Wheat leaf rust resistance protein, resistance gene and application thereof

Abstract

The invention provides a wheat leaf rust resistance protein, a wheat leaf rust resistance gene and application thereof. Wherein the wheat leaf rust resistance protein comprises a DUF1 protein and / or a DUF2 protein, and the DUF1 protein comprises a protein composed of an amino acid sequence as shown in SEQ ID NO: 3 or a protein with 80% or more of homology and leaf rust resistance; the DUF2 protein comprises a protein composed of an amino acid sequence as shown in SEQ ID NO: 5 or a protein with 80% or more of homology and leaf rust resistance. The method can solve the problem of lack of leaf rust resistant protein in the prior art, and is suitable for the field of wheat breeding.

Description

Technical Field

[0001] This invention relates to the field of wheat breeding, and more specifically, to a wheat leaf rust resistance protein, a resistance gene, and their applications. Background Technology

[0002] Wheat is a staple food crop, providing food for about one-third of the world's population. With the continuous growth of the population, it is essential to increase the yield of staple crops such as wheat to meet the increasing demand. Breeding disease-resistant wheat varieties to reduce the damage caused by wheat fungal diseases and minimize yield losses is one of the effective measures to ensure safe wheat production. Wheat leaf rust is an airborne fungal disease caused by Puccinia triticina. In recent years, with climate change, wheat leaf rust has shown a trend of expansion and development, causing increasingly serious damage. Therefore, breeding wheat varieties resistant to leaf rust remains one of the important tasks in wheat breeding.

[0003] Cloning leaf rust resistance genes and breeding leaf rust-resistant wheat varieties is the most economical, safe, and effective solution for controlling this disease. The wheat leaf rust resistance gene Lr51 originates from a close relative of wheat, *Aegilops speltoides* (genome SS). This gene was first identified in a distant hybridization of wheat created by Professor Jan Dvorak at the University of California, Davis, tentatively named LrF7, and located on chromosome 1BL. Subsequently, Professor Jorge Dubcovsky at UC Davis used molecular markers to determine that the translocation line F-7-3 contains a foreign 1S chromosome fragment of approximately 14-32 cM, and developed the CAPS marker S30-13L / AGA7-759R to effectively identify this gene. Studies have shown that Lr51 exhibits high and broad-spectrum resistance to leaf rust fungi from various countries worldwide, and no virulent strains have been reported to date. Although Lr51 exhibits broad-spectrum and high resistance characteristics, it has not yet been widely used in wheat breeding. Therefore, the cloning and breeding of this gene will have significant application prospects.

[0004] Because the wheat genome is extremely large, and over 80% of its sequences are repetitive, research on the isolation and cloning of functional genes in wheat lags far behind that of other crops such as rice and maize. To date, 83 wheat leaf rust resistance genes (Lr1-Lr83) have been officially named internationally. However, only a few of these resistance genes have been successfully cloned, and many of these cloned resistance genes encode nucleotide-linked leucine-rich repetitive proteins (NLRs). However, due to strain evolution and overuse of these genes, some have lost their resistance. Given this situation, cloning the broad-spectrum leaf rust resistance gene Lr51, identifying new types of resistance proteins, and increasing the diversity of resistance gene combinations in major cultivated varieties are particularly important. Summary of the Invention

[0005] The main objective of this invention is to provide a wheat leaf rust resistance protein, a resistance gene, and its application, in order to solve the problem of the lack of leaf rust resistance proteins in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, a wheat leaf rust resistance protein is provided, comprising DUF1 protein and / or DUF2 protein, wherein the DUF1 protein comprises: (a1) a protein having the amino acid sequence shown in SEQ ID NO: 3; or (a2) a protein having one or more amino acids substituted and / or deleted and / or added to the amino acid sequence in (a1) and having resistance to wheat leaf rust; or (a3) ​​a protein having more than 80% homology with the amino acid sequence defined in either (a1) or (a2) and having the same function; the DUF2 protein comprises: (b1) a protein having the amino acid sequence shown in SEQ ID NO: 5; or (b2) a protein having one or more amino acids substituted and / or deleted and / or added to the amino acid sequence in (b1) and having resistance to wheat leaf rust; or (b3) a protein having more than 80% homology with the amino acid sequence defined in either (b1) or (b2) and having the same function.

[0007] To achieve the above objectives, according to a second aspect of the present invention, a wheat leaf rust resistance gene is provided, comprising a DUF1 gene and / or a DUF2 gene; the DUF1 gene comprises: (c1) a nucleotide sequence encoding the DUF1 protein in the wheat leaf rust resistance protein of claim 1; or (c2) a nucleotide sequence encoding the DUF1 protein after hybridization with the DNA molecule defined in (c1) under stringent conditions; or (c3) having the nucleotide sequence shown in SEQ ID NO: 2; or (c4) a gene having more than 70% homology with any of the nucleotide sequences defined in (c1) to (c3) and encoding a protein having the same function; the DUF2 gene comprises: (d1) a nucleotide sequence encoding the DUF2 protein in the wheat leaf rust resistance protein of claim 1; or (d2) a nucleotide sequence encoding the DUF2 protein after hybridization with the DNA molecule defined in (d1) under stringent conditions; or (d3) having the nucleotide sequence shown in SEQ ID NO: 2. NO:4 nucleotide sequence; or a gene that has more than 70% homology with any of the nucleotide sequences defined in (d4) and (d1) to (d3) and encodes a protein with the same function.

[0008] To achieve the above objectives, according to a third aspect of the present invention, an expression cassette is provided, which contains the aforementioned wheat leaf rust resistance gene, including the DUF1 gene and / or the DUF2 gene.

[0009] Furthermore, the expression cassette also includes a regulatory sequence for regulating the expression of wheat leaf rust resistance genes, the regulatory sequence including a promoter; preferably, the promoter includes one or more of the following promoters: constitutive, enhancing, tissue-specific and inducible.

[0010] To achieve the above objectives, according to a fourth aspect of the present invention, a recombinant vector is provided, comprising the aforementioned wheat leaf rust resistance gene or the aforementioned expression cassette; the wheat leaf rust resistance gene includes the DUF1 gene and / or the DUF2 gene; preferably, the recombinant vector includes a translation control signal; preferably, the translation control signal includes an enhancer; preferably, the enhancer includes a translational enhancer and / or a transcriptional enhancer; preferably, the translation control signal is derived from a natural sequence or a synthetically produced sequence; preferably, the recombinant vector includes a plant expression vector; preferably, the plant expression vector includes a binary vector transformed by Agrobacterium and a gene gun bombardment vector; preferably, the plant expression vector includes pCAMBIA1300; preferably, the recombinant vector includes a reporter gene; preferably, the reporter gene includes a resistance gene or a gene expressing an enzyme or luminescent compound that produces a color change; preferably, the resistance gene includes an antibiotic resistance gene or a chemical reagent resistance gene.

[0011] To achieve the above objectives, according to a fifth aspect of the present invention, a non-plant host cell is provided, the host cell being transformed with the above-mentioned recombinant vector; preferably, the host cell comprises Escherichia coli or Agrobacterium rhizogenes; preferably, the Escherichia coli comprises DH5α; preferably, Agrobacterium rhizogenes comprises EHA105.

[0012] To achieve the above objectives, according to a sixth aspect of the present invention, the above-mentioned wheat leaf rust resistance protein, or the above-mentioned wheat leaf rust resistance gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned host cell are provided for use in regulating plant resistance to leaf rust, enhancing or reducing plant resistance to leaf rust, or cultivating transgenic plants with enhanced or reduced resistance to leaf rust.

[0013] To achieve the above objectives, according to a seventh aspect of the present invention, a method for preparing transgenic plants is provided, the method comprising: introducing the above-mentioned wheat leaf rust resistance gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned host cell into a target plant to obtain a transgenic plant resistant to leaf rust.

[0014] Furthermore, the recombinant vector is introduced into the target plant via a plant virus vector, gene gun, or Agrobacterium infection; preferably, the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat; preferably, the wheat leaf rust resistance gene is driven by a constitutive promoter.

[0015] To achieve the above objectives, according to an eighth aspect of the present invention, a method for increasing or decreasing plant resistance to leaf rust is provided, the method comprising: increasing or decreasing any one or more of the copy number, expression level, activity, and content of the aforementioned wheat leaf rust resistance protein in the target plant, thereby enhancing or decreasing the plant's resistance to leaf rust.

[0016] Furthermore, the method is used for plant breeding; the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat; preferably, the leaf rust is caused by a physiological race of leaf rust fungus; preferably, the physiological race of leaf rust fungus is a toxic race of leaf rust fungus prevalent in China, including FHJR, THDB, THTT, FHJL, HCJR, PHQS, PHRT, or PHTT.

[0017] Applying the technical solution of this invention and utilizing the above-mentioned wheat leaf rust resistance protein helps to elucidate the disease resistance mechanism of disease resistance genes and proteins against pathogens, improves the plant's resistance to leaf rust, and provides a reliable and effective source of resistance to leaf rust for plant breeding. Attached Figure Description

[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 The phenotypic results of inoculating leaf rust physiological races FHJR, THDB, THTT, FHJL, HCJR, PHQS, PHRT, or PHTT with near-isogenic lines containing Lr51 and without Lr51 according to Example 1 of the present invention are shown.

[0020] Figure 2 A detailed mapping diagram of the leaf rust resistance gene Lr51 according to Embodiment 2 of the present invention is shown. Wherein, Figure 2 In the middle, 'a' is a schematic diagram of chromosome 1B of the wheat disease-resistant parent material GSTR 441; Figure 2 Figure b is a schematic diagram of the genome-specific molecular markers developed on the exogenous 1S chromosome, with their physical locations referenced from the Chinese Spring 1.0 reference genome: Figure 2 In the middle c, a schematic diagram is shown of partial homologous chromosome recombination of the infiltrated 7S chromosome induced by wheat CSph1b, resulting in key recombinants occurring between 652.4 and 656.1 Mb. Figure 2 The diagram shows the candidate genes in the disease-resistant parent GSTR 441, where d represents the candidate chromosomal region located.

[0021] Figure 3 The figure shows the phenotypic results of the independently susceptible M3 family based on EMS-induced inoculation with the physiological race PHQS of leaf rust fungus according to Example 3 of the present invention.

[0022] Figure 4 The diagram shows the gene structure of the DUF1 and DUF2 genes according to Embodiment 3 of the present invention and a schematic diagram of the base and amino acid changes in the EMS-induced susceptible mutant.

[0023] Figure 5 A schematic diagram of a recombinant vector fragment for transgenic complementation verification according to Embodiment 4 of the present invention is shown.

[0024] Figure 6 The diagram shows the differential phenotypic results of Fielder (the control variety) and some T1 generation transgenic plants after 10 days of inoculation with the physiological race PHQS of leaf rust, according to Example 4 of the present invention. Detailed Implementation

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0026] Terminology Explanation:

[0027] Translation control signals: These are nucleotide sequences, such as enhancers, that exist upstream or downstream of a gene and can regulate the transcription of the target gene, thereby affecting protein translation.

[0028] As mentioned in the background section, overuse of genes in breeding can accelerate the emergence of new, highly virulent leaf rust races, rendering mainstream resistance genes ineffective. Once a virulent race becomes prevalent, it can lead to a sharp decline in wheat yield, severely impacting safe wheat production. Utilizing leaf rust resistance genes in wheat disease resistance breeding is the most economical and effective method for controlling wheat leaf rust, provided that the resistance genes are used correctly in breeding. Currently, 83 formally named wheat leaf rust resistance genes have been reported, but only a few have been successfully isolated and cloned. Therefore, the number of genes that can be selected for breeding using leaf rust resistance genes is limited, and the gene types are also limited. Therefore, in this application, the inventors conducted in-depth research on the leaf rust resistance gene Lr51 from the wheat-closely related plant *Aegilops spp.*, completing the fine mapping, isolation, cloning, and functional verification of Lr51. They discovered that Lr51 encodes a new type of resistance protein, which possesses activity against wheat leaf rust. Based on this, a series of protection schemes are proposed in this application.

[0029] In a first typical embodiment of this application, a wheat leaf rust resistance protein is provided, comprising DUF1 protein and / or DUF2 protein. DUF1 protein comprises: (a1) a protein having the amino acid sequence shown in SEQ ID NO: 3; or (a2) a protein having one or more amino acids substituted and / or deleted and / or added to the amino acid sequence in (a1) and having resistance to wheat leaf rust; or (a3) ​​a protein having more than 80% homology with the amino acid sequence defined in either (a1) or (a2) and having the same function. DUF2 protein comprises: (b1) a protein having the amino acid sequence shown in SEQ ID NO: 5; or (b2) a protein having one or more amino acids substituted and / or deleted and / or added to the amino acid sequence in (b1) and having resistance to wheat leaf rust; or (b3) a protein having more than 80% homology with the amino acid sequence defined in either (b1) or (b2) and having the same function.

[0030] In a preferred embodiment, the DUF1 protein comprises a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology with and having the same function as the amino acid sequence defined in (a1) and (a2); preferably, the DUF2 protein comprises a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology with and having the same function as the amino acid sequence defined in (b1) and (b2).

[0031] The aforementioned wheat leaf rust resistance proteins DUF1 and DUF2 possess activity against wheat leaf rust. Mutating proteins based on the (a1) or (b1) sequence, by substituting and / or deleting and / or adding one or more amino acids, can alter key amino acid binding sites if the mutation occurs at the protein's active site, affecting its activity against wheat leaf rust, leading to increased or decreased activity, or even loss of activity. If the mutation occurs at the protein's inactive site, it may affect the protein's folding pattern, three-dimensional structure, and other properties, thus affecting its physicochemical properties and activity. Proteins with 70%, 80%, 85%, 90%, 95%, or 99% or higher homology and the same function have an active site, active pocket, and activity mechanism that are highly likely identical to the protein provided by the (a1) or (b1) sequence; these are homologous proteins obtained through amino acid mutation. The description of "same function" for homologous proteins in this application refers to activity against wheat leaf rust. Proteins with the same function can be screened using experimental methods commonly used by those skilled in the art.

[0032] The DUF1 and DUF2 proteins work synergistically; the presence of both proteins simultaneously confers resistance to leaf rust in plants, while the presence of a single DUF1 or DUF2 protein does not induce resistance. The plants mentioned in this application include, but are not limited to, wheat and *Aegilops spp.* The synergistic effect of the DUF1 and DUF2 proteins may include, but is not limited to, their independent presence in plants, or their presence as fusion proteins. Fusion proteins include, but are not limited to, direct linkage of DUF1 and DUF2 proteins, or linkage via a linker to form a fusion protein.

[0033] Prior to this study, Lr51 was only known to exist on a translocated exogenous chromosome segment at the end of the wheat 1BL chromosome, and the size of the exogenous segment was unknown. Although CAPS markers can detect this location, precise localization information was lacking. In this application, the inventors first finely localized the gene, and then further cloned the gene, discovering that the specific resistance protein is DUF1 protein + DUF2 protein.

[0034] In this specification, "homology" refers to similarity or identity, particularly identity. "Homology of amino acid sequences" refers to homology relative to the entire amino acid sequence. "Identity" between amino acid sequences refers to the total ratio of the same type of amino acid residues in these sequences. "Similarity" between amino acids refers to the total ratio of the same type of amino acid residues in these sequences plus the ratio of amino acid residues with similar side chain properties. Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool 1) and FASTA.

[0035] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0036] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substituting one another are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0037] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0038] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0039] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0040] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0041] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0042] In a second typical embodiment of this application, a wheat leaf rust resistance gene is provided, comprising a DUF1 gene and / or a DUF2 gene; the DUF1 gene comprises: (c1) a nucleotide sequence encoding the DUF1 protein in the aforementioned wheat leaf rust resistance protein; or (c2) a nucleotide sequence encoding the DUF1 protein after hybridization with a DNA molecule defined by (c1) under stringent conditions; or (c3) having the nucleotide sequence shown in SEQ ID NO: 2; or (c4) a gene having more than 70% homology with any of the nucleotide sequences defined in (c1) to (c3) and encoding a protein with the same function; the DUF2 gene comprises: (d1) a nucleotide sequence encoding the DUF2 protein in the aforementioned wheat leaf rust resistance protein; or (d2) a nucleotide sequence encoding the DUF2 protein after hybridization with a DNA molecule defined by (d1) under stringent conditions; or (d3) having the nucleotide sequence shown in SEQ ID NO: 2. NO:4 nucleotide sequence; or a gene that has more than 70% homology with any of the nucleotide sequences defined in (d4) and (d1) to (d3) and encodes a protein with the same function.

[0043] As used herein, “DNA hybridization under stringent conditions” means that a nucleotide sequence hybridizes specifically with a target sequence in an amount detectably stronger than nonspecific hybridization. Stringent conditions may include, for example, low-salt and / or high-temperature conditions, provided, for example, by approximately 0.02 M to 0.1 M NaCl or equivalent at temperatures of approximately 50 °C to 70 °C.

[0044] In a preferred embodiment, the DUF1 gene comprises a gene having 75% or more, preferably 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with any of the nucleotide sequences defined in (c1) to (c3) and encoding a protein having the same function; preferably, the DUF2 gene comprises a gene having 75% or more, preferably 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with any of the nucleotide sequences defined in (d1) to (d3) and encoding a protein having the same function.

[0045] The aforementioned wheat leaf rust resistance genes may encode proteins with resistance to wheat leaf rust. Mutating nucleotides in the (c1) or (d1) sequence, and hybridizing with DNA molecules defined by (c1) or (d1) under strict conditions without frameshift mutations, can alter key amino acid binding sites in the encoded protein. This can affect the protein's resistance to wheat leaf rust, leading to increased, decreased, or even lost activity. Mutations in nucleotides encoding inactive sites may affect the protein's folding pattern, three-dimensional structure, and other properties, thus impacting its physicochemical properties and activity. Wheat leaf rust resistance genes with 70%, 75%, 85%, 95%, or 99% or higher homology and encoding proteins with the same function likely share the same active sites, active pockets, and activity mechanisms as the genes provided by the (c1) or (d1) sequences, representing homologous genes obtained through nucleotide mutation.

[0046] The resistant parent of this invention is GSTR 441, which carries a foreign chromosome segment from the wheat-related species *Aegilops spp.*, approximately 25 Mb in size, which translocates to chromosome 1B of common wheat *Neepawa*. Studies have shown that this foreign segment contains a broad-spectrum leaf rust resistance gene, Lr51, exhibiting high levels of resistance to leaf rust races worldwide. However, due to the complexity of the wheat genome and the lack of genetic studies and related genomic sequence information, the Lr51 gene has not yet been isolated and cloned in the prior art. This application utilizes a large segregating population for fine mapping, combining third-generation ultra-long read sequencing (ONT) and MutRNASeq methods to clone the Lr51 gene, and functional verification was performed using independent EMS mutants and transgenic complementation experiments.

[0047] In a third typical embodiment of this application, an expression cassette is provided, which contains the aforementioned wheat leaf rust resistance gene, including the DUF1 gene and / or the DUF2 gene.

[0048] In a preferred embodiment, the expression cassette further includes a regulatory sequence for regulating the expression of wheat leaf rust resistance genes, the regulatory sequence including but not limited to a promoter; preferably, the promoter includes one or more of the following promoters: constitutive, enhancing, tissue-specific and inducible.

[0049] The aforementioned expression cassette, or gene expression cassette, consists of a regulatory sequence and the aforementioned wheat leaf rust resistance gene, and may also contain other nucleic acid fragments. The regulatory sequence influences the transcription, translation, and other expression processes of the aforementioned wheat leaf rust resistance gene. The regulatory sequence can be a promoter, enhancer, silencer, regulatory protein attachment site, or other nucleic acid fragments. The promoter can be one or more of constitutive promoters, enhancer promoters, tissue-specific promoters, inducible promoters, or other types of promoters working in combination to regulate gene expression. An expression cassette can contain both the DUF1 and DUF2 genes simultaneously, allowing for the expression of both DUF1 and DUF2 proteins. Alternatively, it can contain only one wheat leaf rust resistance gene (DUF1 or DUF2). Expression of both proteins in wheat can be achieved through mixed transformation of two expression cassettes containing DUF1 and DUF2 genes respectively, or by hybridizing wheat transformed with only the DUF1 gene and wheat transformed with only the DUF2 gene.

[0050] In a fourth typical embodiment of this application, a recombinant vector is provided, which contains the aforementioned wheat leaf rust resistance gene or expression cassette; the wheat leaf rust resistance gene includes the DUF1 gene and / or the DUF2 gene; preferably, the recombinant vector includes a translation control signal; preferably, the translation control signal includes an enhancer; preferably, the enhancer includes a translational enhancer and / or a transcriptional enhancer; preferably, the translation control signal is derived from a natural sequence or a synthetically produced sequence; preferably, the recombinant vector includes a plant expression vector; preferably, the plant expression vector includes a binary vector transformed by Agrobacterium and a gene gun bombardment vector; preferably, the plant expression vector includes pCAMBIA1300; preferably, the recombinant vector includes a reporter gene; preferably, the reporter gene includes a resistance gene or a gene expressing an enzyme or luminescent compound that produces a color change; preferably, the resistance gene includes an antibiotic resistance gene or a chemical reagent resistance gene.

[0051] The recombinant vectors described above contain the wheat leaf rust resistance gene or the aforementioned expression cassette, and may also contain other nucleic acid fragments such as replication origin sites, multiple cloning sites, and translation control signals. Translation control signals derived from natural or synthetic sequences include enhancers, molecular chaperones, and other nucleotide sequences that can affect protein translation. The aforementioned enhancers include translational enhancers and / or transcriptional enhancers, which can be used alone or in combination to regulate protein transcription and translation. The recombinant vectors described above can be plant expression vectors, which can be transformed into plants to express the target gene and produce the target protein, thereby exerting their function. Plant expression vectors include, but are not limited to, binary vectors transformed by Agrobacterium and gene gun bombardment vectors, which can be introduced into plant cells through different transformation methods to improve transformation efficiency. The aforementioned plant expression vectors include, but are not limited to, pCAMBIA1300 used in the examples. Similar to the expression cassettes mentioned above, a recombinant vector can contain both the DUF1 and DUF2 genes, enabling the expression of both DUF1 and DUF2 proteins. Alternatively, it can contain only one wheat leaf rust resistance gene (DUF1 or DUF2), allowing the expression of both proteins through mixed transformation of two recombinant vectors containing the DUF1 and DUF2 genes, respectively.

[0052] The recombinant vectors described above may also include reporter genes; preferably, the reporter genes include, but are not limited to, resistance genes or genes expressing enzymes or luminescent compounds that produce color changes, thereby allowing for the determination of whether the recombinant vector has been successfully transformed and expressed through various methods such as resistance screening, color screening, and fluorescence screening; wherein the resistance genes include, but are not limited to, antibiotic resistance genes or chemical reagent resistance genes, allowing for efficient screening of the transformed parent using antibiotics, chemical reagents, and other drugs to determine whether the recombinant vector has been successfully transformed and expressed. From the perspective of transgenic safety, no reporter gene may be added, and phenotypic screening may be used directly to determine whether the transformation was successful.

[0053] In a fifth typical embodiment of this application, a non-plant host cell is provided, which is transformed with the above-mentioned recombinant vector; preferably, the host cell includes Escherichia coli or Agrobacterium rhizogenes; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium rhizogenes includes EHA105.

[0054] The host cells described above are transformed with recombinant vectors, which can carry the recombinant vectors to perform various functions such as recombinant vector copying, gene expression, and gene integration into chromosomes. The host cells can be various strains such as *Escherichia coli* and *Agrobacterium tumefaciens*, with *Escherichia coli* typically using DH5α and *Agrobacterium tumefaciens* typically using EHA105.

[0055] In a sixth typical embodiment of this application, the above-mentioned wheat leaf rust resistance protein, or the above-mentioned wheat leaf rust resistance gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned host cell are provided for the use of regulating plant resistance to leaf rust, enhancing or reducing plant resistance to leaf rust, or cultivating transgenic plants with enhanced or reduced resistance to leaf rust.

[0056] The above applications utilize wheat leaf rust resistance proteins, genes, expression cassettes, recombinant vectors, or host cells to regulate plant resistance to leaf rust through resistance proteins and proteins encoded by resistance genes; enhance or reduce plant resistance to leaf rust through regulatory sequences and translation control signals; and transform host cells carrying recombinant vectors into parent plants using various transformation methods to cultivate transgenic plants with enhanced or reduced resistance to leaf rust.

[0057] In a seventh typical embodiment of this application, a method for preparing transgenic plants is provided. This method includes introducing the aforementioned wheat leaf rust resistance gene, expression cassette, recombinant vector, or host cell into a target plant to obtain a transgenic plant resistant to leaf rust. Essentially, this involves introducing genes encoding DUF1 and / or DUF2 proteins into the plant.

[0058] In a preferred embodiment, the recombinant vector is introduced into the target plant via a plant virus vector, a gene gun, or Agrobacterium infection; preferably, the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat; preferably, the wheat leaf rust resistance gene is driven by a constitutive promoter.

[0059] In the above methods, the recombinant vector is introduced into the target plant via plant virus vectors, gene guns, or Agrobacterium infection. The above methods for preparing transgenic plants utilize various methods such as plant virus vectors, gene guns, or Agrobacterium infection to introduce wheat leaf rust resistance genes, expression cassettes, recombinant vectors, or host cells into the target plant, resulting in transgenic plants with enhanced resistance to leaf rust. The target plant can be a dicotyledonous or monocotyledonous plant; preferably, the monocotyledonous plant can be wheat, and wheat varieties include, but are not limited to, Fielder wheat. The above methods can also influence the expression, protein activity, or translation of wheat leaf rust resistance genes by establishing mutant libraries, obtaining mutant families, or inducing mutations at nucleotide sites in the resistance gene, thereby obtaining transgenic plants with reduced or enhanced resistance to leaf rust.

[0060] In the eighth typical embodiment of this application, a method for increasing or decreasing plant resistance to leaf rust is provided, the method comprising: increasing or decreasing any one or more of the copy number, expression level, activity or content of the aforementioned wheat leaf rust resistance protein in the target plant, thereby enhancing or decreasing the plant's resistance to leaf rust.

[0061] The above methods can increase or decrease the copy number, expression level, activity, or content of wheat leaf rust resistance proteins in target plants by means of nucleotide sequence mutation, alteration of regulatory sequences, and / or translation control signals, thereby enhancing or reducing the plant's resistance to leaf rust.

[0062] In a preferred embodiment, the method is used for plant breeding; the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat; preferably, the leaf rust is caused by a physiological race of leaf rust fungus; preferably, the physiological race of leaf rust fungus is a toxic race of leaf rust fungus prevalent in China, including FHJR, THDB, THTT, FHJL, HCJR, PHQS, PHRT, or PHTT.

[0063] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0064] Example 1: Resistance spectrum analysis of leaf rust resistance gene Lr51

[0065] Neepawa, a common wheat variety, and GSTR441, a near-isogenic line carrying the Lr51 gene, were cultivated in a plant culture chamber. The plant culture chamber conditions were set as follows: 22℃ during the day, 20℃ at night, 16 hours of light, 8 hours of darkness, and 80-90% humidity. When the wheat seedlings reached the two-leaf stage, they were manually inoculated with eight different physiological races of leaf rust fungus: FHJR, THDB, THTT, FHJL, HCJR, PHQS, PHRT, or PHTT. After inoculation, the plants were kept in darkness and kept moist for 24 hours, followed by at least 2 hours of light. The plant culture chamber was then set to its normal photoperiod. Approximately 10 days after inoculation, leaf rust resistance was assessed and statistically analyzed in the wheat materials, specifically using a 0-4 grade system for leaf rust phenotype classification (i.e., grade 0 for immune; grade 2 for near-immune; grade 1 for highly resistant; grade 2 for moderately resistant; grade 3 for moderately susceptible; and grade 4 for highly susceptible). Figure 1 As shown, near-isogenic lines containing the leaf rust resistance gene Lr51 exhibit high levels of resistance (R), while background materials without Lr51 are susceptible (S).

[0066] Example 2: Fine mapping of leaf rust resistance gene Lr51

[0067] Exogenous 1S chromosomes carrying Lr51 (e.g.) Figure 2 As shown in Figure a), the 1B chromosome of common wheat cannot undergo recombination and exchange. To precisely locate Lr51, we constructed a segregating population using the resistant parent GSTR 441 and the CSph1b mutant, and induced partial recombination and exchange of homologous chromosomes of 1S / 1B using the ph1b mutant. The specific procedures are as follows: Using an indoor plant growth chamber, GSTR 441 was crossed with the susceptible parent CSph1b to obtain F1. F1 plants were self-crossed to obtain F2. Through molecular marker identification, heterozygous plants carrying the ph1b gene and simultaneously carrying the 1S / 1B chromosome were selected from the F2 population and self-crossed to obtain F3. The heterozygous plants carrying the 1S / 1B chromosome in F3 were then self-crossed to harvest F4. Figure 2 As shown, we developed 11 genome-specific molecular markers within the exogenous 1S chromosome segment. Using these markers, we screened 721 F3 and F4 segregating individuals, obtaining individuals exhibiting recombination exchanges within the 25Mb exogenous 1S chromosome, i.e., recombinants. Figure 2 As shown in Figure c, from the obtained recombinants, we selected single plants with a 1S / 1B heterozygous genotype on the reduced exogenous chromosome and a homozygous 1B genotype on the other side. These plants were inoculated with the physiological race PHQS of wheat rust for phenotypic identification. The key recombinants were used to cultivate approximately 25 offspring plants, which were then inoculated with PHQS for phenotypic verification. Through genotype-phenotype combination, Lr51 was finely mapped between molecular markers pku1B4289 and pku1B4309, corresponding to a physical region of 1.4 Mb in the reference genome 1.0 of common wheat (Chinese spring). Figure 2 (b). The candidate region located corresponds to a physical region of 0.79 Mb in the genome of the resistant parent GSTR 441, which does not contain the typical NBS-LRR gene. Figure 2 (d). Figure 2 In this context, Res. indicates high-level resistance, Sus. indicates susceptibility, and Phe. indicates phenotype.

[0068] Example 3: Validation of disease-susceptible EMS mutants and Lr51 candidate genes

[0069] Chemical mutagenesis with ethyl methane sulfonate (EMS) at a concentration of 0.5% was performed on the resistant parent GSTR 441, resulting in 1460 independent M2 mutant families. Phenotypic identification was performed on 460 of these M2 mutant families using an all-weather plant growth chamber. Each family had 25 seedlings, inoculated with the physiological race PHQS of leaf rust fungus. Susceptible individual plants were isolated from 18 families. Genotyping was performed using molecular markers to confirm the presence of the 1S exogenous chromosome in the susceptible individual plants, preventing seed contamination. Subsequently, the susceptible individual plants were transplanted, and M3 seeds were harvested for M3 family development (including…). Figure 3 Phenotypic identification of M1, M6, M8, M9, M16, M22, M26, M43, M50, M57, M58, M61, M63, M71, M73, M90, M97, and M99 confirmed that these families all exhibited the disease-susceptible phenotype. Figure 3 ).

[0070] We first performed Pac-bio sequencing on GSTR 441, assembling contigs containing candidate regions. These contigs were then annotated using the reference genomes of *Aegilops sinensis* and *Aegilops spp.* TS01. We found that none of the finely mapped candidate chromosomal regions contained typical NBS-LRR genes. Using MutRNAseq, we obtained candidate genes for Lr51. Specifically, under leaf rust inoculation conditions, we performed transcriptome sequencing on susceptible mutants of GSTR 441 and performed de novo assembly to obtain transcripts. We then performed local alignment analysis of the Pac-bio assembled sequences of candidate genes within the region against the transcripts. We found 12 mutations in DUF1 and 7 mutations in DUF2 within the mapped region. M57 was mutated in both DUF1 and DUF2, with premature termination occurring in M16 and M57. Figure 4 The above experiments demonstrate that both candidate genes are essential for providing resistance to leaf rust.

[0071] Example 4: Validation of transgenic complementation of the Lr51 candidate gene

[0072] To determine whether candidate genes DUF1 and DUF2 could provide resistance to leaf rust, transgenic complementation of the candidate genes was performed.

[0073] 1. Construction of complementary vector P1300-Lr51

[0074] Based on Pac-bio sequencing assembly data and combined with BLASTN / BLASTX analysis from the NCBI database, the structure of the gene was determined, and the nucleotide sequence shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 3 were obtained, which are the CDS and protein sequences of DUF1; the nucleotide sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 5 were obtained, which are the CDS and protein sequences of DUF2.

[0075] Mutant analysis results indicated that both candidate genes DUF1 and DUF2 are essential for Lr51 to provide leaf rust resistance. Based on this information, to verify transgenic complementation, we constructed a transgenic vector amplified genomic fragments containing DUF1 and DUF2, as shown in SEQ ID NO: 1, including the forward genomic sequence of DUF1 and the reverse genomic sequence of DUF2, consistent with their orientation in the wheat genome. The Lr51 genomic fragment includes 6907 bp upstream of the DUF2 start codon, the full gene length (from ATG to TGA, 2523 bp), and 1502 bp downstream of the DUF2 start codon, as well as a 6 bp restriction enzyme site left over from vector construction, and 2836 bp and 1299 bp 5' uncoding regions upstream of the DUF1 start codon, the full gene length (from ATG to TAG, 2253 bp), and 2764 bp downstream of the DUF1 start codon, totaling 20090 bp. Figure 5 First, PCR amplification was performed using primers p1300-DUF2F1 (SEQ ID NO: 6) and p1300-DUF2R1 (SEQ ID NO: 7), as well as p1300-DUF2F2 (SEQ ID NO: 8) and p1300-DUF2R2 (SEQ ID NO: 9). Then, the amplified DUF2 genomic fragment was recombined into the linearized pCAMBIA1300 vector according to the In-Fusion SnapAssembly Master Mix kit from Baori Biotechnology (Beijing) Co., Ltd., to obtain the p1300-DUF2 plasmid. The genomic fragment of DUF1 was amplified by PCR using primers p1300-DUF2-DUF1F1 (SEQ ID NO: 10) and p1300-DUF2-DUF1R1 (SEQ ID NO: 11), p1300-DUF2-DUF1F2 (SEQ ID NO: 12) and p1300-DUF2-DUF1R2 (SEQ ID NO: 13), as well as p1300-DUF2-DUF1F3 (SEQ ID NO: 14) and p1300-DUF2-DUF1R3 (SEQ ID NO: 15). The amplified fragment was then recombined with the linearized p1300-DUF2 plasmid using In-Fusion SnapAssembly Master Mix to obtain the p1300-DUF2-DUF1 plasmid, which is the complementary vector p1300-Lr51.

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[0082] 2. Obtaining T0 generation transgenic plants

[0083] Using a high-purity plasmid extraction kit (Tiangen Biotech Beijing Co., Ltd.), the plasmid of the complementary vector p1300-Lr51 was extracted and purified, transformed into Agrobacterium strain EHA105, and then transformed into common wheat Fielder by Agrobacterium infection method, resulting in 10 Lr51 complementary T0 generation transgenic plants.

[0084] 3. Resistance identification of transgenic plants (families)

[0085] First, markers were used to identify the positive results of the obtained T0 generation complementary transgenic plants. The results showed that 9 out of 10 transgenic plants were positive. The T0 transgenic plants were then grown in a greenhouse, and T1 generation seeds were harvested through self-pollination. The T1 transgenic families were inoculated with the leaf rust physiological race PHQS, and phenotypic identification was performed 10 days after inoculation. Figure 6 As shown, the T1 generation transgenic plants exhibited high levels of disease resistance phenotypes (2, 3, 4, 5, 6, 7, 8, 9 and 10), while the control Fielder (1) showed high susceptibility.

[0086] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: Based on the preliminary mapping of the leaf rust resistance gene Lr51, the present invention performs fine mapping and rapid cloning (long-read sequencing + MutRNASeq) to obtain the DUF1 and DUF2 genes with wheat leaf rust resistance, as well as the DUF1 and DUF2 proteins encoded by the two genes. These genes possess broad-spectrum resistance and encode novel types of leaf rust resistance proteins, which helps to broaden the types of resistance gene resources and deepen the research on the resistance mechanism of resistance genes against pathogens. Introducing the nucleotide sequences encoding the DUF1 and DUF2 proteins into wheat can improve wheat's resistance to leaf rust, providing a reliable and effective source of leaf rust resistance for wheat molecular breeding.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A wheat leaf rust resistance protein, characterized in that, The wheat leaf rust resistance proteins include DUF1 protein and / or DUF2 protein. The DUF1 protein includes: (a1) A protein having the amino acid sequence shown in SEQ ID NO: 3; or (a2) A protein in which one or more amino acids have been substituted and / or deleted and / or added in the amino acid sequence of (a1) and which has activity against wheat leaf rust; or (a3) is a protein that has more than 80% homology with and has the same function as the amino acid sequence defined in (a1) and (a2); The DUF2 protein includes: (b1) A protein having the amino acid sequence shown in SEQ ID NO: 5; or (b2) A protein in which one or more amino acids have been substituted and / or deleted and / or added in the amino acid sequence of (b1) and which has activity against wheat leaf rust; or (b3) is a protein that has more than 80% homology with the amino acid sequence defined in either (b1) or (b2) and has the same function.

2. A wheat leaf rust resistance gene, characterized in that, The wheat leaf rust resistance genes include the DUF1 gene and / or the DUF2 gene; The DUF1 gene includes: (c1) Encoding the nucleotide sequence of the DUF1 protein in the wheat leaf rust resistance protein of claim 1; or (c2) The nucleotide sequence that hybridizes under stringent conditions with the DNA molecule defined in (c1) and encodes the DUF1 protein; or (c3) has the nucleotide sequence shown in SEQ ID NO: 2; or A gene that has more than 70% homology with any of the nucleotide sequences defined in (c4) and (c1) to (c3) and encodes a protein with the same function; The DUF2 gene includes: (d1) The nucleotide sequence encoding the DUF2 protein in the wheat leaf rust resistance protein of claim 1; or (d2) The nucleotide sequence that hybridizes under stringent conditions with the DNA molecule defined by (d1) and encodes the DUF2 protein; or (d3) has the nucleotide sequence shown in SEQ ID NO: 4; or A gene that has more than 70% homology to any of the nucleotide sequences defined in (d4) and (d1) to (d3) and encodes a protein with the same function.

3. An expression box, characterized in that, The expression cassette contains the wheat leaf rust resistance gene of claim 2, wherein the wheat leaf rust resistance gene includes the DUF1 gene and / or the DUF2 gene.

4. The expression box according to claim 3, characterized in that, The expression cassette also includes a regulatory sequence for regulating the expression of wheat leaf rust resistance genes, the regulatory sequence including a promoter; Preferably, the promoter includes one or more of the following promoters: constitutive, enhancing, tissue-specific, and inducible.

5. A recombinant vector, characterized in that, It contains the wheat leaf rust resistance gene as described in claim 2 or the expression cassette as described in claim 3 or 4; the wheat leaf rust resistance gene includes the DUF1 gene and / or the DUF2 gene; Preferably, the recombinant vector includes a translation control signal; Preferably, the translation control signal includes an enhancer; Preferably, the enhancer includes translational enhancers and / or transcriptional enhancers; Preferably, the translation control signal originates from a natural sequence or an artificially synthesized sequence; Preferably, the recombinant vector includes a plant expression vector; Preferably, the plant expression vector comprises a binary vector transformed by Agrobacterium and a vector bombarded by a gene gun; Preferably, the plant expression vector includes pCAMBIA1300; Preferably, the recombinant vector includes a reporter gene; Preferably, the reporter gene includes an resistance gene or a gene that expresses an enzyme or luminescent compound that produces a color change; Preferably, the resistance gene includes an antibiotic resistance gene or a chemical reagent resistance gene.

6. A non-plant host cell, characterized in that, The host cell transformation includes the recombinant vector as described in claim 5; Preferably, the host cell includes Escherichia coli or Agrobacterium tumefaciens; Preferably, the *Escherichia coli* includes DH5α; Preferably, the Agrobacterium rhizogenes includes EHA105.

7. The use of the wheat leaf rust resistance protein of claim 1, or the wheat leaf rust resistance gene of claim 2, or the expression cassette of claim 3 or 4, or the recombinant vector of claim 5, or the host cell of claim 6 in regulating plant resistance to leaf rust, enhancing or reducing plant resistance to leaf rust, or cultivating transgenic plants with enhanced or reduced resistance to leaf rust.

8. A method for preparing transgenic plants, characterized in that, Will The wheat leaf rust resistance gene as described in claim 2, Or the expression box as described in claim 3 or 4, Or the recombinant vector as described in claim 5, Alternatively, the host cell described in claim 6 may be introduced into the target plant to obtain the transgenic plant resistant to leaf rust.

9. The method according to claim 8, characterized in that, The recombinant vector is introduced into the target plant via a plant virus vector, a gene gun, or Agrobacterium infection. Preferably, the target plant is a dicotyledonous plant or a monocotyledonous plant; Preferably, the target plant is wheat; Preferably, the wheat is Fielder wheat; Preferably, the wheat leaf rust resistance gene is driven by a constitutive promoter.

10. A method for increasing or decreasing plant resistance to leaf rust, characterized in that, The method includes: Increase or decrease any one or more of the copy number, expression level, activity, or content of the wheat leaf rust resistance protein of claim 1 in the target plant, thereby enhancing or reducing the plant's resistance to leaf rust.

11. The method according to claim 10, characterized in that, The method is used for plant breeding; The target plant is a dicotyledonous plant or a monocotyledonous plant; Preferably, the target plant is wheat; Preferably, the wheat is Fielder wheat; Preferably, the leaf rust is caused by a physiological race of leaf rust fungus; Preferably, the physiological race of the leaf rust fungus is a toxic race of the leaf rust fungus prevalent in China, including FHJR, THDB, THTT, FHJL, HCJR, PHQS, PHRT, or PHTT.

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