Wheat stem rust resistance protein, resistance gene and its application

Abstract

The present invention provides a wheat stem rust resistance protein, a resistance gene and an application thereof. The wheat stem rust resistance protein is any one of the following (a)-(c): (a) a protein having an amino acid sequence as shown in SEQ ID NO: 8; or (b) a protein having resistance to wheat stem rust 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 the same function. The present invention can solve the problem of the small number of such 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 stem rust resistance protein, a resistance gene and applications thereof. Background Art

[0002] Wheat is one of the most important food crops in the world, but with the growth of population, the yield of staple crops such as wheat is also in urgent need of improvement. Finding disease-resistant genes and breeding disease-resistant wheat varieties to reduce yield losses are still effective measures to ensure wheat yield and production safety. Wheat stem rust is a type of airborne fungal disease caused by infection with wheat stem rust (Puccinia graminis f. sp.tritici, Pgt). The continuous mutation and evolution of wheat stem rust has led to the emergence of new highly toxic races, such as Ug99 and its variants, TRTTF, TTTTF and other highly toxic physiological races, which have caused the disease resistance genes widely used in breeding to lose resistance. In recent years, stem rust has often broken out in many parts of the world, causing serious wheat yield losses. Therefore, wheat stem rust resistance breeding is still one of the important tasks of wheat breeding.

[0003] Since the highly virulent wheat stem rust race Ug99 was identified, the search for genes that are resistant to Ug99 and its variants has been the focus of global wheat stem rust resistance research. The wheat stem rust resistance gene Sr8155B1 is derived from the tetraploid durum wheat (Triticum turgidum ssp. durum) material 8155-B1. Nirmala et al. reported that although 8155-B1 was susceptible to the original strain of Ug99 TTKSK, 8155-B1 showed near-immune resistance to multiple variants of Ug99, including TTKST, TTKTT, TTTSK and TTKSF+. In addition, Wang et al. found in a 2023 study that the Sr8155B1 gene showed near-immune resistance to six physiological races of stem rust prevalent in China.

[0004] At present, there are 67 stem rust genes officially named internationally (Sr1-Sr67), of which 16 genes have been cloned, but only some of them have been confirmed to be resistant to Ug99 and its variants. Therefore, cloning the stem rust resistance gene Sr8155B1 and determining the corresponding protein sequence can enrich the stem rust resistance gene resources, provide theoretical and material basis for wheat stem rust resistance breeding, and ensure global wheat safety production. Summary of the invention

[0005] The main purpose of the present invention is to provide a wheat stem rust resistance protein, a resistance gene and applications thereof, so as to solve the problem of the shortage of such resistance proteins in the prior art.

[0006] To achieve the above-mentioned object, according to the first aspect of the present invention, there is provided a wheat stem rust resistance protein, which is any one of the following (a)-(c): (a) a protein having an amino acid sequence as shown in SEQ ID NO: 8; or (b) a protein having resistance to wheat stem rust activity 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 the same function.

[0007] Furthermore, a protein having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in any one of (a) and (b) and having the same function.

[0008] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a wheat stem rust resistance gene is provided, which is any one of the following (a)-(d): (a) a nucleotide sequence encoding the above-mentioned wheat stem rust resistance protein; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under stringent conditions and encodes the above-mentioned wheat stem rust resistance protein; or (c) a nucleotide sequence shown in SEQ ID NO: 7; 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 the same function.

[0009] Furthermore, a gene encoding a protein having 75% or more, preferably 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with any of the nucleotide sequences specified in (a) to (c) and having the same function.

[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 a regulatory sequence and the wheat stem rust resistance gene.

[0011] Furthermore, the regulatory sequence includes a promoter; preferably, the promoter includes one or more of the following promoters: constitutive, enhancing, tissue-specific and inducible.

[0012] In order to achieve the above object, according to the fourth aspect of the present invention, a recombinant vector is provided, wherein the recombinant vector comprises the wheat stem rust resistance gene or the 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 transformed by Agrobacterium or a vector bombarded by a gene gun; 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] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, a host cell is provided, wherein the above-mentioned host cell contains the above-mentioned wheat stem rust resistance gene or the above-mentioned expression cassette, or is transformed with the above-mentioned recombinant vector; preferably, the host cell includes a non-plant host cell; 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 above-mentioned wheat stem rust resistance protein, or the above-mentioned wheat stem rust resistance gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned host cell for use in regulating plant resistance to stem rust, enhancing or reducing plant resistance to stem rust, or cultivating transgenic plants with enhanced or reduced resistance to stem rust, or in wheat stem rust resistance breeding.

[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 stem 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 stem 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 stem 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 breeding method for increasing or decreasing the resistance of a plant to stem rust is provided, the method comprising: increasing or decreasing the activity or content of the above-mentioned wheat stem rust resistance protein in the target plant, so that the resistance of the plant to stem rust is enhanced or decreased.

[0019] Further, the target plant is a dicotyledon or a monocotyledon; preferably, the target plant is wheat; preferably, the wheat is Fielder wheat; preferably, the stem rust is caused by a physiological race of stem rust fungus; preferably, the physiological race of stem rust fungus includes 34MKGQM or 34C3RKGQM.

[0020] Beneficial technical effects: By applying the technical scheme of the present invention and using the above-mentioned wheat stem rust resistance protein, it is possible to enhance the resistance of plants to stem rust, and promote in-depth research on the disease resistance mechanism of how resistance proteins resist pathogens, thereby providing a reliable and effective stem rust resistance resource 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 stem rust resistance gene in Examples 1 and 2 according to the present invention is shown. Sr8155B1 Positional cloning of Figure 1 Figure a is a schematic diagram of chromosome 6A of wheat; Figure 1 b is Sr8155B1 Genetic linkage map; Figure 1 In the middle, c is the physical map of the Svevo reference genome corresponding to the localization interval; Figure 1 In the above equation, d means Sr8155B1 The tetraploid hybrid offspring F 3 Physical map of the localized interval in family PKU81.

[0023] Figure 2 The results of the EMS susceptible mutant and Sr8155B1 candidate gene validation experiments in Example 2 of the present invention are shown. Figure 2 Figure a shows the phenotypic identification results of the mutant family. Figure 2 Figure b shows the peak diagram of base mutation sequencing. Figure 2 Middle c shows a schematic diagram of protein translation termination caused by base mutation in the candidate gene.

[0024] Figure 3 The results of candidate gene knockout in Example 3 of the present invention are shown. Figure 3 Figure a shows a schematic diagram of the gRNA target site for gene editing. Figure 3 Middle b shows the sequencing peak diagram of the homozygous editing knockout plant. Figure 3 Middle c shows the results of identifying the disease resistance phenotype of the homozygous edited knockout plants.

[0025] Figure 4The experimental results of transgenic complementation verification of the Sr8155B1 candidate gene in Example 4 of the present invention are shown. Figure 4 Figure a shows a schematic diagram of amplifying the 8155-B1 genome fragment for the construction of a transgenic vector. Figure 4 b shows T 1 The results of the phenotypic identification of the first generation transgenic family against 34MKGQM. Figure 4 Figure c shows T 1 Figure 3. The results of phenotypic identification of the first-generation transgenic lines resistant to 34C3RKGQM. DETAILED DESCRIPTION

[0026] 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.

[0027] Terminology explanation:

[0028] 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.

[0029] As mentioned in the background technology, stem rust subspecies Ug99 and its variants can overcome most of the reported stem rust resistance genes, posing a great threat to the safe production of wheat. Finding new stem rust resistance genes and carrying out disease-resistant breeding is the most economical and effective solution. Currently, only part of the cloned genes are resistant to Ug99 and its variants, so there are fewer genes to choose from during breeding. Therefore, in this application, the inventors conducted in-depth research on the stem rust resistance gene Sr8155B1 against Ug99 variants, completed the fine positioning, cloning and functional verification code of Sr8155B1, and found that Sr8155B1 encodes a typical CC-NBS-LRR disease resistance protein, and the encoded disease resistance protein has anti-wheat stem rust activity. On this basis, a series of protection schemes of this application are proposed.

[0030] In a first typical embodiment of the present application, a wheat stem rust resistance protein is provided, which is any one of the following (a)-(c): (a) a protein having an amino acid sequence as shown in SEQ ID NO: 8; or (b) a protein having resistance to wheat stem rust activity 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 the same function.

[0031] In a preferred embodiment, the protein has 85% or more, preferably 90% or more, more preferably 95% or more, and further preferably 99% or more homology with the amino acid sequence defined in any one of (a) and (b) and has the same function.

[0032] The wheat stem rust resistance protein has the activity of resisting wheat stem rust. Based on the sequence in (a) (SEQ ID NO: 8), the protein is mutated, replaced and / or deleted and / or added with one or several 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 stem rust, causing its activity to increase or decrease or even lose its 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 have a high probability of having the same active site, active pocket, active mechanism, etc. as the protein provided by the sequence (a), and are homologous proteins obtained by amino acid mutation. The description of "same function" of homologous proteins in this application refers to the activity of resisting wheat stem rust. Proteins with the same function can be screened and obtained by experimental means commonly used by those skilled in the art.

[0033] "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) and FASTA.

[0034] 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).

[0035] 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:

[0036] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0037] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0038] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0039] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0040] 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.

[0041] In a second typical embodiment of the present application, a wheat stem rust resistance gene is provided, which is any one of the following (a)-(d): (a) a nucleotide sequence encoding the above-mentioned wheat stem rust resistance protein; or (b) a nucleotide sequence that hybridizes with the DNA molecule defined in (a) under stringent conditions and encodes the above-mentioned wheat stem rust resistance protein; or (c) a nucleotide sequence shown in SEQ ID NO: 7; 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 the same function.

[0042] 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.

[0043] 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 of the nucleotide sequences defined in (a) to (c) and encodes a protein having the same function.

[0044] The wheat stem rust resistance gene is a protein that can encode for resistance to wheat stem rust. The nucleotides are mutated based on the (c1) or (d1) sequence, and hybridize with the DNA molecule defined by (c1) or (d1) under strict conditions without frameshift mutation. 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 stem rust activity of the protein encoded by the gene, resulting in an increase or decrease in its activity or even loss of 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 stem 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 (c1) or (d1) sequence, and are homologous genes obtained by nucleotide mutation.

[0045] The resistant parent of the present invention is 8155-B1. Studies have shown that the material exhibits high resistance to Ug99 variants other than the original strain of Ug99 and domestic stem rust species. The present application first uses a large isolated population for fine positioning, combines transcriptome sequencing results and third-generation PacBio Revio HiFi long-read sequencing to successfully clone the genome sequence of the Sr8155B1 gene (SEQ ID NO: 6), and combines transcriptome and NCBI database BLASTN / BLASTX analysis to obtain a cDNA sequence (SEQ ID NO: 7), and uses EMS mutants, transgenic complementation experiments, and gene editing experiments to perform functional verification.

[0046] In a third typical embodiment of the present application, an expression cassette is provided, wherein the expression cassette comprises a regulatory sequence and the wheat stem rust resistance gene.

[0047] In a preferred embodiment, the regulatory sequence includes a promoter; preferably, the promoter includes one or more of the following promoters: constitutive, enhancing, tissue-specific and inducible.

[0048] The above expression cassette, i.e., gene expression cassette, is composed of a regulatory sequence, the above wheat stem 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 stem 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.

[0049] In a fourth typical embodiment of the present application, a recombinant vector is provided, comprising the above-mentioned wheat stem rust resistance gene or the above-mentioned expression cassette.

[0050] 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 transformed by Agrobacterium or a vector bombarded by a gene gun; 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.

[0051] The above-mentioned recombinant vector, comprising the wheat stem 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 a target gene in the plant, produce a target protein, and thus play a role; the plant expression vector includes but is not limited to a binary vector transformed by Agrobacterium and a vector bombarded by a gene gun, 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. In the embodiment of the present application, pCAMBIA1300 is also referred to as p1300.

[0052] 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.

[0053] In a fifth typical embodiment of the present application, a host cell is provided, wherein the host cell contains the above-mentioned wheat straw rust resistance gene or the above-mentioned expression cassette, or is transformed with the above-mentioned recombinant vector; preferably, the host cell includes a non-plant host cell; preferably, the host cell includes Escherichia coli or Agrobacterium tumefaciens; preferably, Escherichia coli includes DH5α; preferably, Agrobacterium tumefaciens includes EHA105.

[0054] 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. Host cells include but are not limited to various strains such as Escherichia coli and Agrobacterium tumefaciens, among which Escherichia coli includes but is not limited to the commonly used DH5α, and Agrobacterium tumefaciens includes but is not limited to the commonly used EHA105.

[0055] In the sixth typical embodiment of the present application, there is provided the use of the above-mentioned wheat stem rust resistance protein, or the above-mentioned wheat stem rust resistance gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned host cell in regulating plant resistance to stem rust, enhancing or reducing plant resistance to stem rust, or cultivating transgenic plants with enhanced or reduced resistance to stem rust, or in wheat stem rust resistance breeding.

[0056] The above-mentioned application utilizes wheat stem rust resistance proteins, genes, expression cassettes, recombinant vectors or host cells to regulate the plant's resistance to stem rust through resistance proteins, proteins encoded by resistance genes, etc.; enhance or reduce the plant's resistance to stem 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 stem rust.

[0057] In the seventh typical embodiment of the present application, a method for preparing a transgenic plant is provided, the method comprising: introducing the wheat stem rust resistance gene, or the expression cassette, or the recombinant vector, or the host cell into a target plant to obtain a transgenic plant resistant to stem rust.

[0058] 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 stem rust resistance gene is driven by a constitutive promoter.

[0059] 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 a variety of methods such as plant virus vector, gene gun or Agrobacterium infection to introduce wheat stem rust resistance genes, expression cassettes, recombinant vectors or host cells into the target plant to obtain transgenic plants with enhanced resistance to stem 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 stem 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 the resistance gene, thereby obtaining transgenic plants with reduced or enhanced resistance to stem rust.

[0060] In an eighth typical embodiment of the present application, a breeding method for increasing or decreasing a plant's resistance to stem rust is provided, the method comprising: increasing or decreasing the activity or content of the wheat stem rust resistance protein in the target plant, so that the plant's resistance to stem rust is enhanced or decreased.

[0061] The above method can increase or decrease the activity or content of wheat stem 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 stem rust.

[0062] 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; preferably, the stem rust is caused by a physiological race of stem rust fungus; preferably, the physiological race of stem rust fungus includes 34MKGQM or 34C3RKGQM.

[0063] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0064] Example 1: Fine mapping of the stem rust resistance gene Sr8155B1

[0065] In previous studies, our laboratory used molecular markers to construct F sequences in the disease-resistant parent 8155-B1 and the susceptible parent Rusty. 2In the population, Sr8155B1 was located within 0.06 cM between the molecular markers pku2772 and pku43365 (primer sequences see Wang J, LiH, Shen T, et al. High-resolution genetic mapping and identification of candidate genes for the wheat stem rust resistance gene Sr8155B1[J]. CropJournal (2095-5421), 2023, 11(6). DOI:10.1016 / j.cj.2023.09.006.), corresponding to the physical position of Svevo between 2.77-4.34 Mb ( Figure 1 To further fine-tune the location of Sr8155B1, we continued to construct F sequences in the resistant parent 8155-B1 and the susceptible parent Rusty. 2 In the population, 4600 strains of F 2 We screened individual strains and obtained 8 recombinants. We used 9 molecular markers in this interval to perform genetic linkage analysis on these 8 recombinants, and ultimately located the gene in the 0.03 cM interval between molecular markers pku2772 and pku6A3, and co-segregated with molecular marker pku6A2 ( Figure 1 b). The localization interval corresponds to a 295 kb physical interval on the Svevo reference genome. There are 6 high-confidence genes in this interval, including a typical NLR gene (CNL1) and a kinase gene (PK) related to disease resistance ( Figure 1 (c).

[0066] In order to obtain the physical map of the Sr8155B1 candidate interval in the disease-resistant parent 8155-B1, we performed a tetraploid hybridization of the F 3 PacBio HiFi long-read sequencing was performed on family PKU81, and a total of 216 Gb of raw reads were obtained. The data were assembled and 19,847 contigs were obtained, one of which, ptg000390l (https: / ngdc.cncb.ac.cn / bioproject / , accession number: PRJCA032807), contained the Sr8155B1 candidate interval. Sequence annotation showed that the PKU81 physical interval was highly colinear with Svevo, and the interval also contained only 6 high-confidence genes ( Figure 1d). Of these six high-confidence genes, only two were expressed in the leaves of the inoculated resistant parent 8155-B1. In addition, except for CNL1, the sequences of the other five candidate genes were 100% consistent with the Svevo reference genome, and Svevo has been shown to not contain Sr8155B1. Therefore, CNL1 is the most likely candidate gene.

[0067] Example 2: Validation of susceptible EMS mutants and Sr8155B1 candidate gene

[0068] The resistant parent 8155-B1 was treated with ethyl methane sulfonate (EMS) at a concentration of 0.45%, resulting in 480 independent M 2 Mutant families. Using an all-weather plant growth chamber, M 2 The mutant families were phenotypically identified. Twenty seedlings from each family were inoculated with stem rust race 34MKGQM. Only one family had susceptible plants. Figure 2 As shown in a, R indicates resistance and S indicates susceptibility. Subsequently, susceptible plants were transplanted and M 3 Seed, M 3 Single plant ( Figure 2 Phenotypic characterization of m27 in a confirms that this M 3 The family has a susceptible phenotype.

[0069] Using PCR amplification and sequencing, we detected that mutant m27 had a CT base mutation in the candidate gene CNL1 ( Figure 2 b), resulting in a translation termination mutation ( Figure 2 Similarly, we performed PCR amplification and sequencing on the gene regions of the other five candidate genes in the physical interval of Sr8155B1, but no base changes were detected. In summary, we determined CNL1 as a candidate gene.

[0070] Implementation case 3: Candidate gene gene editing knockout verification

[0071] In order to further confirm the function of the candidate gene, gene editing and knockout verification were performed on the resistant parent 8155-B1.

[0072] 1. Construction of gene editing CRISPR / Cas9 vector

[0073] Based on a comprehensive analysis of target specificity and off-target rate, the conserved sequence in the second exon of the CNL1 gene sequence, such as the nucleotide sequence shown in SEQ ID NO: 1, was selected as the gRNA target for gene editing ( Figure 3 (a).

[0074] PCR amplification was performed using primers 8155KO F1 (SEQ ID NO: 2), 8155KO R1 (SEQ ID NO: 3), 8155KO F2 (SEQ ID NO: 4), and 8155KO R2 (SEQ ID NO: 5), respectively. 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-Sr8155B1 plasmid.

[0075] The plasmid of the gene editing vector p1300-Cas9V2-Sr8155B1 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. The plasmid was then transferred into the resistant parent 8155-B1 by Agrobacterium infection, and 143 strains of T were obtained. 0 Generation of transgenic plants.

[0076] gRNA sequence: (SEQ ID NO: 1):

[0077] GAGAAGGTTACGTGACGCCG.

[0078] 8155KO F1: (SEQ ID NO: 2):

[0079] GATTACGAATTCAGGCCTAAAAAAAGCACCGACTCGGTGC.

[0080] 8155KO R1: (SEQ ID NO: 3):

[0081] GAGAAGGTTACGTGACGCCGGTTTCAGAGCTATGCTGGAA.

[0082] 8155KO F2: (SEQ ID NO: 4):

[0083] CGGCGTCACGTAACCTTTCTCTGCTACCTCAGGATGCGCCT.

[0084] 8155KO R2: (SEQ ID NO: 5):

[0085] TTGATTGACAACAGGCCTCATGAATCCAAACCACACGGAGT.

[0086] 2. Resistance identification of gene-edited plants (families)

[0087] First, the obtained T 0 The 143 gene-edited plants were identified by PCR amplification and sequencing. The results showed that 56 of the 143 gene-edited plants were edited at the target site, of which three were homozygous knockout plants, with deletions of "CGTC", "G" and "C" respectively. The sequencing peaks are shown in the figure below. Figure 3 As shown in b. “Reverse” means that when sequencing the PCR product, the sequence is the reverse complementary sequence.

[0088] We inoculated these three homozygous gene-edited knockout plants with stem rust race 34MKGQM and performed phenotypic identification 14 days after inoculation. Figure 3 As shown in c, the gene-edited plants (corresponding to Figure 3 2, 3 and 4) with Rusty (corresponding to Figure 3 The control of c5) showed high sensitivity, while the control 8155-B1 (corresponding to Figure 3 1) in c) shows near immune resistance.

[0089] Example 4: Transgenic complementation verification of Sr8155B1 candidate gene

[0090] To determine whether the candidate gene could provide stem rust resistance, transgenic complementation validation of the candidate gene was performed.

[0091] 1. Construction of complementary vector p1300-Sr8155B1

[0092] Using Pac-bio Revio sequencing data, a contig containing the candidate segment was assembled, and the accuracy of the sequence was verified by PCR amplification and sequencing. Based on the genome 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: 7 and the amino acid sequence shown in SEQ ID NO: 8 were obtained, which are the wheat stem rust resistance gene (CDS) and wheat stem rust resistance protein.

[0093] Based on the above information, in order to verify transgenic complementation, we constructed a transgenic vector amplified with a genomic fragment containing Sr8155B1 as shown in SEQ ID NO: 6, including 2188 bp upstream of the gene start codon, the full length of the gene (from ATG to TGA, 4448 bp) and 3332 bp downstream of the gene, a total of 9968 bp of genomic sequence ( Figure 4PCR amplification was performed using primers 8155VF1 (SEQ ID NO: 9) and 8155VR1 (SEQ ID NO: 10), and 8155VF2 (SEQ ID NO: 11) and 8155VR2 (SEQ ID NO: 12), respectively, and then the candidate gene was recombined into linearized pCAMBIA1300 according to the In-Fusion HD Cloning Kit kit of Bio-Ray Biotechnology (Beijing) Co., Ltd. to obtain p1300-Sr8155B1 plasmid.

[0094] SEQ ID NO: 6:

[0095]

[0096] SEQ ID NO:7:

[0097]

[0098] SEQ ID NO:8:

[0099]

[0100] 8155VF1: (SEQ ID NO: 9): acgacggccagtgccaagcttatctccaggagggtccg.

[0101] 8155VR1: (SEQ ID NO: 10): tcctcaataactagcacacgcaatacgttaaagc.

[0102] 8155VF2: (SEQ ID NO: 11):cgtgtgctagttattgaggattgtgttttctcgg.

[0103] 8155VR2: (SEQ ID NO: 12): catgattacgaattcgagctcgcgctctgtgtacgttgttctc.

[0104] 2. T 0 Obtaining transgenic plants

[0105] The plasmid of the complementary vector p1300-Sr8155B1 was extracted and purified using a plasmid extraction kit (Tiangen Biochemical Technology Beijing Co., Ltd.), and then transformed into the Agrobacterium strain EHA105. The Agrobacterium infection method was then used to transform the plasmid into common wheat Fielder, and 8 Sr8155B1 complementary T strains were obtained. 0 Generation of transgenic plants.

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

[0107] T obtained by using the marker pair 0 The results showed that the 8 transgenic plants obtained were T 0 All transgenic plants are positive plants. 0 Transgenic plants were obtained by self-pollination. 1 We have T 1 The transgenic lines were inoculated with stem rust races 34MKGQM and 34C3RKGQM, and phenotypes were identified 14 days after inoculation. 1 The differential phenotypes of the transgenic complementary plants after inoculation with stem rust physiological races 34MKGQM and 34C3RKGQM for 14 days were as follows: Figure 4 As shown in b and c, T 1 The transgenic lines showed near-immune or even immune phenotypes (corresponding to Figure 4 3, 4, 5, 6 in b and c), the disease resistance phenotype is similar to that of the wild-type parent 8155-B1 (corresponding to Figure 4b and c) are consistent, while the control Fielder (corresponding to Figure 4 2) in b and c showed high susceptibility, indicating that Sr8155B1 still maintained good resistance after being introduced into hexaploid wheat.

[0108] From the above description, it can be concluded that the above-mentioned embodiments of the present invention achieve the following technical effects: the present invention successfully cloned and obtained the stem rust resistance gene Sr8155B1, which is helpful for analyzing the disease resistance mechanism of wheat stem rust resistance gene to pathogens; introducing the nucleotide sequence encoding the Sr8155B1 protein into wheat can improve the resistance of wheat to stem rust, provide a reliable and effective source of stem rust resistance for wheat molecular breeding, and have great application and promotion value for wheat stem rust resistance breeding.

[0109] 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 stem rust resistance protein, characterized in that The wheat stem rust resistance protein is a protein consisting of the amino acid sequence shown in SEQ ID NO:

8.

2. A wheat stem rust resistance gene, characterized in that: As shown in any of the following (a)-(b): (a) a nucleotide sequence encoding the wheat stem rust resistance protein according to claim 1; or (b) The nucleotide sequence shown in SEQ ID NO:

7.

3. An expression cassette, characterized in that The expression cassette comprises a regulatory sequence and the wheat stem rust resistance gene of claim 2.

4. The expression cassette according to claim 3, characterized in that The regulatory sequences include a promoter.

5. A recombinant vector, characterized in that: It comprises the wheat stem rust resistance gene according to claim 2 or the expression cassette according to claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that The recombinant vector includes a translation control signal and / or a reporter gene.

7. A host cell, characterized in that The host cell contains the wheat stem rust resistance gene according to claim 2 or the expression cassette according to claim 3 or 4, or is transformed with the recombinant vector according to claim 5 or 6.

8. Use of the wheat stem rust resistance protein according to claim 1, or the wheat stem rust resistance gene according to claim 2, or the expression cassette according to claim 3 or 4, or the recombinant vector according to claim 5 or 6, or the host cell according to claim 7 in wheat stem rust resistance breeding.

9. The use according to claim 8, characterized in that: The wheat stem rust resistance breeding includes: enhancing the wheat resistance to stem rust, or cultivating transgenic wheat with enhanced resistance to stem rust.

10. A method for preparing transgenic wheat, characterized in that: The method comprises: The wheat stem rust resistance gene according to claim 2, or the expression cassette of claim 3 or 4, or the recombinant vector according to claim 5 or 6, Or the host cell according to claim 7 is introduced into wheat to obtain the transgenic wheat resistant to stem rust.

11. A breeding method for increasing wheat resistance to stem rust, characterized in that: The method comprises: The activity or content of the wheat stem rust resistance protein according to claim 1 in wheat is increased, so that the resistance of the wheat to stem rust is enhanced.

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