Wheat leaf rust resistance protein Lr.ace-4A, resistance gene and application

Abstract

The invention provides a wheat leaf rust resistance protein Lr.ace-4A, a resistance gene and application of the wheat leaf rust resistance protein Lr.ace-4A and the resistance gene. The wheat leaf rust resistance protein Lr.ace-4A comprises: (a) a protein composed of an amino acid sequence represented by SEQ ID NO: 3; or (b) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acids in the amino acid sequence in (a) and has wheat leaf rust resistance activity; or (c) protein which has more than 80% of homology with the amino acid sequence limited by any one of (a) and (b) and has the same function. 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] The present invention relates to the field of wheat breeding, and in particular to a wheat leaf rust resistance protein Lr.ace-4A, a resistance gene and an application thereof. Background Art

[0002] Wheat leaf rust is a type of airborne fungal disease that mainly harms wheat leaves, destroys photosynthesis, and then causes wheat yield reduction, usually causing a 5% to 15% reduction in yield. When the disease is severe, it can cause a reduction of more than 40%. It is an important disease in wheat production and a serious threat to the safe production of wheat. Using disease-resistant genes and breeding disease-resistant wheat varieties is the most economical and effective strategy to control the disease. However, the mutation rate of wheat leaf rust is fast, and new highly toxic subspecies continue to appear, causing many disease-resistant genes to lose resistance. In recent years, with climate change, wheat leaf rust has shown a trend of expansion and development, and the damage is becoming increasingly serious. Therefore, the prevention and control of wheat leaf rust has become an important task in wheat production. However, the genetic basis of the main wheat varieties is very narrow, and effective rust resistance genes are scarce. It is urgent to introduce new disease-resistant genes, enrich the resistance source, and improve the resistance of the main wheat varieties to leaf rust.

[0003] The wheat leaf rust resistance gene Lr.ace-4A comes from the Portuguese durum wheat (T.durum) local variety PI192051. In 2019, North Dakota State University in the United States conducted a population phenotypic analysis and found that Lr.ace-4A showed near-immune resistance to the American leaf rust strain BBBQJ_CA1.2, the Moroccan strain BBBQJ_Mor38-2, the Ethiopian strain EEEEE_Eth50-4, and the Tunisian strain BBBSJ_Tun20-4. Subsequently, using genetic population linkage analysis, the gene was located near the centromere region on chromosome 4A. Lr.ace-4A shows near-immune resistance to leaf rust species from different countries, and will have great application prospects in wheat leaf rust resistance breeding, but it has not yet been successfully cloned and bred.

[0004] Since the wheat genome is very large and more than 80% of the sequences are repetitive sequences, the isolation and cloning of wheat functional genes lags far behind other crops such as rice and corn. So far, there are about 83 wheat leaf rust resistance genes (Lr1-Lr83) that have been officially named internationally, but only about 11 leaf rust resistance genes have been successfully isolated and cloned. Most of these genes come from common wheat, and no leaf rust resistance genes from tetraploid durum wheat have been cloned. Moreover, although some leaf rust resistance genes have been cloned in the prior art, many of them have lost their resistance due to the evolution of strains and excessive use of genes; and some of the existing cloned leaf rust resistance genes come from closely related species of wheat, and there is a linkage drag with wheat, which limits their breeding utilization. In view of this situation, it is of great significance to clone the leaf rust resistance gene Lr.ace-4A, identify new disease resistance proteins, enrich the resistance sources of wheat leaf rust, and improve the diversity of resistance genes. Summary of the invention

[0005] The main purpose of the present invention is to provide a wheat leaf rust resistance protein Lr.ace-4A, a resistance gene and an application, so as to solve the problem of lack of leaf rust resistance protein in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a wheat leaf rust resistance protein Lr.ace-4A is provided, and the wheat leaf rust resistance protein Lr.ace-4A comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 3; or (b) a protein having leaf rust resistance in which the amino acid sequence in (a) is substituted and / or deleted and / or one or more amino acids are added; or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having leaf rust resistance.

[0007] Furthermore, the wheat leaf rust resistance protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in any one of (a) and (b) and has leaf rust resistance.

[0008] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a wheat leaf rust resistance gene is provided, and the wheat leaf rust resistance gene includes the Lr.ace-4A gene; the Lr.ace-4A gene includes: (a) a nucleotide sequence encoding the above-mentioned wheat leaf rust resistance protein Lr.ace-4A; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under strict conditions and encodes the above-mentioned wheat leaf rust resistance protein Lr.ace-4A; or (c) a nucleotide sequence shown in SEQ ID NO: 2; or (d) a gene that has more than 70% homology with any one of the nucleotide sequences defined in (a)-(c) and encodes a protein with leaf rust resistance.

[0009] Furthermore, the wheat leaf rust resistance gene has more than 75%, preferably more than 85%, more preferably more than 95%, and further preferably more than 99% homology with any one of the nucleotide sequences specified in (a) to (c) and encodes a protein with leaf rust resistance.

[0010] In order to achieve the above object, according to the third aspect of the present invention, an expression cassette is provided, the expression cassette comprising the above wheat leaf rust resistance gene.

[0011] Furthermore, the expression cassette also includes a regulatory sequence for regulating the expression of the wheat leaf rust resistance gene, and the regulatory sequence includes a promoter; preferably, the promoter includes one or more of the following promoters: constitutive, enhanced, tissue-specific or inducible.

[0012] In order to achieve the above object, according to the fourth aspect of the present invention, a recombinant vector is provided, which comprises the above wheat leaf rust resistance gene or the above expression cassette.

[0013] Further, the recombinant vector comprises a translation control signal; preferably, the translation control signal comprises an enhancer; preferably, the enhancer comprises a translation enhancer and / or a transcription enhancer; preferably, the translation control signal is derived from a natural sequence or an artificially synthesized sequence; preferably, the recombinant vector comprises a plant expression vector; preferably, the plant expression vector comprises a binary vector for Agrobacterium transformation and a vector for gene gun bombardment; preferably, the plant expression vector comprises pCAMBIA1300; preferably, the recombinant vector comprises a reporter gene; preferably, the reporter gene comprises a resistance gene or a gene expressing an enzyme that produces a color change or a luminescent compound; preferably, the resistance gene comprises an antibiotic resistance gene or a chemical agent resistance gene.

[0014] To achieve the above object, according to the fifth aspect of the present invention, a non-plant host cell is provided, and the host cell is transformed with the above recombinant vector; preferably, the host cell includes Escherichia coli or Agrobacterium tumefaciens; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium tumefaciens includes EHA105.

[0015] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, there is provided an application of 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 in any one or more of the following: regulating the leaf rust resistance of plants, or cultivating transgenic plants with enhanced or reduced leaf rust resistance, or breeding wheat leaf rust resistance; preferably, regulating the leaf rust resistance of plants includes enhancing or reducing the leaf rust resistance of plants; preferably, the leaf rust is leaf rust caused by a physiological race of leaf rust fungus; preferably, the physiological race of leaf rust fungus is a toxic race of the Chinese prevalent leaf rust fungus, and the toxic races of the Chinese prevalent leaf rust fungus include FHJL, PHQS, FHJR, THDB, PHJS, PHST, THSP or HCJR.

[0016] In order to achieve the above-mentioned purpose, according to the seventh aspect of the present invention, a method for preparing a transgenic plant 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 the target plant to obtain a transgenic plant resistant to leaf rust.

[0017] Furthermore, the recombinant vector is introduced into the target plant by 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.

[0018] In order to achieve the above-mentioned purpose, according to the eighth aspect of the present invention, a method for increasing or decreasing the resistance of a plant to leaf rust is provided, the method comprising: increasing or decreasing the activity and / or content of the above-mentioned wheat leaf rust resistance protein Lr.ace-4A in the target plant, so that the resistance of the plant to leaf rust is enhanced or decreased.

[0019] Furthermore, the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat.

[0020] Application of the technical scheme of the present invention and utilization of the above-mentioned wheat leaf rust resistance protein can help analyze the research on the disease resistance mechanism of disease-resistant genes and proteins against pathogens, can improve the resistance of plants to leaf rust, and provide a reliable and effective source of leaf rust resistance for plant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 The figure shows the phenotypic results of the disease-resistant parent PI 192051 containing Lr.ace-4A and the susceptible material Rusty being inoculated with physiological races of leaf rust according to Example 1 of the present invention.

[0023] Figure 2 The schematic diagram of the location of the leaf rust resistance gene Lr.ace-4A according to Example 2 of the present invention is shown. Figure 2 Middle a is a schematic diagram of chromosome 4A; Figure 2 Middle b is a linkage genetic diagram for Lr.ace-4A localization using an F2 segregating population constructed from the susceptible EMS mutant m1 and the wild-type PI 192051; Figure 2 (c) is a schematic diagram of the physical location of the located molecular markers on the durum wheat Svevo1.0 reference genome.

[0024] Figure 3 A schematic diagram of the results of MutISOseq rapid cloning of the Lr.ace-4A candidate gene according to Example 3 of the present invention is shown.

[0025] in, Figure 3 Middle a is a schematic diagram of the phenotypic identification results of the susceptible mutant inoculated with the leaf rust physiological race PHQS; Figure 3 Middle b is a schematic diagram of the candidate gene IGV captured by the MutISOseq rapid cloning method, where the arrows represent the base mutations that occurred in the susceptible mutant; Figure 3 Figure c is a schematic diagram of the Lr.ace-4A gene structure and the base / amino acid changes in the EMS-induced susceptible mutant.

[0026] Figure 4 The results of transgenic complementation and transgenic overexpression verification of the Lr.ace-4A candidate gene according to Example 4 of the present invention are shown. Figure 4 Figure a is a schematic diagram of the Lr.ace-4A genome fragment used for transgenic complementation verification, including 2949 bp upstream of the start codon, 4652 bp of the full gene length (from ATG to TGA) and 1657 bp downstream of the gene; Figure 4 Middle b shows the differential phenotypic results of the control variety Fielder, some T1 generation transgenic complementary plants and the hexaploid introgression line of Lr.ace-4A "Yangmai 21-Lr.ace-4A" BC2F2 plants after inoculation with the physiological race PHQS of leaf rust for 10 days. Figure 4 Middle c shows the differential phenotypic results of the control variety Fielder and some T1 transgenic overexpressing plants after inoculation with the physiological race PHQS of leaf rust for 10 days.

[0027] Figure 5The figure shows the gene editing knockout verification result of the Lr.ace-4A candidate gene according to Example 5 of the present invention. Figure 5 In the middle, a is the structural diagram of the gene; Figure 5 Figure b shows the sequence of sgRNA and the sequencing results of mutation types produced by gene editing; Figure 5 Middle c is the differential phenotypic results of the disease-resistant parent PI 192051, the completely knocked-out T1 transgenic plants and the susceptible control Rusty after inoculation with the physiological race PHQS of leaf rust for 10 days. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0029] Terminology explanation:

[0030] Translation control signal: that is, protein translation control signal, refers to the nucleotide sequence that exists upstream or downstream of the gene and can regulate the transcription of the target gene and thus affect protein translation, such as enhancer.

[0031] As mentioned in the background technology, with climate change and the continuous emergence of new highly toxic leaf rust species, it is difficult for the leaf rust resistance genes in wheat varieties to produce resistance to new toxic species, resulting in the loss of leaf rust resistance in wheat. Once the toxic species becomes popular, it will cause major harm and seriously threaten the safe production of wheat. And using leaf rust resistance genes to cultivate disease-resistant wheat new varieties is the most economical and effective method to control the disease. At present, although there are more than 80 officially named wheat leaf rust resistance genes, only a few of them have been successfully isolated and cloned. Therefore, in this application, the inventors have conducted in-depth research on the leaf rust resistance gene Lr.ace-4A derived from tetraploid durum wheat, completed the positioning of Lr.ace-4A, isolated cloning and functional verification, and found that the resistance protein encoded by Lr.ace-4A has anti-wheat leaf rust activity. On this basis, a series of protection schemes of this application are proposed.

[0032] In a first typical embodiment of the present application, a wheat leaf rust resistance protein Lr.ace-4A (hereinafter referred to as Lr.ace-4A protein) is provided; the Lr.ace-4A protein includes: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 3; or (b) a protein having leaf rust resistance in which the amino acid sequence in (a) is substituted and / or deleted and / or one or more amino acids are added; or (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having leaf rust resistance.

[0033] SEQ ID NO:3:

[0034]

[0035] The wheat leaf rust resistance protein Lr.ace-4A has anti-leaf rust activity. Based on the sequence (a), the protein is mutated, replaced and / or deleted and / or added with one or more amino acids. If the mutation occurs at the active site of the protein, it may cause the key amino acid binding site of the protein to change, affecting the activity of the protein against wheat leaf rust, causing its activity to increase or decrease or even lose activity; if the mutation occurs at the inactive site of the protein, it may affect the folding mode, three-dimensional structure and other properties of the protein, thereby affecting the physicochemical properties and activity of the protein. Proteins with more than 80%, 85%, 90%, 95%, and 99% homology and the same function, whose active sites, active pockets, active mechanisms, etc. are all the same as the proteins provided by the sequence (a) with a high probability, are homologous proteins obtained by amino acid mutation. The description of the "same function" of homologous proteins in this application refers to the activity against wheat leaf rust. Proteins with the same function can be screened by experimental means commonly used by those skilled in the art.

[0036] "Homology" in this specification refers to similarity or identity, and particularly refers to 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 amino acid residues of the same type in these amino acid sequences. "Similarity" between amino acids refers to the total ratio of amino acid residues of the same type in these amino acid sequences and the ratio of amino acid residues with similar properties of side chains. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool).

[0037] As used herein, amino acid residues are abbreviated 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).

[0038] Substitution and replacement rules. Generally speaking, the effects of replacing amino acids with similar properties are similar. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0039] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0040] The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

[0041] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0042] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0043] A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0044] In a preferred embodiment, the wheat leaf rust resistance protein has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in any one of (a) and (b) and is a protein with leaf rust resistance. The above protein variants with homology have similar or identical anti-wheat leaf rust activity to the protein shown in SEQ ID NO: 3.

[0045] In a second typical embodiment of the present application, a wheat leaf rust resistance gene is provided, which includes the Lr.ace-4A gene; the Lr.ace-4A gene includes: (a) a nucleotide sequence encoding the above-mentioned Lr.ace-4A protein; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under strict conditions and encodes the above-mentioned Lr.ace-4A protein; or (c) a nucleotide sequence shown in SEQ ID NO: 2; or (d) a gene that has more than 70% homology with any one of the nucleotide sequences defined in (a)-(c) and encodes a protein with leaf rust resistance.

[0046] SEQ ID NO: 2:

[0047]

[0048] As used herein, the term "DNA molecule hybridization under stringent conditions" means that the nucleotide sequence specifically hybridizes to the target sequence in an amount that is detectably stronger than non-specific hybridization. Stringent conditions can include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalent at a temperature of about 50°C to 70°C.

[0049] In a preferred embodiment, the gene has 75% or more, preferably 85% or more, more preferably 95% or more, and further preferably 99% or more homology with any one of the nucleotide sequences defined in (a) to (c) and encodes a protein conferring resistance to leaf rust.

[0050] The above wheat leaf rust resistance gene can encode a protein with wheat leaf rust resistance activity. Based on the sequence (a), the nucleotides are mutated, hybridized with the DNA molecule specified in (a) under strict conditions, and no frameshift mutation occurs. If the mutation occurs in the nucleotide encoding the active site of the protein, it may cause the key amino acid binding site of the encoded protein to change, affecting the anti-wheat leaf rust activity of the protein encoded by the gene, causing its activity to increase or decrease or even lose its activity; if the mutation occurs in the nucleotide encoding the inactive site of the protein, it may affect the folding mode, three-dimensional structure and other properties of the encoded protein, thereby affecting the physicochemical properties and activity of the protein. Wheat leaf rust resistance genes with 70%, 75%, 85%, 95% or 99% or more homology and encoding proteins with the same function, the active site, active pocket, active mechanism, etc. of the protein encoded by it are most likely the same as the gene provided by the sequence (a), and are homologous genes obtained by nucleotide mutation.

[0051] The resistant parent of the present invention is the durum wheat local variety PI 192051. Studies have shown that the material contains a leaf rust resistance gene Lr.ace-4A, which shows near-immune resistance to leaf rust fungi species around the world. However, due to the complexity of the wheat genome and the lack of genetic research and genome sequence and other related information, the Lr.ace-4A gene has only been located within a larger range on chromosome 4A in the prior art and has not yet been isolated and cloned. This application uses a large number of isolated populations to carry out positioning and independent EMS mutants, combines the MutISOseq method to clone the Lr.ace-4A gene, and uses transgenic complementation, gene overexpression and gene editing knockout experiments to verify the function of the gene.

[0052] In a third typical embodiment of the present application, an expression cassette is provided, the expression cassette comprising the above-mentioned wheat leaf rust resistance gene.

[0053] In a preferred embodiment, the expression cassette also includes a regulatory sequence for regulating the expression of the wheat leaf rust resistance gene, and the regulatory sequence includes but is not limited to a promoter; preferably, the promoter includes but is not limited to one or more of the following promoters: constitutive, enhanced, tissue-specific or inducible.

[0054] The above expression cassette, i.e., gene expression cassette, is composed of a regulatory sequence, the above wheat leaf rust resistance gene, and may also contain other nucleic acid fragments. The regulatory sequence affects the transcription, translation, and other expressions of the above wheat leaf rust resistance gene. The regulatory sequence may be a nucleic acid fragment such as a promoter, an enhancer, a silencer, a regulatory protein attachment site, etc., wherein the promoter may be a constitutive promoter, an enhanced promoter, a tissue-specific promoter, an inducible promoter, or a combination of several other types of promoters to achieve the purpose of regulating gene expression.

[0055] In a fourth typical embodiment of the present application, a recombinant vector is provided, wherein the recombinant vector comprises the above-mentioned wheat leaf rust resistance gene or the above-mentioned expression cassette.

[0056] In a preferred embodiment, the recombinant vector comprises a translation control signal; preferably, the translation control signal comprises an enhancer; preferably, the enhancer comprises a translation enhancer and / or a transcription enhancer; preferably, the translation control signal is derived from a natural sequence or an artificially synthesized sequence; preferably, the recombinant vector comprises a plant expression vector; preferably, the plant expression vector comprises a binary vector for Agrobacterium transformation and a vector for gene gun bombardment; preferably, the plant expression vector comprises pCAMBIA1300; preferably, the recombinant vector comprises a reporter gene; preferably, the reporter gene comprises a resistance gene or a gene expressing an enzyme that produces a color change or a luminescent compound; preferably, the resistance gene comprises an antibiotic resistance gene or a chemical resistance gene.

[0057] The above-mentioned recombinant vector, comprising the wheat leaf rust resistance gene or the above-mentioned expression cassette, may also comprise other nucleic acid fragments such as a replication initiation site, a multiple cloning site, a translation control signal, etc. The translation control signal derived from a natural sequence or an artificially synthesized sequence includes an enhancer, a molecular chaperone, and other nucleotide sequences that can affect protein translation. The above-mentioned enhancer includes a translation enhancer and / or a transcription enhancer, which can be used alone or in combination to regulate protein transcription and translation. The above-mentioned recombinant vector can be a plant expression vector, which can be transformed into a plant, express the target gene in the plant, produce the target protein, and thus play a role; the plant expression vector includes but is not limited to a binary vector for Agrobacterium transformation and a vector for gene gun bombardment, which can be introduced into plant cells by different transformation methods to improve the transformation efficiency, and the above-mentioned plant expression vector includes but is not limited to the pCAMBIA1300 used in the embodiment.

[0058] The above-mentioned recombinant vector may also include a reporter gene; preferably, the reporter gene includes but is not limited to a resistance gene or a gene that expresses an enzyme or luminescent compound that produces a color change, so as to judge whether the recombinant vector is successfully transformed and expressed by various methods such as resistance screening, color screening, and fluorescence screening; wherein the resistance gene includes but is not limited to an antibiotic resistance gene or a chemical agent resistance gene, and antibiotics, chemical agents and other drugs can be used to efficiently screen the transformed mother to judge whether the recombinant vector is successfully transformed and expressed. Considering the safety of transgenics, it is also possible not to add any reporter gene, and directly screen whether the transformation is successful by phenotype.

[0059] In a fifth typical embodiment of the present application, a non-plant host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant vector; preferably, the host cell comprises Escherichia coli or Agrobacterium tumefaciens; preferably, Escherichia coli comprises DH5α; preferably, Agrobacterium tumefaciens comprises EHA105.

[0060] The host cells are transformed with recombinant vectors, which can carry recombinant vectors to perform various functions such as recombinant vector copying, gene expression, gene integration into chromosomes, etc. The host cells can be various strains such as Escherichia coli and Agrobacterium tumefaciens, among which Escherichia coli can be the commonly used DH5α, and Agrobacterium tumefaciens can be the commonly used EHA105.

[0061] In a sixth typical embodiment of the present application, there is provided an application of 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 in any one or more of the following: regulating the leaf rust resistance of plants, or cultivating transgenic plants with enhanced or reduced leaf rust resistance, or breeding wheat leaf rust resistance; preferably, regulating the leaf rust resistance of plants includes enhancing or reducing the leaf rust resistance of plants; preferably, the leaf rust is leaf rust caused by a physiological race of leaf rust fungus; preferably, the physiological race of leaf rust fungus is a toxic race of the Chinese prevalent leaf rust fungus, and the toxic races of the Chinese prevalent leaf rust fungus include FHJL, PHQS, FHJR, THDB, PHJS, PHST, THSP or HCJR.

[0062] The above-mentioned application utilizes wheat leaf rust resistance proteins, genes, expression cassettes, recombinant vectors or host cells to regulate the plant's resistance to leaf rust through resistance proteins, proteins encoded by resistance genes, etc.; enhance or reduce the plant's resistance to leaf rust through regulatory sequences, translation control signals, etc.; and transform host cells carrying recombinant vectors into mother plants using a variety of transformation methods, thereby cultivating transgenic plants with enhanced or reduced resistance to leaf rust.

[0063] In the seventh typical embodiment of the present application, a method for preparing a transgenic plant is provided, wherein 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 is introduced into a target plant to obtain a transgenic plant resistant to leaf rust.

[0064] In a preferred embodiment, the recombinant vector is introduced into the target plant by 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.

[0065] In the above method, the above recombinant vector is introduced into the target plant by plant virus vector, gene gun or Agrobacterium infection. The above method for preparing transgenic plants uses various methods such as plant virus vector, gene gun or Agrobacterium infection to introduce wheat leaf rust resistance gene, expression cassette, recombinant vector or host cell into the target plant to obtain a transgenic plant with enhanced resistance to leaf rust. The target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the monocotyledonous plant can be wheat, and the variety of wheat includes but is not limited to Fielder wheat. The above method can affect the expression of wheat leaf rust resistance gene, protein activity or translation by means of establishing a mutant library, obtaining mutant families, and causing mutations at the nucleotide sites of resistance genes, thereby obtaining transgenic plants with reduced or enhanced resistance to leaf rust.

[0066] In the eighth typical embodiment of the present application, a method for increasing or decreasing a plant's resistance to leaf rust is provided, the method comprising: increasing or decreasing the activity or content of the above-mentioned wheat leaf rust resistance protein in the target plant, so that the plant's resistance to leaf rust is enhanced or decreased.

[0067] The above method can increase or decrease the activity or content of wheat leaf rust resistance protein in the target plant by nucleotide sequence mutation, changing regulatory sequence and / or translation control signal, thereby enhancing or reducing the plant's resistance to leaf rust.

[0068] In a preferred embodiment, the target plant is a dicotyledonous plant or a monocotyledonous plant; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat.

[0069] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0070] Example 1: Analysis of the resistance spectrum of the leaf rust resistance gene Lr.ace-4A

[0071] The durum wheat resistant parent PI 192051 and the durum wheat susceptible parent Rusty were planted in a plant incubator. The plant incubator was set up with the following conditions: 22°C during the day, 20°C at night, 16 hours of light, 8 hours of darkness, and 80%-90% humidity. When the wheat seedlings grew to the two-leaf and one-heart stage, 8 different physiological species of leaf rust FHJL, PHQS, FHJR, THDB, PHJS, PHST, THSP or HCJR were inoculated by manual sweeping method. After inoculation, the seeds were kept in the dark and moisturized for 24 hours. After the dark treatment, the light was kept for more than 2 hours, and then the plant incubator was set to a normal light cycle. About 10 days after inoculation, the wheat materials were identified and counted for leaf rust resistance, and the leaf rust phenotypes were graded according to the grading standard of 0-4 (i.e., 0 is immune; 0; level is near immune; level 1 is highly resistant; level 2 is moderately resistant; level 3 is moderately susceptible; level 4 is highly susceptible). The grading results are as follows. Figure 1 As shown, the resistant parent PI 192051 containing the leaf rust resistance gene Lr.ace-4A exhibited a nearly immune resistance phenotype (R), while the control durum wheat material Rusty without Lr.ace-4A exhibited a high susceptibility (S).

[0072] Example 2: Localization of the leaf rust resistance gene Lr.ace-4A

[0073] The resistant parent PI 192051 and its susceptible EMS mutant family m1 were hybridized to obtain F1, and the obtained F1 was self-pollinated to obtain the F2 segregating population. At the same time, transcriptome sequencing of PI 192051 and m1 was performed, and bioinformatics analysis was used to identify single nucleotide polymorphism (SNP) sites between the parents. Combined with the tetraploid durum wheat variety Svevo reference genome, CAPS markers were developed, such as Figure 2 As shown, Figure 2a is a schematic diagram of chromosome 4A, where "Meriem et al. 2019" refers to the preliminary positioning of the Lr.ace-4A gene between the 4A chromosome molecular markers IWA232 (145.2Mb) and IWA1793 (562.8Mb; Svevo genome v1.0) by Meriem et al. in 2019, which is a larger positioning interval (Meriem Aoun, James A Kolmer et al., Mapping of novel leaf rust and stem rust resistance genes in the Portuguese durum wheat landrace PI 192051, G3 Genes|Genomes|Genetics, Volume 9, Issue 8, 1 August 2019, Pages 2535–2547); Using the obtained molecular markers, we screened 105 F2 plants and, combined with the phenotypic identification of the obtained recombinants, located the Lr.ace-4A gene between the molecular markers pkus8123 and pku4169, and co-segregated with the molecular markers pku5601, pku7069, pku0374, and pku0332 (e.g. Figure 2 (b), where "cM" refers to the unit of genetic distance "centimorgan" and "Marker" refers to molecular markers). The candidate interval located is consistent with the results of previous studies, and the corresponding physical interval in the Svevo reference genome is approximately 435.5Mb (e.g. Figure 2 Further recombinant screening revealed that it was difficult to obtain recombinants between molecular markers pku8123 and pku4169, and the localization interval could not be further narrowed.

[0074] Example 3: Screening of susceptible EMS mutants and rapid cloning of Lr.ace-4A by MutISOseq

[0075] The schematic diagram of MutISOseq rapid cloning of Lr.ace-4A candidate gene results is shown in Figure 3As shown. The resistant parent PI192051 was subjected to ethyl methane sulfonate (EMS) chemical mutagenesis treatment with an EMS concentration of 0.6%, and 2000 independent M2 mutant families were obtained. Using an all-weather plant growth chamber, about 500 M2 mutant families were phenotypically identified. Twenty seedlings were planted in each family and inoculated with the physiological race PHQS of leaf rust. Susceptible plants were isolated from 7 families. Molecular markers were used for genotyping to ensure that the seed genotype was consistent with that of the parent PI 192051 to prevent seed contamination. Subsequently, susceptible M2 plants were transplanted, M3 seeds were harvested, and M3 families (including Figure 3 The phenotypic identification of m13, m12, m22, m1, m23, m2, and m10 shown in a confirms that these families are susceptible to the disease. The phenotypic identification results of the susceptible mutants inoculated with the leaf rust physiological race PHQS are shown in the figure. Figure 3 As shown in a.

[0076] The candidate gene of Lr.ace-4A was obtained by using the MutISOseq method. The specific operation is as follows: Under the condition of leaf rust inoculation, the full-length transcriptome sequencing (ISO-seq) of PI 192051 was performed, and the original sequencing data was polished and duplicates were removed to obtain high-quality full-length transcripts, and the obtained high-quality full-length transcripts were used as reference sequences. At the same time, the transcriptome sequencing of the 7 EMS-susceptible mutant families was performed, and they were aligned to the reference sequence. Analysis found that one transcript transcript / 32998 had a typical EMS-caused SNP change from G to A or C to T in all 7 mutants. The schematic diagram of the candidate gene IGV captured by the MutISOseq rapid cloning method is shown in the figure below. Figure 3 As shown in b, the arrows indicate the base mutations that occur in the susceptible mutant.

[0077] The transcript transcript / 32998 was annotated and found to contain a typical CC-NBS-LRR gene. Subsequently, the mutants were amplified by PCR and confirmed that the 7 EMS-susceptible mutants obtained all had base mutations in this transcript. These mutations were all located in the gene coding region and caused amino acid changes. The schematic diagram of the Lr.ace-4A gene structure and the base / amino acid changes in the EMS-induced susceptible mutants is shown in Figure 3 As shown in c. The homologous gene of the Svevo reference genome corresponding to transcript / 32998 is TRITD4Av1G167080, and the protein encoded by this gene has only 3 amino acid differences between Svevo and PI 192051. Based on the above experiments, it is speculated that this candidate gene is necessary for providing leaf rust resistance.

[0078] Example 4: Transgenic complementation and transgenic overexpression of the Lr.ace-4A candidate gene

[0079] To determine whether this candidate gene could provide leaf rust resistance, genetic complementation validation of the candidate gene was performed:

[0080] 1. Construction of complementary vector p1300-Lr.ace-4A

[0081] In order to obtain the introns, upstream and downstream sequences of the candidate gene, the resequencing data and transcriptome data of the disease-resistant parent PI 192051 were used to splice a genomic sequence containing the candidate gene, and the accuracy of the sequence was verified by PCR amplification and sequencing. Based on the genomic sequence and transcript sequence, combined with NCBI database BLASTN / BLASTX analysis, 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 wheat leaf rust resistance gene (CDS) and wheat leaf rust resistance protein.

[0082] The results of transgenic complementation and transgenic overexpression verification of the Lr.ace-4A candidate gene are shown in the figure Figure 4 According to the above information, in order to verify the transgenic complementation, we constructed a transgenic vector amplified with the Lr.ace-4A genomic fragment as shown in SEQ ID NO: 1, including 2949 bp upstream of the gene start codon, the full length of the gene (from ATG to TGA, 4652 bp) and 1657 bp downstream of the gene, a total of 9258 bp of genomic sequence (schematic diagram of the Lr.ace-4A genomic fragment used for transgenic complementation verification is shown in Figure 4 PCR amplification was performed using primers p1300-Lr.ace-4AF1 (SEQ ID NO: 4) and p1300-Lr.ace-4AR1 (SEQ ID NO: 5), and p1300-Lr.ace-4AF2 (SEQ ID NO: 6) and p1300-Lr.ace-4AR2 (SEQ ID NO: 7), respectively. Then, the candidate gene was recombined into linearized pCAMBIA1300 according to the method of the In-Fusion HD Cloning Kit kit of Bio-Tech (Beijing) Co., Ltd. to obtain the p1300-Lr.ace-4A plasmid.

[0083] SEQ ID NO: 1:

[0084]

[0085] p1300-Lr.ace-4AF1: (SEQ ID NO: 4):

[0086] tgaccatgattacgaattcgagctcctcctaggggaaagaggccag.

[0087] p1300-Lr.ace-4AR1: (SEQ ID NO: 5):

[0088] cacaatttgtgacctcaggatggtagcagc.

[0089] p1300-Lr.ace-4AF2: (SEQ ID NO: 6):

[0090] tcctgaggtcacaaattgtgtgcatcagggct.

[0091] p1300-Lr.ace-4AR2: (SEQ ID NO: 7):

[0092] acgacggccagtgccaagcttctgtcgtgcacttctggagtg.

[0093] 2. Obtaining T0 generation transgenic plants

[0094] The plasmid of the complementary vector p1300-Lr.ace-4A was extracted and purified using a plasmid extraction kit (Tiangen Biochemical Technology Beijing Co., Ltd.), and then transferred into the Agrobacterium strain EHA105. The plasmid was then transferred into common wheat Fielder by Agrobacterium infection, and 60 Lr.ace-4A complementary T0 generation transgenic plants were obtained.

[0095] 3. Resistance identification of transgenic plants (families)

[0096] The obtained T0 generation complementary transgenic plants were positively identified by using markers, and the results showed that 58 of the obtained T0 transgenic plants were positive plants. T0 transgenic plants were planted in the greenhouse, and T1 generation seeds were harvested by self-pollination. We inoculated the T1 transgenic family with the leaf rust physiological race PHQS, and performed phenotypic identification 10 days after inoculation. The schematic diagram of the differential phenotypic results of the control variety Fielder, some T1 generation transgenic complementary plants, and the hexaploid introgression line "Yangmai 21-Lr.ace-4A" BC2F2 plants with the leaf rust physiological race PHQS after inoculation and culture for 10 days is shown in Figure 4As shown in middle b, the T1 transgenic plants showed high or medium resistance phenotypes (2, 3, 4, 5, ), and the disease resistance phenotype was consistent with the phenotype of the hexaploid introgression line of Lr.ace-4A "Yangmai 21-Lr.ace-4A (6)", while the control Fielder (1) showed high sensitivity. This indicates that after Lr.ace-4A was introduced into hexaploid wheat, the resistance was weakened to a certain extent.

[0097] At the same time, we carried out transgenic overexpression verification:

[0098] 1. Construction of overexpression vector p1300-Ubi-Lr.ace-4A.

[0099] We constructed a transgenic vector containing the Lr.ace-4A sequence as shown in SEQ ID NO: 2, and the CDS from the start codon ATG to the stop codon TGA is 3525 bp in length. An overexpression vector driven by the Ubi promoter from corn was constructed. PCR amplification was performed using primers p1300-Ubi-Lr.ace-4AF1 (SEQ ID NO: 8) and p1300-Ubi-Lr.ace-4AR1 (SEQ ID NO: 9), and then the candidate gene CDS was recombined into the linearized p1300-Ubi-CDS according to the In-Fusion HD Cloning Kit of Bio-Tech (Beijing) Co., Ltd. to obtain the p1300-Ubi-Lr.ace-4A plasmid.

[0100] p1300-Ubi-Lr.ace-4AF1: (SEQ ID NO: 8):

[0101] ggtgttatcttctgcaaagcttatggcggacctcgcggtg.

[0102] p1300-Ubi-Lr.ace-4AR1: (SEQ ID NO: 9):

[0103] attcccgggttacttgtacactattcctcctgctccaaa.

[0104] 2. Obtaining T0 generation transgenic plants

[0105] The plasmid of the overexpression vector p1300-Ubi-Lr.ace-4A was extracted and purified using a plasmid extraction kit (Tiangen Biochemical Technology Beijing Co., Ltd.), and then transferred into the Agrobacterium strain EHA105. The plasmid was then transferred into common wheat Fielder by Agrobacterium infection, and 30 p1300-Ubi-Lr.ace-4A overexpressing T0 generation transgenic plants were obtained.

[0106] 3. Resistance identification of transgenic plants (families)

[0107] First, the obtained T0 generation complementary transgenic plants were positively identified using markers. The results showed that 27 of the obtained T0 transgenic plants were positive plants. T0 transgenic plants were planted in the greenhouse and T1 generation seeds were harvested by self-pollination. We inoculated the T1 transgenic families with the leaf rust physiological race PHQS and performed phenotypic identification 10 days after inoculation. The schematic diagram of the differential phenotypic results of the control variety Fielder and some T1 generation transgenic overexpressing plants after inoculation with the leaf rust physiological race PHQS for 10 days is shown in the figure. Figure 4 As shown in Figure c, the T1 transgenic plants showed a high resistance phenotype (2, 3, 4, 5), while the control Fielder (1) showed a high sensitivity. The disease resistance of the overexpressing transgenic plants was stronger than that of the hexaploid introgression line of Lr.ace-4A, "Yangmai 21-Lr.ace-4A (6)", suggesting that the disease resistance phenotype of this gene is related to its transgenic expression level.

[0108] Example 5: Gene editing knockout verification of Lr.ace-4A candidate gene

[0109] To further identify this candidate gene, we then carried out gene editing knockout verification.

[0110] 1. Construction of gene editing CRISPR / Cas9 vector

[0111] According to the results of comprehensive analysis of conditions such as target specificity and off-target rate, the conserved sequence on the second exon of the Lr.ace-4A gene sequence, such as the nucleotide sequence shown in SEQ ID NO: 10, was selected as the gene editing gRNA target.

[0112] PCR amplification was performed using primers p1300-Cas9V2-Lr.ace-4AF1 (SEQ ID NO: 11) and p1300-Cas9V2-Lr.ace-4AR1 (SEQ ID NO: 12), respectively, and then the target site sequence was assembled into the linearized p1300-Cas9V2 vector according to the In-Fusion HD Cloning Kit of Bio-Rad Biotechnology (Beijing) Co., Ltd. to obtain the p1300-Cas9V2-Lr.ace-4A plasmid.

[0113] The plasmid of the gene editing vector p1300-Cas9V2-Lr.ace-4A was extracted and purified using a plasmid extraction kit (purchased from Tiangen Biochemical Technology Beijing Co., Ltd.), and then transferred into the Agrobacterium strain EHA105. It was then transferred into the resistant parent PI 192051 by Agrobacterium infection, and 33 T0 generation transgenic plants were obtained.

[0114] gRNA sequence: (SEQ ID NO: 10):

[0115] gccaatgagactattaaccg.

[0116] p1300-Cas9V2-Lr.ace-4AR1: (SEQ ID NO: 11):

[0117] gccaatgagactattaaccggtttcagagctatgctggaa.

[0118] p1300-Cas9V2-Lr.ace-4AR1: (SEQ ID NO: 12):

[0119] cggttaatagtctcattggctgctacctcaggatgcgcct.

[0120] 2. Phenotypic identification of gene-edited plants

[0121] Schematic diagram of the gene editing knockout verification results of the Lr.ace-4A candidate gene Figure 5 As shown. Among them, Figure 5 Figure a is a schematic diagram of the structure of the Lr.ace-4A gene. The results of sequencing and identification of gene-edited plants showed that 30 transgenic plants were positive materials, and a complete knockout family was found. This family inserted a base A, causing a frameshift mutation (sgRNA sequence and the sequencing results of the mutated fragments generated by gene editing are shown in the figure below). Figure 5 As shown in b, "WT" represents the wild type, SEQ ID NO: 13 is the sgRNA sequence of the wild type, and SEQ ID NO: 14 is the sequence of the T1-6-1, T1-6-2, and T1-6-3 edited mutants mutated at the sgRNA position), and T1 generation seeds were harvested by self-pollination. The T1 transgenic family was inoculated with the leaf rust physiological race PHQS, and the phenotype was identified 10 days after inoculation. Figure 5 As shown in c, the T1 generation fully edited transgenic plants have lost their resistance. Figure 5 T1-6-1, T1-6-2, and T1-6-3 in medium C showed high sensitivity, which was consistent with the phenotype of the susceptible control Rusty; Figure 5 The 1 in C is the resistant parent PI 192051, which shows near immunity.

[0122] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention successfully cloned and obtained the leaf rust resistance gene Lr.ace-4A, which is helpful for analyzing the disease resistance mechanism of wheat leaf rust resistance genes to pathogens; introducing the nucleic acid sequence encoding the Lr.ace-4A protein into wheat can improve the resistance of wheat to leaf rust, providing a reliable and effective leaf rust resistance source for wheat molecular breeding.

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A wheat leaf rust resistance protein Lr.ace-4A, characterized in that: The wheat leaf rust resistance protein Lr.ace-4A comprises: (a) a protein having the amino acid sequence shown in SEQ ID NO: 3; or (b) a protein having leaf rust resistance in which the amino acid sequence in (a) is substituted and / or deleted and / or one or more amino acids are added; or (c) A protein having 80% or more homology to the amino acid sequence defined in any one of (a) and (b) and having leaf rust resistance.

2. The wheat leaf rust resistance protein Lr.ace-4A according to claim 1, characterized in that The wheat leaf rust resistance protein Lr.ace-4A has more than 85%, preferably more than 90%, more preferably more than 95%, and further preferably more than 99% homology with the amino acid sequence defined in either (a) or (b) and has the same function.

3. A wheat leaf rust resistance gene, characterized in that: The wheat leaf rust resistance gene includes the Lr.ace-4A gene; The Lr.ace-4A gene includes: (a) a nucleotide sequence encoding the wheat leaf rust resistance protein Lr.ace-4A according to claim 1 or 2; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under stringent conditions and encodes the wheat leaf rust resistance protein Lr.ace-4A according to claim 1 or 2; or (c) having the nucleotide sequence shown in SEQ ID NO: 2; or (d) A gene having 70% or more homology with any one of the nucleotide sequences defined in (a) to (c) and encoding a protein conferring resistance to leaf rust.

4. The gene according to claim 3, characterized in that The wheat leaf rust resistance gene has 75% or more, preferably 85% or more, more preferably 95% or more, and further preferably 99% or more homology with any one of the nucleotide sequences specified in (a) to (c) and encodes a protein with leaf rust resistance.

5. An expression cassette, characterized in that The expression cassette comprises the wheat leaf rust resistance gene according to claim 3 or 4.

6. The expression cassette according to claim 5, characterized in that The expression cassette also includes a regulatory sequence for regulating the expression of the wheat leaf rust resistance gene, and the regulatory sequence includes a promoter; Preferably, the promoter comprises one or more of the following promoters: constitutive, enhanced, tissue-specific or inducible.

7. A recombinant vector, characterized in that: The recombinant vector comprises the wheat leaf rust resistance gene according to claim 3 or 4 or the expression cassette according to claim 5 or 6.

8. The recombinant vector according to claim 7, characterized in that The recombinant vector includes a translation control signal; Preferably, the translational control signal comprises an enhancer; Preferably, the enhancer comprises a translation enhancer and / or a transcription enhancer; Preferably, the translation control signal is derived from a natural sequence or an artificially synthesized sequence; Preferably, the recombinant vector comprises a plant expression vector; Preferably, the plant expression vector includes a binary vector for Agrobacterium transformation and a vector for gene gun bombardment; Preferably, the plant expression vector comprises pCAMBIA1300; Preferably, the recombinant vector comprises a reporter gene; Preferably, the reporter gene comprises a resistance gene or a gene expressing an enzyme or luminescent compound that produces a color change; Preferably, the resistance gene comprises an antibiotic resistance gene or a chemical agent resistance gene.

9. A non-plant host cell, characterized in that The host cell is transformed with the recombinant vector according to claim 7 or 8; Preferably, the host cell comprises Escherichia coli or Agrobacterium tumefaciens; Preferably, the Escherichia coli comprises DH5α; Preferably, the Agrobacterium tumefaciens comprises EHA105.

10. Use of the wheat leaf rust resistance protein Lr.ace-4A according to claim 1 or 2, or the wheat leaf rust resistance gene according to claim 3 or 4, or the expression cassette according to claim 5 or 6, or the recombinant vector according to claim 7 or 8, or the host cell according to claim 9 in any one or more of the following: regulating the leaf rust resistance of plants, or cultivating transgenic plants with enhanced or reduced leaf rust resistance, or breeding wheat leaf rust resistance; Preferably, regulating the leaf rust resistance of the plant comprises enhancing or reducing the leaf rust resistance of the plant; Preferably, the leaf rust is caused by a physiological species of Puccinia repens; Preferably, the physiological race of leaf rust is a toxic race of the Chinese prevalent leaf rust, and the toxic race of the Chinese prevalent leaf rust includes FHJL, PHQS, FHJR, THDB, PHJS, PHST, THSP or HCJR.

11. A method for preparing a transgenic plant, characterized in that: The method comprises: The wheat leaf rust resistance gene according to claim 3 or 4, or the expression cassette of claim 5 or 6, or the recombinant vector according to claim 7 or 8, Or the host cell described in claim 9 is introduced into a target plant to obtain the transgenic plant resistant to leaf rust.

12. The method according to claim 11, characterized in that The recombinant vector is introduced into the target plant by plant virus vector, gene gun or Agrobacterium infection; Preferably, the target plant is a dicotyledon or a monocotyledon; 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.

13. A method for increasing or decreasing plant resistance to leaf rust, characterized in that: The method comprises: Increasing or decreasing the activity and / or content of the wheat leaf rust resistance protein Lr.ace-4A according to claim 1 or 2 in the target plant, so that the resistance of the plant to leaf rust is enhanced or decreased.

14. The method according to claim 13, characterized in that The target plant is a dicotyledon or a monocotyledon; Preferably, the target plant is wheat; Preferably, the wheat is Fielder wheat.

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