WO2024077531A1 - 来自烟草的抗斑萎病基因rtsw及其应用 - Google Patents

来自烟草的抗斑萎病基因rtsw及其应用 Download PDF

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WO2024077531A1
WO2024077531A1 PCT/CN2022/124977 CN2022124977W WO2024077531A1 WO 2024077531 A1 WO2024077531 A1 WO 2024077531A1 CN 2022124977 W CN2022124977 W CN 2022124977W WO 2024077531 A1 WO2024077531 A1 WO 2024077531A1
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plant
virus
seq
tnl1
nucleotide sequence
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PCT/CN2022/124977
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French (fr)
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黄昌军
刘勇
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云南省烟草农业科学研究院
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to gene separation and plant improvement, in particular to a tobacco spotted wilt resistance gene RTSW and application thereof.
  • Orthotospoviruses belong to the Bunyavirales family. With their wide host range (infecting more than 1,090 plant species in 84 families) and huge economic losses, they have become a huge threat to agricultural production. They are recognized as one of the most destructive plant viruses and are ranked second in the list of the world's most important plant viruses.
  • Tobacco spotted wilt disease (TSWD) is a serious disease caused by infection with orthotospoviruses. TSWD causes tobacco plants to become dwarfed, leaves to shrink, and small necrotic rings to appear on the leaves. These rings often merge into large spots to form irregular necrotic areas.
  • Sw-5b and Tsw genes Disease-resistant gene resources have been reported in tomatoes and peppers, namely the Sw-5b and Tsw genes, both of which are CC-NBS-LRR structural type disease-resistant genes.
  • the Sw-5b gene is resistant to multiple American orthotospoviruses, but has no resistance to the Eurasian orthotospoviruses that cause serious damage.
  • the Tsw gene is only resistant to TSWV. In addition to these two genes, no reports have been found about orthotospoviruses resistance genes or resistance loci in other crops.
  • Disease-resistant breakthrough virus strains targeting the Sw-5b and Tsw genes have been reported in many countries, so new disease-resistant gene resources are urgently needed.
  • N.alata has good resistance to TSWV. After inoculation with TSWV, N.alata only showed mild allergic necrosis symptoms on the inoculated leaves, and the virus could not be detected in the systemic leaves. Using N.otophora as a bridge parent, Gajos et al.
  • the inventor's research group obtained spotted wilt-resistant tobacco plants without linkage drag through genetic locus analysis, molecular marker development and large-scale screening, see the international patent application with application number PCT/CN2021/129382, invention name “Spotted wilt-resistant tobacco plants without linkage drag and breeding methods thereof”, and the Chinese patent application with application number 202111311707.0, invention name “Molecular markers for screening spotted wilt-resistant tobacco plants without linkage drag and their applications”, all of which are incorporated herein by reference.
  • the RTSW gene that confers TSWV resistance to tobacco has not been cloned, which limits the research on the disease resistance mechanism and breeding application of the RTSW gene.
  • the breeding of other crops for resistance to spotted wilt also has the problem of limited available resistance gene resources. Therefore, the cloning and use of the RTSW gene will also be beneficial to the prevention and control of spotted wilt in other crops.
  • the present invention relates to the isolation of plant resistance genes, and specifically to the isolation of RTSW genes that confer resistance to at least one virus of the genus Tomato spotted wilt virus to plants (especially Solanaceae plants).
  • the RTSW gene is derived from N.alata, a winged tobacco plant, and is isolated from the non-linked drag-resistant tobacco plant previously created by the inventor.
  • the nucleotide sequence of the RTSW gene of the present invention includes, but is not limited to, the nucleotide sequence of the wild-type RTSW gene (including the natural promoter and the 3' adjacent region containing the coding region), the cDNA sequence, and the nucleotide sequence containing only the coding region.
  • Examples of the nucleotide sequence of the RTSW gene include the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 and variants thereof.
  • the RTSW gene encodes the RTSW protein.
  • the RTSW protein can confer resistance to at least one virus of the genus Tomato spotted wilt virus to plants (especially Solanaceae plants) containing the RTSW protein.
  • Examples of the amino acid sequence of the RTSW protein include the amino acid sequence shown in SEQ ID NO: 3 and variants thereof.
  • RTSW is not only resistant to various American-type tomato spotted wilt viruses, but also resistant to the Eurasian-type tomato spotted wilt viruses that cause serious damage.
  • Transgenic experiment results show that the expression of the RTSW gene alone in Nicotiana benthamiana, tomato and potato can confer broad-spectrum resistance to tomato spotted wilt viruses in the plants. Therefore, RTSW is a valuable disease-resistant gene resource that can be utilized.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence of a RTSW gene or a variant thereof, wherein the variant is a naturally occurring or non-naturally occurring variant.
  • the nucleic acid molecule of the present invention comprises a nucleotide sequence selected from the following (a1)-(a5):
  • nucleic acid molecule (a3) a nucleotide sequence having at least 90% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, wherein the nucleic acid molecule is capable of conferring resistance to at least one virus of the genus Tomato spotted wilt virus on a plant comprising the nucleic acid molecule;
  • nucleic acid molecule A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the nucleic acid molecule is capable of conferring resistance to at least one tomato spotted wilt virus on a plant containing the nucleic acid molecule.
  • the nucleic acid molecules of the present invention include polynucleotide derivatives or variants that are substantially similar in sequence and function similarly to natural polynucleotides (e.g., polynucleotides shown in SEQ ID NO: 1 or 2 or polynucleotides encoding the amino acid sequence shown in SEQ ID NO: 3).
  • the natural polynucleotides contain naturally occurring nucleotide sequences.
  • polynucleotide derivatives or variants include: polynucleotides that have one or more nucleotides deleted and/or added at the 5' and/or 3' ends of the natural polynucleotides; polynucleotides that have one or more nucleotides deleted and/or added at one or more internal sites of the natural polynucleotides; polynucleotides that have one or more nucleotides substituted at one or more sites of the natural polynucleotides; and/or polynucleotides that encode one of the RTSW proteins of the present invention due to the degeneracy of the genetic code.
  • the polynucleotide derivatives or variants include naturally occurring polynucleotides, such as naturally occurring allelic variants, which can be identified using well-known molecular biology techniques (e.g., polymerase chain reaction and hybridization techniques); and synthetic polynucleotides, such as polynucleotides generated by site-directed mutagenesis that still encode the RTSW proteins of the present invention.
  • the polynucleotide derivative or variant has at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the natural polynucleotide.
  • the amino acid sequence encoded by the polynucleotide derivative or variant has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and has the same anti-disease function.
  • the present invention relates to a polynucleotide construct comprising a nucleic acid molecule of the present invention and one or more regulatory elements that are operably linked for carrying out gene transcription and expression in a prokaryotic or eukaryotic host cell.
  • the polynucleotide construct can be a plasmid, a cosmid, a phage or an expression vector, a transformation vector, a shuttle vector or a cloning vector. It can be double-stranded or single-stranded, linear or circular, and can be integrated into a genome or present in an extrachromosomal manner to transform a prokaryotic or eukaryotic host cell.
  • the present invention provides an expression cassette, which comprises a nucleic acid molecule of the present invention and an operably linked promoter.
  • the promoter may be a natural promoter of the RTSW gene, such as a promoter having a nucleotide sequence as shown in SEQ ID NO: 4; or a heterologous promoter, such as a constitutively induced promoter (such as a 35S promoter derived from a "cauliflower mosaic virus” known in the art), or a pathogen-induced promoter (such as a PR1 gene promoter).
  • the present invention provides a vector comprising the nucleic acid molecule or expression cassette of the present invention.
  • nucleic acid molecules, expression cassettes or vectors of the present invention can be used to transform any plant, including but not limited to monocotyledonous plants and dicotyledonous plants.
  • the plant is preferably a plant of the Solanaceae family.
  • the present invention provides a host cell transformed with a nucleic acid molecule, expression cassette or vector of the present invention.
  • the host cell may be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a yeast cell or a plant cell).
  • the host cell is an Agrobacterium transformed with a nucleic acid molecule or vector of the present invention, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, or a plant cell.
  • Methods for obtaining the host cell include, but are not limited to, methods known in the art such as chemical drug induction, thermal transformation, gene gun transformation, Agrobacterium-mediated transformation, protoplast transfection, transduction, vacuum infiltration or electroporation.
  • the nucleic acid molecule, expression cassette or vector of the present invention is introduced into Agrobacterium by methods such as chemical drug induction, thermal transformation or electroporation; the nucleic acid molecule, expression cassette or vector of the present invention is introduced into a plant cell by methods such as gene gun transformation, Agrobacterium-mediated transformation, protoplast transfection, transduction or plant virus-mediated gene introduction.
  • the present invention relates to transgenic plants or parts thereof comprising transgenic plant cells (plant cells transformed with nucleic acid molecules, expression cassettes or vectors of the present invention).
  • the parts here can be cells, tissues, organs or a combination of several cells, tissues or organs, such as flowers, leaves, tubers, fruits or seeds.
  • the transgenic plants, especially Solanaceae plants, have higher resistance to multiple tomato spotted wilt virus viruses than non-transgenic plants.
  • the present invention provides a transgenic plant comprising a heterologous polynucleotide construct stably incorporated into its genome, wherein the polynucleotide construct comprises a nucleotide sequence selected from the group consisting of (a1) to (a5):
  • nucleic acid molecule (a3) a nucleotide sequence having at least 90% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, wherein the nucleic acid molecule is capable of conferring resistance to at least one virus of the genus Tomato spotted wilt virus on a plant comprising the nucleic acid molecule;
  • nucleic acid molecule A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the nucleic acid molecule is capable of conferring resistance to at least one tomato spotted wilt virus on a plant containing the nucleic acid molecule.
  • the polynucleotide construct further comprises an operably linked promoter for expressing the nucleotide sequence in a plant.
  • a suitable promoter may be a natural promoter of the RTSW gene, such as a promoter having a nucleotide sequence as shown in SEQ ID NO: 4.
  • the transgenic plant is a Solanaceae plant, which includes but is not limited to cultivated Solanaceae plants.
  • the Solanaceae plant is tobacco, potato or tomato.
  • the transgenic plant comprises resistance to at least one tomato spotted wilt virus relative to a control plant.
  • the control plant refers to a starting plant for obtaining the transgenic plant that does not contain the polynucleotide construct.
  • the transgenic plants comprise resistance to Tomato Spotted Wilt Virus (TSWV), Impatiens Necrotic Spot Virus (INSV), Peanut Ringspot Virus (GRSV), Chrysanthemum Stem Necrosis Virus (CSNV), Tomato Ring Spot Virus (TZSV), Peanut Bud Necrosis Virus (GBNV), Mulberry Vein Band-Associated Virus (MVBaV), Pepper Chlorotic Virus (CaCV), and Calla Lily Chlorotic Spot Virus (CCSV), relative to control plants.
  • TSWV Tomato Spotted Wilt Virus
  • INSV Impatiens Necrotic Spot Virus
  • GRSV Resistive Ringspot Virus
  • CSNV Chrysanthemum Stem Necrosis Virus
  • TZSV Tomato Ring Spot Virus
  • GBNV Mulberry Vein Band-Associated Virus
  • CaCV Pepper Chlorotic Virus
  • CCSV Calla Lily Chlorotic Spot Virus
  • Methods for obtaining the transgenic plants of the present invention include, but are not limited to, artificial de novo gene synthesis methods, transgenic technology, protoplast regeneration, hybrid breeding methods, and genome editing methods.
  • the genome editing methods include CRISPR/Cas9 technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, etc.
  • the present invention provides seeds from any of the transgenic plants, wherein the seeds comprise the nucleic acid molecule of the present invention.
  • the present invention provides a method for conferring or enhancing resistance to at least one virus of the genus Tomato wilt virus in a plant, comprising introducing a heterologous polynucleotide construct into at least one plant cell, wherein the polynucleotide construct comprises a nucleotide sequence selected from the group consisting of (a1) to (a5):
  • nucleic acid molecule (a3) a nucleotide sequence having at least 90% sequence identity with at least one of the nucleotide sequences shown in SEQ ID NOs: 1 and 2, wherein the nucleic acid molecule is capable of conferring resistance to at least one virus of the genus Tomato spotted wilt virus on a plant comprising the nucleic acid molecule;
  • nucleic acid molecule A nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, wherein the nucleic acid molecule is capable of conferring resistance to at least one tomato spotted wilt virus on a plant containing the nucleic acid molecule.
  • the polynucleotide construct further comprises an operably linked promoter for expressing the nucleotide sequence in a plant, and the polynucleotide construct is stably incorporated into the genome of the plant cell.
  • the plant cell is regenerated into a plant comprising the polynucleotide construct in its genome. Regeneration methods known in the art can be used.
  • the plant is tobacco, potato or tomato.
  • the plant comprises resistance to at least one Tomato spotted wilt virus relative to control plants.
  • the plant comprises resistance to Tomato Spotted Wilt Virus (TSWV), Impatiens Necrotic Spot Virus (INSV), Peanut Ringspot Virus (GRSV), Chrysanthemum Stem Necrosis Virus (CSNV), Tomato Ring Spot Virus (TZSV), Peanut Bud Necrosis Virus (GBNV), Mulberry Vein Band-Associated Virus (MVBaV), Pepper Chlorotic Virus (CaCV), and Calla Lily Chlorotic Spot Virus (CCSV) relative to control plants.
  • TSWV Tomato Spotted Wilt Virus
  • INSV Impatiens Necrotic Spot Virus
  • GRSV Resistive Ringspot Virus
  • CSNV Chrysanthemum Stem Necrosis Virus
  • TZSV Tomato Ring Spot Virus
  • GBNV Mulberry Vein Band-Associated Virus
  • CaCV Pepper Chlorotic Virus
  • CCSV Calla Lily Chlorotic Spot Virus
  • the present invention provides a plant produced by the method of the present invention, or a fruit, tuber, leaf or seed of the plant, wherein the fruit, tuber, leaf or seed comprises the polynucleotide construct.
  • the present invention provides a method for identifying a plant comprising conferred or enhanced resistance to at least one Tomato mottled wilt virus, comprising detecting the presence of RTSW in said plant or in at least one part or cell thereof.
  • the plant is tobacco, potato or tomato.
  • the presence of the RTSW is detected by detecting at least one marker in the RTSW.
  • the present invention provides primers for detecting RTSW gene and a kit comprising the primers, wherein the nucleotide sequences of the primers are shown in SEQ ID NOs: 22 and 23.
  • the method for detecting RTSW gene using the primers comprises: isolating nucleic acid from a plant or germplasm to be tested; and performing PCR using the primers in the isolated nucleic acid.
  • the present invention provides a polypeptide comprising an amino acid sequence selected from the following (B1)-(B4):
  • polypeptides of the present invention can be produced in a variety of ways, including but not limited to in vitro de novo synthesis, prokaryotic cell expression, and eukaryotic cell expression.
  • polypeptides of the present invention include polypeptide derivatives or variants that are substantially similar in sequence and functionally similar to natural proteins (e.g., proteins with amino acid sequences such as those shown in SEQ ID NO: 3).
  • natural proteins e.g., proteins with amino acid sequences such as those shown in SEQ ID NO: 3
  • the natural proteins contain naturally occurring amino acid sequences.
  • the polypeptide derivatives or variants may be derived from genetic polymorphisms or artificial manipulations.
  • the polypeptide derivatives or variants may be produced in a variety of ways known in the art, including amino acid substitutions, deletions, and insertions.
  • the polypeptide derivatives or variants are obtained by deleting one or more amino acids at the N-terminus and/or C-terminus of the natural protein, deleting and/or adding one or more amino acids at one or more internal sites of the natural protein, or replacing one or more amino acids at one or more sites of the natural protein.
  • the polypeptide derivatives or variants have a sequence identity of at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with the amino acid sequence of the natural protein.
  • the polypeptide derivative or variant may differ from the native protein by only 1-15, 1-10 (e.g., 6-10), 5, 4, 3, 2 or even 1 amino acid residue.
  • the polypeptide derivative or variant has the same anti-disease function as the native protein.
  • the viruses of the genus Tomato spotted wilt virus described herein include but are not limited to American viruses: Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), Chrysanthemum stem necrosis virus (CSNV); Eurasian viruses: Tomato zonate spot virus (TZSV), Calla lily chlorotic spot virus (CCSV), Tomato chlorotic spot virus (TCSV), Mulberry vein banding associated virus (MVBaV), Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (CSNV); bud necrosis virus (GBNV), Tomato necrotic spot virus (TNSV), pepper chlorotic spot virus (PCSV), Tomato necrotic ringspot virus (TNRV), Polygonum ringspot virus (PolRSV) and Hippeastrum chlorotic ringspot virus (HCRV).
  • TSWV Tomato spotted wilt virus
  • INSV Impatiens necrotic spot virus
  • the Solanaceae plants described herein refer to members of the Solanaceae family, including domesticated and non-domesticated members.
  • the Solanaceae family includes, but is not limited to, Nicotiana spp., tomato (Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), petunia (Petunia spp.), pepper (Capsicum spp.), Physalis spp., woody Solanaceae [Solanum dulcamara], horticultural blueberry (Solanum scabrum), eggplant (Solanum macrocarpon), Solanum demissum, Solanum stoloniferum, Solanum papita, Solanum bulbocastanum, Solanum edinense, Solanum schenckii, Solanum hjertingii, Solanum venturi, Solanum mochiquense, Solanum chacoense and currant tomato
  • the tobacco described herein may be a wild species or a cultivated species (Nicotiana tabacum.L).
  • Wild tobacco species include, but are not limited to, N. benthamiana, N. alata, N. glauca, N. gossei, N. kawakamii, N. knightiana, N. langsdorffi, N. otophora, N. sylvestris, N. tomentosa, N. tomentosiformis, N. undulate, N. x sanderae, N. rustica, N. suaveolens, N. attenuate.
  • Cultivated tobacco species include, but are not limited to, burley type, dark type, flue-cured type, Maryland type, oriental type, or cigar.
  • the cultivated tobacco of the present invention can be selected from a plurality of common tobacco varieties, including but not limited to K326, Yunyan 87, Yunyan 97, Yunyan 85, Yunyan 116, Yunyan 121, NC89, Zhongyan 100, Honghua Dajinyuan, Cuibi No.
  • RTSW gene refers to a gene derived from the N. alata genome and which confers resistance to spotted wilt disease on plants.
  • RTSW locus or "TSWV resistance locus” refers to a DNA fragment derived from the N.alata genome, comprising the RTSW gene, which confers resistance to spotted wilt in plants in either the heterozygous or homozygous state.
  • RTSW introduced fragment refers to a genomic DNA fragment of N.alata comprising the RTSW gene.
  • tobacco plants containing the "RTSW introduced fragment” have a tobacco spotted wilt resistance trait.
  • short RTSW introduced fragment refers to a DNA fragment in which the non-target gene components linked to the RTSW gene (referred to as linked drag gene components) on the long RTSW introduced fragment are partially or completely deleted, and the complete RTSW gene function is retained.
  • introduction refers to the transfer of a desired allele of a genetic locus from one genetic background to another.
  • allele generally refers to a pair of genes that are located at the same position on a pair of homologous chromosomes and control opposite traits. Most traits are determined by two or more alleles. If the alleles are the same, the individual is homozygous for the trait. If the alleles are different, the individual is heterozygous for the trait. In a heterozygous pair, the dominant allele prevents the trait of the recessive allele from being expressed. In the present invention, the RTSW locus is a dominant allele.
  • line generally refers to a group of plants used non-commercially, such as for plant research.
  • a “line” typically displays very little overall variation among individuals in one or more traits of interest, although there may be some variation among individuals in other traits.
  • cell necrosis hypersensitive response is a typical disease resistance reaction of rapid cell necrosis after plant-pathogen incompatible interaction. It is a disease resistance mechanism of plants, accompanied by programmed cell death. It is specifically manifested as the emergence of cellular reactive oxygen species, the rapid response of related disease resistance marker genes, and local cell death.
  • avirulent gene or "avirulent gene NSm” means that according to the gene-for-gene hypothesis, for any host disease resistance gene, the pathogenic species has a corresponding avirulent gene. Only when the pathogen carrying the avirulent gene infects the host plant carrying the corresponding resistance gene, will the plant be induced to produce resistance, otherwise the plant will be infected and become diseased.
  • the avirulent gene NSm can produce a cell necrosis hypersensitive response (HR) in the host plant carrying the RTSW gene, and can be used to detect tobacco's resistance to spotted wilt.
  • HR hypersensitive response
  • the Chinese patent with patent number ZL201710414755.X discloses a method for identifying tobacco resistance using the NSm gene of tomato spotted wilt virus.
  • transgenic refers to the introduction of artificially isolated and modified genes into the genome of an organism.
  • the expression of the introduced genes causes heritable modifications to the traits of the organism, and this technology is called transgenic technology.
  • the commonly used terms “genetic engineering”, “genetic engineering” and “genetic transformation” are all synonyms for transgenic.
  • plant includes plants at any stage of maturity or development, and any cell, tissue or organ (plant part) derived from any of the plants.
  • Plant parts include, but are not limited to, seeds, fruits, stems, tubers, roots, flowers, ovules, stamens, leaves, embryos, meristem regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, etc.
  • transgenic plant and “transformed plant” are equivalent terms, which refer to "plants” as described above, wherein the plant comprises a heterologous nucleic acid molecule, a heterologous polynucleotide, or a heterologous polynucleotide construct introduced into the plant by, for example, any stable transformation method and transient transformation method disclosed elsewhere herein or otherwise known in the art.
  • transgenic plant and “transformed plant” also refer to, for example, a plant into which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct is first introduced, as well as any of its progeny plants comprising the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.
  • CRISPR/Cas is an adaptive immune system in bacteria and archaea that can specifically degrade the DNA of invading phages or exogenous plasmids.
  • CRISPR is the abbreviation of "clustered and regularly interspaced short palindromic sequences", and Cas refers to the protein that binds to CRISPR RNA.
  • gRNA small RNA molecule
  • the 20 nt (nucleotides) guide sequence (called Spacer) at the 5' end of the gRNA must be complementary to the sequence of the target DNA site (called Protospacer);
  • the target site must have a PAM (Protospacer-adjacent motif), of which the most widely used PAM sequence of Streptococcus pyogenes Cas9 is 5'-NGG-3'.
  • Pol II type II RNA polymerase
  • Pol III type III RNA polymerase
  • codon or “genetic code” refers to the triplets of three nucleotides starting from the start codon AUG on the messenger RNA molecule from the 5' end to the 3' end.
  • the genetic code determines the synthesis order of each amino acid on the peptide chain, as well as the start, extension and termination of protein synthesis.
  • the genetic code is a set of rules that translates the DNA or RNA sequence into the amino acid sequence of the protein in groups of three nucleotides for protein synthesis. Almost all organisms use the same genetic code, which is called the standard genetic code.
  • intron refers to intervening sequences in the DNA of eukaryotic cells. During the process of DNA transcription into RNA, introns on DNA will be transcribed into the precursor RNA, but introns on RNA will be spliced out before the RNA leaves the cell nucleus for translation. The part of the gene retained in the mature mRNA is called "exon”. The genes of eukaryotic organisms contain exons and introns, which is one of the characteristics that distinguish them from prokaryotes.
  • FIG. 1 shows the location, cloning, gene structure and protein characteristics of the RTSW gene.
  • A The image shows that the candidate disease resistance genes were narrowed down to the 0.8 Mb interval of 59.2Mb-60Mb on chromosome 3 of N.alata using the susceptible No. 16 and the resistant No. 12. The candidate genes contained in it are indicated by arrows.
  • B The gene structure of two candidate genes TNL1 and TNL2: It contains four exon structures and encodes TIR-NBS-LRR proteins.
  • TNL1 encodes a full-length TIR-NBS-LRR protein with a total length of 1089aa.
  • TNL2 encodes a truncated TIR-NBS-LRR protein with a total length of 817aa.
  • FIG. 2 shows the results of identifying TNL1 and TNL2 using NSm-induced HR.
  • A is a schematic diagram of the TNL expression cassette.
  • 35S represents the 35S promoter from "cauliflower mosaic virus";
  • TNLs represents any one of the full-length TNL1 gene, the full-length TNL1 CDs, the full-length TNL2 gene, and the full-length TNL2 CDs;
  • NOS represents the Nos terminator used for transcription termination.
  • TNLs+TSWV_NSm The left figure (TNLs+TSWV_NSm) shows the results of co-infiltration of EHA105-35S-TNLs and EHA105-35S-TSWV_NSm into susceptible tobacco K326 leaves (represented as 35S-TNL1, 35S-TNL1_CDs, 35S-TNL2, 35S-TNL2_CDs in the figure), and co-infiltration of EHA105-35S-Sw-5b and EHA105-35S-TSWV_NSm into leaves was used as a control (represented as 35S-Sw5b in the figure);
  • the right figure shows the results of co-infiltration of EHA105-35S-TNLs and EHA105-35S-TSWV_NSm into susceptible tobacco K326 leaves (represented as 35S-TNL1, 35S-TNL1_CDs, 35S-TNL2, 35S-TNL2_CDs in the figure), and co-infiltration of EHA105-35S-Sw
  • 35S-TNL1 and 35S-TNL1_CDs could induce HR by co-infiltration with TSWV_NSm or TZSV_NSm, while 35S-TNL2 or 35S-TNL2_CDs could not induce HR by co-infiltration with TSWV_NSm or TZSV_NSm.
  • 35S-TNL1 only could not induce HR, indicating that the HR phenotype was activated after co-infiltration of TNL1 and NSm.
  • Figure 3 shows gene editing of TNL1 and TNL2.
  • A shows the design of gRNA based on the conserved sequences of TNL1 and TNL2 and its construction into CRISPR/Cas9 vector.
  • B shows 21 independent T0 generation TNL1 and TNL2 editing events. The wild type without editing events is represented by wt; i represents the inserted base, and the number after i represents the number of inserted bases; d represents the deleted base, and the number after d represents the number of deleted bases.
  • C shows the induction of HR in leaves of T0 generation plants with edited TNL1 and/or TNL2 after infiltration with TSWV_NSm or TZSV_NSm.
  • Figure 4 shows the phenotype of proTNL1:TNL1 transgenic K326 tobacco after inoculation with some avirulent genes and viruses.
  • A is a schematic diagram of the proTNL1:TNL1 expression cassette.
  • proTNL1 represents the natural promoter of the TNL1 gene
  • TNL1 represents the full length of the TNL1 gene
  • NOS is the terminator.
  • B is the detection of avirulent gene-induced HR in the proTNL1:TNL1 T0 generation transgenic K326 positive line (#1).
  • the NSm genes of TSWV, INSV, TZSV and CCSV can all induce obvious HR, while the NSm genes of TNSV and PCSV cannot induce HR.
  • the co-infiltration of the tomato Sw-5b gene and TSWV_NSm (represented by Sw-5b+TSWV_NSm) was used as a positive control, and the infiltration of the empty vector (represented by EV) was used as a negative control.
  • Figure C shows two randomly selected proTNL1:TNL1 transgenic K326 positive lines (#1 and #2) inoculated with TSWV and TZSV. Photos were taken 21 days after inoculation, and the non-transgenic K326 showed obvious TSWV and TZSV virus symptoms, while the proTNL1:TNL1 transgenic line was not infected by the virus.
  • Figure 5 shows the phenotype of proTNL1:TNL1 transgenic N. benthamiana after inoculation with TSWV and TZSV.
  • Two proTNL1:TNL1 transgenic N. benthamiana positive lines (#1 and #4) and non-transgenic N. benthamiana (denoted by Nb) were inoculated with TSWV and TZSV.
  • Photos were taken 21 days after inoculation.
  • Non-transgenic N. benthamiana showed obvious TSWV and TZSV virus symptoms, while the proTNL1:TNL1 transgenic N. benthamiana line was not infected by the virus and did not show any virus symptoms.
  • Figure 6 shows the phenotype of proTNL1:TNL1 transgenic tomatoes after inoculation with TSWV and TZSV.
  • Two proTNL1:TNL1 transgenic tomato positive lines (#1 and #2) and non-transgenic tomatoes (denoted by S1) were inoculated with TSWV and TZSV.
  • Photos were taken 21 days after inoculation, and non-transgenic tomatoes showed obvious TSWV and TZSV virus symptoms, while the two proTNL1:TNL1 transgenic tomato lines were not infected by the virus and did not show any virus symptoms.
  • Figure 7 shows the phenotype of proTNL1:TNL1 transgenic potatoes after inoculation with TSWV and TZSV.
  • Two proTNL1:TNL1 transgenic potato positive lines (#1 and #2) and non-transgenic potatoes (indicated by St) were inoculated with TSWV and TZSV. Leaves were taken and photographed 21 days after inoculation. Non-transgenic potatoes showed obvious TSWV and TZSV virus symptoms, while the two proTNL1:TNL1 transgenic potato lines were not infected by the virus and did not show any virus symptoms.
  • nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three letter codes for amino acids.
  • the nucleotide sequences follow the standard convention of starting at the 5' end and proceeding toward the 3' end. Only one strand of each nucleotide sequence is shown, it being understood that the complementary strand of the strand shown is also included.
  • the amino acid sequences follow the standard convention of starting at the amino terminus of the sequence and proceeding toward the carboxyl terminus.
  • SEQ ID NO: 1 shows the nucleotide sequence of the RTSW gene (TNL1).
  • a stop codon e.g., TGA, TAG, TAA
  • TGA, TAG, TAA can be operably linked to the 3' end of the nucleic acid molecule comprising SEQ ID NO: 1.
  • SEQ ID NO: 2 shows the nucleotide sequence of the coding region of RTSW cDNA (TNL1 cDNA). If desired, a stop codon (e.g., TGA, TAG, TAA) can be operably linked to the 3' end of the nucleic acid molecule comprising SEQ ID NO: 2.
  • a stop codon e.g., TGA, TAG, TAA
  • SEQ ID NO: 3 shows the amino acid sequence of the RTSW protein (TNL1 protein) encoded by the RTSW gene (TNL1).
  • SEQ ID NO: 4 shows the nucleotide sequence of the natural promoter of the RTSW gene (TNL1).
  • SEQ ID NO: 5 shows the nucleotide sequence of TNL2 gene.
  • SEQ ID NO: 6 shows the nucleotide sequence of the coding region of TNL2 cDNA.
  • SEQ ID NO: 7 shows the amino acid sequence of TNL2 protein encoded by the TNL2 gene.
  • SEQ ID NO: 8 shows the nucleotide sequence of the expression cassette (proTNL1: TNL1) containing the RTSW gene and its natural promoter.
  • SEQ ID NO: 9 shows the nucleotide sequence of NaChr3_59.2M marker.
  • SEQ ID NO: 10 shows the nucleotide sequence of NaChr3_59.7M marker.
  • SEQ ID NO: 11 shows the nucleotide sequence of primer NaChr3_59.2MF.
  • SEQ ID NO: 12 shows the nucleotide sequence of primer NaChr3_59.2MR.
  • SEQ ID NO: 13 shows the nucleotide sequence of primer NaChr3_59.7MF.
  • SEQ ID NO: 14 shows the nucleotide sequence of primer NaChr3_59.7MR.
  • SEQ ID NO: 15 shows the nucleotide sequence of primer TNL1_35SF.
  • SEQ ID NO: 16 shows the nucleotide sequence of primer TNL1_35SR.
  • SEQ ID NO: 17 shows the nucleotide sequence of primer TNL2_35SF.
  • SEQ ID NO: 18 shows the nucleotide sequence of primer TNL2_35SR.
  • SEQ ID NO: 19 shows the nucleotide sequence of gRNA.
  • SEQ ID NO: 20 shows the nucleotide sequence of primer gRNA-F.
  • SEQ ID NO: 21 shows the nucleotide sequence of primer gRNA-R.
  • SEQ ID NO: 22 shows the nucleotide sequence of primer TNL1editTestF.
  • SEQ ID NO: 23 shows the nucleotide sequence of primer TNL1editTestR.
  • SEQ ID NO: 24 shows the nucleotide sequence of primer TNL2editTestF.
  • SEQ ID NO: 25 shows the nucleotide sequence of primer TNL2editTestR.
  • SEQ ID NO: 26 shows the nucleotide sequence of primer TNL1_NatProF.
  • 'Polata' is a TSWV-resistant tobacco material containing RTSW sites, described in non-patent literature (Laskowska D, A,2010.TSWV resistance in DH lines of tobacco(Nicotiana tabacum L)obtained from a hybrid between'Polalta'and Plant Breeding 129, 731-3.).
  • K326 is a TSWV-susceptible tobacco cultivar that does not contain the RTSW locus, which is described in non-patent literature (Edwards et al., 2017, A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448.). The public can obtain its reference genome sequence from the website https://solgenomics.net/organism/Nicotiana_tabacum/genome.
  • N.alata is a wild tobacco that is resistant to TSWV, which is recorded in non-patent literature (Laskowska et al., 2013, A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus (TSWV). Euphytica 193, 207-19.).
  • the accession number of N.alata in the American Tobacco Germplasm Bank is PI42334.
  • the 18 tobacco plants with no linkage drag and shortened RTSW introduced fragments were obtained by the applicant in the previous experiments, using ‘Polata’ as the male parent and ‘K326’ as the female parent for hybridization, and using K326 as the recurrent parent for multiple generations of backcrossing, and were screened by molecular marker detection and TSWV resistance detection.
  • the screening process is recorded in the international patent application with application number PCT/CN2021/129382, the invention name of “Tobacco plants resistant to spotted wilt without linkage drag and breeding methods thereof” and the Chinese patent application with application number 202111311707.0, the invention name of “Molecular markers for screening tobacco plants resistant to spotted wilt without linkage drag and their applications”, and the entire contents of the patent applications are hereby incorporated herein by reference.
  • K326 RTSW is a tobacco variety containing the RTSW locus. It is a tobacco variety resistant to tobacco spotted wilt obtained by hybridizing 'Polata' as the male parent and 'K326' as the female parent.
  • the public can obtain the above tobacco materials from tobacco germplasm resource preservation units or Yunnan Tobacco Agricultural Science Research Institute.
  • Tomato spotted wilt virus Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), tomato ring spot virus (TZSV), Calla lily chlorotic spot virus (CCSV), pepper chlorotic virus (CaCV), tomato necrotic spot virus (TNSV), pepper chlorotic spot virus (PCSV) and Amaryllis chlorotic ring spot virus (HCRV).
  • TSWV Tomato spotted wilt virus
  • INSV Impatiens necrotic spot virus
  • TZSV tomato ring spot virus
  • CCSV CSV
  • pepper chlorotic virus CaCV
  • tomato necrotic spot virus TNSV
  • PCSV pepper chlorotic spot virus
  • HCRV Amaryllis chlorotic ring spot virus
  • the expression vector pCambia1300-NSm-YFP containing the orthotospoviruses NSm gene was constructed in our laboratory and stored in the Yunnan Tobacco Agricultural Science Research Institute.
  • the 14 orthotospoviruses NSm genes are specifically the American TSWV NSm gene (NCBI accession number: JF960236.1), CSNV NSm gene (NCBI accession number: AF213675), GRSV NSm gene (NCBI accession number: KY350137) and INSV NSm gene (NCBI accession number: NC_003616), as well as the Eurasian TZSV NSm gene (NCBI accession number: KM374588), CCSV NSm gene (NCBI accession number: KT004454), MVbaV NSm gene (NCBI accession number: NC_003616).
  • NSm gene fragments of 14 viruses were cloned into the pCambia1300-YFP expression vector to obtain the recombinant plasmid pCambia1300-NSm-YFP.
  • the NSm gene is located downstream of the 35S promoter and forms a complete reading frame with the YFP gene, which can express the NSm-YFP fusion protein, in which the YFP tag is used to detect the protein expression level.
  • pCambia1300-NSm-YFP was introduced into Agrobacterium tumefaciens EHA105 to obtain the EHA105-pCambia1300-NSm-YFP series of strains.
  • pK2-35S-TSWV_NSm is a pK2GW7 plant expression vector with TSWV NSm gene (NCBI accession number: JF960236.1), which is a pK2-35S-NSm vector disclosed in Chinese patent No. ZL201710414755.X, and the invention name is "A method for identifying tobacco resistance using NSm gene of tomato spotted wilt virus", and the entire contents of the patent are incorporated herein by reference.
  • EHA105-35S-TSWV_NSm is a strain obtained by introducing pK2-35S-TSWV_NSm vector into Agrobacterium EHA105, and is stored in Yunnan Tobacco Agricultural Science Research Institute.
  • pK2-35S-TZSV_NSm is a pK2GW7 plant expression vector with the TZSV NSm gene (NCBI accession number: KM374588). Its construction method is the same as the above-mentioned pK2-35S-NSm vector, see Chinese patent ZL201710414755.X.
  • the TZSV NSm gene was cloned from the cDNA of TZSV.
  • EHA105-35S-TZSV_NSm is a strain obtained by introducing the pK2-35S-TZSV_NSm vector into Agrobacterium EHA105, and is stored in the Yunnan Tobacco Agricultural Science Research Institute.
  • p2300-35S-Sw-5b is a vector obtained by inserting the full length of tomato Sw-5b gene (NCBI accession number: AY007366) into the pCambia2300 plasmid.
  • the Sw-5b gene was cloned from the genomic DNA of tomato cultivar 43419.
  • the construction method of the p2300-35S-Sw-5b vector is recorded in the literature "Zhao W, Jiang L, Feng Z, Chen X, Huang Y, Xue F, Huang C, Liu Y, Li F, Liu Y et al. Plasmodesmata targeting and intercellular trafficking of Tomato spotted wilt tospovirus movement protein NSm is independent of its function in HR induction.
  • EHA105-35S-Sw-5b is a strain obtained by introducing p2300-35S-Sw-5b into Agrobacterium EHA105 and is stored in Yunnan Tobacco Agricultural Science Research Institute.
  • pRGEB31-PF is a CRISPR/Cas9 gene editing vector constructed by the applicant in the early stage and is stored in the Yunnan Tobacco Agricultural Science Research Institute.
  • the construction method of pRGEB31-PF is recorded in the Chinese invention patent with patent number ZL201811347308.8 and the invention name is "Recombinant vector for rapidly obtaining non-transgenic gene-edited plants and method of use", and the entire content of the patent is incorporated herein by reference.
  • Pos means positive
  • Neg means negative
  • HR means cell necrosis hypersensitive reaction
  • with HR means resistance to spotted wilt
  • without HR means no resistance to spotted wilt.
  • Typical resistance genes in plants i.e., resistance genes that conform to the "gene-for-gene” hypothesis, generally have a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) structure (Moffett, Advances in virus research, 2009, vol. 75, 1-33, 228-229).
  • NBS nucleotide binding site
  • LRR leucine-rich repeat
  • the inventors previously demonstrated that NSm is a non-toxic gene of the RTSW gene (see Chinese patent No. ZL201710414755.X), indicating that the RTSW gene is very likely a typical NBS-LRR resistance gene.
  • both YC03G182780 and YC03G182790 contain typical conserved domains such as TIR, NB-ARC and LRR, and are Toll-like receptor-nucleotide binding site-leucine-rich repeat (Toll/interleukin-1 receptor nucleotide-binding site leucine-rich repeat, TIR-NBS-LRR, TNL) type resistance genes (Table 4).
  • YC03G182790 and YC03G182780 were named TNL1 and TNL2, respectively, as candidate genes.
  • genomic DNA from N.alata and K326 RTSW tobacco leaves were extracted using a plant genomic DNA extraction kit (Tiangen Biochemical, Catalog No.: DP360) according to the product instructions.
  • the obtained genomic DNA was used as a template to amplify the full length of TNL1 gene using TNL1_35SF and TNL1_35SR primers, and the full length of TNL2 gene was amplified using TNL2_35SF and TNL2_35SR primers.
  • PCR system 2 ⁇ Phanta Max Buffer (Vazyme, Catalog No.: P505) 25 ⁇ l, dNTP Mix (10mM each) 1 ⁇ l, upstream primer (10 ⁇ M) 2 ⁇ l, downstream primer (10 ⁇ M) 2 ⁇ l, Phanta Max Super-Fidelity DNA Polymerase 1 ⁇ l, template DNA 2 ⁇ l, add ddH 2 O to 50 ⁇ L.
  • the PCR reaction program was: 95°C pre-denaturation for 3min; then 35 cycles: 95°C denaturation for 15s, 60°C annealing for 15s, 72°C extension for 4min; after the cycle, 72°C extension for 5min; and storage at 4°C.
  • the PCR products were cloned and sequenced, and sequence comparison showed that the TNL1 sequence sequenced from N.alata and K326 RTSW clones was consistent, and the TNL2 sequence was also consistent, and was completely consistent with the gene sequence assembled in the genome.
  • RNA from N.alata tobacco leaves was extracted using a plant total RNA extraction kit (Qiagen, catalog number: 74904) according to the product instructions.
  • the extracted RNA was then reverse transcribed using a cDNA one-strand synthesis kit (Vazyme, catalog number: R312-01) to obtain cDNA.
  • the reverse transcription system and procedure refer to the kit instructions.
  • TNL1_35SF and TNL1_35SR primers were used to amplify the full-length CDs of TNL1 (coding region of cDNA), and TNL2_35SF and TNL2_35SR primers were used to amplify the full-length CDs of TNL2.
  • the PCR system and PCR procedure were the same as above.
  • the PCR products were sequenced to obtain the full-length sequence of CDs.
  • the full-length sequence of CDs was compared with the N.alata genome sequence, and the transcript structure of the gene was analyzed.
  • Primer name Primer sequences serial number TNL1_35SF catttggagaggacacgctcgagATGGATACTCAATTAGTTAGAGG SEQ ID NO:15 TNL1_35SR tctcattaaagcaggactctagaGATTTGGCCAAGGGAAAAAGATTACG SEQ ID NO:16 TNL2_35SF catttggagaggacacgctcgagATGGATACTCAATTAGTTAGAGTAG SEQ ID NO: 17 TNL2_35SR tctcattaaagcaggactctagaGTTAGGATTGGTTGGGTGGACTA SEQ ID NO:18
  • the amplification products of TNL1_35SF and TNL1_35SR primers contain 230bp of 3’UTR region.
  • the amplification products of TNL2_35SF and TNL2_35SR primers contain 150bp of 3’UTR region.
  • the above primers contain overlap sequences (indicated by lowercase letters) at both ends of phellsgate8 linearization for recombinant cloning.
  • TNL1 and TNL2 have a structure of 4 exons and 3 introns (Figure 1B).
  • the full length of the TNL1 gene is 4532bp (the gene sequence without the stop codon is shown in SEQ ID NO: 1); the full length of CDs is 3270bp (the CDs sequence without the stop codon is shown in SEQ ID NO: 2), encoding a protein sequence of 1089 amino acids (SEQ ID NO: 3).
  • the full length of the TNL2 gene is 4630bp (SEQ ID NO: 5); the full length of CDs is 2454bp (SEQ ID NO: 6), encoding a protein sequence of 817 amino acids (SEQ ID NO: 7).
  • TNL2 and TNL1 are similar, TNL2 has a premature stop codon in the fourth exon, resulting in a truncated CDs.
  • TNL1 and TNL2 are RTSW genes.
  • a preliminary verification was carried out using the disease resistance gene identification system established by the inventors in the early stage (see Chinese invention patent No. ZL201710415015.8, invention name "A method for screening disease resistance genes using NSm gene of tomato spotted wilt virus", the entire content of which is hereby incorporated by reference into this article).
  • the phellsgate8 empty plasmid was double-digested with XhoI and XbaI restriction endonucleases to obtain a linearized phellsgate8 plasmid.
  • the full-length TNL1 gene, full-length CDs of TNL1, full-length TNL2 gene, and full-length CDs of TNL2 obtained in Example 2 were cloned into the phellsgate8 plasmid according to the product instructions using the ClonExpress one-step cloning kit (Vazyme, catalog number C112-01) to obtain recombinant plasmids 35S-TNL1, 35S-TNL1_CDs, 35S-TNL2, and 35S-TNL2_CDs.
  • 35S-TNL1 expresses the full-length TNL1 gene
  • 35S-TNL1_CDs expresses the full-length CDs of TNL1
  • 35S-TNL2 expresses the full-length TNL2 gene
  • 35S-TNL2_CDs expresses the full-length CDs of TNL2.
  • the expression of these genes is driven by the 35S promoter.
  • 35S-TNL1, 35S-TNL1_CDs, 35S-TNL2 and 35S-TNL2_CDs were transformed into Escherichia coli DH5 ⁇ by heat shock method and sequenced to obtain recombinant plasmids with accurate insertion sequences.
  • 35S-TNL1, 35S-TNL1_CDs, 35S-TNL2 and 35S-TNL2_CDs were introduced into Agrobacterium tumefaciens EHA105 strain, respectively, to obtain recombinant bacteria EHA105-35S-TNL1, EHA105-35S-TNL1_CDs, EHA105-35S-TNL2 and EHA105-35S-TNL2_CDs.
  • infiltration buffer 10mmol/L MgCl2 , 10mmol/L MES, 200 ⁇ mol/L acetosyringone
  • EHA105-35S-TSWV_NSm bacterial suspension and EHA105-35S-TZSV_NSm bacterial suspension were mixed with EHA105-35S-TNL1, EHA105-35S-TNL1_CDs, EHA105-35S-TNL2 or EHA105-35S-TNL2_CDs bacterial suspensions at a volume ratio of 1:1 to obtain 8 test bacterial suspensions.
  • EHA105-35S-TSWV_NSm bacterial suspension and EHA105-35S-Sw-5b bacterial suspension were mixed at a volume ratio of 1:1 as a positive control.
  • the OD600 of the control bacterial suspension was controlled to be 0.5.
  • Inoculation of tobacco leaves Use a sterile syringe without a needle to inject 9.5-10.5 microliters of the test bacterial suspension from the back of the leaf of the susceptible tobacco 'K326' into the leaf veins to form a visible infiltration spot; place the inoculated tobacco in an environment of 20-28°C and 80% humidity, alternately expose to continuous light for 16 hours and continuous darkness for 8 hours, and observe for a total of 72 hours. In the same way, inoculate the positive control and the single EHA105-35S-TNL1 bacterial suspension on the leaves of the susceptible tobacco 'K326'.
  • test bacterial suspension can induce a cell necrosis hypersensitive response (HR) on the leaves of the susceptible tobacco 'K326', it indicates that the candidate gene in the test bacterial suspension is a disease-resistant gene of the non-toxic gene NSm in the test bacterial suspension.
  • HR cell necrosis hypersensitive response
  • the EHA105-35S-TNL1 bacterial suspension alone could not induce HR in ‘K326’ tobacco, indicating that the HR phenotype was produced by the co-activation of the TNL1 gene and the NSm gene ( Figure 2B). Therefore, it can be preliminarily determined that the candidate gene TNL1 is the RTSW gene.
  • TNL2 and TNL1 are only about 4 Kb apart and have similar gene structures, TNL2 is not related to the HR stimulated by NSm and may not play a role in resistance to tobacco spotted wilt.
  • TNL1 and TNL2 are RTSW genes
  • the recombinant vector used in the gene editing system is a CRISPR/Cas9 vector for plant gene editing that carries PAP1 and NtFT expression elements (PF Cassete). Since the NtFT expression element produces a protein that promotes early flowering of plants, and the PAP1 expression element produces a protein that promotes the biosynthesis of anthocyanins in plants, the color of the T0 transgenic plant can be used to determine whether an editing event has occurred. If the plant contains a transgenic element, it will appear purple, and if it does not contain a transgenic element, it will appear green.
  • TNL1 and TNL2 have high homology, so a gRNA with a conserved sequence can be used to edit TNL1 and TNL2 separately or simultaneously.
  • the target sequence was designed based on the conserved sequence in the first exon of TNL1 and TNL2: CTATGATGTTGATCCTTCTG (SEQ ID NO: 19).
  • the 3' of the gRNA is adjacent to the Cas9 nuclease PAM sequence of "AGG" ( Figure 3A).
  • the two primers for synthesizing the gRNA are as follows:
  • gRNA-F 5’-GGCACTATGATGTTGATCCTTCTG-3’ (SEQ ID NO: 20)
  • gRNA-R 3’-CAGAAGGATCAACATCATAGCAAA-5’ (SEQ ID NO: 21)
  • the construction method of the pRGEB31-PF vector is described in Chinese patent No. ZL201811347308.8.
  • the target sequence is inserted into the pRGEB31-PF vector through two adjacent BsaI sites on the vector, so the upstream sequence (gRNA-F) needs to add a GGCA linker, and the downstream sequence (gRNA-R) needs to add an AAAC linker.
  • the primers gRNA-F and gRNA-R are annealed to form a dimer structure. Annealing system: gRNA-F 20 ⁇ l, gRNA-R 20 ⁇ l, 10 ⁇ Annealing buffer 5 ⁇ l, H 2 O 5 ⁇ l.
  • Annealing program 95°C5min, 90°C1min, 80°C1min, 70°C1min, 60°C1min, 50°C1min, 40°C1min, 30°C1min, 20°C1min, 10°C1min.
  • the gRNA is inserted into the pRGEB31-PF vector after BsaI digestion to obtain a ligation product.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ competent cells, the plasmid was extracted and sequenced to obtain the recombinant plasmid Cas9-PF-gRNA with the correct insertion sequence.
  • Cas9-PF-gRNA was electroporated into Agrobacterium EHA105, and an Agrobacterium-mediated tobacco transformation experiment was performed.
  • K326 RTSW tobacco with a genotype of RTSW heterozygous (RTSW/rtsw) was used as a material to induce callus tissue and used in tobacco transformation experiments.
  • RTSW/rtsw genotype of RTSW heterozygous
  • only one allele needs to be edited to completely disable the gene to be verified and produce a phenotype.
  • resistant callus differentiation and regeneration obtained T0 generation transgenic positive plants.
  • Primer name Primer sequences serial number TNL 1editTestF AGGTGAAATTATGGCGAAAGTG SEQ ID NO:22 TNL 1editTestR CTTCAGTAAGTGCAGCTCTCC SEQ ID NO:23 TNL2editTestF CTGGTCATCTTTATTCCAAATTAGA SEQ ID NO:24 TNL2editTestR TTGAATCAAGAATATGTACCCGTCAG SEQ ID NO:25
  • the genomic DNA of 30 T0 transgenic positive plants was extracted using a plant genomic DNA extraction kit (Tiangen Biochemical, Cat. No.: DP360) according to the product instructions.
  • a plant genomic DNA extraction kit Tiangen Biochemical, Cat. No.: DP360
  • the sequence containing the TNL1 editing site was amplified using TNL1editTestF and TNL1editTestR primers
  • the sequence containing the TNL2 editing site was amplified using TNL2editTestF and TNL2editTestR primers.
  • the PCR system and procedure are the same as those in Example 2.
  • the PCR products were sequenced by Thermo Fisher Scientific (Guangzhou), and the sequencing primers were TNL1editTestF and TNL2editTestF.
  • the sequencing products were analyzed to determine whether base substitution, deletion or insertion occurred at the target site, that is, 3 bp upstream of PAM, to determine whether an editing event occurred.
  • the results showed that among the 30 T0 transgenic positive plants, 21 plants were edited at the TNL1 or TNL2 target site, with an editing efficiency of 70%. Among them, 5 plants were edited with TNL1 alone, 4 plants were edited with TNL2 alone, and the remaining 12 plants were edited with TNL1 and TNL2 at the same time (Figure 3B).
  • TSWV resistance detection can be directly performed on T0 plants.
  • the NSm gene was used to detect TSWV resistance in T0 generation plants.
  • an infiltration buffer (10mmol/L MgCl 2 , 10mmol/L MES, 200 ⁇ mol/L acetosyringone
  • EHA105-35S-TZSV_NSm was inoculated on tobacco leaves in the same way.
  • TSWV_NSm or TZSV_NSm can induce necrosis hypersensitive response (HR) on the leaves of T0 generation plants, it indicates that the T0 generation plants are resistant to TSWV or TZSV and their RTSW gene is functional; if TSWV_NSm or TZSV_NSm cannot induce HR on the leaves of T0 generation plants, it indicates that the T0 generation plants are not resistant to TSWV or TZSV and their RTSW gene is nonfunctional.
  • HR necrosis hypersensitive response
  • TSWV_NSm and TZSV_NSm could not induce HR in 5 plants with TNL1 edited alone and 12 plants with TNL1 and TNL2 edited simultaneously; TSWV_NSm and TZSV_NSm could induce obvious HR in 4 plants with TNL2 edited alone and 9 plants with neither TNL1 nor TNL2 edited (Table 7, Figure 3C).
  • T0 generation plant number Edit Type TSWV_NSm TZSV_NSm 1 TNL1 alone is edited No HR No HR 2 TNL1 and TNL2 are edited simultaneously No HR No HR 3 Neither TNL1 nor TNL2 was edited HR HR 4 TNL2 is edited alone HR HR 5 TNL1 alone is edited No HR No HR 6 TNL1 and TNL2 are edited simultaneously No HR No HR 7 TNL1 and TNL2 are edited simultaneously No HR No HR 8 Neither TNL1 nor TNL2 was edited HR HR 9 TNL1 alone is edited No HR No HR 10 TNL1 and TNL2 are edited simultaneously No HR No HR No HR 11 TNL1 alone is edited No HR No HR No HR 12 TNL2 is edited alone HR HR 13 Neither TNL1 nor TNL2 was edited HR HR 14 TNL1 and TNL2 are edited simultaneously No HR No HR 15 TNL2 is edited alone HR HR 16 Neither TNL1 nor TNL2 was edited HR HR 17 TNL1 and TNL2 are edited simultaneously No HR No HR 18 Neither TNL1 nor TNL2 was edited HR HR
  • T1 generation seeds were sown in a light culture room and T1 generation plants were cultivated by conventional methods. Since the Cas9-PF-gRNA vector carries anthocyanin visualization markers, if the T1 generation plants contain transgenic elements, they will appear purple, and if they do not contain transgenic elements, they will appear green. When the T1 generation plants have 4-5 leaves, completely green plants are picked, and the genomic DNA of each plant is extracted for genotype and editing homozygosity detection. Using genomic DNA as a template, PCR reactions were performed using primer pairs TNL1editTestF/TNL1editTestR and TNL2editTestF/TNL2editTestR, respectively.
  • PCR result is negative, it indicates that the genotype of the plant is rtsw/rtsw, and the plant is an isolated plant that does not contain the RTSW introduced fragment. Therefore, only plants with positive PCR results are analyzed in the next step.
  • the sequencing results showed that the T1 generation plants with positive PCR results all contained expected mutants, indicating that the editing event is heritable and has all been homozygous mutations.
  • TSWV and TZSV diseased leaf sap were inoculated on T1 generation plants with positive PCR results. The TSWV and TZSV disease incidence of the plants was investigated 14 days and 21 days after inoculation (Table 8).
  • the phellsgate8 empty plasmid was double-digested with SacI and XbaI restriction endonucleases to obtain a linearized phellsgate8 plasmid.
  • the PCR product was cloned into the phellsgate8 plasmid using the ClonExpress one-step cloning kit (Vazyme, catalog number C112-01) according to the product instructions to obtain the recombinant plasmid phellsgate8-proTNL1:TNL1.
  • the recombinant plasmid phellsgate8-proTNL1:TNL1 was transformed into Escherichia coli DH5 ⁇ , and positive clones were selected for sequencing.
  • the sequencing results showed that the amplified proTNL1:TNL1 was completely consistent with the gene sequence assembled in the genome, and the proTNL1:TNL1 sequence inserted in the recombinant plasmid was accurate and had no mutations.
  • the phellsgate8-proTNL1:TNL1 was introduced into the EHA105 strain of Agrobacterium tumefaciens to obtain the recombinant bacterium EHA105-phellsgate8-proTNL1:TNL1.
  • EHA105-phellsgate8-proTNL1:TNL1 was cultured in the LB medium of Agrobacterium tumefaciens at 28°C for 24 hours, and the cells were collected by centrifugation.
  • Susceptible tobacco ‘K326’ was used as the material to induce callus tissue, and EHA105-phellsgate8-proTNL1:TNL1 was used to infect K326 callus tissue. After kanamycin resistance screening, resistant callus tissue was obtained. Resistant callus tissue was differentiated and regenerated to obtain T0 transgenic strains. Genomic DNA of T0 transgenic plants was extracted, and PCR reaction was performed using TNL1editTestF/TNL1editTestR primer pair. Plants with positive PCR results were proTNL1:TNL1 transgenic positive plants.
  • TNL1 transgenic plants In order to prove the resistance of TNL1 transgenic plants to viruses, two T0 generation proTNL1:TNL1 transgenic positive plants (proTNL1:TNL1-#1 and proTNL1:TNL1-#2) were randomly selected and self-crossed to obtain T1 generation seeds. T1 generation seeds were sown separately in a light culture room and T1 plants were cultivated by conventional methods. When the T1 plants had 4-5 leaves, the genomic DNA of each plant was extracted and transgenic detection was performed using TNL1editTestF/TNL1editTestR primers. T1 plants with positive test results were selected and inoculated with TSWV, INSV, TZSV, CCSV, CaCV, TNSV, PCSV and HCRV diseased leaf sap. The disease condition of the plants was investigated 21 days after inoculation (Table 10).
  • the NSm gene of orthotospoviruses is the Avr gene corresponding to the resistance gene RTSW.
  • the NSm infiltration of the virus can be used to detect the orthotospovirus resistance of RTSW plants.
  • the detection method can be found in the Chinese patent No. ZL201710414755.X.
  • NSm expression vectors of 14 orthotospoviruses including American TSWV, CSNV, GRSV and INSV, and Eurasian TZSV, CCSV, MVBaV, CaCV, GBNV, TNSV, PCSV, TNRV, PolRSV and HCRV.
  • the NSm gene fragments of the 14 viruses were cloned into the pCambia1300-YFP expression vector to obtain the recombinant plasmid pCambia1300-NSm-YFP.
  • pCambia1300-NSm-YFP the NSm gene is located downstream of the 35S promoter and forms a complete reading frame with the YFP gene, which can express the NSm-YFP fusion protein, in which the YFP tag is used to detect the protein expression level.
  • pCambia1300-NSm-YFP was introduced into Agrobacterium tumefaciens EHA105 to obtain EHA105-pCambia1300-NSm-YFP.
  • pCambia1300-YFP was introduced into Agrobacterium tumefaciens EHA105 to obtain EHA105-pCambia1300-YFP as an empty vector control strain.
  • the NSm genes of all four tested American types TSWV, CSNV, GRSV and INSV, and the five tested Eurasian types TZSV, CCSV, MVBaV, CaCV and GBNV could induce obvious HR like the positive control, while the NSm genes of TNSV, PCSV, TNRV, PolRSV and HCRV could not induce HR like the empty vector control.
  • the NSm genes of all viruses could not induce any degree of HR (Table 11).
  • Plants with positive PCR were proTNL1:TNL1 transgenic positive plants.
  • N. benthamiana and tomato 2-3 T0 transgenic positive plants were randomly selected and self-crossed to obtain T1 seeds.
  • T0 generation tubers were directly harvested.
  • T1 generation seeds or T0 generation tubers of transgenic positive plants were sown in a light culture room and the plants were cultivated by conventional methods. When the plants had 4-5 leaves, the genomic DNA of each plant was taken for transgenic element detection.
  • Plants with positive test results were selected and inoculated with TSWV and TZSV diseased leaf juice, respectively. The TSWV and TZSV incidence of the plants were investigated 14 days and 21 days after inoculation, respectively (Table 12).

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Abstract

本发明涉及基因分离和植物改良，具体涉及来自烟草的抗斑萎病基因RTSW及其应用。提供用于赋予或增强植物对正番茄斑萎病毒属病毒的抗性的核酸分子，以及包含所述核酸分子的表达盒、载体、宿主细胞、植物和种子。所述核酸分子编码RTSW基因产物或其变体。还提供用于赋予或增强植物对正番茄斑萎病毒属病毒的抗性的方法，其包括将所述核酸分子导入植物细胞中。

Description

来自烟草的抗斑萎病基因RTSW及其应用 技术领域
本发明涉及基因分离和植物改良，具体涉及来自烟草的抗斑萎病基因RTSW及其应用。
背景技术
正番茄斑萎病毒属病毒(Orthotospoviruses)属于布尼亚病毒目番茄斑萎病毒科，以其广泛的寄主范围(能侵染超过84个科1090种植物)和造成的巨大经济损失已经成为农业生产的巨大威胁，被公认是植物病毒中最具破坏性的病毒之一，在全球最重要的植物病毒名单中排名第二。烟草斑萎病(Tobacco spotted wilt disease，TSWD)是由orthotospoviruses侵染所造成的严重病害。TSWD造成烟株矮化、叶片皱缩、在叶片上可密布小的坏死环，这些环常常合并为大斑，形成不规则的坏死区；病害从发病部位迅速扩张至烟株顶端，顶芽萎蔫下垂，最终导致整株坏死，完全失去经济价值。已有的报道表明，多种orthotospoviruses均可以侵染烟草造成TSWD，主要包括美洲型病毒：番茄斑萎病毒(Tomato spotted wilt virus，TSWV)和欧亚型病毒：番茄环纹斑点病毒(Tomato zonate spot virus，TZSV)等。目前在栽培烟草(Nicotiana tabacum L.)中没有抗TSWD的烤烟品种。选育广谱抗orthotospoviruses的品种是最经济、最有效的防治手段，可以从根本上满足农业绿色防控的需求。
番茄和辣椒中已经报道有抗病基因资源，分别为Sw-5b和Tsw基因，均为CC-NBS-LRR结构类型的抗病基因。Sw-5b基因对美洲型的多个orthotospoviruses有抗性，但对于危害严重的欧亚型orthotospoviruses没有抗性。Tsw基因仅对TSWV有抗性。除了这两个基因，还未在其他作物中发现有关orthotospoviruses抗性基因或抗性位点的报道。针对Sw-5b和Tsw基因的抗病突破病毒株系已经在多国报道，因此急需新的抗病基因资源。
前期的研究表明，具翼烟草(Nicotiana alata)对TSWV具有很好的抗性。具翼烟草(N.alata)接种TSWV后仅在接种叶上表现轻微过敏性坏死症状，在系统叶中检测不到病毒的存在。利用耳状烟草(N.otophora)作为桥梁亲本，Gajos等人成功地将TSWV抗性位点(该抗性位点命名为RTSW位点，其中RTSW是 Resistance to  TSWV的简称)从N.alata转育至栽培烟草中，产生了包含长RTSW导入片段的育种材料‘Polalta’(Laskowska D，
Figure PCTCN2022124977-appb-000001
A，2010.TSWV resistance in DH lines of tobacco(Nicotiana tabacum L.)obtained from a hybrid between‘Polalta’and
Figure PCTCN2022124977-appb-000002
Plant Breeding 129，731-3.)。
野生近缘种中往往同时存在许多对农艺性状不利的基因，如果这些基因和要转移的目标基因紧密连锁，就会导致连锁累赘，增加优异基因利用的难度。抗斑萎病烟草‘Polalta’及其他包含长RTSW导入片段的栽培烟草都表现出比较严重的连锁累赘。遗传关系表明，这些连锁累赘来源于长RTSW导入片段上与RTSW位点紧密连锁或共分离的连锁累赘基因组分。发明人课题组通过遗传位点分析、分子标记开发及大规模筛选获得了无连锁累赘的抗斑萎病烟草植物，参见申请号为PCT/CN2021/129382，发明名称为“无连锁累赘的抗斑萎病烟草植物及其选育方法”的国际专利申请，以及申请号为202111311707.0，发明名称为“用于筛选无连锁累赘的抗斑萎病烟草植物的分子标记及其应用”的中国专利申请，通过引用将其全部内容 合并到本文中。
到目前为止，赋予烟草TSWV抗性的RTSW基因尚未克隆，限制了RTSW基因的抗病机制研究及育种利用。除烟草以外，其他作物的抗斑萎病育种也存在可用抗性基因资源少的问题，因此RTSW基因的克隆和利用也将有利于其他作物的斑萎病防治。
发明内容
本发明涉及植物抗性基因的分离，具体涉及赋予植物(特别是茄科植物)对至少一种正番茄斑萎病毒属病毒的抗性的RTSW基因的分离。所述RTSW基因来源于具翼烟草N.alata，分离自发明人前期创制的无连锁累赘的抗斑萎病烟草。本发明的RTSW基因的核苷酸序列包括但不限于野生型RTSW基因(包含天然启动子及含编码区的3’相邻区)的核苷酸序列、cDNA序列和仅包含编码区的核苷酸序列。所述RTSW基因的核苷酸序列的实例包括SEQ ID NO：1和SEQ ID NO：2所示的核苷酸序列及其变体。所述RTSW基因编码RTSW蛋白。所述RTSW蛋白能够赋予包含所述RTSW蛋白的植物(特别是茄科植物)对至少一种正番茄斑萎病毒属病毒的抗性。所述RTSW蛋白的氨基酸序列的实例包括SEQ ID NO：3所示的氨基酸序列及其变体。
经实验证明，本发明克隆的RTSW基因不仅对美洲型的多种正番茄斑萎病毒属病毒有抗性，对于危害严重的欧亚型正番茄斑萎病毒属病毒也有抗性。转基因实验结果显示，在本氏烟、番茄和马铃薯中单独表达RTSW基因就可以赋予植株对正番茄斑萎病毒属病毒的广谱抗性。因此RTSW是可以利用的宝贵的抗病基因资源。
本发明提供包含RTSW基因或其变体的核苷酸序列的核酸分子，其中所述变体为天然存在的或非天然存在的变体。在一些实施例中，本发明的核酸分子包含选自以下(a1)-(a5)的核苷酸序列：
(a1)SEQ ID NO：1所示的核苷酸序列；
(a2)SEQ ID NO：2所示的核苷酸序列；
(a3)与SEQ ID NO：1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
(a4)编码SEQ ID NO：3所示的氨基酸序列的核苷酸序列；
(a5)编码与SEQ ID NO：3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
本发明的核酸分子包括与天然多核苷酸(例如SEQ ID NO：1或2所示的多核苷酸或编码SEQ ID NO：3所示的氨基酸序列的多核苷酸)的序列基本相似、功能类似的多核苷酸衍生物或者变体。所述天然多核苷酸包含天然存在的核苷酸序列。所述多核苷酸衍生物或者变体包括：在天然多核苷酸的5’和/或3’端缺失和/或添加一个或多个核苷酸的多核苷酸；在天然多核苷酸的一个或多个内部位点缺失和/或添加一个或多个核苷酸的多核苷酸；在天然多核苷酸的一个或多个位点取代一个或多个核苷酸的多核苷酸；和/或因遗传密码的简并性而编码本发明的RTSW蛋白之一的多核苷酸。所述多核苷酸衍生物或者变体包括天然存在的多核苷酸， 例如天然存在的等位基因变体，可使用众所周知的分子生物学技术(例如聚合酶链式反应和杂交技术)进行鉴定；以及合成的多核苷酸，例如通过定点突变生成的仍编码本发明的RTSW蛋白的多核苷酸。在本发明的一些实施例中，所述多核苷酸衍生物或者变体与所述天然多核苷酸具有至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列一致性。所述多核苷酸衍生物或者变体编码的氨基酸序列与SEQ ID NO：3所示的氨基酸序列具有至少90％的序列一致性，并具有一样的抗病功能。
本发明涉及多核苷酸构建体，其包含本发明的核酸分子以及可操作连接的用于在原核或真核宿主细胞中进行基因转录和表达的一个或多个调控元件。所述多核苷酸构建体可以是质粒、粘粒、噬菌体或者表达载体、转化载体、穿梭载体或克隆载体。它可以是双链的或者单链的，线性的或者环状的，可以通过整合入基因组或存在于染色体外的方式转化原核或真核宿主细胞。
本发明提供一种表达盒，其包含本发明的核酸分子和可操作连接的启动子。所述启动子可以是RTSW基因的天然启动子，例如核苷酸序列如SEQ ID NO：4所示的启动子；也可以是异源启动子，例如组成型诱导的启动子(例如本领域已知的“花椰菜花叶病毒”来源的35S启动子)，或病原体诱导的启动子(例如PR1基因启动子)。
本发明提供一种载体，其包含本发明的核酸分子或表达盒。
本发明的核酸分子、表达盒或载体可用于转化任何植物，包括但不限于单子叶植物和双子叶植物。所述植物优选为茄科植物。
本发明提供一种宿主细胞，其转化有本发明的核酸分子、表达盒或载体。所述宿主细胞可以是原核细胞(例如细菌细胞)或真核细胞(例如酵母细胞或植物细胞)。在本发明的一些实施例中，所述宿主细胞为转化有本发明的核酸分子或载体的农杆菌，例如根癌农杆菌(Agrobacterium tumefaciens)或毛根农杆菌(Agrobacterium rhizogenes)，或植物细胞。获得所述宿主细胞的方法包括但不限于本领域熟知的化学药物诱导、热转化、基因枪转化、农杆菌介导的转化、原生质体转染、转导、真空渗透或电穿孔的方法。例如，通过化学药物诱导、热转化或电穿孔等方法将本发明的核酸分子、表达盒或载体导入农杆菌；通过基因枪转化、农杆菌介导的转化、原生质体转染、转导或植物病毒介导的基因导入等方法将本发明的核酸分子、表达盒或载体导入植物细胞。
本发明涉及包含转基因植物细胞(转化有本发明的核酸分子、表达盒或载体的植物细胞)的转基因植物或其部分。这里的部分可以是细胞、组织、器官或若干细胞、组织或器官的组合，例如花、叶、块茎、果实或种子。所述转基因植物，特别是茄科植物，与非转基因植物相比，具有对多种正番茄斑萎病毒属病毒更高的抗性。
本发明提供一种转基因植物，其包含稳定并入其基因组中的异源多核苷酸构建体，所述多核苷酸构建体包含选自以下(a1)-(a5)的核苷酸序列：
(a1)SEQ ID NO：1所示的核苷酸序列；
(a2)SEQ ID NO：2所示的核苷酸序列；
(a3)与SEQ ID NO：1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
(a4)编码SEQ ID NO：3所示的氨基酸序列的核苷酸序列；
(a5)编码与SEQ ID NO：3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
在本发明的一些实施例中，所述多核苷酸构建体还包含可操作连接的用于在植物中表达所述核苷酸序列的启动子。合适的启动子可以是RTSW基因的天然启动子，例如核苷酸序列如SEQ ID NO：4所示的启动子。
在本发明的一些实施例中，所述转基因植物为茄科植物。所述茄科植物包括但不限于栽培的茄科植物。
在本发明的一些实施例中，所述茄科植物为烟草、马铃薯或番茄。
在本发明的一些实施例中，相对于对照植物，所述转基因植物包含对至少一种正番茄斑萎病毒属病毒的抗性。所述对照植物是指不含有所述多核苷酸构建体的用于获得所述转基因植物的出发植物。
在本发明的一些实施例中，相对于对照植物，所述转基因植物包含对番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)、花生环斑病毒(GRSV)、菊花茎坏死病毒(CSNV)、番茄环纹斑点病毒(TZSV)、花生芽坏死病毒(GBNV)、桑脉带相关病毒(MVBaV)、辣椒褪绿病毒(CaCV)和马蹄莲褪绿斑病毒(CCSV)的抗性。
获得本发明的转基因植物的方法包括但不限于人工从头基因合成方法、转基因技术、原生质体再生、杂交育种方法、基因组编辑方法。所述基因组编辑方法包括CRISPR/Cas9技术、锌指核酸内切酶(ZFN)技术、类转录激活因子效应物核酸酶(TALEN)技术等。
本发明提供来自任一所述的转基因植物的种子，所述种子包含本发明的核酸分子。
本发明提供用于赋予或增强植物对至少一种正番茄斑萎病毒属病毒的抗性的方法，其包括将异源多核苷酸构建体导入至少一个植物细胞中，所述多核苷酸构建体包含选自以下(a1)-(a5)的核苷酸序列：
(a1)SEQ ID NO：1所示的核苷酸序列；
(a2)SEQ ID NO：2所示的核苷酸序列；
(a3)与SEQ ID NO：1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
(a4)编码SEQ ID NO：3所示的氨基酸序列的核苷酸序列；
(a5)编码与SEQ ID NO：3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
在本发明的一些实施例中，所述多核苷酸构建体还包含可操作连接的用于在植物中表达所述核苷酸序列的启动子，并且所述多核苷酸构建体被稳定并入所述植物细胞的基因组中。
在本发明的一些实施例中，使所述植物细胞再生成在其基因组中包含所述多核苷酸构建体的植物。可以采用本领域己知的再生方法。
在本发明的一些实施例中，所述植物为烟草、马铃薯或番茄。
在本发明的一些实施例中，相对于对照植物，所述植物包含对至少一种正番茄斑萎病毒属病毒的抗性。
在本发明的一些实施例中，相对于对照植物，所述植物包含对番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)、花生环斑病毒(GRSV)、菊花茎坏死病毒(CSNV)、番茄环纹斑点病毒(TZSV)、花生芽坏死病毒(GBNV)、桑脉带相关病毒(MVBaV)、辣椒褪绿病毒(CaCV)和马蹄莲褪绿斑病毒(CCSV)的抗性。
本发明提供通过本发明的方法产生的植物、或所述植物的果实、块茎、叶子或种子，其中所述果实、块茎、叶子或种子包含所述多核苷酸构建体。
本发明提供用于鉴定包含赋予的或增强的对至少一种正番茄斑萎病毒属病毒的抗性的植物的方法，其包括检测所述植物中或其至少一个部分或细胞中RTSW的存在。
在本发明的一些实施例中，所述植物为烟草、马铃薯或番茄。
在本发明的一些实施例中，通过检测RTSW中的至少一个标记来检测RTSW的存在。
本发明提供用于检测RTSW基因的引物以及包含所述引物的试剂盒，所述引物的核苷酸序列如SEQ ID NO：22和23所示。使用所述引物检测RTSW基因的方法包括：从待测植物或种质中分离核酸；在分离的核酸中利用所述引物进行PCR。
本发明提供一种多肽，其包含选自以下(B1)-(B4)的氨基酸序列：
(B1)由SEQ ID NO：1所示的核苷酸序列编码的氨基酸序列；
(B2)由SEQ ID NO：2所示的核苷酸序列编码的氨基酸序列；
(B3)SEQ ID NO：3所示的氨基酸序列；
(B4)与SEQ ID NO：3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列，其中包含所述氨基酸序列的多肽能够赋予包含所述多肽的植物对至少一种正番茄斑萎病毒属病毒的抗性。
本发明的多肽可以由多种方式产生，包括但不限于体外从头合成、原核细胞表达和真核细胞表达。
本发明的多肽包括与天然蛋白质(例如氨基酸序列如SEQ ID NO：3所示的蛋白质)的序列基本相似、功能类似的多肽衍生物或者变体。所述天然蛋白质包含天然存在的氨基酸序列。所述多肽衍生物或者变体可来自遗传多态性或人工操作。所述多肽衍生物或者变体可通过本领域已知的多种方式产生，包括氨基酸取代、缺失和插入。例如，通过在天然蛋白质的N-端和/或C-端缺失一个或多个氨基酸，在天然蛋白质的一个或多个内部位点缺失和/或添加一个或多个氨基酸，或者在天然蛋白质的一个或多个位点取代一个或多个氨基酸来获得所述多肽衍生物或者变体。所述多肽衍生物或者变体与所述天然蛋白质的氨基酸序列具有至少约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列一致性。所述多肽衍生物或者变体可因仅1-15个、1-10个(例如6-10个)、5个、4个、3个、2个乃至1个氨基酸残基的不同而与所述天然蛋白质不同。所述多肽衍生物或者变体与所述天然蛋白质具有一样的抗病功能。
本文所述的正番茄斑萎病毒属病毒包括但不限于美洲型病毒：番茄斑萎病毒(Tomato spotted wilt virus，TSWV)、凤仙花坏死斑病毒(Impatiens necrotic spot virus，INSV)、花生环斑病毒(Groundnut ringspot virus，GRSV)、菊花茎坏死病毒(Chrysanthemum stem necrosis virus， CSNV)；欧亚型病毒：番茄环纹斑点病毒(Tomato zonate spot virus，TZSV)、马蹄莲褪绿斑病毒(Calla lily chlorotic spot virus，CCSV)、番茄褪绿病毒(Tomato chlorotic spot virus，TCSV)、桑脉带相关病毒(Mulberry vein bandingassociated virus，MVBaV)、辣椒褪绿病毒(Capsicum chlorosis virus，CaCV)、花生芽坏死病毒(Groundnut bud necrosis virus，GBNV)、番茄坏死斑点病毒(Tomato necrotic spot virus，TNSV)、辣椒褪绿斑点病毒(pepper chlorotic spot virus，PCSV)、番茄坏死环斑病毒(Tomato necrotic ringspot virus，TNRV)、蓼属轮斑病毒(Polygonum ringspot virus，PolRSV)和朱顶红褪绿环斑病毒(Hippeastrum chlorotic ringspot virus，HCRV)。
本文所述的茄科植物是指茄科(Solanaceae)家族的成员，包括驯化成员和非驯化成员。所述茄科植物包括但不限于烟草属(Nicotiana spp.)、番茄(Solanum lycopersicum)、马铃薯(Solanum tuberosum)、茄子(Solanum melongena)、矮牵牛属(Petunia spp.)、辣椒属(Capsicum spp.)、酸浆属(Physalis spp.)、木本茄属植物[欧白英(Solanum dulcamara)]、园艺越橘类植物(Solanum scabrum)、茄子(Solanum macrocarpon)、Solanum demissum、Solanum stoloniferum、Solanum papita、Solanum bulbocastanum、Solanum edinense、Solanum schenckii、Solanum hjertingii、Solanum venturi、Solanum mochiquense、Solanum chacoense和醋栗番茄(Solanum pimpinellifolium)。优选的，所述茄科植物为在农业中栽培的茄科植物，包括但不限于烟草、马铃薯、番茄、茄子、辣椒和矮牵牛。更优选的，所述茄科植物为烟草、马铃薯和番茄。
本文所述的烟草可以是野生种或栽培种(Nicotiana tabacum.L)。烟草野生种包括但不限于本氏烟(N.benthamiana)、具翼烟草(N.alata)、粉蓝烟草(N.glauca)、哥西氏烟草(N.gossei)、卡瓦卡米氏烟草(N.kawakamii)、耐特氏烟草(N.knightiana)、朗氏烟草(N.langsdorffi)、耳状烟草(N.otophora)、林烟草(N.sylvestris)、域毛烟草(N.tomentosa)、域毛状烟草(N.tomentosiformis)、波叶烟草(N.undulate)、花烟草(N.x sanderae)、黄花烟草(N.rustica)、香甜烟草(N.suaveolens)、渐狭叶烟草(N.attenuate)。烟草栽培种包括但不限于白肋烟型、深色型、烤烟型、马里兰型、东方型或雪茄烟。本发明所述的栽培烟草可选自多个普通烟草品种，包括但不限于K326、云烟87、云烟97、云烟85、云烟116、云烟121、NC89、中烟100、红花大金元、翠碧1号、BU 64、CC 101、CC 200、CC 13、CC 27、CC 33、CC 35、CC 37、CC65、CC 67、CC 301、CC 400、CC 500、CC 600、CC 700、CC 800、CC 900、CC 1063、Coker 176、Coker 319、Coker 371Gold、Coker 48、CU 263、DF911、Galpao烟草、GL 26H、GL 338、GL350、GL 395、GL 600、GL 737、GL 939、GL 973、GF 157、GF 318、RJR 901、HB 04P、K149、K326、K 346、K 358、K394、K 399、K 730、NC 196、NC 37NF、NC 471、NC 55、NC 92、NC2326、NC95、NC 925、PVH 1118、PVH 1452、PVH 2110、PVH2254、PVH 2275、VA116、VA119、KDH 959、KT200、KT204LC、KY 10、KY 14、KY 160、KY 17、KY 171、KY 907、KY907LC、KTY14 x L8LC、Little Crittenden、McNair 373、McNair 944、msKY 14xL8、Narrow Leaf Madole、NC 100、NC 102、NC 2000、NC 291、NC 297、NC 299、NC 3、NC 4、NC 5、NC 6、NC7、NC 606、NC 71、NC72、NC 810、NC BH 129、NC 2002、Neal Smith Madole、OXFORD 207、‘Perique’烟草、PVH03、PVH09、PVH19、PVH50、PVH51、R 610、R 630、R 7-11、R 7-12、RG 17、RG 81、RG H51、RGH 4、RGH 51、RS 1410、Speight 168、Speight 172、Speight 179、Speight 210、Speight 220、Speight 225、Speight 227、 Speight 234、Speight G-28、Speight G-70、Speight H-6、Speight H20、Speight NF3、TI 1406、TI 1269、TN 86、TN86LC、TN 90、TN90LC、TN 97、TN97LC、TN D94、TN D950、TR(Tom Rosson)Madole、VA 309或VA359。
定义
除非另外定义，本文使用的所有技术和科学术语具有与本披露主题所属领域的普通技术人员通常所理解的意义相同的意义。尽管认为以下术语可以很好地为本领域的普通技术人员所理解，但是提出以下定义是为了使本披露主题更容易理解。
术语“RTSW基因”是指来源于N.alata基因组并使植物具有抗斑萎病性状的基因。
术语“RTSW位点”或“TSWV抗性位点”是指来源于N.alata基因组，包含RTSW基因的DNA片段，该位点在杂合或者纯合状态都赋予植株抗斑萎病性状。
术语“RTSW导入片段”是指包含RTSW基因的N.alata的基因组DNA片段。通常，含有“RTSW导入片段”的烟草植株具有抗烟草斑萎病性状。
术语“短RTSW导入片段”是指长RTSW导入片段上与RTSW基因连锁的非目标基因组分(简称连锁累赘基因组分)部分缺失或全部缺失，并保留完整RTSW基因功能的DNA片段。
术语“导入”是指将遗传基因座的所需等位基因从一个遗传背景传递到另一个遗传背景。
术语“等位基因”通常是指位于一对同源染色体的相同位置上控制着相对性状的一对基因。多数性状由两个或两个以上的等位基因决定。如果等位基因相同，则该个体对该性状来说是纯合子。如果等位基因不同，则该个体对该性状来说是杂合子。在杂合子配对中，显性等位基因使隐性等位基因的性状得不到表现。在本发明中，RTSW基因座是显性等位基因。
术语“基因型”是指个体(或群体)在一个或多个与可观察的和/或可检测的和/或所表现的性状(表型)相关的遗传基因座处的遗传组成。基因型由个体遗传自其亲本的一个或多个已知基因座的一个或多个等位基因定义。“基因型”可以指单个或多个基因座的遗传组成，也可以指基因组中所有基因的遗传组成。可以使用标记来间接表征基因型和/或通过核酸测序来直接表征基因型。
术语“系”通常是指非商业化使用，例如用于植物研究的一组植物。“系”通常在个体间的一个或多个目标性状显示非常小的总体差异，尽管在个体间的其他性状可能存在一些差异。
术语“细胞坏死过敏反应”或“HR”是植物-病原物不亲和互作后发生的一种细胞快速坏死的典型抗病反应，是植物的一种抗病机制，伴随细胞的程序性死亡。具体表现为细胞活性氧进发，相关抗病标记基因的快速响应以及局部的细胞死亡。
术语“无毒基因”或“无毒基因NSm”是指根据基因对基因假说，对于任何一个寄主抗病基因，病原物种都有一个与之相对应的无毒基因。只有当携带无毒基因的病原菌感染携带有相对应的抗性基因的寄主植物时，才会诱导植物产生抗性，否则就会导致植物被感染致病。无毒基因NSm能够在携带RTSW基因的寄主植物中产生细胞坏死过敏反应(HR)，可用于检测烟草的斑萎病抗性。专利号为ZL201710414755.X的中国专利公开了一种利用番茄斑萎病毒NSm基因鉴定烟草抗性的方法。
术语“转基因”是指将人工分离和修饰过的基因导入生物体基因组中。由于导入基因的 表达，引起生物体的性状的可遗传的修饰，这一技术称之为转基因技术。人们常说的“遗传工程”、“基因工程”、“遗传转化”均为转基因的同义词。
术语“植物”包括处于成熟或发育的任何阶段的植物，以及来源于任何所述植物的任何细胞、组织或器官(植物部分)。植物部分包括但不限于种子、果实、茎、块茎、根、花、胚珠、雄蕊、叶、胚、分生组织区、愈伤组织、花药培养物、配子体、孢子体、花粉、小孢子、原生质体等。
术语“转基因植物”和“转化植物”为等同的术语，其是指如上所述的“植物”，其中所述植物包含通过例如在本文别处公开的或在本领域中以其他方式得知的任何的稳定转化法和瞬时转化法引入植物中的异源核酸分子、异源多核苷酸或异源多核苷酸构建体。所述“转基因植物”和“转化植物”亦指，例如将所述异源核酸分子、异源多核苷酸或异源多核苷酸构建体首先引入其中的植物，以及包含所述异源核酸分子、异源多核苷酸或异源多核苷酸构建体的任何的其子代植物。
术语“基因编辑”或“基因组编辑”或“基因组DNA编辑”，通常是指利用CRISPR/Cas系统进行的基因编辑。CRISPR/Cas是细菌和古细菌中的一种适应性免疫系统，能特异地降解入侵噬菌体或外源质粒的DNA，其中CRISPR是“成簇和规律间隔的短回文序列”的简称，而Cas是指与CRISPR RNA结合的蛋白。在2012年，Jinek等解开了化脓链球菌Streptococcus pyogenes的II型CRISPR/Cas9系统作用机制，并证明Cas9核酸酶(本文专指Strepcococcus pyogenes的Cas9)可以在一个人造的小RNA分子(称为gRNA，即Guide RNA)的引导下去靶向切割DNA双链。利用Cas9/gRNA标靶特定的DNA位点需要满足2个条件：(1)gRNA的5’端20nt(核苷酸)的引导序列(称为Spacer)与靶DNA位点的序列(称为Protospacer)互补匹配；(2)靶位点必需存在PAM(Protospacer-adjacentmotif)，其中使用最广的化脓链球菌Cas9的PAM序列为5’-NGG-3’。在使用CRISPR/Cas9进行基因组编辑时，一般用Pol II(II型RNA聚合酶)启动子表达含核定位信号的Cas9，用Pol III(III型RNA聚合酶)启动子表达gRNA，Cas9/gRNA复合体识别靶DNA后在PAM前面的第3个和第4个脱氧核酸之间切割DNA双链，形成DSB(双链DNA断裂)。
术语“密码子”或者“遗传密码子”指信使RNA分子上从5’端到3’端方向，由起始密码子AUG开始，每三个核苷酸组成的三联体。遗传密码子决定肽链上每一个氨基酸和各氨基酸的合成顺序，以及蛋白质合成的起始、延伸和终止。遗传密码子是一组规则，将DNA或RNA序列以三个核苷酸为一组的密码子转译为蛋白质的氨基酸序列，以用于蛋白质合成。几乎所有的生物都使用同样的遗传密码子，称为标准遗传密码。
术语“内含子”是真核生物细胞DNA中的间插序列。在DNA转录到RNA的过程中，DNA上的内含子会被转录到前体RNA中，但RNA上的内含子会在RNA离开细胞核进行转译前被剪除。保留在成熟mRNA中的基因部分被称为“外显子”。真核生物的基因含有外显子和内含子，是其区别原核生物的特征之一。
附图说明
图1显示了RTSW基因定位、克隆、基因结构及编码的蛋白特点。A，图示利用感病的16号单株和抗病的12号单株将候选抗病基因缩小至N.alata 3号染色体59.2Mb-60Mb的0.8 Mb区间中，其中包含的候选基因用箭头表示。B，图示两个候选基因TNL1和TNL2的基因结构：包含四个外显子结构，编码TIR-NBS-LRR类蛋白。TNL1编码全长的TIR-NBS-LRR蛋白，全长1089aa。TNL2编码一个截短的TIR-NBS-LRR蛋白，全长817aa。
图2显示了利用NSm诱导HR对TNL1和TNL2进行鉴定的结果。A为TNL表达盒示意图。35S代表“花椰菜花叶病毒”来源的35S启动子；TNLs代表TNL1基因全长、TNL1 CDs全长、TNL2基因全长和TNL2 CDs全长中的任意一种；NOS代表用于转录终止的Nos终止子。B：左图(TNLs+TSWV_NSm)为使用EHA105-35S-TNLs与EHA105-35S-TSWV_NSm共浸润感病烟草K326叶片的结果(图中表示为35S-TNL1、35S-TNL1_CDs、35S-TNL2、35S-TNL2_CDs)，以EHA105-35S-Sw-5b和EHA105-35S-TSWV_NSm共浸润叶片作为对照(图中表示为35S-Sw5b)；右图(TNLs+TZSV_NSm)为使用EHA105-35S-TNLs与EHA105-35S-TZSV_NSm共浸润感病烟草K326叶片的结果(图中表示为35S-TNL1、35S-TNL1_CDs、35S-TNL2、35S-TNL2_CDs)，以EHA105-35S-Sw-5b和EHA105-35S-TZSV_NSm共浸润叶片作为对照(图中表示为35S-Sw5b)；图中35S-TNL1 only表示单独接种EHA105-35S-TNL1作为阴性对照。浸润后3天观察表型：35S-TNL1和35S-TNL1_CDs均可以和TSWV_NSm或者TZSV_NSm共浸润诱导产生HR，35S-TNL2或35S-TNL2_CDs不能和TSWV_NSm或者TZSV_NSm共浸润诱导产生HR。而作为阴性对照，35S-TNL1 only不能诱导HR，表明HR表型是TNL1和NSm共浸润后激活产生的。另一对照35S-Sw5b仅能和TSWV_NSm共浸润产生HR，而不能和TZSV_NSm共浸润产生HR，和报道的Sw5b能抗美洲型而不能抗亚洲型Orthotospovirus的结果相符。
图3显示了对TNL1和TNL2的基因编辑。A图示根据TNL1和TNL2的保守序列设计gRNA并构建至CRISPR/Cas9载体。B图示21个独立的T0代TNL1和TNL2编辑事件。没有编辑事件的野生型用wt表示；i表示插入碱基，i后面的数字表示插入的碱基数量；d表示缺失碱基，d后面的数字表示缺失的碱基数量。C图示TNL1和/或TNL2被编辑的T0代植株的叶片浸润TSWV_NSm或者TZSV_NSm后诱导HR的情况。TNL1单独被编辑(用tnl1表示)和TNL1/TNL2同时被编辑(用tnl1/tn12表示)均不能诱导HR，而TNL2单独被编辑(用tnl2表示)或者TNL1/TNL2未被编辑(用TNL1/TNL2表示)能诱导典型的HR；图片下方的比值表示各编辑事件中产生HR的植株数/植株总数。
图4显示了proTNL1：TNL1转基因K326烟草接种部分无毒基因和病毒后的表型。A为proTNL1：TNL1表达盒示意图。proTNL1表示TNL1基因的天然启动子，TNL1表示TNL1基因全长，NOS为终止子。B为proTNL1：TNL1T0代转基因K326阳性株系(#1)进行无毒基因诱导HR的检测。与含有RTSW位点的抗斑萎病烟草株系一样，在proTNL1：TNL1转基因阳性的株系中，TSWV、INSV、TZSV及CCSV的NSm基因均可以诱导明显的HR，而TNSV和PCSV的NSm基因不能诱导HR。番茄的Sw-5b基因和TSWV_NSm(用Sw-5b+TSWV_NSm表示)共浸润作为阳性对照，空载体(用EV表示)浸润作为阴性对照。C图示随机选择的两个proTNL1：TNL1转基因K326阳性株系(#1和#2)接种TSWV和TZSV。接种后21天拍照，非转基因K326表现明显的TSWV和TZSV病毒症状，而proTNL1：TNL1转基因株系未被病毒侵染。
图5显示了proTNL1：TNL1转基因本氏烟接种TSWV和TZSV后的表型。两个 proTNL1：TNL1转基因本氏烟阳性株系(#1和#4)和非转基因本氏烟(用Nb表示)均接种TSWV和TZSV。接种后21天拍照，非转基因本氏烟表现明显的TSWV和TZSV病毒症状，而proTNL1：TNL1转基因本氏烟株系未被病毒侵染，没有任何病毒症状。
图6显示了proTNL1：TNL1转基因番茄接种TSWV和TZSV后的表型。两个proTNL1：TNL1转基因番茄阳性株系(#1和#2)和非转基因番茄(用Sl表示)均接种TSWV和TZSV。接种后21天拍照，非转基因番茄表现明显的TSWV和TZSV病毒症状，而两个proTNL1：TNL1转基因番茄株系未被病毒侵染，没有任何病毒症状。
图7显示了proTNL1：TNL1转基因马铃薯接种TSWV和TZSV后的表型。两个proTNL1：TNL1转基因马铃薯阳性株系(#1和#2)和非转基因马铃薯(用St表示)均接种TSWV和TZSV。接种后21天取叶片拍照，非转基因马铃薯表现明显的TSWV和TZSV病毒症状，而两个proTNL1：TNL1转基因马铃薯株系未被病毒侵染，没有任何病毒症状。
序列表说明
在随附的序列表中列举的核苷酸序列和氨基酸序列使用用于核苷酸碱基的标准字母缩写以及用于氨基酸的三字母密码来显示。所述核苷酸序列遵循从5’端开始并朝着3’端前进的标准惯例。仅显示了各核苷酸序列的一条链，应理解的是，所显示的那条链的互补链也包含在内。所述氨基酸序列遵循从所述序列的氨基端开始并朝着羧基端前进的标准惯例。
SEQ ID NO：1显示了RTSW基因(TNL1)的核苷酸序列。需要时，可将终止密码子(例如TGA、TAG、TAA)与包含SEQ ID NO：1的核酸分子的3’端可操作连接。
SEQ ID NO：2显示了RTSWcDNA(TNL1cDNA)的编码区的核苷酸序列。需要时，可将终止密码子(例如TGA、TAG、TAA)与包含SEQ ID NO：2的核酸分子的3’端可操作连接。
SEQ ID NO：3显示了RTSW基因(TNL1)编码的RTSW蛋白(TNL1蛋白)的氨基酸序列。
SEQ ID NO：4显示了RTSW基因(TNL1)的天然启动子的核苷酸序列。
SEQ ID NO：5显示了TNL2基因的核苷酸序列。
SEQ ID NO：6显示了TNL2 cDNA的编码区的核苷酸序列。
SEQ ID NO：7显示了TNL2基因编码的TNL2蛋白的氨基酸序列。
SEQ ID NO：8显示了包含RTSW基因及其天然启动子的表达盒(proTNL1：TNL1)的核苷酸序列。
SEQ ID NO：9显示了NaChr3_59.2M标记的核苷酸序列。
SEQ ID NO：10显示了NaChr3_59.7M标记的核苷酸序列。
SEQ ID NO：11显示了引物NaChr3_59.2MF的核苷酸序列。
SEQ ID NO：12显示了引物NaChr3_59.2MR的核苷酸序列。
SEQ ID NO：13显示了引物NaChr3_59.7MF的核苷酸序列。
SEQ ID NO：14显示了引物NaChr3_59.7MR的核苷酸序列。
SEQ ID NO：15显示了引物TNL1_35SF的核苷酸序列。
SEQ ID NO：16显示了引物TNL1_35SR的核苷酸序列。
SEQ ID NO：17显示了引物TNL2_35SF的核苷酸序列。
SEQ ID NO：18显示了引物TNL2_35SR的核苷酸序列。
SEQ ID NO：19显示了gRNA的核苷酸序列。
SEQ ID NO：20显示了引物gRNA-F的核苷酸序列。
SEQ ID NO：21显示了引物gRNA-R的核苷酸序列。
SEQ ID NO：22显示了引物TNL1editTestF的核苷酸序列。
SEQ ID NO：23显示了引物TNL1editTestR的核苷酸序列。
SEQ ID NO：24显示了引物TNL2editTestF的核苷酸序列。
SEQ ID NO：25显示了引物TNL2editTestR的核苷酸序列。
SEQ ID NO：26显示了引物TNL1_NatProF的核苷酸序列。
具体实施方式
下面结合实施例对本发明作进一步的详细描述。本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过购买获得的常规产品。
以下实施例中使用的烟草材料：
‘Polata’是包含RTSW位点的抗TSWV的烟草材料，记载在非专利文献(Laskowska D，
Figure PCTCN2022124977-appb-000003
A，2010.TSWV resistance in DH lines of tobacco(Nicotiana tabacum L)obtained from a hybrid between‘Polalta’and
Figure PCTCN2022124977-appb-000004
Plant Breeding 129，731-3.)中。
‘K326’为未包含RTSW位点的感TSWV的烟草栽培品种，记载在非专利文献(Edwards et al.，2017，A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency.BMC Genomics 18，448.)中。公众可从网站https：//solgenomics.net/organism/Nicotiana_tabacum/genome获得其参考基因组序列。
N.alata是一种抗TSWV的野生烟草，记载在非专利文献(Laskowska et al.，2013，A survey of Nicotiana germplasm for resistance to Tomato spotted wilt virus(TSWV).Euphytica 193，207-19.)中。N.alata在美国烟草种质库中的登录号为PI42334。
18株无连锁累赘且RTSW导入片段缩短的烟草植株是本申请人在前期实验中，以‘Polata’为父本、以‘K326’为母本进行杂交，并以K326为轮回亲本进行多代回交，通过分子标记检测和TSWV抗性检测筛选得到。筛选过程记载在申请号为PCT/CN2021/129382，发明名称为“无连锁累赘的抗斑萎病烟草植物及其选育方法”的国际专利申请和申请号为202111311707.0，发明名称为“用于筛选无连锁累赘的抗斑萎病烟草植物的分子标记及其应用”的中国专利申请中，在此通过引用将所述专利申请的全部内容并入本文。
K326 RTSW是含有RTSW位点的烟草，是以‘Polata’为父本、以‘K326’为母本进行杂交得到的具有烟草斑萎病抗性的烟草。
公众可从烟草种质资源保存单位或云南省烟草农业科学研究院获得以上烟草材料。
以下实施例中使用的病毒：
番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)、番茄环纹斑点病毒(TZSV)、马 蹄莲褪绿斑病毒(CCSV)、辣椒褪绿病毒(CaCV)、番茄坏死斑点病毒(TNSV)、辣椒褪绿斑点病毒(PCSV)和朱顶红褪绿环斑病毒(HCRV)。病毒的毒源保存于云南省烟草农业科学研究院。
以下实施例中使用的载体和菌株：
包含orthotospoviruses NSm基因的表达载体pCambia1300-NSm-YFP为本实验室构建，并保存于云南省烟草农业科学研究院。14种orthotospoviruses NSm基因具体为美洲型TSWV NSm基因(NCBI登录号：JF960236.1)，CSNVNSm基因(NCBI登录号：AF213675)，GRSV NSm基因(NCBI登录号：KY350137)和INSV NSm基因(NCBI登录号：NC_003616)，以及欧亚型TZSV NSm基因(NCBI登录号：KM374588)，CCSV NSm基因(NCBI登录号：KT004454)，MVbaV NSm基因(NCBI登录号：NC_026618)，CaCV NSm基因(NCBI登录号：NC_008303)，GBNV NSm基因(NCBI登录号：KY006470)，TNSV NSm基因(NCBI登录号：KT984753)，PCSVNSm基因(NCBI登录号：KY315810)，TNRVNSm基因(NCBI登录号：FJ947152)，PolRSVNSm基因(NCBI登录号：EU271753)和HCRVNSm基因(NCBI登录号：KY363497)。将14种病毒的NSm基因片段分别克隆至pCambia1300-YFP表达载体中，获得重组质粒pCambia1300-NSm-YFP。在pCambia1300-NSm-YFP中，NSm基因位于35S启动子的下游，与YFP基因组成完整的读码框，可表达NSm-YFP融合蛋白，其中的YFP标签用于蛋白表达水平的检测。将pCambia1300-NSm-YFP导入根癌农杆菌EHA105中，获得EHA105-pCambia1300-NSm-YFP系列菌株。
pK2-35S-TSWV_NSm是带有TSWV NSm基因(NCBI登录号：JF960236.1)的pK2GW7植物表达载体，是专利号为ZL201710414755.X，发明名称为“一种利用番茄斑萎病毒NSm基因鉴定烟草抗性的方法”的中国专利中公开的pK2-35S-NSm载体，通过引用将该专利的全部内容并入本文。EHA105-35S-TSWV_NSm是将pK2-35S-TSWV_NSm载体导入农杆菌EHA105得到的菌株，保存于云南省烟草农业科学研究院。在中国专利ZL201710414755.X中已证明N.alata和‘Polata’烟草的叶片接种EHA105-35S-TSWV_NSm后均产生明显的细胞坏死过敏反应，说明N.alata和‘Polata’烟草中的RTSW基因与TSWV互作的无毒基因为NSm。
pK2-35S-TZSV_NSm是带有TZSV NSm基因(NCBI登录号：KM374588)的pK2GW7植物表达载体，其构建方法同上述pK2-35S-NSm载体，参见中国专利ZL201710414755.X。其中TZSV NSm基因从TZSV的cDNA中克隆得到。EHA105-35S-TZSV_NSm是将pK2-35S-TZSV_NSm载体导入农杆菌EHA105得到的菌株，保存于云南省烟草农业科学研究院。
p2300-35S-Sw-5b是将番茄Sw-5b基因(NCBI登录号：AY007366)全长插入pCambia2300质粒得到的载体。其中Sw-5b基因从番茄栽培品种43419的基因组DNA中克隆得到。p2300-35S-Sw-5b载体的构建方法记载在文献“Zhao W，Jiang L，Feng Z，Chen X，Huang Y，Xue F，Huang C，Liu Y，Li F，Liu Y et al.Plasmodesmata targeting and intercellular trafficking of Tomato spotted wilt tospovirus movement protein NSm is independent of its function in HR induction.Journal of General Virology(2016)，97，1-8.”中。EHA105-35S-Sw-5b是将p2300-35S-Sw-5b导入农杆菌EHA105得到的菌株，保存于云南省烟草农业科学研究院。
pRGEB31-PF是申请人前期构建的CRISPR/Cas9基因编辑载体，保存于云南省烟草农业 科学研究院。pRGEB31-PF的构建方法记载在专利号为ZL201811347308.8，发明名称为“快速获得无转基因基因编辑植株的重组载体及使用方法”的中国发明专利中，通过引用将该专利的全部内容并入本文。
实施例1.RTSW基因的精细定位及生物信息学分析
在前期筛选无连锁累赘的抗斑萎病烟草的实验中，我们获得了18株无连锁累赘且RTSW导入片段缩短的烟草植株，其中5个单株(1号、4号、11号、12号、17号)在瞬时表达NSm基因时能够产生细胞坏死过敏反应(HR)，具有斑萎病抗性；其余13个单株(2号、3号、5号、6号、7号、8号、9号、10号、13号、14号、15号、16号、18号)在瞬时表达NSm基因时没有HR。分子标记检测结果表明单株12号中的RTSW导入片段最短，仅有NaChr3_59M标记为阳性，其他标记均为阴性(参见申请号为PCT/CN2021/129382的国际专利申请和申请号为202111311707.0的中国专利申请)。
我们以单株12号为对照，利用前期开发的NaChr3_59M和NaChr3_60M标记(参见申请号为PCT/CN2021/129382的国际专利申请和申请号为202111311707.0的中国专利申请)对上述18株烟草植株中没有HR的13个单株进行检测。结果显示除了作为阳性对照的供体亲本‘Polata’，14个单株的NaChr3_60M标记检测均为阴性(表2)。其中没有HR的单株2号、9号、13号、14号、16号和18号以及有HR的单株12号的NaChr3_59M标记检测为阳性，因此推测RTSW基因位于NaChr3_59M和NaChr3_60M标记之间。
为了精细定位RTSW基因，我们在NaChr3_59M和NaChr3_60M标记之间开发了分子标记NaChr3_59.2M(SEQ ID NO：9)和NaChr3_59.7M(SEQ ID NO：10)，并对单株12号和上述13个单株进行了标记检测。结果显示，仅有单株12号和单株16号的NaChr3_59.2M标记检测为阳性，并且仅有单株12号的NaChr3_59.7M标记检测为阳性。
表1分子标记引物
Figure PCTCN2022124977-appb-000005
表2分子标记检测结果
Figure PCTCN2022124977-appb-000006
Figure PCTCN2022124977-appb-000007
注：表中Pos表示阳性，Neg表示阴性；HR表示细胞坏死过敏反应，“有HR”表示具有斑萎病抗性，“无HR”表示不具有斑萎病抗性。
结果表明，无HR(即不含有RTSW基因)的单株的NaChr3_59.7M标记均为阴性。无HR的具有最长RTSW导入片段的单株16号的NaChr3_59.2M标记检测为阳性，而有HR的具有最短RTSW导入片段的12号单株的NaChr3_60M标记检测为阴性，因此将RTSW基因定位在N.alata基因组3号染色体59201149-59996790bp(约0.8Mb)的区间中(图1A)。
植物典型的抗性基因，即符合“基因对基因”假说的抗病基因，一般都具有核苷酸结合位点(NBS，nucleotide-binding site)和亮氨酸富集重复(LRR，leucine-rich repeat)的结构(Moffett，Advances in virus research，2009，vol.75，1-33，228-229)。发明人在前期证明了NSm是RTSW基因的无毒基因(参见专利号为ZL201710414755.X的中国专利)，表明RTSW基因极有可能是典型的NBS-LRR类的抗性基因。据此，我们对N.alata基因组3号染色体59201149-59996790bp区间进行生物信息学分析，发现在该区间只有2个NBS-LRR类抗性基因，分别是YC03G182780和YC03G182790(表3，图1B)。
表3候选基因组区间的基因预测
Figure PCTCN2022124977-appb-000008
Figure PCTCN2022124977-appb-000009
通过保守结构域在线软件(https：//www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)分析，YC03G182780和YC03G182790均含有典型的TIR、NB-ARC和LRR等保守的结构域，为Toll样受体-核苷酸结合位点-亮氨酸富集重复(Toll/interleukin-1 receptor nucleotide-binding site leucine-rich repeat，TIR-NBS-LRR，TNL)型抗性基因(表4)。将YC03G182790和YC03G182780分别命名为TNL1和TNL2，作为候选基因。
表4 TNL1和TNL2基因的保守结构域分析
Figure PCTCN2022124977-appb-000010
实施例2.候选基因的克隆与结构分析
根据基因组信息分别设计TNL1和TNL2基因全长的特异性引物(表5)。使用植物基因组DNA提取试剂盒(天根Tiangen生化，货号：DP360)按照产品说明书分别提取N.alata和K326 RTSW烟草叶片的基因组DNA。以获得的基因组DNA为模板，使用TNL1_35SF和TNL1_35SR引物扩增TNL1的基因全长，使用TNL2_35SF和TNL2_35SR引物扩增TNL2的基因全长。PCR体系：2×Phanta Max Buffer(Vazyme，货号：P505)25μl，dNTP Mix(10mM each)1μl，上游引物(10μM)2μl，下游引物(10μM)2μl，Phanta Max Super-Fidelity DNA Polymerase 1μl，模板DNA 2μl，加ddH 2O至50μL。PCR反应程序为：95℃预变性3min；然后进入35个循环：95℃变性15s，60℃退火15s，72℃延伸4min；循环结束后72℃延伸5min；4℃保存。对PCR产物进行克隆测序，序列比对发现从N.alata和K326 RTSW克隆测序的TNL1序列一致，TNL2序列也一致，并且和基因组组装的基因序列完全一致。
使用植物总RNA提取试剂盒(Qiagen，货号：74904)按照产品说明书提取N.alata烟草叶片的RNA。然后使用cDNA一链合成试剂盒(Vazyme，货号：R312-01)对提取的RNA 进行反转录，获得cDNA。反转录体系和程序参照试剂盒说明书。以获得的cDNA为模板，使用TNL1_35SF和TNL1_35SR引物扩增TNL1的CDs全长(cDNA的编码区)，使用TNL2_35SF和TNL2_35SR引物扩增TNL2的CDs全长。PCR体系和PCR程序同上。对PCR产物进行测序，得到CDs全长序列。将CDs全长序列与N.alata基因组序列进行比对，并分析基因的转录本结构。
表5 TNL1和TNL2的扩增引物
引物名称 引物序列 序列号
TNL1_35SF catttggagaggacacgctcgagATGGATACTCAATTAGTTAGAGG SEQ ID NO：15
TNL1_35SR tctcattaaagcaggactctagaGATTTGGCCAAGGGAAAAAGATTACG SEQ ID NO：16
TNL2_35SF catttggagaggacacgctcgagATGGATACTCAATTAGTTAGAGTAG SEQ ID NO：17
TNL2_35SR tctcattaaagcaggactctagaGTTAGGATTGGTTGGGTGGACTA SEQ ID NO：18
注：TNL1_35SF和TNL1_35SR引物的扩增产物包含230bp的3’UTR区。TNL2_35SF和TNL2_35SR引物的扩增产物包含150bp的3’UTR区。上述引物包含phellsgate8线性化两端的overlap序列(用小写字母表示)，用于重组克隆。
结果显示，TNL1和TNL2都具有4个外显子和3个内含子的结构(图1B)。TNL1的基因全长4532bp(不含终止密码子的基因序列如SEQ ID NO：1所示)；CDs全长3270bp(不含终止密码子的CDs序列如SEQ ID NO：2所示)，编码1089个氨基酸的蛋白质序列(SEQ ID NO：3)。TNL2的基因全长4630bp(SEQ ID NO：5)；CDs全长2454bp(SEQ ID NO：6)，编码817个氨基酸的蛋白质序列(SEQ ID NO：7)。虽然TNL2和TNL1的基因结构类似，但TNL2在第四个外显子上有一个提前的终止密码子，因此造成一个截短的CDs。
实施例3.利用无毒基因共浸润方法对候选基因的功能进行初步验证
为了明确TNL1和TNL2是否是RTSW基因，利用发明人前期建立的抗病基因鉴定体系(参见专利号为ZL201710415015.8，发明名称为“一种利用番茄斑萎病毒NSm基因进行抗病基因筛选的方法”的中国发明专利，在此通过引用将该专利的全部内容并入本文)进行初步验证。
用XhoI和XbaI限制性内切酶对phellsgate8空质粒进行双酶切，得到线性化phellsgate8质粒。使用ClonExpress一步法克隆试剂盒(Vazyme，货号C112-01)按照产品说明书将实施例2中获得的TNL1基因全长、TNL1的CDs全长、TNL2基因全长和TNL2的CDs全长分别克隆至phellsgate8质粒中，得到重组质粒35S-TNL1、35S-TNL1_CDs、35S-TNL2和35S-TNL2_CDs。35S-TNL1表达TNL1基因全长；35S-TNL1_CDs表达TNL1的CDs全长；35S-TNL2表达TNL2基因全长；35S-TNL2_CDs表达TNL2的CDs全长。这些基因的表达由35S启动子驱动。将35S-TNL1、35S-TNL1_CDs、35S-TNL2和35S-TNL2_CDs通过热激法分别转化大肠杆菌DH5α并进行测序鉴定，获得插入序列准确的重组质粒。
将35S-TNL1、35S-TNL1_CDs、35S-TNL2和35S-TNL2_CDs分别导入根癌农杆菌EHA105菌株中，获得重组菌EHA105-35S-TNL1、EHA105-35S-TNL1_CDs、EHA105-35S-TNL2和EHA105-35S-TNL2_CDs。将获得的重组菌在LB培养基中于28℃培养24小时，离心收集菌 体，再用浸润缓冲液(10mmol/L MgCl 2，10mmol/L MES，200μmol/L acetosyringone)重悬菌体，得到OD600＝0.5的菌体悬液。使用同样的方法制备EHA105-35S-TSWV_NSm、EHA105-35S-TZSV_NSm和EHA105-35S-Sw-5b的菌体悬液(OD600＝0.5)。
将EHA105-35S-TSWV_NSm菌体悬液和EHA105-35S-TZSV_NSm菌体悬液分别与EHA105-35S-TNL1、EHA105-35S-TNL1_CDs、EHA105-35S-TNL2或EHA105-35S-TNL2_CDs的菌体悬液按照1：1的体积比混合，得到8份待测菌体悬液。将EHA105-35S-TSWV_NSm菌体悬液和EHA105-35S-Sw-5b菌体悬液按照1：1的体积比混合，作为阳性对照。控制菌体悬液的OD600＝0.5。接种烟草叶片：用无菌的去掉针头的注射器将9.5-10.5微升待测菌体悬液从感病烟草‘K326’的叶背注渗入叶脉间，形成一个可见的浸润斑；将接种后的烟草置于20-28℃及80％湿度的环境中，交替进行连续光照16小时及连续黑暗8小时，共观察72小时。按照同样的方法在感病烟草‘K326’的叶片上分别接种阳性对照和单独的EHA105-35S-TNL1菌体悬液。若待测菌体悬液能够在感病烟草‘K326’的叶片上诱导产生细胞坏死过敏反应(HR)，则表明该待测菌体悬液中的候选基因为该待测菌体悬液中的无毒基因NSm的抗病基因。
结果显示，EHA105-35S-TNL1和EHA105-35S-TSWV_NSm的混合菌体悬液、EHA105-35S-TNL1和EHA105-35S-TZSV_NSm的混合菌体悬液、EHA105-35S-TNL1_CDs和EHA105-35S-TSWV_NSm的混合菌体悬液、EHA105-35S-TNL1_CDs和EHA105-35S-TZSV_NSm的混合菌体悬液均可以诱导‘K326’烟草产生HR；而EHA105-35S-TNL2和EHA105-35S-TNL2_CDs均不能与EHA105-35S-TSWV_NSm或EHA105-35S-TZSV_NSm共同诱导‘K326’烟草产生HR。作为对照，单独的EHA105-35S-TNL1菌体悬液不能诱导‘K326’烟草产生HR，表明HR表型是TNL1基因和NSm基因共同激活产生的(图2B)。因此，可以初步确定候选基因TNL1是RTSW基因。虽然TNL2和TNL1相距仅4Kb左右并且拥有相似的基因结构，但TNL2与NSm激发的HR无关，可能在抗烟草斑萎病中没有作用。
实施例4.通过基因敲除进一步验证候选基因的功能
为了进一步明确TNL1和TNL2是否是RTSW基因，利用发明人前期建立的基因编辑系统(参见中国专利ZL201811347308.8)在含有RTSW位点的烟草中敲除TNL1和TNL2。所述基因编辑系统使用的重组载体是携带有PAP1和NtFT表达元件(PF Cassete)的用于植物基因编辑的CRISPR/Cas9的载体。由于NtFT表达元件产生促进植物早花的蛋白，PAP1表达元件产生促进植物花青素生物合成的蛋白，因此可以利用T0转基因植株的颜色来判断是否发生编辑事件。如果植株含有转基因元件则表现为紫色，不含有转基因元件则表现为绿色。
TNL1和TNL2同源性较高，因此利用一个保守序列的gRNA可以分别编辑TNL1和TNL2，或同时编辑TNL1和TNL2。基于TNL1和TNL2的第一个外显子中保守的序列设计得到靶标序列(gRNA)：CTATGATGTTGATCCTTCTG(SEQ ID NO：19)。该gRNA的3’紧邻着“AGG”的Cas9核酸酶PAM序列(图3A)。合成gRNA的两条引物如下：
gRNA-F：5’-GGCACTATGATGTTGATCCTTCTG-3’(SEQ ID NO：20)
gRNA-R：3’-CAGAAGGATCAACATCATAGCAAA-5’(SEQ ID NO：21)
pRGEB31-PF载体的构建方法参见专利号为ZL201811347308.8的中国专利。通过载体上两个相邻的BsaI位点将靶标序列插入pRGEB31-PF载体，所以上游序列(gRNA-F)需添加GGCA接头，下游序列(gRNA-R)需添加AAAC接头。将引物gRNA-F和gRNA-R通过退火反应形成二聚体结构。退火体系：gRNA-F 20μl，gRNA-R 20μl，10×Annealing buffer 5μl，H 2O 5μl。退火程序：95℃5min，90℃1min，80℃1min，70℃1min，60℃1min，50℃1min，40℃1min，30℃1min，20℃1min，10℃1min。然后将gRNA插入经BsaI酶切后的pRGEB31-PF载体，得到连接产物。将所述连接产物转化大肠杆菌DH5α感受态细胞，提取质粒并进行测序鉴定，得到插入序列正确的重组质粒Cas9-PF-gRNA。将Cas9-PF-gRNA电击转化农杆菌EHA105，并进行农杆菌介导的烟草转化实验。为了在T0代获得更高的编辑效率并在T1代获得纯合突变体，以基因型为RTSW杂合型(RTSW/rtsw)的K326 RTSW烟草为材料诱导愈伤组织并用于烟草转化实验。在RTSW位点杂合型的背景下，只需要编辑一个等位基因就能完全使待验证基因失去功能而出现表型。经过潮霉素抗性筛选，抗性愈伤组织分化再生获得T0代转基因阳性植株。
根据目的基因，在靶标序列的上游和下游分别设计特异引物(表6)，用于检测转基因烟草中TNL1和TNL2基因突变体。
表6 TNL1和TNL2基因突变体检测引物
引物名称 引物序列 序列号
TNL 1editTestF AGGTGAAATTATGGCGAAAGTG SEQ ID NO：22
TNL 1editTestR CTTCAGTAAGTGCAGCTCTCC SEQ ID NO：23
TNL2editTestF CTGGTCATCTTTATTCCAAATTAGA SEQ ID NO：24
TNL2editTestR TTGAATCAAGAATATGTACCCGTCAG SEQ ID NO：25
使用植物基因组DNA提取试剂盒(天根Tiangen生化，货号：DP360)按照产品说明书分别提取30株T0代转基因阳性植株的基因组DNA。以每个植株的基因组DNA为模板，利用TNL1editTestF和TNL1editTestR引物扩增包含TNL1编辑位点的序列，利用TNL2editTestF和TNL2editTestR引物扩增包含TNL2编辑位点的序列。PCR体系和程序与实施例2中的相同。由赛默飞世尔科技公司(广州)对PCR产物进行测序，测序引物为TNL1editTestF和TNL2editTestF。
分析测序产物是否在靶标位点，即在PAM上游3bp处，出现碱基替换、缺失或插入来判定是否有编辑事件发生。结果表明，30株T0代转基因阳性植株中，21株植株在TNL1或TNL2靶标位点发生了编辑，编辑效率为70％。其中5株为TNL1单独被编辑，4株为TNL2单独被编辑，其余12株为TNL1和TNL2同时被编辑(图3B)。由于烟草转化实验中使用的是基因型为RTSW/rtsw的烟草的诱导愈伤组织，该愈伤组织中的TNL1或TNL2均只含有一个等位基因，所以任何针对TNL1或TNL2的编辑事件均会影响相应基因的功能，等同于纯合突变体的效果。因此，可直接在T0代植株上进行TSWV抗性检测。
利用NSm基因检测T0代植株的TSWV抗性，检测方法参见专利号为ZL201710414755.X的中国专利。具体为：将EHA105-35S-TSWV_NSm在LB培养基中于28℃培养24小时，离心收集菌体，再用浸润缓冲液(10mmol/L MgCl 2，10mmol/L MES，200μmol/L acetosyringone) 重悬菌体，得到OD600＝0.5的菌体悬液。用无菌的去掉针头的注射器将9.5-10.5微升菌体悬液从烟草的叶背注渗入叶脉间，形成一个可见的浸润斑；将接种后的烟草置于20-28℃及80％湿度的环境中，交替进行连续光照16小时及连续黑暗8小时，共观察72小时。按照同样的方法在烟草叶片上接种EHA105-35S-TZSV_NSm。若TSWV_NSm或TZSV_NSm能够在T0代植株的叶片上诱导产生细胞坏死过敏反应(HR)，则表明该T0代植株具有TSWV或TZSV抗性，其RTSW基因具有功能；若TSWV_NSm或TZSV_NSm不能在T0代植株的叶片上诱导产生HR，则表明该T0代植株没有TSWV或TZSV抗性，其RTSW基因失去功能。
结果显示，在30株T0代转基因阳性植株中，有21个TNL1和/或TNL2被编辑的事件。无毒基因TSWV_NSm和TZSV_NSm在5株TNL1单独被编辑的植株和12株TNL1和TNL2同时被编辑的植株上均不能诱导HR；TSWV_NSm和TZSV_NSm在4株TNL2单独被编辑和9株TNL1和TNL2均未被编辑的植株上均能诱导明显的HR(表7，图3C)。
表7 T0代植株的TSWV抗性检测
T0代植株编号 编辑类型 TSWV_NSm TZSV_NSm
1 TNL1单独被编辑 无HR 无HR
2 TNL1和TNL2同时被编辑 无HR 无HR
3 TNL1和TNL2均未被编辑 HR HR
4 TNL2单独被编辑 HR HR
5 TNL1单独被编辑 无HR 无HR
6 TNL1和TNL2同时被编辑 无HR 无HR
7 TNL1和TNL2同时被编辑 无HR 无HR
8 TNL1和TNL2均未被编辑 HR HR
9 TNL1单独被编辑 无HR 无HR
10 TNL1和TNL2同时被编辑 无HR 无HR
11 TNL1单独被编辑 无HR 无HR
12 TNL2单独被编辑 HR HR
13 TNL1和TNL2均未被编辑 HR HR
14 TNL1和TNL2同时被编辑 无HR 无HR
15 TNL2单独被编辑 HR HR
16 TNL1和TNL2均未被编辑 HR HR
17 TNL1和TNL2同时被编辑 无HR 无HR
18 TNL1和TNL2均未被编辑 HR HR
19 TNL1和TNL2同时被编辑 无HR 无HR
20 TNL1和TNL2同时被编辑 无HR 无HR
21 TNL1和TNL2均未被编辑 HR HR
22 TNL1和TNL2同时被编辑 无HR 无HR
23 TNL2单独被编辑 HR HR
24 TNL1和TNL2均未被编辑 HR HR
25 TNL1和TNL2同时被编辑 无HR 无HR
26 TNL1和TNL2均未被编辑 HR HR
27 TNL1单独被编辑 无HR 无HR
28 TNL1和TNL2同时被编辑 无HR 无HR
29 TNL1和TNL2均未被编辑 HR HR
30 TNL1和TNL2同时被编辑 无HR 无HR
为了证明TNL1和TNL2的编辑事件是可遗传的，同时为了排除编辑载体的转基因元件对于编辑事件的干扰，选择2株TNL1单独被编辑的植株(基因型为tnl1 CasKO/tnl1，TNL2/tnl2)，2株TNL2单独被编辑的植株(基因型为TNL1/tnl1，tnl2 CasKO/tnl2)，2株TNL1和TNL2同时被编辑的植株(基因型为tni1 CasKO/tnl1，tnl2 CasKO/tnl2)和2株TNL1和TNL2均未被编辑的植株(基因型为TNL1/tnl1，TNL2/tnl2)，分别自交，获得T1代种子。在光照培养室内分别播种T1代种子，以常规方法培育T1代植株。由于Cas9-PF-gRNA载体带有花青素可视化标记，如果T1代植株含有转基因元件则表现为紫色，不含有转基因元件则表现为绿色。在T1代植株具有4-5片叶时，挑取完全绿色的植株，提取每株植物的基因组DNA进行基因型和编辑纯合检测。以基因组DNA为模板，分别利用TNL1editTestF/TNL1editTestR和TNL2editTestF/TNL2editTestR引物对进行PCR反应。如果PCR结果为阴性，则表明该植株的基因型为rtsw/rtsw，该植株为不含有RTSW导入片段的分离单株。因此，仅对PCR结果为阳性的植株进行下一步分析。测序结果显示，PCR结果为阳性的T1代植株均含有符合预期的突变型，表明编辑事件是可遗传的，并且都已经纯合突变。在PCR结果为阳性的T1代植株上分别接种TSWV和TZSV病叶汁液。接种后14d和21d分别调查植株的TSWV和TZSV发病情况(表8)。
表8基因被编辑的植株对TSWV和TZSV的抗性
Figure PCTCN2022124977-appb-000011
如表8所示，接种后14天TNL2单独被编辑以及TNL1和TNL2均未被编辑的T1代植株 的发病率为0％，表现为高抗。而TNL1单独被编辑或者TNL1和TNL2同时被编辑的T1代植株的发病率达到100％，表现为感病。以上结果表明，只要TNL1被编辑就能使含有RTSW位点的植株完全丧失抗斑萎病的功能，而单独编辑TNL2对植株的抗性没有影响。
实施例5.携带TNL1的稳定转化烟草的广谱斑萎病抗性鉴定
为了检测单一的TNL1是否能充分发挥RTSW位点的功能，也为了鉴定TNL1是否具有广谱抗斑萎病的功能，我们克隆了带有天然TNL1启动子(SEQ ID NO：4)的全长基因proTNL1：TNL1(SEQ ID NO：8)。我们在TNL1的读码框的起始密码子ATG上游2Kb左右设计得到引物TNL1_NatProF(表9)。以N.alata的基因组DNA为模板，使用TNL1_NatProF和TNL1_35SR引物扩增proTNL1：TNL1。PCR体系和程序与实施例2中的相同。获得6.6Kb的特异性条带。用SacI和XbaI限制性内切酶对phellsgate8空质粒进行双酶切，得到线性化phellsgate8质粒。使用ClonExpress一步法克隆试剂盒(Vazyme，货号C112-01)按照产品说明书将PCR产物克隆至phellsgate8质粒中，得到重组质粒phellsgate8-proTNL1：TNL1。将重组质粒phellsgate8-proTNL1：TNL1转化大肠杆菌DH5α，挑选阳性克隆进行测序。测序结果表明，扩增的proTNL1：TNL1和基因组组装的基因序列完全一致，重组质粒中插入的proTNL1：TNL1序列准确无突变。
表9引物TNL1_NatProF
Figure PCTCN2022124977-appb-000012
将phellsgate8-proTNL1：TNL1导入根癌农杆菌EHA105菌株中，得到重组菌EHA105-phellsgate8-proTNL1：TNL1。将EHA105-phellsgate8-proTNL1：TNL1在根癌农杆菌LB培养基中于28℃培养24小时，离心收集菌体。
以感病烟草‘K326’为材料诱导愈伤组织，并使用EHA105-phellsgate8-proTNL1：TNL1侵染K326愈伤组织。经过卡纳霉素抗性筛选，得到抗性愈伤组织。抗性愈伤组织分化再生获得T0代转基因株系。提取T0代转基因植株的基因组DNA，并利用TNL1editTestF/TNL1editTestR引物对进行PCR反应。PCR结果为阳性的植株即为proTNL1：TNL1转基因阳性植株。
为了证明TNL1转基因植株对病毒的抗性，随机选择2个T0代proTNL1：TNL1转基因阳性植株(proTNL1：TNL1-#1和proTNL1：TNL1-#2)，自交收种获得T1代种子。在光照培养室内分别播种T1代种子，以常规方法培育T1植株。当T1植株具有4-5片叶时，提取每株植物的基因组DNA，利用TNL1editTestF/TNL1editTestR引物进行转基因检测。挑选检测结果为阳性的T1植株，分别接种TSWV、INSV、TZSV、CCSV、CaCV、TNSV、PCSV和HCRV病叶汁液。接种后21d调查植株的发病情况(表10)。
表10转基因植株接种orthotospoviruses的发病情况
Figure PCTCN2022124977-appb-000013
病毒挑战接种结果显示，2个独立的proTNL1：TNL1转基因植物均表现出对美洲型病毒TSWV和INSV以及欧亚型病毒TZSV和CCSV的抗性。在所有的转基因植株中，在系统叶中均不能检测到病毒的存在(图4C)。对于欧亚型CaCV，相对于非转基因对照，在proTNL1：TNL1转基因植物中病毒的发病率显著下降，病毒的症状明显减轻，表现出一定的抗病作用。作为对照，非转基因的普通K326栽培烟草是高度易感的。病毒接种实验结果表明，单独表达TNL1的转基因烟草能对5种orthotospoviruses产生抗性，具有广谱抗性。上述结果表明单独转基因表达TNL1就足以发挥RTSW抗性位点的功能，因此确认是TNL1赋予了烟草斑萎病抗性，TNL1即为RTSW基因。
前期发明人已经证明，Orthotospoviruses的NSm基因是抗性基因RTSW对应的Avr基因，为了进一步证明RTSW的广谱抗性，可以利用病毒的NSm浸润检测RTSW植株的orthotospoviruses抗性，检测方法参见专利号为ZL201710414755.X的中国专利。通过从病毒基因组cDNA中特异性扩增NSm或人工合成NSm基因的方法，我们共构建了14种orthotospoviruses(包括美洲型TSWV，CSNV，GRSV和INSV，以及欧亚型TZSV，CCSV，MVBaV，CaCV，GBNV，TNSV，PCSV，TNRV，PolRSV和HCRV)的NSm表达载体。将14种病毒的NSm基因片段分别克隆至pCambia1300-YFP表达载体中，获得重组质粒pCambia1300-NSm-YFP。在pCambia1300-NSm-YFP中，NSm基因位于35S启动子的下游，与YFP基因组成完整的读码框，可表达NSm-YFP融合蛋白，其中的YFP标签用于蛋白表达水平的检测。将pCambia1300-NSm-YFP导入根癌农杆菌EHA105中，获得EHA105-pCambia1300-NSm-YFP。将pCambia1300-YFP导入根癌农杆菌EHA105中，得到EHA105-pCambia1300-YFP作为空载体对照菌株。
将EHA105-35S-TSWV_NSm菌体悬液(OD600＝0.5)和EHA105-35S-Sw-5b菌体悬液(OD600＝0.5)按照1：1的体积比混合，作为阳性对照菌液。将EHA105-pCambia1300-YFP菌体悬液(OD600＝0.5)作为阴性对照菌液。用2mL注射器分别浸润选取的2个T1代转基因阳性植株和野生型对照K326植株的最大的2片叶片，每张叶片注射8孔，其中1孔接种阳性对照菌液，1孔接种阴性对照菌液，其余6孔每孔分别接种6种病毒的EHA105-pCambia1300-NSm-YFP菌体悬液(OD600＝0.5)，设置三个重复，接种后将植株放在20-28℃的光照培养室中培养72h，观察植株是否产生细胞坏死过敏反应(HR)。
结果显示，阳性对照菌液能够在所有T1代proTNL1：TNL1转基因阳性植株的叶片上诱导 产生HR，而阴性对照菌液在所有T1代proTNL1：TNL1转基因阳性植株的叶片上都不能诱导产生HR。在2个独立的proTNL1：TNL1转基因阳性植株中，单独表达病毒NSm和病毒挑战接种表现高度一致的抗性结果。所有4个待测美洲型TSWV，CSNV，GRSV和INSV，以及5个待测欧亚型TZSV，CCSV，MVBaV，CaCV和GBNV的NSm基因均可以和阳性对照一样诱导明显的HR，而TNSV，PCSV，TNRV，PolRSV和HCRV的NSm基因和空载体对照一样不能诱导HR。在非转基因的普通K326栽培烟草中，所有病毒的NSm基因均不能诱导任何程度的HR(表11)。
表11浸润EHA105-pCambia1300-NSm-YFP检测转基因植株的orthotospoviruses抗性
Figure PCTCN2022124977-appb-000014
以上实验证明，单独敲除RTSW基因可导致抗性植株失去斑萎病抗性，并且单独RTSW转基因足以赋予植株产生斑萎病的广谱抗性，因此RTSW基因对于斑萎病抗性是充分且必要的。
实施例6.RTSW基因在本氏烟、番茄及马铃薯中的功能验证
为了证明RTSW基因的广适性，我们选取了茄科的其他作物进行了RTSW转基因实验。以普通感病材料本氏烟烟草(N.benthamiana)，栽培马铃薯(Solanum tuberosum L)和栽培番茄(S.lycopersicum)为材料诱导愈伤组织。将EHA105-phellsgate8-proTNL1：TNL1分别转入本氏烟烟草、马铃薯和番茄的愈伤组织中。经过卡纳霉素抗性筛选，得到抗性愈伤组织。抗性愈伤组织分化再生获得T0代转基因植株。提取T0代转基因植株的基因组DNA，并利用TNL1editTestF/TNL1editTestR引物对进行PCR反应。PCR阳性的植株即为proTNL1：TNL1转基因阳性植株。针对本氏烟和番茄，随机选择2-3个T0代转基因阳性植株，自交收种获得T1代种子。对于块茎无性繁殖的马铃薯，直接收获T0代的薯块。在光照培养室内播种转基因阳性植株的T1代种子或T0代薯块，以常规方法培育植株。当植株具有4-5片叶时，取每 株植物的基因组DNA进行转基因元件检测。挑选检测结果为阳性的植株，分别接种TSWV和TZSV病叶汁液。接种后14d和21d分别调查植株的TSWV和TZSV发病情况(表12)。
表12转基因本氏烟、番茄和马铃薯的抗病性检测
Figure PCTCN2022124977-appb-000015
病毒挑战接种结果显示，在本氏烟(图5)、番茄(图6)和马铃薯(图7)中，不同的独立的proTNL1：TNL1转基因植物均表现出对TSWV和TZSV的抗性反应。在所有的转基因植株的系统叶中均不能检测到病毒的存在。作为对照，非转基因的本氏烟、番茄和马铃薯对TSWV和TZSV是高度易感的。
序列1-10
＞SEQ ID NO：1 TNL1基因
Figure PCTCN2022124977-appb-000016
Figure PCTCN2022124977-appb-000017
＞SEQ ID NO：2 TNL1 cDNA的编码区
Figure PCTCN2022124977-appb-000018
Figure PCTCN2022124977-appb-000019
＞SEQ ID NO：3 TNL1蛋白
Figure PCTCN2022124977-appb-000020
＞SEQ ID NO：4 TNL1基因的天然启动子
Figure PCTCN2022124977-appb-000021
Figure PCTCN2022124977-appb-000022
＞SEQ ID NO：5 TNL2基因
Figure PCTCN2022124977-appb-000023
Figure PCTCN2022124977-appb-000024
＞SEQ ID NO：6 TNL2 cDNA的编码区
Figure PCTCN2022124977-appb-000025
Figure PCTCN2022124977-appb-000026
＞SEQ ID NO：7 TNL2蛋白
Figure PCTCN2022124977-appb-000027
Figure PCTCN2022124977-appb-000028
＞SEQ ID NO：8 proTNL1：TNL1
Figure PCTCN2022124977-appb-000029
Figure PCTCN2022124977-appb-000030
Figure PCTCN2022124977-appb-000031
＞SEQ ID NO：9分子标记NaChr3_59.2M
Figure PCTCN2022124977-appb-000032
＞SEQ ID NO：10分子标记NaChr3_59.7M
Figure PCTCN2022124977-appb-000033

Claims (20)

  1. 一种核酸分子，其包含选自以下(a1)-(a5)的核苷酸序列：
    (a1)SEQ ID NO:1所示的核苷酸序列；
    (a2)SEQ ID NO:2所示的核苷酸序列；
    (a3)与SEQ ID NO:1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
    (a4)编码SEQ ID NO:3所示的氨基酸序列的核苷酸序列；
    (a5)编码与SEQ ID NO:3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
  2. 一种表达盒，其包含权利要求1所述的核酸分子和可操作连接的启动子。
  3. 一种载体，其包含权利要求1所述的核酸分子或权利要求2所述的表达盒。
  4. 一种宿主细胞，其转化有权利要求1所述的核酸分子、权利要求2所述的表达盒或权利要求3所述的载体。
  5. 一种转基因植物，其包含稳定并入其基因组中的异源多核苷酸构建体，所述多核苷酸构建体包含选自以下(a1)-(a5)的核苷酸序列：
    (a1)SEQ ID NO:1所示的核苷酸序列；
    (a2)SEQ ID NO:2所示的核苷酸序列；
    (a3)与SEQ ID NO:1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
    (a4)编码SEQ ID NO:3所示的氨基酸序列的核苷酸序列；
    (a5)编码与SEQ ID NO:3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
  6. 根据权利要求5所述的转基因植物，其中所述多核苷酸构建体还包含可操作连接的用于在植物中表达所述核苷酸序列的启动子。
  7. 根据权利要求5所述的转基因植物，其中所述转基因植物为茄科植物。
  8. 根据权利要求7所述的转基因植物，其中所述茄科植物为烟草、马铃薯或番茄。
  9. 根据权利要求5-8任一所述的转基因植物，其中相对于对照植物，所述转基因植物包含对至少一种正番茄斑萎病毒属病毒的抗性。
  10. 根据权利要求9所述的转基因植物，其中相对于对照植物，所述转基因植物包含对番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)、花生环斑病毒(GRSV)、菊花茎坏死病毒(CSNV)、番茄环纹斑点病毒(TZSV)、花生芽坏死病毒(GBNV)、桑脉带相关病毒(MVBaV)、辣椒褪绿病毒(CaCV)和马蹄莲褪绿斑病毒(CCSV)的抗性。
  11. 用于赋予或增强植物对至少一种正番茄斑萎病毒属病毒的抗性的方法，其包括将异源多核苷酸构建体导入至少一个植物细胞中，所述多核苷酸构建体包含选自以下(a1)-(a5)的核苷酸序列：
    (a1)SEQ ID NO:1所示的核苷酸序列；
    (a2)SEQ ID NO:2所示的核苷酸序列；
    (a3)与SEQ ID NO:1和2所示的核苷酸序列中的至少一个具有至少90％序列一致性的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性；
    (a4)编码SEQ ID NO:3所示的氨基酸序列的核苷酸序列；
    (a5)编码与SEQ ID NO:3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列的核苷酸序列，其中所述核酸分子能够赋予包含所述核酸分子的植物对至少一种正番茄斑萎病毒属病毒的抗性。
  12. 根据权利要求11所述的方法，其中所述多核苷酸构建体还包含可操作连接的用于在植物中表达所述核苷酸序列的启动子，并且所述多核苷酸构建体被稳定并入所述植物细胞的基因组中。
  13. 根据权利要求12所述的方法，其中使所述植物细胞再生成在其基因组中包含所述多核苷酸构建体的植物。
  14. 根据权利要求13所述的方法，其中所述植物为烟草、马铃薯或番茄。
  15. 根据权利要求13所述的方法，其中相对于对照植物，所述植物至少包含对番茄斑萎病毒(TSWV)、凤仙花坏死斑病毒(INSV)、花生环斑病毒(GRSV)、菊花茎坏死病毒(CSNV)、番茄环纹斑点病毒(TZSV)、花生芽坏死病毒(GBNV)、桑脉带相关病毒(MVBaV)、辣椒褪绿病毒(CaCV)和马蹄莲褪绿斑病毒(CCSV)的抗性。
  16. 由权利要求11-15任一所述的方法产生的植物、或所述植物的果实、块茎、叶子或种子，其中所述果实、块茎、叶子或种子包含所述多核苷酸构建体。
  17. 用于鉴定包含赋予的或增强的对至少一种正番茄斑萎病毒属病毒的抗性的植物的方法，其包括检测所述植物中或其至少一个部分或细胞中RTSW的存在。
  18. 根据权利要求17所述的方法，其中所述植物为烟草、马铃薯或番茄。
  19. 根据权利要求17或18所述的方法，其中通过检测RTSW中的至少一个标记来检测RTSW的存在。
  20. 一种多肽，其包含选自以下(B1)-(B4)的氨基酸序列：
    (B1)由SEQ ID NO:1所示的核苷酸序列编码的氨基酸序列；
    (B2)由SEQ ID NO:2所示的核苷酸序列编码的氨基酸序列；
    (B3)SEQ ID NO:3所示的氨基酸序列；
    (B4)与SEQ ID NO:3所示的氨基酸序列具有至少90％序列一致性的氨基酸序列，其中包含所述氨基酸序列的多肽能够赋予包含所述多肽的植物对至少一种正番茄斑萎病毒属病毒的抗性。
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