Wheat leaf rust resistance protein Lr.ace-4A, resistance genes and applications

Abstract

The present invention provides a wheat leaf rust resistance protein Lr.ace-4A, a resistance gene, and an application. 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 wheat leaf rust resistance activity after substitution and / or deletion and / or addition of one or more amino acids in the amino acid sequence in (a); or (c) a protein having more than 80% homology with the amino acid sequence defined in either (a) or (b) and having the same function. The present invention can solve the problem of the lack of leaf rust resistance proteins in the prior art and is applicable to 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 an airborne fungal disease that primarily damages wheat leaves, disrupting photosynthesis and leading to yield losses of typically 5% to 15%, and in severe cases, over 40%. It is a major wheat disease and a serious threat to safe production. Utilizing disease-resistance genes to breed disease-resistant wheat varieties is the most economical and effective strategy for controlling this disease. However, wheat leaf rust fungi mutate rapidly, with new, highly virulent races constantly emerging, resulting in the loss of many resistance genes. In recent years, with climate change, wheat leaf rust has shown a trend of expansion and devastation, becoming increasingly severe. Therefore, controlling wheat leaf rust has become a crucial task in wheat production. However, the genetic base of major wheat varieties is very narrow, and effective rust-resistance genes are scarce. There is an urgent need to introduce new resistance genes to enrich the resistance pool and improve the resistance of major 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 on chromosome 4A near the centromere region. Lr.ace-4A shows near-immune resistance to leaf rust races from different countries, and will have significant application prospects in wheat leaf rust resistance breeding, but it has not yet been successfully cloned and bred.

[0004] Because the wheat genome is extremely large and over 80% of its sequences are repetitive, research on the isolation and cloning of wheat's functional genes lags far behind that of other crops, such as rice and corn. To date, approximately 83 wheat leaf rust resistance genes (Lr1-Lr83) have been officially named internationally. However, only about 11 have been successfully isolated and cloned. Most of these genes originate from common wheat, and no genes from tetraploid durum wheat have yet been cloned. Furthermore, although some leaf rust resistance genes have been cloned, many have lost their resistance due to bacterial evolution and gene overuse. Furthermore, some of the cloned leaf rust resistance genes originate from closely related species of wheat, creating linkage drag with wheat and limiting their potential for breeding. Given this situation, cloning the leaf rust resistance gene Lr.ace-4A, identifying novel resistance proteins, enriching wheat's resistance to leaf rust, and increasing the diversity of resistance genes are of great significance. 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 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 either (a) or (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 even more 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, which 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 stringent 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 a homology of more than 75%, preferably more than 85%, more preferably more than 95%, and even more preferably more than 99% with any one of the nucleotide sequences specified in (a) to (c) and encodes a gene that confers leaf rust resistance.

[0010] In order to achieve the above object, according to a third aspect of the present invention, an expression cassette is provided, which includes the above-mentioned 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 a 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 includes a translation control signal; preferably, the translation control signal includes an enhancer; preferably, the enhancer includes 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 includes 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 includes pCAMBIA1300; preferably, the recombinant vector includes a reporter gene; preferably, the reporter gene includes a resistance gene or a gene that expresses an enzyme that produces a color change or a luminescent compound; preferably, the resistance gene includes an antibiotic resistance gene or a chemical agent resistance gene.

[0014] To achieve the above objectives, according to the fifth aspect of the present invention, a non-plant host cell is provided, which 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 the use 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 object, according to the seventh aspect of the present invention, a method for preparing a transgenic plant is provided, which comprises: 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.

[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 a plant's resistance 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 plant's resistance 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] The application of the technical solution of the present invention and the use of the above-mentioned wheat leaf rust resistance protein will help to 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 accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying 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 mapping using an F2 segregating population constructed from the susceptible EMS mutant m1 and the wild type PI 192051; Figure 2 Figure 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 showing 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 indicate 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 induced by EMS in susceptible mutants.

[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 Middle a is a schematic diagram of the Lr.ace-4A genomic fragment used for transgenic complementation verification, including 2949 bp upstream of the start codon, the full gene length of 4652 bp (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 complemented 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 generation 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 generated by gene editing; Figure 5 Middle c shows 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 with reference to the embodiments.

[0029] Explanation of terms:

[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 races, the leaf rust resistance genes in wheat varieties are difficult to produce resistance to the new toxic races, resulting in the loss of wheat leaf rust resistance. Once the toxic races become popular, they will cause major harm and seriously threaten the safe production of wheat. Utilizing leaf rust resistance genes to cultivate disease-resistant wheat new varieties is the most economical and effective method to control this 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, isolation, cloning and functional verification of Lr.ace-4A, 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 the present 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 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 either (a) or (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 to replace and / or delete and / or add one or more amino acids. If the mutation occurs in the protein's active site, it may cause changes in the protein's key amino acid binding sites, affecting the protein's anti-wheat leaf rust activity, resulting in increased, decreased, or even inactive activity. If the mutation occurs in the protein's inactive site, it may affect the protein's folding structure, three-dimensional structure, and other properties, thereby affecting the protein's physicochemical properties and activity. Proteins with 80%, 85%, 90%, 95%, or 99% or more homology and identical functions are likely to have the same active site, active pocket, and active mechanism as the protein provided by sequence (a). These proteins are homologous proteins obtained through amino acid mutation. The description of "identical function" of homologous proteins in this application refers to anti-wheat leaf rust activity. Proteins with identical functions can be screened and obtained through commonly used experimental methods used by those skilled in the art.

[0036] "Homology" in this specification refers to similarity or identity, and particularly refers to identity. "Amino acid sequence homology" refers to the homology relative to the entire amino acid sequence. "Identity" between amino acid sequences refers to the sum of the ratios of amino acid residues of the same type in these amino acid sequences. "Similarity" between amino acids refers to the sum of the ratios of amino acid residues of the same type in these amino acid sequences and the ratios of amino acid residues with similar properties in their 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 refer to the fact that amino acids with similar properties will have similar effects when substituted with each other. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" 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] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

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

[0043] Those 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 is a protein that has greater than 85%, preferably greater than 90%, more preferably greater than 95%, and even more preferably greater than 99% homology to the amino acid sequence defined in either (a) or (b) and confers leaf rust resistance. Such homologous protein variants have similar or identical wheat leaf rust resistance activity to the protein set forth 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 stringent 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 hybridization under stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence in an amount that is detectably stronger than nonspecific hybridization. Stringent conditions can include, for example, low salt and / or high temperature conditions, such as provided by about 0.02 M to 0.1 M 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 to any one of the nucleotide sequences defined in (a) to (c) and encodes a protein conferring resistance to leaf rust.

[0050] The wheat leaf rust resistance gene described above can encode a protein with wheat leaf rust resistance activity. Based on the sequence (a), nucleotides are mutated. Under stringent conditions, hybridization with the DNA molecule defined in (a) is performed without frameshift mutations. If the mutation occurs in a nucleotide encoding the protein's active site, it may alter the key amino acid binding site of the encoded protein, affecting the wheat leaf rust resistance of the protein encoded by the gene, resulting in an increase, decrease, or even loss of activity. If the mutation occurs in a nucleotide encoding an inactive site of the protein, it may affect the folding mode and three-dimensional structure of the encoded protein, thereby affecting the protein's physicochemical properties and activity. Wheat leaf rust resistance genes with 70%, 75%, 85%, 95%, or 99% or more homology and encoding proteins with the same function are likely to have the same active site, active pocket, and active mechanism as the gene provided by the sequence (a), and are homologous genes obtained through 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 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 functionally verify the gene.

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

[0053] In a preferred embodiment, the expression cassette further comprises a regulatory sequence for regulating the expression of the wheat leaf rust resistance gene, the regulatory sequence including but 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 aforementioned expression cassette, i.e., a gene expression cassette, consists of regulatory sequences and the aforementioned wheat leaf rust resistance gene, and may also contain other nucleic acid fragments. The regulatory sequences influence the transcription, translation, and other expression of the aforementioned wheat leaf rust resistance gene. The regulatory sequences may be nucleic acid fragments such as promoters, enhancers, silencers, and regulatory protein attachment sites. The promoter may be a constitutive promoter, an enhancing promoter, a tissue-specific promoter, an inducible promoter, or a combination of 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, which 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 origin site, a multiple cloning site, and a translation control signal. Translation control signals derived from natural or synthetic sequences include enhancers, molecular chaperones, and other nucleotide sequences that can affect protein translation. The above-mentioned enhancers include translation enhancers and / or transcription enhancers, 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 that can be transformed into plants to express the target gene in the plant and produce the target protein to exert its effect; plant expression vectors include but are not limited to binary vectors for Agrobacterium transformation and vectors for gene gun bombardment, which can be introduced into plant cells through different transformation methods to improve transformation efficiency. The above-mentioned plant expression vectors include but are not limited to pCAMBIA1300 used in the examples.

[0058] The above-mentioned recombinant vectors may also include reporter genes. Preferably, reporter genes include, but are not limited to, resistance genes or genes that express enzymes or luminescent compounds that produce color changes. This allows for successful transformation and expression of the recombinant vector to be determined through various methods, such as resistance screening, color screening, and fluorescence screening. Resistance genes include, but are not limited to, antibiotic resistance genes or chemical resistance genes. Transformed cells can be efficiently screened for antibiotics, chemicals, and other drugs to determine successful transformation and expression. For transgenic safety considerations, it is also possible to omit any reporter genes and directly screen for transformation success based on phenotypic analysis.

[0059] In a fifth typical embodiment of the present 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 tumefaciens; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium tumefaciens includes EHA105.

[0060] The host cells are transformed with recombinant vectors and can carry out various functions such as recombinant vector replication, gene expression, and gene integration into chromosomes. The host cells can be various strains such as Escherichia coli and Agrobacterium tumefaciens. Among them, Escherichia coli can be the commonly used DH5α strain, and Agrobacterium tumefaciens can be the commonly used EHA105 strain.

[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 a plant, or cultivating a transgenic plant with enhanced or reduced leaf rust resistance, or breeding wheat leaf rust resistance; preferably, regulating the leaf rust resistance of a plant includes enhancing or reducing the leaf rust resistance of a plant; preferably, the leaf rust is a 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 applications utilize 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 various 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 means of a plant virus vector, a gene gun or Agrobacterium infection. The above method for preparing transgenic plants utilizes a variety of methods such as plant virus vectors, a gene gun or Agrobacterium infection to introduce wheat leaf rust resistance genes, expression cassettes, recombinant vectors or host cells into the target plant to obtain a transgenic plant with enhanced resistance to leaf rust. The target plant is a dicot or a monocot; preferably, the monocot can be wheat, and wheat varieties include but are not limited to Fielder wheat. The above method can affect the expression of wheat leaf rust resistance genes, protein activity or translation by, for example, 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 an 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 with reference to specific embodiments.

[0070] Example 1: Resistance spectrum analysis of 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 leaf rust physiological species 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 on 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. Specifically, the leaf rust phenotype was graded according to the grading standard of 0-4 (i.e., level 0 is immune; level 0 is nearly 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 was hybridized with its susceptible EMS mutant family m1 to obtain F1, which was then self-pollinated to obtain an F2 segregating population. Simultaneously, transcriptome sequencing was performed on PI 192051 and m1, 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 2In the figure, a is a schematic diagram of chromosome 4A. “Meriem et al. 2019” refers to the preliminary positioning of the Lr.ace-4A gene to the range between the molecular markers IWA232 (145.2 Mb) and IWA1793 (562.8 Mb; Svevo genome v1.0) on chromosome 4A by Meriem et al. in 2019, which is a relatively large 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 (As shown in (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 (as shown in (b), where "cM" refers to the unit of genetic distance "centimorgan" and "Marker" refers to molecular markers). 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 using MutISOseq

[0075] The schematic diagram of the results of MutISOseq rapid cloning of Lr.ace-4A candidate genes is shown in Figure 3As shown. The resistant parent PI192051 was subjected to chemical mutagenesis treatment with ethyl methane sulfonate (EMS) at an EMS concentration of 0.6%, and 2,000 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 leaf rust physiological race PHQS. 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) confirmed that these families were all 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 MutISOseq method was used to obtain the candidate gene of Lr.ace-4A. The specific operation was as follows: Under the condition of leaf rust inoculation, full-length transcriptome sequencing (ISO-seq) was performed on PI 192051. The raw sequencing data was polished and duplicates were removed to obtain high-quality full-length transcripts, which were used as reference sequences. At the same time, transcriptome sequencing was performed on the 7 EMS-susceptible mutant families obtained and aligned to the reference sequence. Analysis found that one transcript, transcript / 32998, had a typical EMS-induced 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 below. Figure 3 As shown in b, the arrows indicate the base mutations that occur in the susceptible mutant.

[0077] Transcript / 32998 was annotated and found to contain a typical CC-NBS-LRR gene. Subsequently, PCR amplification of the mutants confirmed that all seven EMS-susceptible mutants had base mutations in this transcript. These mutations were all located in the gene coding region and resulted in amino acid changes. A schematic diagram of the Lr.ace-4A gene structure and the base / amino acid changes in EMS-induced susceptible mutants is shown below. Figure 3 As shown in Figure 3c, transcript / 32998 corresponds to the homologous gene TRITD4Av1G167080 in the Svevo reference genome. The protein encoded by this gene differs by only three amino acids between Svevo and PI 192051. Based on these experiments, it is speculated that this candidate gene is essential for conferring 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 of the candidate gene was performed:

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

[0081] To obtain the introns, upstream, and downstream sequences of the candidate gene, resequencing data and transcriptome data from the disease-resistant parent, PI 192051, were used to construct a genomic sequence containing the candidate gene. The sequence accuracy was verified by PCR amplification and sequencing. Based on this genomic and transcript sequence, combined with BLASTN / BLASTX analysis using the NCBI database, the gene structure was determined, resulting in the nucleotide sequence shown in SEQ ID NO:2 and the amino acid sequence shown in SEQ ID NO:3, 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 Based on the above information, in order to verify the complementation of transgenes, we constructed a transgenic vector to amplify the genomic fragment containing Lr.ace-4A 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 (the 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), as well as primers p1300-Lr.ace-4AF2 (SEQ ID NO: 6) and p1300-Lr.ace-4AR2 (SEQ ID NO: 7), respectively. The candidate gene was then recombined into linearized pCAMBIA1300 using the In-Fusion HD Cloning Kit from 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 complementation vector p1300-Lr.ace-4A was extracted and purified using a plasmid extraction kit (Tiangen Biochemical Technology Beijing Co., Ltd.), and then transformed into Agrobacterium strain EHA105. The plasmid was then transformed into common wheat Fielder by Agrobacterium infection, and 60 Lr.ace-4A complemented 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 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 after inoculation with the leaf rust physiological race PHQS for 10 days is shown in the figure below. Figure 4As shown in middle b, the T1 transgenic plants showed high or moderate resistance phenotypes (2, 3, 4, 5). The disease resistance phenotype was consistent with that of the hexaploid introgression line of Lr.ace-4A, "Yangmai 21-Lr.ace-4A (6)", while the control, Fielder (1), showed high susceptibility. 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 (SEQ ID NO: 2). The CDS, from the start codon ATG to the stop codon TGA, is 3525 bp long. An overexpression vector driven by the maize Ubi promoter 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), respectively. The candidate gene CDS was then recombined into the linearized p1300-Ubi-CDS using the In-Fusion HD Cloning Kit from Bio-Ray Biotechnology (Beijing) Co., Ltd., generating 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 transformed into Agrobacterium strain EHA105. The plasmid was then transformed into common wheat Fielder by Agrobacterium infection, and 30 T0 generation transgenic plants overexpressing p1300-Ubi-Lr.ace-4A 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. The T0 transgenic plants were grown in the greenhouse and self-pollinated to harvest T1 generation seeds. We inoculated the T1 transgenic families with the leaf rust physiological race PHQS and performed phenotypic analysis 10 days after inoculation. The differential phenotypic results between the control variety Fielder and some T1 generation transgenic overexpressing plants after inoculation with the leaf rust physiological race PHQS are shown in the figure below. 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 performed gene editing knockout validation.

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

[0111] Based on the results of a comprehensive analysis of conditions such as target specificity and off-target rate, a 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. Then, the target site sequence was incorporated into the linearized p1300-Cas9V2 vector according to the In-Fusion HD Cloning Kit of Bio-Ray Biotechnology (Beijing) Co., Ltd. to obtain the p1300-Cas9V2-Lr.ace-4A plasmid.

[0113] A plasmid extraction kit (purchased from Tiangen Biochemical Technology Beijing Co., Ltd.) was used to extract and purify the plasmid of the gene editing vector p1300-Cas9V2-Lr.ace-4A, which was then transformed into the Agrobacterium strain EHA105. The transgenic plant was then transformed into the resistant parent PI 192051 through 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. Sequencing of gene-edited plants revealed that 30 transgenic plants were positive, and a complete knockout lineage was found. This lineage had a single base A inserted, resulting in a frameshift mutation (the sgRNA sequence and the sequencing results of the mutant fragments generated by gene editing are shown in the figure). Figure 5 As shown in b, where "WT" represents the wild type, SEQ ID NO: 13 is the wild type sgRNA sequence, and SEQ ID NO: 14 is the sequence of the T1-6-1, T1-6-2, and T1-6-3 edited mutants with mutations at the sgRNA position), T1 generation seeds were harvested by self-pollination. The T1 transgenic family was inoculated with the leaf rust physiological race PHQS, and the phenotypic identification was performed 10 days after inoculation. Figure 5 As shown in middle 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 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 showed near immunity.

[0122] From the above description, it can be seen that the above-mentioned 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 against 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 source of leaf rust resistance for wheat molecular breeding.

[0123] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. 1) Wheat leaf rust resistance protein Lr.ace-4A, or 2) A gene encoding the wheat leaf rust resistance protein Lr.ace-4A in 1), or 3) an expression cassette containing the gene in 2) above, or 4) A recombinant vector containing the gene in 2) or the expression cassette in 3) above, or 5) Host cells containing the recombinant vector described in 4) Application in any one or more of the following: regulating wheat leaf rust resistance, or cultivating transgenic wheat with enhanced or reduced leaf rust resistance, or breeding wheat for leaf rust resistance; The wheat leaf rust resistance protein Lr.ace-4A is a protein consisting of the amino acid sequence shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that The regulating the leaf rust resistance of a plant includes increasing or decreasing the leaf rust resistance of a plant.

3. The use according to claim 1, characterized in that The leaf rust disease is caused by a physiological species of Puccinia revoluta.

4. The use according to claim 3, characterized in that The leaf rust physiological race is a toxic race of the leaf rust fungus prevalent in China, and the toxic race of the leaf rust fungus prevalent in China includes FHJL, PHQS, FHJR, THDB, PHJS, PHST, THSP or HCJR.

5. The use according to claim 1, characterized in that The gene encoding the wheat leaf rust resistance protein Lr.ace-4A has a nucleotide sequence as shown in SEQ ID NO:

2.

6. The use according to claim 1, characterized in that The expression cassette further comprises a regulatory sequence for regulating the expression of the wheat leaf rust resistance gene, wherein the regulatory sequence comprises a promoter.

7. The use according to claim 6, characterized in that The promoter includes one or more of the following promoters: constitutive, enhanced, tissue-specific or inducible.

8. The use according to claim 1, characterized in that The recombinant vector includes translational control signals.

9. The use according to claim 8, characterized in that The translational control signals include enhancers.

10. The use according to claim 9, characterized in that The enhancers include translation enhancers and / or transcription enhancers.

11. The use according to claim 8, characterized in that The translation control signal is derived from a natural sequence or an artificially synthesized sequence.

12. The use according to claim 8, characterized in that The recombinant vector includes a plant expression vector.

13. The use according to claim 12, characterized in that The plant expression vectors include binary vectors for Agrobacterium transformation and vectors for gene gun bombardment.

14. The use according to claim 13, characterized in that The plant expression vector includes pCAMBIA1300.

15. The use according to claim 1, characterized in that The recombinant vector includes a reporter gene.

16. The use according to claim 15, characterized in that The reporter gene includes a resistance gene or a gene that expresses an enzyme that produces a color change or a luminescent compound.

17. The use according to claim 16, characterized in that The resistance gene includes an antibiotic resistance gene or a chemical agent resistance gene.

18. The use according to claim 1, characterized in that The host cell includes Escherichia coli or Agrobacterium tumefaciens.

19. The use according to claim 18, characterized in that The Escherichia coli includes DH5α.

20. The use according to claim 18, characterized in that The Agrobacterium tumefaciens includes EHA105.

21. A method for preparing a transgenic plant, characterized in that: The method comprises: Gene encoding wheat leaf rust resistance protein Lr.ace-4A, or the expression cassette in any one of claims 1, 6 or 7, Or the recombinant vector in the application of any one of claims 1 or 8 to 17, or the host cell used in any one of claims 1 or 18 to 20 is introduced into a target plant to obtain the transgenic plant resistant to leaf rust; The wheat leaf rust resistance protein Lr.ace-4A is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; The target plant is wheat.

22. The method according to claim 21, characterized in that The recombinant vector is introduced into the target plant through plant virus vector, gene gun or Agrobacterium infection.

23. The method according to claim 21, characterized in that The wheat is Fielder wheat.

24. The method according to claim 21, characterized in that The wheat leaf rust resistance gene is driven by a constitutive promoter.

25. A method for increasing or decreasing resistance of a plant 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 in a target plant, so that the plant's resistance to leaf rust is enhanced or decreased; The wheat leaf rust resistance protein Lr.ace-4A is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3; The target plant is wheat.

26. The method according to claim 25, characterized in that The wheat is Fielder wheat.

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