thaw methods, etc.

[0068] The present invention also provides a method for cultivating plants resistant to root-knot nematode disease, the method comprising increasing the content and / or activity of the protein SlWRKY30 in the target plant to obtain a root-knot nematode-resistant plant with higher resistance to root-knot nematode disease than the target plant.

[0069] In the above method, increasing the content and / or activity of the protein SlWRKY30 in the target plant can be achieved by increasing the expression level of the gene encoding the protein SlWRKY30 in the target plant.

[0070] In the above method, increasing the expression level of the gene encoding the protein SlWRKY30 in the target plant can be achieved by introducing the gene encoding the protein SlWRKY30 into the target plant.

[0071] In the above method, the gene encoding the protein can be any of the following:

[0072] F1) The coding sequence is the DNA molecule at positions 268-1236 of SEQ ID No. 3;

[0073] F2) is the DNA molecule with nucleotide sequence from position 268 to 1236 of SEQ ID No. 3.

[0074] Furthermore, the enhancement of the expression level of the gene encoding the protein SlWRKY30 in the target plant can be achieved by introducing the DNA molecule shown at positions 268-1236 of SEQ ID No. 3 into tomato.

[0075] The method for cultivating plants resistant to root-knot nematode disease may include the following steps:

[0076] (1) Construct a recombinant vector containing the DNA molecule shown in positions 268-1236 of SEQ ID No. 3;

[0077] (2) Introduce the recombinant vector constructed in step (1) into the target plant (such as a crop);

[0078] (3) The plants resistant to root-knot nematode disease were obtained through screening and identification.

[0079] In the above method, the recombinant vector can be the recombinant vector flag-SlWRKY30-OE described herein.

[0080] Furthermore, the introduction refers to recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, or in-planta techniques.

[0081] In the above method, the plant may be any of the following:

[0082] G1) Monocotyledonous or dicotyledonous plants;

[0083] G2) Solanaceae plants;

[0084] G3) Solanaceae plants;

[0085] G4) Tomato.

[0086] Furthermore, the tomato may specifically be the tomato variety Solanum lycopersicum cv Castlemart (CM).

[0087] In this article, the plants resistant to root-knot nematode disease can be those with increased (upregulated) resistance to root-knot nematode disease.

[0088] The root-knot nematodes mentioned in this article may be southern root-knot nematodes, Javan root-knot nematodes, peanut root-knot nematodes, or northern root-knot nematodes.

[0089] Increased (upregulated) resistance to root-knot nematode disease is manifested in the reduction (decreased) root-knot index of the plant after increasing the expression level and / or activity of the SlWRKY30 protein in the recipient plant (target plant).

[0090] In the above method, the plant resistant to root-knot nematode disease can be a plant resistant to southern root-knot nematode disease.

[0091] Furthermore, the plant resistant to root-knot nematode disease may be a tomato resistant to root-knot nematode disease, specifically a tomato resistant to southern root-knot nematode disease.

[0092] This invention also provides the application of the method for cultivating plants resistant to root-knot nematodes in the creation of plants resistant to root-knot nematodes and / or in plant breeding.

[0093] In this article, the plant may be a crop (such as an agricultural crop).

[0094] The plant breeding described in this article can be for the breeding of crops to resist root-knot nematode disease, specifically for the breeding of tomatoes to resist southern root-knot nematode disease. The purpose of this breeding is to select tomatoes with improved resistance to southern root-knot nematode disease.

[0095] The plant breeding described in this article can be a molecular breeding method that utilizes the SlWRKY30 gene and / or protein SlWRKY30 described in this invention to improve the resistance of plants (such as crops) to root-knot nematode disease.

[0096] The regulation of plant root-knot nematode resistance described in this article can either increase (upregulate) or decrease (downregulate) plant root-knot nematode resistance.

[0097] The resistance to root-knot nematode disease described in this article refers to resistance to southern root-knot nematode (Meloidogyne incognita) disease.

[0098] The resistance to root-knot nematodes can be resistance to southern root-knot nematodes, resistance to Javanese root-knot nematodes, resistance to peanut root-knot nematodes, or resistance to northern root-knot nematodes.

[0099] The root-knot nematode disease described in this article can be southern root-knot nematode disease, Javanese root-knot nematode disease, peanut root-knot nematode disease, or northern root-knot nematode disease, specifically southern root-knot nematode (Meloidogyne incognita) disease.

[0100] Specifically, the regulation of plant resistance to root-knot nematodes described in this article includes increasing (upregulating) or decreasing (downregulating) the plant's resistance to root-knot nematodes. Further, the regulation of plant resistance to root-knot nematodes described in this article can be the regulation of resistance to southern root-knot nematodes, including increasing (upregulating) or decreasing (downregulating) the plant's resistance to southern root-knot nematodes.

[0101] The regulation of plant resistance to root-knot nematode disease can be achieved by overexpressing the tomato SlWRKY30 gene.

[0102] In this document, the term "root-knot nematode-resistant plants" is understood to include not only first-generation transgenic plants obtained by transforming the target plant with the SlWRKY30 gene, but also its progeny. This gene can be propagated within the species, or transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The term "root-knot nematode-resistant plants" includes seeds, callus tissue, intact plants, and cells.

[0103] This invention introduces the SlWRKY30 gene, derived from tomato (Solanum lycopersicum), which regulates resistance to root-knot nematodes, into wild-type tomato (WT) as a recipient plant, resulting in overexpressing plants (flag-SlWRKY30-OE tomatoes). Further analysis of the difference in resistance to WT before and after SlWRKY30 gene overexpression in wild-type CM tomato plants was conducted by inoculating with the southern root-knot nematode (Meloidogyne incognita) to identify the resistance of SlWRKY30 gene overexpressing plants. Experiments showed that after WT stress, compared with the non-transgenic recipient control, the transgenic tomatoes overexpressing the SlWRKY30 gene (flag-SlWRKY30-OE overexpressing plants) exhibited reduced sensitivity to WT and a significantly decreased root knot index, indicating that overexpression of the SlWRKY30 gene can significantly improve the resistance of tomatoes to WT.

[0104] The SlWRKY30 gene and its encoded SlWRKY30 protein identified in this invention can regulate plant resistance to root-knot nematodes (e.g., increase or decrease plant resistance to root-knot nematodes). Increasing the content and / or activity of the SlWRKY30 protein in the target plant (e.g., overexpression of the SlWRKY30 gene) can significantly improve the root-knot nematode resistance of the target plant. Conversely, decreasing the content and / or activity of the SlWRKY30 protein in the target plant (e.g., inhibiting the expression of the SlWRKY30 gene) can significantly decrease the root-knot nematode resistance of the target plant.

[0105] To breed tomatoes with enhanced resistance to southern root-knot nematode disease and increase yield under biotic stress, this invention, for the first time, utilizes the SlWRKY30 gene in tomato breeding for resistance to southern root-knot nematode disease. This provides a valuable gene resource for tomato breeding against the disease, opens up a new field for the application of the SlWRKY30 gene, and enriches the gene pool related to regulating resistance to southern root-knot nematode disease in tomatoes. The SlWRKY30 gene and its encoded protein, which regulate plant resistance to root-knot nematode disease, have broad application prospects in tomato breeding and potential value in ensuring high and stable tomato yields. Attached Figure Description

[0106] Figure 1 To detect the expression level of the SlWRKY30 gene in flag-SlWRKY30-OE overexpressing plants using quantitative real-time PCR (qRT-PCR), CM represents wild-type tomato plants; flag-SlWRKY30-OE represents SlWRKY30 overexpressing plants. Different lowercase letters indicate significant differences.

[0107] Figure 2 Acid fuchsin staining results of roots of different tomato lines 7 days after inoculation with southern root-knot nematodes. CM represents wild-type tomato plants; flag-SlWRKY30-OE represents plants overexpressing the SlWRKY30 gene.

[0108] Figure 3 Statistical graph of root knot index 7 days after inoculation of different tomato lines with Southern root-knot nematode. CM represents wild-type tomato plants; flag-SlWRKY30-OE represents plants overexpressing the SlWRKY30 gene. Detailed Implementation

[0109] 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.

[0110] 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.

[0111] The tomato variety *Solanum lycopersicum* cv Castlemart (CM) and the modified pCAMBIA1300 vector in the following examples are described in the following literature: Huang Huang, Wenchao Zhao, Hui Qiao, Chonghua Li, Lulu Sun, Rui Yang, Xuechun Ma, Jilin Ma, Susheng Song, and Shaohui Wang. SlWRKY45 interacts with jasmonate-ZIM domain proteins tonegatively regulate defense against the root-knot nematode *Meloidogyneincognita* in tomato. 2022. Horticulture Research, 9: uhac197. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention and may not be used for any other purpose.

[0112] In the following examples, KT stands for kinetin, 2,4-D stands for 2,4-dichlorophenoxyacetic acid, IAA stands for indole-3-acetic acid, and ZT stands for zeatin.

[0113] In the following examples, Kan refers to kanamycin, Rif refers to rifampicin, and Hyg refers to hygromycin B.

[0114] In the following examples, A1 medium is MS solid medium containing 1 mg / L IAA and 1.75 mg / L ZT, A2 resistance medium is MS solid medium containing 1.0 mg / L IAA, 1.75 mg / L ZT and 5 mg / L Hyg, A3 medium is MS solid medium containing 1.0 mg / L IAA, 1.75 mg / L ZT and 5 mg / L Hyg, and A4 medium is MS solid medium containing 5 mg / L Hyg.

[0115] The sequences involved in the following embodiments are shown in Table 1.

[0116] Table 1. SlWRKY30 gene-related sequences

[0117]

[0118]

[0119]

[0120] Example 1: Obtaining and identifying plants overexpressing the SlWRKY30 gene

[0121] The inventors of this application, through extensive and in-depth research, isolated and cloned a tomato gene from the wild-type (WT) tomato variety *Solanumlycopersicum* cv Castlemart (CM), naming it SlWRKY30. The nucleotide sequence of the SlWRKY30 genome is shown in SEQ ID No. 2, with a full length of 2663 bp. The SlWRKY30 cDNA sequence is shown in SEQ ID No. 3, with a length of 1252 bp. The coding sequence (CDS) of the SlWRKY30 gene is shown at positions 268-1236 of SEQ ID No. 3, with a length of 969 bp, encoding 322 amino acids, the amino acid sequence of which is shown in SEQ ID No. 1. The protein encoded by the SlWRKY30 gene is named SlWRKY30 protein (SEQ ID No. 1).

[0122] I. Construction of the flag-SlWRKY30-OE recombinant vector

[0123] RNA was extracted from wild-type tomato CM using the Trizol method and reverse transcribed into cDNA using the TransGen AT311 reverse transcription kit. Using the cDNA as a template, SlWRKY30 gene cloning primers (SlWRKY30-F and SlWRKY30-R) were designed to amplify the DNA fragment containing the 969 bp coding sequence of the SlWRKY30 gene (serial positions 268-1236 of SEQ ID No. 3) by PCR. The amplified fragment was detected by 1% agarose gel electrophoresis and purified for later use. The obtained amplified fragment had Sal I and Spe I restriction endonuclease recognition sites added to the 5′ and 3′ ends of the 969 bp SlWRKY30 gene coding sequence, respectively. The primer sequences are shown below:

[0124] Primer SlWRKY30-F: 5'-acgc gtcgac atgtctgataataacccttttaatc-3' (the underlined sequence is the Sal I restriction endonuclease site),

[0125] Primer SlWRKY30-R: 5'-cgg actagt tcatggctctcttttgttgatc-3' (The underlined sequence is the restriction endonuclease Spe I site).

[0126] The pCAMBIA1300 vector, modified by double digestion with Sal I and Spe I, was recovered by 1% agarose gel electrophoresis and agarose gel DNA purification and recovery kit.

[0127] The amplified fragment obtained above was ligated to the linearized vector backbone, and the sequence was verified by sequencing by Sangon Biotech (Shanghai) Co., Ltd., confirming successful vector ligation. The recombinant plasmid (recombinant vector) showing the insertion of the DNA fragment shown at positions 268-1236 of SEQ ID No. 3 between the Sal I and Spe I restriction sites of the modified pCAMBIA1300 vector was named flag-SlWRKY30-OE.

[0128] The recombinant vector flag-SlWRKY30-OE is obtained by replacing the fragment (small fragment) between the Sal I and Spe I recognition sites of the modified pCAMBIA1300 vector with the DNA fragment whose nucleotide sequence is shown at positions 268-1236 of SEQ ID No. 3 in the sequence listing, while keeping the other nucleotide sequences of the modified pCAMBIA1300 vector unchanged. The recombinant vector flag-SlWRKY30-OE expresses the SlWRKY30 protein with the amino acid sequence shown in SEQ ID No. 1.

[0129] II. Transformation of Agrobacterium

[0130] The successfully ligated vector plasmid flag-SlWRKY30-OE was transformed into Agrobacterium tumefaciens strain GV3101 to obtain recombinant Agrobacterium GV3101 / flag-SlWRKY30-OE. After PCR verification showed the presence of the expected band (969 bp), the bacterial culture was mixed with 60% glycerol in an equal ratio and stored at -80°C.

[0131] III. Genetic Transformation of Tomato

[0132] 1. Sowing

[0133] Disinfect the seeds with 75% ethanol for 4-5 minutes. Rinse with sterile water 4-5 times, then disinfect with 2-3% sodium hypochlorite for 8 minutes. Rinse with sterile water 8-10 times. Sow the seeds on 1 / 2 MS medium and culture for 5-7 days until the cotyledons are fully expanded and no true leaves have emerged.

[0134] 2. Explant cutting

[0135] Cut off both ends of the cotyledons to obtain 1cm. 2 After removing the explants from both sides, soak them in MS solution (with 1 mg / L KT and 1 mg / L 2,4-D added) for 40 min. Then transfer the explants face up to A1 medium (MS solid medium + 1 mg / L IAA + 1.75 mg / L ZT) and pre-culture for 1-2 days. This operation is performed in a sterile environment throughout.

[0136] 3. Preparation of inoculated bacterial solution

[0137] Single-clone positive colonies (recombinant Agrobacterium GV3101 / flag-SlWRKY30-OE) were picked and placed in 2 ml of YEB liquid medium containing antibiotics (50 mg / L Kan and 50 mg / L Rif), and cultured with shaking (200 rpm) at 28°C. On the day of infection, the bacterial suspension (approximately OD600 = 1) was poured into 20 ml of YEB liquid medium containing antibiotics (50 mg / L Kan and 50 mg / L Rif), and cultured with shaking (200 rpm) at 28°C for about 4 hours to obtain Agrobacterium suspension with OD600 = 1-2.

[0138] 4. Infection of explants

[0139] The Agrobacterium tumefaciens bacterial suspension obtained in step 3 was centrifuged at 4000 rpm for 10 min at room temperature, the supernatant was discarded, the bacterial cells were collected and resuspended in YEB liquid medium, centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in MS salt solution. The OD600 was adjusted to 0.3-0.5, the explants were immersed in the resuspension and cultured for 15 minutes. After blotting off the excess liquid with sterile filter paper, the explants were transferred to A1 medium with the back side facing up and cultured in the dark for 2 days.

[0140] 5. Cultivate

[0141] After two days of co-culture (dark culture), the cotyledons were transferred to A2 resistance medium (MS solid medium + 1.0 mg / L IAA + 1.75 mg / L ZT + 5 mg / L Hyg) and cultured at 26℃ (16h light) / 18℃ (8h dark). The A2 resistance medium was replaced weekly until callus formation. After callus formation, the plantlets were transferred to A3 medium (MS solid medium + 1.0 mg / L IAA + 1.75 mg / L ZT + 5 mg / L Hyg) to induce budding and seedling development. The seedlings were then transferred to A4 medium (MS solid medium + 5 mg / L Hyg) for root selection. Finally, T0 generation transgenic tomato plants (i.e., SlWRKY30 gene overexpressing tomatoes) were obtained.

[0142] 6. Identification

[0143] DNA was extracted from leaves of T0 generation transgenic tomato plants, amplified by PCR, and then sent to Sangon Biotech for sequencing to screen for positive transformation seedlings (i.e., flag-SlWRKY30-OE positive seedlings). The identification primers are as follows.

[0144] flag-SlWRKY30-F:5'-GAACACGGGGGACTCTAGA-3';

[0145] SlWRKY30-R: 5'-CGGACTAGTTCATGGCTCTCTTTTGTTGATC-3'.

[0146] The PCR amplification product containing the target fragment (1000bp) was identified as a flag-SlWRKY30-OE positive seedling (flag-SlWRKY30-OE tomato).

[0147] IV. Detection of SlWRKY30 gene expression level in flag-SlWRKY30-OE positive seedlings using qRT-PCR

[0148] Leaves from uniformly growing, positive seedlings were collected, RNA was extracted, and then reverse transcribed into cDNA. The expression level of the SlWRKY30 gene in the sampled plants was detected by qRT-PCR using primers SlWRKY30RT-F and SlWRKY30RT-R. The control internal reference gene was Sl-Actin2, with primers Sl-Actin2-F and Sl-Actin2-R.

[0149] SlWRKY30RT-F: 5'-GGTCAGAAGGCAGTGAAGAACAG-3';

[0150] SlWRKY30RT-R: 5'-GTGATCACAATTGATGGGTCTTC-3'.

[0151] Sl-Actin2-F: 5'-TTGCTGACCGTATGAGCAAG-3';

[0152] Sl-Actin2-R: 5'-GGACAATGGATGGACCAGAC-3'.

[0153] The results are as follows Figure 1 As shown, compared with the control group (CM), the expression level of the SlWRKY30 gene in the experimental group (flag-SlWRKY30-OE) tomato plants was significantly increased.

[0154] Example 2: Identification of resistance to southern root-knot nematode disease in plants overexpressing the SlWRKY30 gene

[0155] Tomatoes tested: control group (CM) tomatoes obtained in Example 1 and experimental group flag-SlWRKY30-OE tomatoes that were positive by qRT-PCR (i.e., SlWRKY30 overexpression lines).

[0156] The root-knot nematode is the southern root-knot nematode (Meloidogyne incognita).

[0157] 1. Inoculation of different strains with root-knot nematodes

[0158] Once the seedlings have grown to the "four leaves and one heart" stage, they are inoculated with root-knot nematodes. Before inoculation, keep the substrate in the seedling pot moist. Make three holes around the tomato roots with a radius of 1.5 cm. Use a pipette to evenly inject a pre-counted suspension of southern root-knot nematodes (M. incognita) into the three holes. Each plant is inoculated with 300 southern root-knot nematodes.

[0159] 2. Determination of root knot index

[0160] Seven days after inoculation, samples were taken, the fresh weight of the roots of the experimental seedlings was measured, and the total number of root knots for each plant was counted.

[0161] Calculate the root knot index using the following formula:

[0162] Gallindex = Number of root knots / Fresh weight of roots (grams)

[0163] The results are as follows Figure 2 and Figure 3 As shown, the root knot index of the experimental group (flag-SlWRKY30-OE tomato) was significantly lower than that of the control group (CM), indicating that overexpression of the SlWRKY30 gene can significantly improve the resistance of tomatoes to southern root-knot nematode disease.

[0164] In summary, the SlWRKY30 gene and its encoded SlWRKY30 protein identified in this invention can regulate plant resistance to root-knot nematodes (e.g., increase or decrease plant resistance to root-knot nematodes). Increasing the content and / or activity of the SlWRKY30 protein in the target plant (e.g., overexpression of the SlWRKY30 gene) can significantly improve the root-knot nematode resistance of the target plant. Conversely, decreasing the content and / or activity of the SlWRKY30 protein in the target plant (e.g., inhibiting the expression of the SlWRKY30 gene) can significantly decrease the root-knot nematode resistance of the target plant.

[0165] 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. Use of a protein, characterized in that, The application is any one of the following: A1) application in improving tomato resistance to root-knot nematode; A2) application in preparing a product for improving tomato resistance to root-knot nematode; A3) application in cultivating tomato resistant to root-knot nematode; A4) application in preparing a product for cultivating tomato resistant to root-knot nematode; The amino acid sequence of the protein is shown in SEQ ID No. 1; the application is achieved by increasing the content of the protein in tomato.

2. Use according to claim 1, characterized in that, The protein is derived from tomato.

3. Use of a biomaterial associated with the protein as claimed in claim 1 or 2, characterised in that, The application is any one of the following: C1) application in improving tomato resistance to root-knot nematode; C2) application in preparing a product for improving tomato resistance to root-knot nematode; C3) application in cultivating tomato resistant to root-knot nematode; C4) application in preparing a product for cultivating tomato resistant to root-knot nematode; The biological material is any one of the following: D1) a nucleic acid molecule encoding the protein in claim 1 or 2; D2) an expression cassette containing the nucleic acid molecule in D1); D3) a recombinant vector containing the nucleic acid molecule in D1), or a recombinant vector containing the expression cassette in D2); D4) a recombinant microorganism containing the nucleic acid molecule in D1), or a recombinant microorganism containing the expression cassette in D2), or a recombinant microorganism containing the recombinant vector in D3); D5) a recombinant host cell containing the nucleic acid molecule in D1), or a recombinant host cell containing the expression cassette in D2), or a recombinant host cell containing the recombinant vector in D3); D6) transgenic plant tissue containing the nucleic acid molecule in D1), or transgenic plant tissue containing the expression cassette in D2); D7) transgenic plant organ containing the nucleic acid molecule in D1), or transgenic plant organ containing the expression cassette in D2); The application is achieved by increasing the content of the protein in tomato.

4. Use according to claim 3, characterized in that, The nucleic acid molecule in D1) is a DNA molecule with a coding sequence of 268-1236 of SEQ ID No.

3.

5. A method of breeding a nematode-resistant tomato plant, comprising, The method comprises increasing the content of the protein in claim 1 or 2 in the target tomato, to obtain tomato resistant to root-knot nematode with higher resistance to root-knot nematode than the target tomato.

6. The method of claim 5, wherein, The increase of the content of the protein in claim 1 or 2 in the target tomato is achieved by increasing the expression amount of the gene encoding the protein in the target tomato.

7. The method of claim 6, wherein, The increase of the expression amount of the gene encoding the protein in the target tomato is achieved by introducing the gene encoding the protein in claim 1 or 2 into the target tomato.

8. The method of claim 7, wherein, The nucleotide sequence of the gene encoding the protein is shown in 268-1236 of SEQ ID No.

3.

9. The method according to any one of claims 5-8, characterized in that, The tomato resistant to root-knot nematode is tomato resistant to southern root-knot nematode.

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