Asian soybean rust resistance gene and use thereof

Abstract

The present invention relates to the field of biotechnology engineering, and specifically relates to an Asian soybean rust resistance gene and the use thereof. The resistance gene is RppGM12H3 or RppGM12H4, which has characteristics such as a low cytotoxicity. The obtained transgenic material exhibits efficient and broad-spectrum control effects against different Phakopsora pachyrhizi strains.

Description

A gene for resistance to soybean rust in Asia and its application Technical Field

[0001] This invention relates to the field of biotechnology engineering, and more specifically, to an Asian soybean rust resistance gene and its application. Background Technology

[0002] Soybeans are an important grain and oil crop, serving as a significant source of protein for feed and consumption, as well as a crucial raw material for vegetable oils. Soybean rust is a major disease affecting soybean production worldwide. Infection with rust typically results in yield reductions of 10% to 30%, and in severe cases, yield losses can exceed 50%. Currently, 39 countries and regions globally, including China, the United States, Australia, and Brazil, are major areas affected by soybean rust.

[0003] Soybean rust is mainly caused by Phakopsora pachyrhizi, a highly infectious fungus with a wide host range, affecting 150 species across 53 genera of leguminous plants, including soybeans, common beans, kidney beans, and broad beans. Soybean rust primarily damages the leaves, but can also infect petioles and stems. In the early stages of infection, small grayish-brown spots can be seen on the leaves when held up to the light. These spots then enlarge, becoming yellowish-brown, reddish-brown, or purplish-brown. When the lesions are dense, they form necrotic spots, slightly raised on the leaf surface containing a powdery mass of fungal spores. Upon rupture, these spores release a rust-colored powdery substance. Currently, most soybean varieties lack resistance to soybean rust, and chemical control methods are primarily used to manage the disease in production. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, this invention provides an Asian soybean rust resistance gene and its application.

[0005] The technical solution adopted in this invention is as follows:

[0006] The present invention provides a protein comprising an amino acid sequence having at least 97%, at least 98%, at least 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0007] In one specific embodiment, the amino acid sequence of the protein is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0008] The present invention also provides an isolated nucleic acid molecule having a nucleic acid sequence selected from the following:

[0009] (1) The nucleic acid sequence encoding the protein or its complementary sequence;

[0010] (2) A nucleic acid sequence or its complementary sequence as shown in SEQ ID NO:3 or 4;

[0011] (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; or

[0012] (4) A nucleic acid sequence that encodes the same protein as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

[0013] The present invention also provides a gene expression cassette comprising the aforementioned nucleic acid molecule.

[0014] The present invention also provides a composition comprising an effective amount of the protein described herein, the composition being effective in controlling Asian soybean rust.

[0015] The present invention also provides a DNA construct comprising a nucleic acid molecule expressing the protein, or comprising the nucleic acid molecule or the gene expression cassette.

[0016] The present invention also provides a host cell comprising the aforementioned nucleic acid molecule, the aforementioned gene expression cassette, or the aforementioned DNA construct.

[0017] In one specific embodiment, the host cell is a plant cell.

[0018] The present invention also provides a method for controlling Asian soybean rust, comprising introducing the nucleic acid molecule, the gene expression cassette, or the DNA construct into a plant for expression to generate resistance to Asian soybean rust.

[0019] The present invention also provides a method for cultivating transgenic plants resistant to Asian soybean rust, comprising the aforementioned plant cell regeneration plant.

[0020] The present invention also provides plants produced by the method.

[0021] The present invention also provides the application of the described protein, the described nucleic acid molecule, the described gene expression cassette, or the described DNA construct in resistance to Asian soybean rust.

[0022] In one specific embodiment, the plant is a legume.

[0023] In another specific embodiment, the plant is soybean.

[0024] Some of the terms used in this specification are defined as follows.

[0025] “Nucleic acid” means deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the essential properties of natural nucleotides in that they hybridize with single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.

[0026] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” or “nucleic acid sequence” are used interchangeably to refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The nucleotide name “R” indicates a purine, such as guanine or adenine; “Y” indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); “M” indicates adenine or cytosine; “K” indicates guanine or thymine; and “W” indicates adenine or thymine.

[0027] The term "protein" refers to a polymer of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids, as well as to polymers containing natural amino acids.

[0028] As used herein, the term “coding” or “encoding” and its grammatical variations are used to indicate that a nucleic acid contains the desired information that guides the translation of a nucleotide sequence (e.g., a legume sequence) into a specific protein using codons. Nucleic acids encoding proteins may contain untranslated sequences (e.g., introns) within the translated regions of the nucleic acid, or may lack such intermediate untranslated sequences (e.g., as in cDNA).

[0029] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0030] In the case of two nucleic acid or polypeptide sequences, the terms “sequence identity” or “identity” as used herein can refer to the same residues in both sequences when comparing maximum correspondences on a specified comparison window.

[0031] The sequence alignment method used for comparison is well known to those skilled in the art and can be determined using the BLAST algorithm (Altschul et al., 1990, Mol.Biol. 215:403-10) obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) with default parameters.

[0032] Various procedures and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. Detailed considerations for sequence alignment methods and homology calculations can be found, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0033] The Basic Local Alignment Search Tool (BLASTTM; Altschul et al. (1990)) of the National Center for Biotechnology Information (NCBI), including the NCBI (Bethesda, MD), and is available on the Internet, and can be used in conjunction with several sequence analysis programs. Descriptions of how to use this program to determine sequence identity can be found in the "Help" section of BLASTTM on the Internet. For comparing nucleic acid sequences, the "Blast 2 Sequence" function of the BLASTTM (Blastn) program with default parameters can be used. When evaluated using this method, nucleic acid sequences with greater similarity to the reference sequence will show an increased percentage of identity.

[0034] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitution, where an amino acid is replaced by another amino acid belonging to the same group, falls within the scope of this invention, provided that the substitution does not impair the protein's biological activity. Furthermore, this invention also covers mutant proteins that also include one or more other nonconservative substitutions, provided that the nonconservative substitution does not significantly affect the desired function and biological activity of the protein of this invention.

[0035] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of mutant proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a bioactive fragment of a mutant protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0036] The term "expression" and its grammatical variations refer to the biosynthesis or process that produces polynucleotides, including the transcription and / or translation of gene products. For example, nucleic acid molecules disclosed in this invention can be transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the transcribed mRNA can be subsequently translated into a protein. The term "gene product" and its grammatical variations can refer to, for example, transcripts and encoded proteins. The inhibition (or enhancement) of the expression or function of a gene product (i.e., the target gene product) can be in a comparative context between any two plants, for example, the expression or function of a gene product in a genetically modified plant relative to the expression or function of a gene product in a corresponding but susceptible wild-type plant or other susceptible plant. The expression level of the gene product may not be present in wild-type plants. For example, a "wild-type" plant can be a plant, plant cell, or plant part that does not express a foreign resistance gene.

[0037] A “DNA construct” is a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs can be used for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation purposes, i.e., introducing heterologous DNA into host cells to produce transgenic plants and cells, and can thus be contained in plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. Components of a DNA construct or a vector containing a DNA construct include one or more gene expression elements operatively linked to a transcribed DNA sequence, such as: operatively linked DNA, operatively linked protein-coding DNA molecules, and a promoter for operatively linked expression of a 3' untranslated region (UTR). Gene expression elements that can be used in the practice of this invention include, but are not limited to, one or more of the following types of elements: promoters, 5'UTRs, enhancers, leader sequences, cis-acting elements, introns, target sequences, 3'UTRs, and one or more selective marker transgenes.

[0038] An "expression cassette" refers to DNA capable of expressing proteins in a host cell. This DNA includes not only promoters that initiate transcription of the protein-encoding gene but also terminators that terminate transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: cauliflower mosaic virus constitutive promoter 35S; a wound-inducible promoter from tomato leucine aminopeptidase (“LAP”) (Chao et al., (1999) Plant Physiol 120:979-992); a chemically induced promoter from tobacco pathogenesis-associated 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both induced by methyl jasmonate); heat shock promoters; tetracycline-inducible promoters; seed-specific promoters, such as the millet seed-specific promoter pF128; seed storage protein-specific promoters (e.g., bean protein, rapeseed protein, oleic acid protein, and soybean β-conglycinin promoters) (Beachy et al., (1985) EMBO J. [Journal of the European Society for Molecular Biology] 4:3047-3053). They can be used alone or in combination with other plant promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al., (1985) Nature, 313:810; Rosenberg et al., (1987) Gene, 56:125; Guerineau et al., (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al., Genes Dev., 5:141; Mogen et al., (1990) Plant Cell, 2:1261; Munroe et al., (1990) Gene, 91:151; Ballad et al., (1989) Nucleic Acids Res., 17:7891; Joshi et al., (1987) Nucleic Acid Res., 15:9627.

[0039] “Vector” means any recombinant DNA molecule that can be used for bacterial or plant transformation purposes, containing any of a variety of nucleic acids containing one or more desired sequences to be delivered to and / or expressed in cells. Vectors are typically made of DNA, but RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phage particles, viral genomes, synthetic chromosomes, etc. Recombinant DNA molecules, as listed in the sequence listing, can be inserted into vectors as part of a construct having a recombinant DNA molecule operatively linked to a gene expression element that functions in plants to influence the expression of an engineered protein encoded by the recombinant DNA molecule. General methods for manipulating DNA molecules for the preparation and use of recombinant DNA constructs and plant transformation vectors are well known in the art and are described in detail, for example, in manuals and laboratory guides including MR Green and J Sambrook, “Molecular Cloning: A Laboratory Manual” (4th edition), ISBN: 978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012).

[0040] "Host cell" should be understood as any single-celled or multi-celled organism that can introduce mutant protein-encoding nucleic acids, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0041] The term "transformation" and its grammatical variations are used in this invention to refer, for example, the transfer of a nucleic acid fragment into the genome of a host organism, thereby acquiring genetically stable inheritance. A host organism containing the transformed nucleic acid fragment is called a "transgenic" organism. The term "host cell" and its grammatical variations refer to the cell in which the transformation of the recombinant DNA construct occurs, and may include yeast cells, bacterial cells, and / or plant cells.

[0042] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" as used herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., autofertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

[0043] In gene introgression or backcrossing, the "donor" parent is the parental plant that possesses the desired gene or locus for introgression. The "recipient" parent (used once or multiple times) or "recurrent" parent (used twice or more times) is the parental plant into which the gene or locus is introgressed. The initial hybridization produces the F1 generation.

[0044] The term "gene editing" generally refers to a technique that involves the insertion, deletion, modification, or replacement of DNA in the genome. Many gene editing systems suitable for use in the methods of this invention are known in the art, including, but not limited to, zinc finger nuclease (ZFN) systems, transcription activation-like effector nuclease (TALEN) systems, and CRISPR / Cas systems.

[0045] The terms “variant,” “mutation,” and their grammatical variations refer to substantially similar sequences. For nucleic acid molecules, variants include the deletion and / or addition of one or more nucleotides at one or more sites in the native nucleic acid molecule, and / or the substitution of one or more nucleotides at one or more sites in the native nucleic acid molecule.

[0046] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, particularly monocotyledonous or dicotyledonous plants. Plants of this invention include inbred lines, hybrids, open-pollinated varieties, double-haploid plants, or plants from segregating populations. The term "plant" and its grammatical variations can refer to the whole plant, any part thereof, or cell or tissue cultures derived from a plant. Thus, the term "plant" can refer to any of the following: whole plant, plant part or organ, plant tissue, seed, and / or plant cell. A plant cell is a cell of a plant obtained directly from a seed or plant, or derived from a cell culture obtained from a plant. Progeny, variants, and mutants of regenerated plants are within the scope of this invention, provided that these parts contain an introduced resistance gene. Thus, the term "soybean plant" can refer to the whole soybean plant, one or more parts of a soybean plant (e.g., roots, root tips, stems, leaves, buds, flowers, pods, seeds, cotyledons, etc.), soybean plant cells, soybean plant protoplasts, and / or soybean plant callus.

[0047] The term "plant part" includes, but is not limited to, embryos, pollen, seeds, leaves, flowers (including but not limited to anthers, ovules, etc.), fruits, stems or branches, roots, root tips, cells (including intact cells in plants and / or plant parts), protoplasts, plant cell tissue cultures, plant callus, plant masses, etc. Therefore, plant parts include soybean tissue cultures that can regenerate soybean plants. Furthermore, "plant cell" refers to the structural and physiological unit of a plant, including the cell wall, and may also refer to the protoplast. The plant cells of this invention can be in the form of isolated single cells, or can be cultured cells, or can be part of a higher-level tissue unit (e.g., plant tissue or plant organ). In this invention, "plant cell" should be understood as any cell derived from or found in a plant capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, plants, or seeds.

[0048] In plant contexts, the term “introduction” refers to any method of introduction, including but not limited to: gene introgression, transgenesis, regular clustering of short palindromic repeats (CRISPR), transcription activator-like effector nucleases (TALENs) (Feng et al. 2013, Joung and Sander 2013), large-scale nucleases, or zinc finger nucleases (ZFNs).

[0049] The term "soybean" refers to soybeans and any plant varieties cultivated or grown using soybeans (including plants of the genus "wild soybean").

[0050] The terms “increase,” “enhance,” “enhance,” etc., and their grammatical variations, are used to refer to any promotion or gain or increase in the expression, function, or activity of a target gene (e.g., a resistance gene) product compared to a susceptible plant, thereby providing enhanced resistance to one or more pathogens (e.g., *Laminaria*) or diseases (e.g., rust). Furthermore, as used herein, the terms “lead to” or “increase,” and their grammatical variations, can refer to higher expression of a target gene product such that the level is increased by 10% or more, 50% or more, or 100% relative to cells or plants lacking the target gene or protein disclosed herein.

[0051] The term "immunity" or "immunity" refers to the absence of any macroscopically visible disease symptoms. As used herein, the term "partial resistance" refers to the presence of macroscopically visible lesions (with little or no spore formation) and / or a reduction in the extent or degree of any disease symptoms and / or a delay in the progression of any disease symptoms, and may, for example, exhibit a reduction in the number of lesions or lesions in which spore formation is reduced. As used herein, with respect to rust, the term "susceptibility" or "lack of resistance" refers to lesions occurring at spore formation levels equal to or higher than those observed in reference standards (e.g., Williams 82 or the Peking variety).

[0052] The term "resistance" refers to the absence or inconspicuousness of symptoms of one or more diseases caused by plant pathogens in a plant. Resistance may mean fewer disease symptoms, such as a lower number of infected plants, milder disease severity, fewer leaf spots or defoliation, and lower or no yield loss, compared to susceptible plants or plants that do not contain genes that effectively reduce the symptoms of one or more diseases. Furthermore, resistance may include prevention or delay of pathogen proliferation. Generally, the term "resistance" includes both immunocompetence and partial resistance as defined above.

[0053] As used herein, the terms “phenotype,” “phenotypic trait,” or “trait” refer to one or more characteristics and / or manifestations of an organism. A phenotype is a manifestation that can be observed with the naked eye or by any other evaluative means known in the art (e.g., microscopy, biochemical analysis, or electromechanical measurement).

[0054] Alternatively, inhibition (or enhancement) of the expression or function of a target gene product can be achieved in settings where comparisons are made between plant cells, organelles, tissues, or plant parts within the same or different plants, and including comparisons between developmental stages or time periods of the same or different plants. Any method or composition that downregulates the expression of a target gene product or downregulates its functional activity at the transcriptional or translational level can be used to achieve inhibition of the expression or function of a target gene product. Similarly, any method or composition that induces or upregulates the expression of a target gene product, or increases or activates or upregulates its functional activity at the transcriptional or translational level, can be used to achieve increased expression or function of a target gene or protein. Methods for inhibiting or enhancing gene expression are well known in the art.

[0055] The genes and nucleic acid molecules disclosed in this invention include naturally occurring sequences and their mutant or modified forms. The proteins disclosed in this invention also include naturally occurring proteins and their variants, fragments, and modified forms. Such variants and fragments will still possess the ability to confer or enhance plant resistance to fungal pathogens. In one embodiment, mutations generated in the DNA encoding the variant or fragment generally do not place the sequence outside the reading frame and preferably do not create complementary regions that could generate secondary mRNA structures.

[0056] This invention relates to proteins associated with rust resistance in plants, nucleic acid sequences encoding such proteins, and their uses. The proteins, their encoding genes, and methods disclosed in this invention can be used to protect plants from rust pathogens. In some embodiments, the rust is a legume rust disease.

[0057] In other embodiments, the legume rust is soybean rust. The pathogen of soybean rust may be *Solanum melilotus* or *Solanum spp.* As used below and in the claims, every reference to soybean rust includes Asian soybean rust.

[0058] In a specific embodiment of the present invention, the pathogen of soybean rust is *Potamogeton crispus*.

[0059] In a preferred embodiment, the present invention relates to proteins associated with rust resistance in legumes.

[0060] Leguminosae can include plants of the genera *Glycine* (wild soybean), *Glycine*, *Pistacia*, *Lysimachia*, *Alfalfa*, *Phaseolus*, *Vicia*, *Phyllostachys*, *Trifolium*, or *Vigna*.

[0061] Plants in the genus *Glycine* can include *Glycine arenaria*, *Glycine argyrea*, wild soybean (*Glycine cyrtoloba*), grey soybean (*Glycine canescens*), Penghu soybean (*Glycine clandestine*), curved soybean (*Glycine curvata*), sickle-leaved soybean (*Glycine falcata*), broadleaf soybean (*Glycine latifolia*), small-leaved soybean (*Glycine microphylla*), *Glycine pescadrensis*, *Glycine stenophita*, *Glycine syndetica*, saline-land wild soybean (*Glycine soja Seib. et Zucc.*), *Glycine max(L.) Merrill.*, tobacco soybean (*Glycine tabacina*), or short-haired wild soybean (*Glycine tomentella*).

[0062] Chickpea species can include chickpea (Cicer arietinum), chickpea echinospermum, reticulate chickpea (Cicer reticulatum), or long-leaved chickpea (Cicer pinnatifidum).

[0063] Plants in the genus *Lycopus* can be *Lablab purpureus*.

[0064] Alfalfa plants can be either alfalfa or clover.

[0065] Plants in the genus *Phaseolus* can include common bean (*Phaseolus vulgaris*), cotton bean (*Phaseolus lunatus*), pointed-leaf bean (*Phaseolus acutifolius*), or purslane bean (*Phaseolus coccineus*).

[0066] Plants in the genus *Pisum* can include Abyssinian pea (*Pisum abyssinicum*), Asian pea (*Pisum sativum*), tall pea (*Pisum elatius*), velvety-leaved pea (*Pisum fulvum*), transcaucasian pea (*Pisum transcaucasium*), or dwarf pea (*Pisum humile*).

[0067] Plants of the genus *Pueraria* can include kudzu root.

[0068] Plants in the genus Trifolium can be yellow trifolium (Trifolium aureum) or western trifolium (Trifolium occidentale).

[0069] Plants in the genus Vigna can include cowpea (Vigna unguiculata), Vigna dalzelliana, long-leaved cowpea (Vigna oblongifolia), short-fruited cowpea (Vigna parkeri), narrow-leaved cowpea (Vigna filicaulis), Korki's cowpea (Vigna kirkii), yellow cowpea (Vigna luteola), radiate cowpea (Vigna radiata), three-leaved cowpea (Vigna trilobata), yellow cowpea (Vigna luteola), or Mungo cowpea (Vigna mungo).

[0070] In addition, legumes can be any of the following: soybean, alfalfa, clover, pea, common bean, lentil, lupin, bean tree, carob, soybean, peanut, or tamarind.

[0071] In a specific embodiment of the present invention, the plant is the soybean variety Williams 82 (Glycine max (L.) Merrill).

[0072] The proteins, their encoding genes, and methods disclosed in this invention can be used to protect plants from rust pathogens. The methods disclosed in this invention can increase, enhance, or improve the resistance of soybeans to the obligate parasitic fungi *Ipomoea spp.* (a major rust pathogen) or *Ipomoea spp.*. For example, increased or enhanced resistance to rust pathogens can be compared to the effects of the pathogens on susceptible plants. The extent of increased or enhanced resistance may vary but is correlated with disease symptoms (e.g., lesion color) and the incidence observed on the plant or plant tissue (e.g., leaves). Values ​​for immunity, resistance, and susceptibility can be given. For example, a resistance value indicates the degree of resistance of a plant to a plant disease (e.g., rust). These values ​​can also be used to compare the degree of resistance between, for example, target plants (e.g., transgenic legumes) and susceptible plants (e.g., Williams 82 or Tianlong 1) or reference standards.

[0073] The proteins, their encoding genes, and methods disclosed in this invention involve isolating resistance genes from leguminous plants and subsequently transferring these resistance genes into recipient plants, such as soybeans, to provide or enhance resistance to *Solanum rust*. One embodiment of the application involves transferring the resistance gene into sexually compatible or incompatible species to produce resistance. The resistance genes of this invention can be used alone or in combination with other resistance genes or with non-resistance genes to provide or enhance resistance to rust in recipient species.

[0074] Therefore, the transgenic method disclosed in this invention can be used alone or in combination with other strategies to generate or confer rust resistance in plants. Other available strategies include, but are not limited to, blocking the functional activity of effectors, inhibiting the uptake of pathogens or pathogenic factors (e.g., fungi) into host cells (e.g., plant cells), and / or conventional resistance breeding.

[0075] The methods disclosed in this invention can provide or enhance rust resistance in plants, preventing rust pathogens from reproducing or significantly reducing their reproduction rate. Therefore, the methods of this invention can alleviate one or more symptoms (i.e., disease symptoms) of rust in legumes compared to plants susceptible to or tolerant of *Strombyx mori* rust. The plants referred to in this invention also include transgenic legumes (e.g., soybeans) in which disease resistance genes or proteins have been introduced through genetic engineering methods to enhance their resistance to legume rust upon exposure.

[0076] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Unless otherwise expressly stated herein, the terms “a,” “an,” and “the” as used in the foregoing include their plural forms. The terms “comprises” and / or “comprising,” or “includes” and / or “including” as used herein specifically refer to the presence of the features, factors, and / or ingredients described herein, without excluding the presence and addition of one or more other features, factors, and ingredients. The term “and / or” as used above includes all or one of the items in the list of combinations.

[0077] This invention has been described in detail through a series of embodiments, but the invention is not limited to the disclosed embodiments. Any variations, substitutions, or replacements that fall within the scope of this invention, not described herein, may be modified according to public needs.

[0078] This invention addresses the increasingly serious Asian soybean rust disease in production by providing an Asian soybean rust resistance gene and its application. The RppGM12H3 or RppGM12H4 resistance gene has the characteristics of low cytotoxicity, and the obtained transgenic material has a highly efficient and broad-spectrum control effect against different soybean and potato rust strains. Attached Figure Description

[0079] Figure 1. Resistance identification results of each transgenic material to strain KA:Php:2 - leaf disease status.

[0080] sequence list Detailed Implementation

[0081] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention, and these embodiments are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent or imply that the experiments described below are all or only the experiments performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention shown in specific aspects without departing from the spirit or scope broadly described herein. Therefore, this document is to be considered illustrative rather than restrictive in all aspects.

[0082] Example 1: Design and Construction of Transgenic Vectors

[0083] The expression vector pCambia1301 was linearized by double digestion with restriction endonucleases HindIII and BstEII. Then, the disease resistance genes Rpp6907-7, RppGM12H3, and RppGM12H4 were introduced into the linearized expression vector pCambia1301 using seamless cloning, yielding the final transgenic vectors P1, P2, and P3, respectively. The nucleotide sequences of these three genes are SEQ ID NO:6, SEQ ID NO:3, and SEQ ID NO:4, respectively, and the amino acid sequences of their encoded proteins are SEQ ID NO:5, SEQ ID NO:1, and SEQ ID NO:2, respectively.

[0084] Example 2: Soybean genetic transformation

[0085] By using Agrobacterium-mediated soybean genetic transformation, expression vectors containing the aforementioned disease resistance genes were introduced into the transgenic soybean recipient Williams 82, while the empty vector pCambia1301 was transformed as a control. The transformation positivity rate of each vector was calculated (transgenic positivity rate = number of transgenic positive seedlings / number of infected explants × 100): the transgenic positivity rate of the blank control vector pCambia1301 was 11.4%, and the transgenic positivity rates of P1, P2, and P3 were 5.3%, 10.0%, and 9.3%, respectively. The results showed that the transgenic vectors carrying the disease resistance genes RppGM12H3 and RppGM12H4 had a significantly higher transgenic positivity rate than the transgenic vector carrying Rpp6907-7, indicating that the disease resistance proteins RppGM12H3 and RppGM12H4 have lower cytotoxicity to soybean.

[0086] Example 3: Inoculation and Resistance Identification of Soybean Rust

[0087] Two soybean rust fungus strains (numbered KA:Php:1 and KA:Php:2, respectively) were used to inoculate transgenic materials of soybean wild-type control Williams 82 and pCambia1301, P1, P2, and P3. The inoculation method for soybean rust is as follows:

[0088] 1. Collection of urediniospores

[0089] Gently brush the soybean leaves with a soft brush to collect the urediniospores of the soybean rust fungus. Resuspend the spores in sterile water containing 0.1% Tween 80 and adjust the spore concentration to 10. 5 ~10 6 per ml.

[0090] 2. Spray inoculation

[0091] Spray inoculation was carried out when the first compound leaf of soybean unfolded. The amount of spore solution inoculated should be such that all leaves are just covered with spore solution. Wild-type control Williams 82 and transgenic materials pCambia1301, P1, P2 and P3 were sprayed inoculated separately. At least 30 plants of each material were inoculated. After spray inoculation, all materials were placed in a dark room with a humidity of more than 95% and cultured for 24 hours, and then carried out conventional culture.

[0092] 3. Methods for investigating the condition

[0093] Disease survey was conducted 15 days after inoculation. Twenty plants were randomly selected from each material sample for investigation. The leaves with the most severe disease on each plant were selected as the investigation subjects. Disease severity was recorded and disease index was calculated. The resistance of the materials was evaluated based on the disease index. The methods for disease severity judgment and disease index calculation are shown in Tables 1 and 2.

[0094] Disease index = [Σ(disease grade × number of seedlings with that disease grade) × 100] / (highest disease grade value × total number of leaves surveyed).

[0095] Table 1 Criteria for Determining Disease Level

[0096] Table 2 Disease Index Grading Standards

[0097] 4. Disease index and result analysis

[0098] The disease index and resistance data of the transgenic materials of wild-type control Williams 82 and pCambia1301, P1, P2, and P3 inoculated with two strains of *Potamogeton crispus* are shown in Tables 3 and 4. The disease status of representative leaves is shown in Figure 1.

[0099] Table 3. Results of the investigation of inoculated strain KA:Php:1

[0100] Table 4. Results of the investigation of inoculated strain KA:Php:2

[0101] The survey results showed that the transgenic materials P1, P2, and P3 all had significantly improved resistance to soybean rust compared to the wild-type material Williams 82 and the negative control pCambia1301, and the resistance levels of the transgenic materials P2 and P3 were significantly stronger than those of the transgenic material P1.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A protein, characterized in that, The protein comprises an amino acid sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The protein of claim 1, wherein, The amino acid sequence of the protein is shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. An isolated nucleic acid molecule, comprising, It has a nucleic acid sequence selected from the following: (1) A nucleic acid sequence or its complementary sequence encoding the protein as described in claim 1 or 2; (2) A nucleic acid sequence or its complementary sequence as shown in SEQ ID NO:3 or 4; (3) A nucleic acid sequence that hybridizes to the sequence shown in (1) or (2) under stringent conditions; or (4) A nucleic acid sequence that encodes the same protein as the sequence shown in (1) or (2) due to the degeneracy of the genetic code, or its complementary sequence.

4. A gene expression cassette comprising the nucleic acid molecule as described in claim 3.

5. A composition comprising an effective amount of the protein as described in claim 1 or 2, the composition being effective in controlling Asian soybean rust.

6. A DNA construct comprising a nucleic acid molecule expressing the protein of claim 1 or 2, or comprising a nucleic acid molecule of claim 3 or a gene expression cassette of claim 4.

7. A host cell comprising the nucleic acid molecule of claim 3, the gene expression cassette of claim 4, or the DNA construct of claim 6; preferably, the host cell is a plant cell.

8. A method for controlling Asian soybean rust, comprising introducing a nucleic acid molecule as described in claim 3, a gene expression cassette as described in claim 4, or a DNA construct as described in claim 6 into a plant for expression to generate resistance to Asian soybean rust; preferably, the plant is a legume; more preferably, the plant is soybean.

9. A method for cultivating transgenic plants resistant to Asian soybean rust and the plants produced by said method, comprising regenerating plants from the plant cells of claim 7; preferably, said plant is a legume; more preferably, said plant is soybean.

10. The application of the protein as described in claim 1 or 2, the nucleic acid molecule as described in claim 3, the gene expression cassette as described in claim 4, or the DNA construct as described in claim 6 in resistance to Asian soybean rust; preferably, the plant is a legume; more preferably, the plant is soybean.

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