WO2024060538A1 - 水稻抗病基因及其用途 - Google Patents

水稻抗病基因及其用途 Download PDF

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WO2024060538A1
WO2024060538A1 PCT/CN2023/081851 CN2023081851W WO2024060538A1 WO 2024060538 A1 WO2024060538 A1 WO 2024060538A1 CN 2023081851 W CN2023081851 W CN 2023081851W WO 2024060538 A1 WO2024060538 A1 WO 2024060538A1
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gene
flotillin1
plant
rice
seq
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PCT/CN2023/081851
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English (en)
French (fr)
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崔峰
葛盘泮
卢虹
马永焕
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中国科学院动物研究所
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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
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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
    • C12N15/63—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 application relates to the field of biotechnology, specifically to the use of rice Flotillin1 gene and Importin ⁇ 4 gene, and the use of molecules that inhibit the transcription or translation of Flotillin1 gene and Importin ⁇ 4 gene in the preparation of kits.
  • the present application also relates to a method of obtaining plants capable of resisting pathogens.
  • Plant virus infection has become the second largest disease in agricultural production, causing huge economic losses worldwide every year.
  • Rice virus is one of the important groups of plant viruses, which seriously threatens rice production in East Asia, causing a 16% reduction in yield at the least and a total crop failure at the worst.
  • the most serious outbreaks include rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), rice black-streaked dwarf virus (RBSDV), rice ragged stunt virus (RRSV) and rice grassy stunt virus (RGSV). Therefore, blocking the spread of viruses in plant hosts and cultivating excellent varieties with broad-spectrum resistance to viruses are important ways to deal with viral diseases.
  • Rice stripe virus is a single-stranded RNA virus. Its genome includes four RNA strands (RNA1, RNA2, RNA3, RNA4) and encodes a total of seven proteins (RdRp, NS2, NSvc2, NS3, NP, SP, NSvc4 ).
  • Current research on RSV and the rice host mostly focuses on the interaction between the protein encoded by RSV itself and rice host factors, affecting the virus's own replication or affecting the gene silencing, immunity, and autophagy pathways of plant host cells (Fu et al., 2018; Zheng et al., 2017; Zhao et al., 2016; Kong et al., 2014).
  • the receptor of RSV in the rice host remains unclear.
  • the present application provides a method for obtaining a plant capable of resisting pathogens, the method comprising: reducing or inhibiting the transcription or translation of the Flotillin1 gene and the Importin ⁇ 4 gene in the plant, thereby reducing or inhibiting the The expression levels of proteins encoded by Flotillin1 gene and Importin ⁇ 4 gene respectively in plants.
  • the plant is a grass plant. In certain embodiments, the plant is selected from wheat, barley, corn, rice, sorghum.
  • the pathogen is a virus. In certain embodiments, the pathogen is rice streak virus.
  • amino acid sequence of the protein encoded by the Importin ⁇ 4 gene is shown in SEQ ID NO: 3.
  • amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO: 1.
  • reducing or inhibiting the expression of the plant Flotillin1 gene and Importin ⁇ 4 gene is by any one of the following methods: substitution, deletion or addition of one or several nucleotides (e.g. 1, 2 or 3 nucleotide substitutions, deletions, or additions), site-specific mutagenesis, ethyl mesylate mutagenesis, targeted induction of localized mutations in the genome, or gene editing (e.g., sgRNA-guided gene knockout).
  • substitution, deletion or addition of one or several nucleotides e.g. 1, 2 or 3 nucleotide substitutions, deletions, or additions
  • site-specific mutagenesis e.g. 1, 2 or 3 nucleotide substitutions, deletions, or additions
  • ethyl mesylate mutagenesis e.g., targeted induction of localized mutations in the genome
  • gene editing e.g., sgRNA-guided gene knockout
  • sgRNAs targeting the Flotillin1 gene and the Importin ⁇ 4 gene are designed to guide Cas9 to edit the target site.
  • each copy of the Flotillin1 gene and the Importin ⁇ 4 gene all become defective.
  • sgRNA sequences targeting the rice Flotillin1 gene and Importin ⁇ 4 gene were designed respectively, and corresponding vectors containing sgRNA and cas9 protein were constructed.
  • Agrobacterium as a gene manipulation tool to carry the vector, the sgRNA sequences targeting the rice Flotillin1 gene and Importin ⁇ 4 gene were efficiently introduced into the rice cell genome, reducing the expression levels of the rice Flotillin1 gene and Importin ⁇ 4 gene, and significantly down-regulating the Flotillin1 gene. and the expression of Importin ⁇ 4 at the mRNA level and protein level.
  • the method is carried out by steps (a) to (f) of:
  • step (e) converting the plant cells of step (c) or (d) into plants;
  • the sgRNA1 and sgRNA2 are constructed in the same vector or different vectors. In certain embodiments, the sgRNA1 and sgRNA2 are constructed in the pYLCRISPR/Cas9P ⁇ bi-H vector.
  • the Agrobacterium is Agrobacterium EHA105.
  • RNA of the plant is extracted, the RNA is reverse transcribed into cDNA, and the nucleotide fragments targeted by sgRNA1 and sgRNA2 contained in the cDNA are amplified by primers , for screening; or, extract the genomic DNA of the plant, and use primers to amplify the nucleotide fragments targeted by sgRNA1 and sgRNA2 contained in the DNA for screening.
  • nucleotide sequence of sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.
  • nucleotide sequence of sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.
  • this application provides the use of a molecule that specifically inhibits the transcription or translation of the Flotillin1 gene and the Importin ⁇ 4 gene or specifically inhibits the expression level of the proteins encoded by the Flotillin1 gene and the Importin ⁇ 4 gene respectively in the preparation of a kit,
  • the kit is used to obtain plants capable of resisting pathogens, or to improve the ability of plants to resist pathogens.
  • the molecules include but are not limited to nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, proteins or interfering lentiviruses.
  • the nucleic acid molecules include but are not limited to: antisense oligonucleotides, bis stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), or guide RNA (sgRNA).
  • dsRNA bis stranded RNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • sgRNA guide RNA
  • the nucleic acid molecule contains at least 2, at least 3, at least 4, at least 5, at least 6 sgRNAs.
  • the nucleic acid molecule includes sgRNA1 and sgRNA2, the sgRNA1 is capable of targeting the Flotillin1 gene or a fragment thereof, and the sgRNA2 is capable of targeting the Importin ⁇ 4 gene or a fragment thereof.
  • nucleotide sequence of sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.
  • nucleotide sequence of sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.
  • the kit further includes: a vector, Agrobacterium tumefaciens, a medium and/or reagent for culturing plant cells or tissues, a reagent for extracting plant DNA or RNA, and amplifying sgRNA1 and sgRNA2. Primer sets targeting fragments separately, or any combination thereof.
  • the vector is pYLCRISPR/Cas9P ⁇ bi-H.
  • the sgRNA1 and sgRNA2 are or are not included in the vector.
  • the Agrobacterium is Agrobacterium EHA105.
  • the plant is a grass plant. In certain embodiments, the plant is selected from wheat, barley, corn, rice, sorghum.
  • the pathogen is a virus. In certain embodiments, the pathogen is rice streak virus.
  • amino acid sequence of the protein encoded by the Importin ⁇ 4 gene is shown in SEQ ID NO: 3.
  • amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO: 1.
  • the application provides a kit comprising any one or more of the following (1) to (6):
  • the kit includes all combinations of (1) to (6).
  • the nucleic acid molecule is selected from an antisense oligonucleotide, a dsRNA, a siRNA, a shRNA, or a sgRNA.
  • the nucleic acid molecule comprises sgRNA1 and sgRNA2, the sgRNA1 is capable of targeting the Flotillin1 gene or a fragment thereof, and the sgRNA2 is capable of targeting the Importin ⁇ 4 gene or a fragment thereof.
  • the kit is used to obtain plants capable of resisting pathogens, or to improve the ability of plants to resist pathogens.
  • the plant is a grass plant. In certain embodiments, the plant is selected from wheat, barley, corn, rice, sorghum.
  • the pathogen is a virus. In certain embodiments, the pathogen is rice streak virus.
  • amino acid sequence of the protein encoded by the Importin ⁇ 4 gene is shown in SEQ ID NO: 3.
  • amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO: 1.
  • nucleotide sequence of sgRNA1 is SEQ ID NO:5 or SEQ ID NO:6.
  • nucleotide sequence of sgRNA2 is SEQ ID NO:7 or SEQ ID NO:8.
  • the vector is pYLCRISPR/Cas9P ⁇ bi-H.
  • the Agrobacterium is Agrobacterium EHA105.
  • the term "defective" refers to a cell or plant in which the level of a protein encoded by a gene is reduced compared to a wild-type plant. The reduction may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (i.e., complete inhibition of protein levels).
  • the term "protein encoded by the Flotillin1 gene” refers to the naturally occurring, biologically active Flotillin1 protein. Flotillin1 protein exists in a variety of grass plants, and the amino acid sequence of this protein can be easily obtained from various public databases (eg, GenBank database). In certain embodiments, the amino acid sequence of the protein encoded by the Flotillin1 gene is shown in SEQ ID NO: 1.
  • the term "protein encoded by the Importin ⁇ 4 gene” refers to a naturally occurring, biologically active Importin ⁇ 4 protein.
  • Importin ⁇ 4 protein exists in a variety of grass plants, and the amino acid sequence of this protein can be easily obtained from various public databases (eg, GenBank database).
  • the amino acid sequence of the protein encoded by the Importin ⁇ 4 gene is shown in SEQ ID NO: 3.
  • Flotillin1 gene refers to any nucleic acid encoding a Flotillin1 protein, including DNA (eg, genomic DNA), RNA (eg, mRNA).
  • DNA eg, genomic DNA
  • RNA eg, mRNA
  • the nucleotide sequence of the Flotillin1 gene is set forth in SEQ ID NO: 2.
  • the term "Importin ⁇ 4 gene” refers to any nucleic acid encoding an Importin ⁇ 4 protein, including DNA (eg, genomic DNA), RNA (eg, mRNA).
  • DNA eg, genomic DNA
  • RNA eg, mRNA
  • the nucleotide sequence of the Importin ⁇ 4 gene is set forth in SEQ ID NO: 4.
  • pathogen refers to all organisms capable of causing disease in plants, including fungi, nematodes, bacteria and viruses. In certain embodiments, the pathogen is rice streak virus.
  • pathogen-resistant plant refers to a plant that can resist the infection and/or spread of the pathogen to a certain extent, which is specifically manifested in that after the pathogen infects the plant, it is similar to a wild-type plant. Compared with this, the disease incidence of the plants is reduced.
  • codons are degenerate. That is, during the translation of proteins, each amino acid may correspond to one or more codons, for example, up to six codons. There are great differences in the degenerate codons that encode a certain amino acid, and there are different preferences. This preference phenomenon is called "codon preference”. Therefore, as used herein, the term "codon preference” refers to the situation in which a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules based on codon preference is particularly advantageous in some cases, for example, it may help to increase the expression level of the protein encoded by the nucleic acid molecule.
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids (e.g., naked plasmids); phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived Artificial chromosomes (PAC); phages such as lambda phage or M13 phage and viral vectors, etc.
  • plasmids e.g., naked plasmids
  • phagemids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived Artificial chromosomes (PAC)
  • phages such as lambda phage or M13 phage and viral vectors, etc.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Tumor vacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • Tumor vacuolating viruses such as SV40
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • sgRNA small guide RNA
  • the Cas9 protein performs site-specific editing of the target nucleic acid.
  • sgRNA targeting the target nucleic acid is designed to guide the Cas9 nuclease to target specific sites of the target nucleic acid. Not every sgRNA has the same cleavage efficiency. In view of this inconsistency, it is necessary to screen multiple sgRNAs to find the one with the highest cutting efficiency.
  • nucleotide sequence of the sgRNA targeting the Flotillin1 gene is SEQ ID NO:5 or SEQ ID NO:6. In certain embodiments, the nucleotide sequence of the sgRNA targeting the Importin ⁇ 4 gene is SEQ ID NO:7 or SEQ ID NO:8.
  • the effect of the single knockout mutant was not ideal; further, the applicant unexpectedly found that the rice mutant with simultaneous knockout of Flotillin1 and Importin ⁇ 4 had significant It inhibited the spread rate of RSV in rice (the spread rate was inhibited by at least 50% compared with WT), and also significantly reduced the incidence of RSV in rice (the incidence rate was reduced by at least 40% compared with WT).
  • this mutant has no effect on rice plant height, thousand-grain weight and germination rate. Therefore, this mutant
  • the knockout combination ie, knockout of Flotillin1 gene and Importin ⁇ 4 gene
  • Figure 1 shows the verification results of the transcript level and protein level of the rice Flotillin1 mutant.
  • a in Figure 1 represents the expression of Flotillin1 transcript level in wild-type rice (WT) and Flotillin1 mutant;
  • B in Figure 1 represents Expression of Flotillin1 protein levels in wild-type rice (WT) and Flotillin1 mutant. Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 2 shows the effect of knocking out Flotillin1 on rice plant height, thousand-grain weight and germination rate.
  • a in Figure 2 and B in 2 represent the changes in rice plant height after knocking out Flotillin1.
  • the scale bar is 10cm;
  • Figure 2 C in and D in 2 represent the changes in rice 1,000-kernel weight after Flotillin1 is knocked out, and the scale bar is 5cm.
  • Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 3 shows the effect of knocking out Flotillin1 on the diffusion rate of RSV in rice.
  • a in Figure 3 and B in Figure 3 indicate that in the Flotillin1 mutant, RSV infected the leaves in epidermal cells and fiber tissue 12 hours after inoculation. , the diffusion rate of mesophyll cells, bundle sheath cells, sieve tubes and companion cells, the scale bar is 20nm;
  • C in Figure 3 and D in Figure 3 indicate that in the Flotillin1 mutant, 1 day after RSV infects the systemic leaves, the growth rate of the epidermal cells in the system leaves , the diffusion rate of fibrous tissue, mesophyll cells, bundle sheath cells, sieve tubes and companion cells, the scale bar is 20nm.
  • Figure 4 shows the effect of knocking out Flotillin1 on the expression of RSV NP in rice.
  • A, B, and C in Figure 4 respectively represent the NP after RSV infects rice system leaves 1d, 4d, and 7d in the Flotillin1 mutant. changes in expression. Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 5 shows the effect of knocking out Flotillin1 on rice disease incidence, where A in Figure 5 indicates when the insect population density When the insect population density is 10, the effect of RSV infection on the incidence of rice in Flotillin1 mutant. B in Figure 5 shows the effect of RSV infection on the incidence of Flotillin1 mutant rice when the insect population density is 2. Values are expressed as mean ⁇ standard error. Asterisks indicate significant differences between treatments, *P ⁇ 0.05, **P ⁇ 0.01. Different lowercase letters indicate significant differences in gene expression between treatments (P ⁇ 0.05).
  • Figure 6 shows the effect of knocking out Importin ⁇ 4 on rice plant height and thousand-grain weight.
  • a and B in Figure 6 represent the changes in rice plant height after knocking out Importin ⁇ 4.
  • the scale bar is 10cm;
  • C in Figure 6 represents knocking out Importin ⁇ 4.
  • changes in the thousand-grain weight of rice Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 7 shows the effect of knocking out Importin ⁇ 4 on the expression of RSV NP in rice.
  • Figure 7A, B, and C respectively show the expression of NP in the Importin ⁇ 4 mutant after RSV infected the leaves of the rice system for 1d, 4d and 7d. Variety. Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 8 shows the effect of knocking out Importin ⁇ 4 on the incidence of rice.
  • a in Figure 8 shows the effect of RSV infection on the incidence of rice in the Importin ⁇ 4 mutant when the insect population density is 10.
  • B in Figure 8 shows the effect of RSV infection on the incidence of Importin ⁇ 4 mutant rice when the insect population density is 2.
  • Values are expressed as mean ⁇ standard error. Asterisks indicate significant differences between treatments, *P ⁇ 0.05, **P ⁇ 0.01. Different lowercase letters indicate significant differences in gene expression between treatments (P ⁇ 0.05).
  • Figure 9 shows the effect of knocking out Flotillin1-Importin ⁇ 4 on rice plant height and thousand-grain weight.
  • a and B in Figure 9 represent the changes in rice plant height after knocking out Flotillin1-Importin ⁇ 4.
  • the scale bar is 10cm;
  • C in Figure 9 Indicates the changes in 1000-grain weight of rice after Flotillin1-Importin ⁇ 4 is knocked out. Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 10 shows the effect of knocking out Flotillin1-Importin ⁇ 4 on the expression of RSV NP in rice.
  • A, B, and C in Figure 10 respectively indicate that in the Flotillin1-Importin ⁇ 4 mutant, RSV infected the rice system leaves on 1d and 4d. and changes in NP expression after 7 days. Values are expressed as mean ⁇ standard error, and different lowercase letters indicate significant differences in gene expression in different treatments (P ⁇ 0.05).
  • Figure 11 shows the effect of knocking out Flotillin1-Importin ⁇ 4 on the incidence of rice.
  • a in Figure 11 shows the effect of RSV infection on the incidence of rice in the Flotillin1-Importin ⁇ 4 mutant when the insect population density is 10.
  • B in Figure 11 shows the effect of RSV infection on the incidence of Flotillin1-Importin ⁇ 4 mutant rice when the insect population density is 2.
  • Values are expressed as mean ⁇ standard error. Asterisks indicate significant differences between treatments, *P ⁇ 0.05, **P ⁇ 0.01. Different lowercase letters indicate significant differences in gene expression between treatments (P ⁇ 0.05).
  • Test insects The strains of Laodelphax striatell ⁇ s with and without the virus used in this article were mainly collected from Hai'an, Jiangsu province and domesticated for a long time.
  • the Laodelphax striatell ⁇ s were raised in glass bottles containing 2-3 cm rice seedlings and sealed with nylon nets. The rearing temperature was maintained at around 25 degrees Celsius, and the photoperiod was 16h:8h (light: dark). In order to ensure adequate nutrition, the rice seedlings were replaced once a week.
  • the Laodelphax striatell ⁇ s with and without the virus were raised in different greenhouses with the same environmental conditions, and the virus load was detected and screened by dot-linked immunosorbent assay every three months.
  • Blocking Add 10mL of 1% skim milk (prepared with 1x PBST buffer) to the incubation box containing the dried nitrocellulose membrane, place it on a shaker, and block for 30 minutes at room temperature.
  • Incubate the secondary antibody Use 1% skim milk to dilute the goat anti-mouse secondary antibody (CW0102S) conjugated with horseradish peroxidase (HRP) at a ratio of 1:10000, and add 10 mL Put it into the incubation box, place it on a shaker, and incubate at room temperature for 1 hour.
  • CW0102S goat anti-mouse secondary antibody conjugated with horseradish peroxidase
  • the target vector was transformed into Agrobacterium EHA105, and the Agrobacterium-mediated method was transferred into the embryogenic callus of Nipponbare rice. Targeting the Flotillin1 gene, more than 3 homozygous Flotillin1 gene knockout mutants were identified.
  • primers F1 and R1 for targeting effect detection are as follows: forward primer F1:5 (nucleotide sequence is shown in SEQ ID NO:15), reverse primer R1:5 (nucleotide sequence is shown in SEQ ID NO:16).
  • Vector construction Design two sections of Importin ⁇ 4 (its amino acid sequence is shown in SEQ ID NO: 3, nucleoside The targeting sgRNAs (Table 3) whose acid sequence is shown in SEQ ID NO: 4) were sequentially linked into the CRISPR/Cas9 binary vector pYLCRISPR/Cas9P ⁇ bi-H (Wuhan Boyuan Biotechnology).
  • the target vector was transformed into Agrobacterium EHA105, and the Agrobacterium-mediated method was introduced into the embryogenic callus of Nipponbare rice. Targeting the Importin ⁇ 4 gene, more than 3 homozygous Importin ⁇ 4 gene knockout mutants were identified.
  • (2) Molecular identification of Importin ⁇ 4 mutant rice extract the DNA of the transgenic plant, design primers at both ends of the targeting sgRNAs, perform PCR to expand the fragments containing the targeting region, and sequence to analyze the targeting effect.
  • the sequences of the targeting effect detection primers F1 and R1 are as follows: forward primer F1:6 (the nucleotide sequence is shown in SEQ ID NO:19), and reverse primer R1:6 (the nucleotide sequence is shown in SEQ ID NO:20 ).
  • TargetsgRNAs of Flotillin1 (its amino acid sequence is shown in SEQ ID NO:1, and its nucleotide sequence is shown in SEQ ID NO:2) (the specific sequence is the same as in Table 2 above) and two segments
  • the targeting sgRNAs of OsImportin ⁇ 4 (its amino acid sequence is shown in SEQ ID NO:3, and its nucleotide sequence is shown in SEQ ID NO:4) (the specific sequence is the same as in Table 3 above), and is sequentially connected into the CRISPR/Cas9 binary vector pYLCRISPR/ Cas9P ⁇ bi-H.
  • the target vector was transformed into Agrobacterium EHA105, and Agrobacterium-mediated transformation was carried out into Nipponbare rice. Targeting the Importin ⁇ 4 gene and Flotillin1 gene, homozygous Importin ⁇ 4-Flotillin1 gene knockout mutants were identified.
  • RNA extraction RNA from rice leaves was extracted using the Trizol method (Ambion, 15596018). The specific steps are as follows:
  • cDNA synthesis was performed on RNA using the M-MLV reverse transcription system (Promega, MSA). Specific steps are as follows.
  • RNA solution into a 0.2 mL PCR tube, use double-distilled water (without RNase) to fill the volume to 12 ⁇ L, and add 1 ⁇ L of random primers (if you want to clone or detect viral genes, you need to use random primers.
  • Oligo-dT primers can be used), mix by pipetting, centrifuge to the bottom of the tube, incubate at 70°C for 60 minutes, and then incubate at 4°C for 10 minutes; add 5 ⁇ L 5x MLV buffer, 5 ⁇ L dNTPs, and 1 ⁇ L reverse transcriptase to the above tube , 1 ⁇ L of RNase inhibitor, mix well by pipetting, centrifuge to the bottom of the tube, incubate at 42°C for 60 minutes, and incubate at 75°C for 15 minutes to terminate the reaction; dilute the reverse transcription product according to experimental requirements and store it in a -20°C refrigerator.
  • Rice protein was extracted using Kangwei Reagent's RIPA lysis buffer kit (CW2333). First, add 5ml Plant protein extraction reagent (containing 1% Protease inhibitor cocktail) according to the ratio of 1g tissue, mechanical homogenization and lysis; incubate on ice for 20-30 minutes; 4°C13, 400g, centrifuge for 20 minutes; collect the supernatant -20°C save.
  • Plant protein extraction reagent containing 1% Protease inhibitor cocktail
  • Each plant is inoculated with an average of 10 and 2 poisonous bovine planthoppers, and they are clamped with microscopic insect cages. 2 days after exposure to the poison, the poisonous bovine planthoppers and microscopic insect cages were removed, and the incidence rate was calculated. There were 6 biological replicates for each treatment.
  • the rice leaves treated with poison were fixed with 4% paraformaldehyde (Coolab, SL18301) at 4°C overnight; the tissues were transferred to hard gelatin capsules (Electron Microscopy Sciences, 70102), and the embedding agent (Japanese Sakura , SAKMRA 4583) embedded, drained of air, and quick-frozen at -20°C for 5 minutes; cut into 10 ⁇ m sections with a freezing microtome (Leica), placed on adhesive slides, and dried in the air; place the sections in a solution containing 10% Incubate in goat serum (Beyotime, C0265) for 1 hour at room temperature to block non-specific binding sites; elute twice with PBST (containing 0.1% Tween 20); dilute the primary antibody (RSV NP mouse monoclonal antibody 1:500) with PBST , incubate overnight at 4°C, elute three times with PBST, and start the operation in the dark; dilute the secondary antibody (abberior, STORANG

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Abstract

一种Flotillin1基因和Importinα4基因的用途，以及抑制Flotillin1基因和Importinα4基因的转录或翻译的分子在制备试剂盒中的用途。还涉及一种获得能够抵抗病原体的植物的方法。

Description

水稻抗病基因及其用途 技术领域
本申请涉及生物技术领域，具体涉及水稻Flotillin1基因和Importinα4基因的用途，以及抑制Flotillin1基因和Importinα4基因的转录或翻译的分子在制备试剂盒中的用途。本申请还涉及一种获得能够抵抗病原体的植株的方法。
背景技术
植物病毒感染已经成为农业生产中的第二大病害，每年在全球造成巨大的经济损失。水稻病毒是植物病毒的重要类群之一，严重威胁着东亚地区的水稻产量，轻则导致减产16％，重则绝收。近年来爆发较为严重的主要包括水稻条纹病毒(rice stripe virus，RSV)，南方水稻黑条矮缩病毒(southern rice black-streaked dwarf virus，SRBSDV)，水稻黑条矮缩病毒(rice black-streaked dwarf virus,RBSDV)，水稻齿矮病毒(rice ragged stunt virus，RRSV)以及水稻草矮病毒(rice grassy stunt virus，RGSV)。因此，阻断病毒在植物宿主内的传播，培育广谱抗病毒的优良品种，是应对病毒病的重要途径。
水稻条纹病毒(rice stripe virus,RSV)是单链RNA病毒，基因组包括四条RNA链(RNA1、RNA2、RNA3、RNA4)，共编码七个蛋白(RdRp、NS2、NSvc2、NS3、NP、SP、NSvc4)。目前有关RSV与水稻宿主的研究多集中在RSV自身编码的蛋白与水稻宿主因子互作，影响病毒自身复制或者影响植物宿主细胞的基因沉默，免疫，自噬通路等(Fu et al.,2018；Zheng et al.,2017；Zhao et al.,2016；Kong et al.,2014)。但RSV在水稻宿主中的受体仍不清楚。
综上所述，植物病毒侵染严重威胁着全球经济安全和食品安全，但是有关阻断病毒传播的研究比较匮乏。需要提供一种抗病基因，以应对水稻的病毒侵染。
发明内容
本申请的发明人经过大量实验和反复摸索，发现了植物中的Flotillin1基因和Importinα4对植物病原体的抗性的起负调控作用。因此，制备了敲除Flotillin1和Importinα4的水稻突变体，并意外的发现该突变体显著的抑制了RSV在水稻中的扩散率，还显著的降低了RSV在水稻中的发病率。并且，该突变体对水稻株高，千粒重及萌 发率没有影响。因此，敲除Flotillin1基因和Importinα4基因的突变体植株在植物抗病中具有较好的应用潜能。
因此，在第一方面，本申请提供了一种获得能够抵抗病原体的植株的方法，所述方法包括：减少或抑制所述植株中Flotillin1基因和Importinα4基因的转录或翻译，从而减少或抑制所述植株中Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平。
在某些实施方案中，所述植物是禾本科植物。在某些实施方案中，所述植物选自小麦，大麦，玉米，水稻，高粱。
在某些实施方案中，所述病原体是病毒。在某些实施方案中，所述病原体是水稻条纹病毒。
在某些实施方案中，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示。
在某些实施方案中，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
在某些实施方案中，减少或抑制所述植物Flotillin1基因和Importinα4基因的表达是通过下列的任意一项方法：一个或几个核苷酸的置换、缺失或添加(例如1个，2个或3个核苷酸的置换、缺失或添加)，位点特异性诱变，甲磺酸乙酯诱变，定向诱导基因组局部突变，或基因编辑(例如，由sgRNA引导的基因敲除)。
在某些实施方案中，通过设计靶向Flotillin1基因和Importinα4基因的sgRNA，以引导Cas9对靶向位点进行编辑。
在某些实施方案中，当所述植物细胞中存在Flotillin1基因和Importinα4基因的几个(例如，1个，2个，3个，4个)拷贝时，使Flotillin1基因和Importinα4基因的每个拷贝均变成有缺陷的。
在本发明的具体实施方案中，分别设计了针对水稻Flotillin1基因和Importinα4基因的sgRNA序列，构建相应的含有sgRNA和cas9蛋白的载体。使用农杆菌作为基因操作工具携带所述载体靶向地将针对水稻Flotillin1基因和Importinα4基因的sgRNA序列高效导入水稻细胞基因组中，降低了水稻Flotillin1基因和Importinα4基因的表达水平，显著的下调了Flotillin1基因和Importinα4在mRNA水平和蛋白水平的表达。
因此，在某些实施方案中，所述方法通过以下步骤(a)至(f)实现：
(a)构建含有sgRNA1和sgRNA2的载体，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段；
(b)将所述载体转化农杆菌；
(c)将所述农杆菌侵染植物细胞；
(d)任选地，选择具有缺陷的Flotillin1基因和Importinα4基因的植物细胞；
(e)将步骤(c)或(d)的植物细胞生成植物；
(f)任选地，筛选具有缺陷的Flotillin1基因和Importinα4基因的植物，以获得能够抵抗病原体的植物。
在某些实施方案中，所述sgRNA1和sgRNA2构建于同一载体或不同的载体中。在某些实施方案中，所述sgRNA1和sgRNA2构建于pYLCRISPR/Cas9Pμbi-H载体中。
在某些实施方案中，所述农杆菌是EHA105农杆菌。
在某些实施方案中，在步骤(f)中，提取所述植物的RNA，将所述RNA逆转录为cDNA，通过引物扩增所述cDNA中包含的sgRNA1和sgRNA2靶向的核苷酸片段，以进行筛选；或者，提取所述植物的基因组DNA，通过引物扩增所述DNA中包含的sgRNA1和sgRNA2靶向的核苷酸片段，以进行筛选。
在某些实施方案中，所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6。
在某些实施方案中，所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
在第二方面，本申请提供了一种特异性抑制Flotillin1基因和Importinα4基因的转录或翻译或特异性抑制Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平的分子在制备试剂盒中的用途，所述试剂盒用于获得能够抵抗病原体的植株，或用于提高植株抵抗病原体的能力。
在本发明的一个实施方案中，其中所述的分子包括但不限于核酸分子、碳水化合物、脂类、小分子化学药、抗体药、多肽、蛋白或干扰慢病毒。
在本发明的一个实施方案中，其中所述核酸分子包括但不限于：反义寡核苷酸，双 链RNA(dsRNA)，小干扰RNA(siRNA)，短发夹RNA(shRNA)，或向导RNA(sgRNA)。
在某些实施方案中，所述核酸分子包含至少2个，至少3个，至少4个，至少5个，至少6个sgRNA。
在某些实施方案中，所述核酸分子包含sgRNA1和sgRNA2，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段。
在某些实施方案中，所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6。
在某些实施方案中，所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
在某些实施方案中，所述试剂盒还包含：载体，农杆菌，用于培养植物细胞或组织的培养基和/或试剂，用于提取植物DNA或RNA的试剂，扩增包含sgRNA1和sgRNA2分别靶向的片段的引物组，或其任何组合。
在某些实施方案中，所述载体是pYLCRISPR/Cas9Pμbi-H。
在某些实施方案中，所述sgRNA1和sgRNA2包含或不包含于所述载体中。
在某些实施方案中，所述农杆菌是EHA105农杆菌。
在某些实施方案中，所述植物是禾本科植物。在某些实施方案中，所述植物选自小麦，大麦，玉米，水稻，高粱。
在某些实施方案中，所述病原体是病毒。在某些实施方案中，所述病原体是水稻条纹病毒。
在某些实施方案中，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示。
在某些实施方案中，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
在第三方面，本申请提供了一种试剂盒，其包含以下(1)至(6)的任意一项或多项：
(1)特异性抑制Flotillin1基因和Importinα4基因的转录或翻译或特异性抑制 Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平的核酸分子；
(2)包含所述核酸分子的载体；
(3)包含所述载体的农杆菌；
(4)用于培养植物细胞或组织的培养基和/或试剂；
(5)用于提取植物DNA或RNA的试剂；
(6)用于扩增包含Flotillin1基因和Importinα4基因的核苷酸片段的引物组。
在某些实施方案中，所述试剂盒包含(1)至(6)的全部组合。
在某些实施方案中，所述核酸分子选自反义寡核苷酸，dsRNA，siRNA，shRNA，或sgRNA。
在某些实施方案中，所述核酸分子包含sgRNA1和sgRNA2，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段。
在某些实施方案中，其中，所述试剂盒用于获得能够抵抗病原体的植株，或用于提高植株抵抗病原体的能力。
在某些实施方案中，所述植物是禾本科植物。在某些实施方案中，所述植物选自小麦，大麦，玉米，水稻，高粱。
在某些实施方案中，所述病原体是病毒。在某些实施方案中，所述病原体是水稻条纹病毒。
在某些实施方案中，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示。
在某些实施方案中，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
在某些实施方案中，所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6。
在某些实施方案中，所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
在某些实施方案中，所述载体是pYLCRISPR/Cas9Pμbi-H。
在某些实施方案中，所述农杆菌是EHA105农杆菌。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的分子遗传学、核酸化学、化学、分子生物学、生物化学、细胞培养、微生物学、细胞生物学、基因组学和重组DNA等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“有缺陷的”是指细胞或植物中的基因所编码的蛋白的水平与野生型植物相比有所降低，降低可以为约10％，20％，30％，40％，50％，60％，70％，80％，90％，或100％(即，完全抑制蛋白的水平)。
如本文中所使用的，术语“Flotillin1基因所编码的蛋白质”是指天然存在的，具有生物学活性的Flotillin1蛋白。Flotillin1蛋白存在于多种禾本科植物中，可方便地从各种公共数据库(例如，GenBank数据库)获得该蛋白的氨基酸序列。在某些实施方案中，Flotillin1基因所编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
如本文中所使用的，术语“Importinα4基因所编码的蛋白质”是指天然存在的，具有生物学活性的Importinα4蛋白。Importinα4蛋白存在于多种禾本科植物中，可方便地从各种公共数据库(例如，GenBank数据库)获得该蛋白的氨基酸序列。在某些实施方案中，Importinα4基因所编码的蛋白质的氨基酸序列如SEQ ID NO:3所示。
如本文中所使用的，术语“Flotillin1基因”是指编码Flotillin1蛋白的任何核酸，包括DNA(例如，基因组DNA)，RNA(例如，mRNA)。在某些实施方案中，Flotillin1基因的核苷酸序列如SEQ ID NO:2所示。
如本文中所使用的，术语“Importinα4基因”是指编码Importinα4蛋白的任何核酸，包括DNA(例如，基因组DNA)，RNA(例如，mRNA)。在某些实施方案中，Importinα4基因的核苷酸序列如SEQ ID NO:4所示。
如本文中所使用的，术语“病原体”是指能够引起植物发病的所有生物，包括真菌，线虫，细菌和病毒。在某些实施方案中，所述病原体是水稻条纹病毒。
如本文中所使用的，术语“能够抵抗病原体的植株”是指植株能够一定程度的抵抗所述病原体的侵染和/或扩散，具体表现为所述病原体侵染植株后，与野生型植株相比，所述植株的发病情况减轻。
如本领域技术人员所知晓的，密码子存在简并性。即，在蛋白质的翻译过程中，每个氨基酸可对应1种或多种密码子，例如可对应多达6种密码子。不同的物种在使用编 码某一氨基酸的简并密码子时存在着很大的差异，有着不同的偏好。这种偏好现象即被称为“密码子偏好性”。因此，如本文中所使用的，术语“密码子偏好性”是指某一物种偏爱使用某些特定的密码子来编码氨基酸的情况。根据密码子偏好性来优化核酸分子的序列在某些情况下是特别有利的，例如，可能有助于提高核酸分子所编码的蛋白质的表达水平。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒(例如裸质粒)；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及病毒载体等。可用作载体的病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“sgRNA(small guide RNA)”是向导RNA用于靶向目标核酸，在sgRNA的引导下Cas9蛋白对目标核酸进行定点编辑。通常，在CRISPR/Cas9基因组编辑技术中，通过设计靶向目标核酸的sgRNA，以引导Cas9核酸酶靶向目标核酸的特定位点。并非每条sgRNA都具有同等的切割效率。鉴于这种不一致性，对多条sgRNA进行筛选，从中找到切割效率最高的一条是很有必要的。在某些实施方案中，靶向Flotillin1基因的sgRNA的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6。在某些实施方案中，靶向Importinα4基因的sgRNA的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
发明的有益效果
申请人分别制备了单敲除Importinα4或Flotillin1的水稻突变体，通过实验测试发现单敲除的突变体效果并不理想；进一步的，申请人意外的发现同时敲除Flotillin1和Importinα4的水稻突变体显著的抑制了RSV在水稻中的扩散率(与WT相比扩散率抑制至少50％)，还显著的降低了RSV在水稻中的发病率(与WT相比发病率降低至少40％)。并且，该突变体对水稻株高，千粒重及萌发率均没有影响。因此，该突变体以 及其进行的敲除组合(即，敲除Flotillin1基因和Importinα4基因)在植物抗病中具有较好的应用潜能。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了水稻Flotillin1突变体转录水平和蛋白水平的验证结果，其中，图1中的A表示野生型水稻(WT)和Flotillin1突变体中，Flotillin1转录水平的表达量；图1中的B表示野生型水稻(WT)和Flotillin1突变体中，Flotillin1蛋白水平的表达量。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图2显示了敲除Flotillin1对水稻株高，千粒重及萌发率的影响结果，其中，图2中的A和2中的B表示敲除Flotillin1后，水稻株高的变化，标尺为10cm；图2中的C和2中的D表示敲除Flotillin1后，水稻千粒重的变化，标尺为5cm。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图3显示了敲除Flotillin1对RSV在水稻中扩散率的影响，其中，图3中的A和3中的B表示在Flotillin1突变体中，RSV侵染接种叶12h后，在表皮细胞，纤维组织，叶肉细胞，维管束鞘细胞，筛管及伴胞的扩散率，标尺为20nm；图3中的C和3中的D表示在Flotillin1突变体中，RSV侵染系统叶1d后，在表皮细胞，纤维组织，叶肉细胞，维管束鞘细胞，筛管及伴胞的扩散率，标尺为20nm。
Epi，表皮细胞，Fib，纤维组织，Mes，叶肉细胞，Bs，维管束鞘细胞，SE，筛管，CC，伴胞。蓝色荧光表示胞间连丝染色信号，红色荧光表示RSV NP的信号。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图4显示了敲除Flotillin1对RSV NP在水稻中表达量的影响，其中，图4中的A，B，C分别表示在Flotillin1突变体中，RSV侵染水稻系统叶1d，4d及7d后NP表达量的变化。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图5显示了敲除Flotillin1对水稻发病率的影响，其中，图5中的A表示当虫口密度 为10头时，在Flotillin1突变体中，RSV侵染对水稻发病率的影响，图5中的B表示当虫口密度为2头时RSV侵染对Flotillin1突变体水稻发病率的影响。值用平均值±标准误表示，星号表示处理间有显著差异，*P<0.05，**P<0.01，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图6显示了敲除Importinα4对水稻株高及千粒重的影响，其中，图6中的A和B表示敲除Importinα4后，水稻株高的变化，标尺为10cm；图6中的C表示敲除Importinα4后，水稻千粒重的变化。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图7显示了敲除Importinα4对RSV NP在水稻中表达量的影响，其中，图7A，B，C分别表示在Importinα4突变体中，RSV侵染水稻系统叶1d，4d及7d后NP表达量的变化。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图8显示了敲除Importinα4对水稻发病率的影响，图8中的A表示当虫口密度为10头时，在Importinα4突变体中，RSV侵染对水稻发病率的影响。图8中的B表示当虫口密度为2头时，RSV侵染对Importinα4突变体水稻发病率的影响。值用平均值±标准误表示，星号表示处理间有显著差异，*P<0.05，**P<0.01，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图9显示了敲除Flotillin1-Importinα4对水稻株高及千粒重的影响，其中，图9中的A和B表示敲除Flotillin1-Importinα4后，水稻株高的变化，标尺为10cm；图9中的C表示敲除Flotillin1-Importinα4后，水稻千粒重的变化。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图10显示了敲除Flotillin1-Importinα4对RSV NP在水稻中表达量的影响，其中，图10中的A，B，C分别表示在Flotillin1-Importinα4突变体中，RSV侵染水稻系统叶1d，4d及7d后NP表达量的变化。值用平均值±标准误表示，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
图11显示了敲除Flotillin1-Importinα4对水稻发病率的影响，图11中的A表示当虫口密度为10头时，在Flotillin1-Importinα4突变体中，RSV侵染对水稻发病率的影响。图11中的B表示当虫口密度为2头时，RSV侵染对Flotillin1-Importinα4突变体水稻发病率的影响。值用平均值±标准误表示，星号表示处理间有显著差异，*P<0.05，**P<0.01，不同小写字母表示不同处理的基因表达差异显著(P<0.05)。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述


具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。例如，本发明中所使用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等常规技术，可参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学 方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR 2：实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.供试昆虫与供试植物
供试昆虫：本文中用到的带毒和不带毒的灰飞虱(Laodelphax striatellμs)品系主要是从江苏海安采集并经过长期驯化而来的。灰飞虱被饲养在装有2-3cm水稻幼苗的玻璃瓶中，并用尼龙网进行封口。饲养温度维持在25摄氏度左右，光照周期为16h:8h(光照：黑暗)。为了保证充足的营养，每周更换一次水稻苗。带毒品系和无毒品系的灰飞虱分别在环境条件相同的不同温室饲养，并且每三个月通过斑点酶联免疫进行病毒量的检测和筛选。
带毒灰飞虱的筛选：为了获得一个高带毒率的灰飞虱品系，我们定期对带毒种群进行筛选。取待产的雌性灰飞虱40-50只，分瓶饲养。待产的幼虫孵出，每瓶随机取5只幼虫，采用斑点酶联免疫的方法对灰飞虱幼虫的病毒量进行检测。将检测的幼虫全为带毒的雌性灰飞虱后代混合扩繁，作为带毒品系的种群。具体的斑点酶联免疫的操作步骤如下：
(1)将待测灰飞虱放入0.2mL PCR管中，每管一只，加入5μL 0.05M碳酸盐缓冲液，使用顶端处理过的枪头进行研磨。
(2)取3μL研磨后的样品硝酸纤维素膜上，放在孵育盒中，置于通风橱中晾干。
(3)封闭：向盛有晾干的硝酸纤维素膜的孵育盒中加入10mL 1％脱脂牛奶(用1x PBST缓冲液配制)，放置于摇床上，室温下封闭30min。
(4)孵育一抗：倒出封闭液，用1％脱脂牛奶按照1:5000的比例稀释RSV NP单抗(Zhao,et al.,2016)，加入10mL到孵育盒内，置于摇床上，室温孵育2h。
(5)清洗一抗：倒出一抗稀释液，加入1x PBST缓冲液，在摇床上洗涤15min，重复2-3次。
(6)孵育二抗：用1％脱脂牛奶按照1：10000的比例稀释连有辣根过氧化物酶(Horseradish Peroxidase，HRP)的羊抗鼠的二抗(康为世纪，CW0102S)，加入10mL到孵育盒内，置于摇床上，室温孵育1h。
(7)清洗二抗：倒出二抗稀释液，加入1x PBST缓冲液，在摇床上洗涤15min，重复2-3次。
(8)将清洗干净的膜放入显色液(10mL 2mM PBS溶液，2mL无水乙醇，6μg4-氯-1-萘酚，7μL 30％的H2O2混合而成)中，置于摇床上，室温显色2h。
(9)显色后，使用自来水冲洗三次，室温晾干，呈现蓝紫色斑点的表示该样品带毒。
供试植物：日本晴水稻(Oryza sativa Japonica Groμp)置于人工培养架中培养，温度为26℃/24℃，光周期为L：D＝14：10，空气相对湿度为70％。培养45天后用于实验。
实施例2.水稻突变体的制备
1.水稻Flotillin1突变体
(1)载体构建：设计两段Flotillin1(其氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示)的打靶sgRNAs(表2)，顺序连入CRISPR/Cas9双元载体pYLCRISPR/Cas9Pμbi-H(武汉伯远生物)中。
表2.Flotillin1的sgRNA序列
(2)目的载体转化农杆菌EHA105，农杆菌介导法转入日本晴水稻胚性愈伤组织中，针对Flotillin1基因，鉴定3株以上Flotillin1基因敲除纯合的突变体。
(3)Flotillin1突变体水稻分子鉴定：提取转基因植物DNA，在打靶sgRNAs两端设计引物，做PCR扩展包含打靶区域的片段，测序分析打靶效果。打靶效果检测引物F1和R1序列如下：正向引物F1:5(核苷酸序列如SEQ ID NO:15所示)，反向引物R1:5(核苷酸序列如SEQ ID NO:16所示)。
2.水稻Importinα4突变体
(1)载体构建：设计两段Importinα4(其氨基酸序列如SEQ ID NO:3所示，核苷 酸序列如SEQ ID NO:4所示)的打靶sgRNAs(表3)，顺序连入CRISPR/Cas9双元载体pYLCRISPR/Cas9Pμbi-H(武汉伯远生物)中。
表3.OsImportinα4的sgRNA序列
(2)目的载体转化农杆菌EHA105，农杆菌介导法转入日本晴水稻胚性愈伤组织中，针对Importinα4基因，鉴定3株以上Importinα4基因敲除纯合的突变体。
(3)Importinα4突变体水稻分子鉴定：提取转基因植物DNA，在打靶sgRNAs两端设计引物，做PCR扩展包含打靶区域的片段，测序分析打靶效果。打靶效果检测引物F1和R1序列如下：正向引物F1:6(核苷酸序列如SEQ ID NO:19所示)，反向引物R1:6(核苷酸序列如SEQ ID NO:20所示)。
3.水稻Importinα4-Flotillin1突变体
(1)载体构建：设计两段Flotillin1(其氨基酸序列如SEQ ID NO:1所示，核苷酸序列如SEQ ID NO:2所示)的打靶sgRNAs(具体序列同上述表2)及两段OsImportinα4(其氨基酸序列如SEQ ID NO:3所示，核苷酸序列如SEQ ID NO:4所示)的打靶sgRNAs(具体序列同上述表3)，顺序连入CRISPR/Cas9双元载体pYLCRISPR/Cas9Pμbi-H中。
(2)目的载体转化农杆菌EHA105，农杆菌介导法转入日本晴水稻中，针对Importinα4基因和Flotillin1基因，鉴定Importinα4-Flotillin1基因敲除纯合的突变体。
(3)Importinα4突变体水稻分子鉴定：提取转基因植物DNA，在打靶sgRNAs两端设计引物，做PCR扩展包含打靶区域的片段，测序分析打靶效果。
实施例3.水稻突变体的验证与测试步骤
1.对水稻的RNA进行提取，cDNA合成及qPCR定量
(1)RNA提取：水稻叶片RNA的提取采用Trizol法(Ambion，15596018)，具体步骤如下：
取200mg的水稻叶片于1.5mL的离心管中，加入1mL Trizol试剂(Ambion，15596018)把样品研磨充分；室温静置5min使样品充分裂解，4℃，12000rpm离心5 min，弃沉淀；加入200μL氯仿，剧烈振荡15s，室温放置15min，4℃，12000rpm离心15min，样品分为三层；取上层水相至新的1.5mL离心管中，加入0.5mL异丙醇，上下轻轻颠倒混匀，室温放置5min，4℃，12000rpm离心10min，弃上清；离心管中加入1mL 75％的乙醇，温和震荡，悬浮沉淀，4℃，8000g离心5min，弃上清；1mL 75％的乙醇重复洗涤一次；将离心管开盖置于超净台，室温晾干5min，加入30μL RNase-free ddH2O溶解RNA沉淀；取1μL于Nanodrop 2000上，测定RNA浓度，选取没有被蛋白质、酚类、无机盐、碳水化合物等杂质污染且浓度大于125ng/μL的样品进行后续实验。
(2)cDNA合成：利用M-MLV反转录系统(Promega，ΜSA)对RNA进行cDNA合成。具体步骤如下。
将1μg的RNA溶液加入0.2mL PCR管中，使用双蒸水(无RNA酶)补齐体积至12μL，加入1μL随机引物(如果要克隆或检测病毒基因，则需要使用随机引物，如果只克隆真核基因，可以使用Oligo-dT引物)，吹打混匀，离心至管底，70℃孵育60min，再4℃孵育10min；向上述管中加入5μL 5x MLV缓冲液，5μL dNTP，1μL反转录酶，1μL RNA酶抑制剂，充分吹打混匀，离心至管底，42℃孵育60min，75℃孵育15min终止反应；根据实验需求对反转录产物进行稀释，并放入-20℃冰箱进行保存。
(3)引物设计：各基因定量引物设计见(表4)
表4.目的基因的引物序列
(4)qPCR定量：使用天根的Talent qPCR PreMix(FP209-01)进行qPCR反应，反应体系为见(表5)。采用两步法PCR反应程序进行反应，反应程序见(表6)。贴 好封口膜，离心，使得反应混合物离至管底。将反应体系置于荧光定量PCR仪(Thermo Pikoreal 96 Real-Time PCR System)，开始反应。
表5.qPCR反应体系
表6.qPCR反应程序
2.水稻蛋白提取
对水稻的蛋白进行提取，采用康为试剂的RIPA裂解液试剂盒(CW2333)提取。首先按照1g组织加入5ml Plant protein extraction reagent(含1％Protease inhibitor cocktail)的比例，机械匀浆裂解；冰上孵育20-30分钟；4℃13，400g，离心20分钟；收集上清-20℃保存。
3.Western实验
配制10％分离胶和5％浓缩胶；80V跑30min，待样品进入浓缩胶后，120V跑1h，当溴酚蓝跑至绿线后停止；配制1L转膜液(200ml无水甲醇，甘氨酸11.25g，Tris3.025g，加超纯水定容至1L)；将膜在甲醇中活化1min左右；然后转移至转膜液:转膜液倒入托盘中，黑板朝下，按照黑板-海绵-滤纸-胶-膜-滤纸-海绵-白板的顺序排好，每加一层用滚轮拍一次气泡，安装好之后放入转膜槽，黑板对黑侧，白板对红侧；转膜槽置于冰水混合物中，100V，1h:5％脱脂牛奶(百奥易杰，BE6250)封闭1h，摇床转速60rpm/min:倒掉牛奶，加入5ml新的牛奶，加入1.5μl一抗(1:3000)室温2h或4℃过夜，PBST洗3次，每次5min；加入5ml牛奶，加入1μl二抗(1:5000)，室温1h，PBST洗3次，每次5-15min；ECL发光液(Thermo，A38556)，A，B液1:1混匀，每张膜加入400μl，照胶仪成像。
7.水稻接种RSV实验
待水稻苗长到2.5叶龄时接毒，每株平均接种30头带毒灰飞虱，并用微虫笼夹住。带毒灰飞虱取食叶片为接种叶，其余水稻叶片为系统叶，接毒2d后去除带毒灰飞虱及微虫笼。分别与去虫后1d，4d，7d取系统叶及接种叶进行qPCR定量检测侵染率，每个处理8个生物学重复，并于12h取接种叶，1d取系统叶进行植物免疫荧光实验检测病毒扩散率，每个处理9-12个生物学重复。水稻发病率接毒两个体系，分别为每株平均接种10头及2头带毒灰飞虱，并用微虫笼夹住。接毒2d后去除带毒灰飞虱及微虫笼，统计发病率，每个处理6个生物学重复。
8.水稻叶片的免疫荧光实验
接毒处理后的水稻叶片用4％多聚甲醛(酷来搏，SL18301)于4℃固定过夜；将组织转移至硬质的明胶胶囊(Electron Microscopy Sciences，70102)中，包埋剂(日本樱花，SAKΜRA 4583)包埋，排出空气，-20℃速冻5min；用冰冻切片机(Leica)切成10μm切片，置于粘附型载玻片，在空气中晾干；将切片置于含有10％山羊血清(Beyotime，C0265)中室温孵育1h以封闭非特异性的结合位点；PBST(含0.1％吐温20)洗脱二次；用PBST将一抗稀释(RSV NP鼠单抗1:500)，4℃孵育过夜，用PBST洗脱三次，开始避光操作；用PBST将二抗(abberior，STORANGE-1002/1001)稀释(1:1000)，室温下孵育3h，用PBST洗脱3次；每个切片加入20μl浓度为0.25mg/mL的Aniline Blμe Flμorochrome(Biosμpplies，100-1)，并加入100μl抗荧光淬灭剂(BOSTER，AR1109)，室温30min染胞间连丝；指甲油封片，激光共聚焦显微镜拍摄(Aniline Blμe Flμorochrome的激发光波长390nm，散发波长480nm，STORANGE-1002的)；每个处理至少6个生物学重复。
实施例4.水稻Flotillin1突变体的研究
1.转录水平和蛋白水平
结果表明(图1)，在转录水平上，与野生型水稻WT相比，Flotillin1突变体水稻中Flotillin1基因的表达量显著降低；在蛋白水平上，与野生型水稻WT相比，Flotillin1突变体水稻中Flotillin1蛋白的表达量显著下降。
2.敲除Flotillin1基因对水稻株高，千粒重及萌发率的影响
与WT相比，Flotillin1突变体中，水稻的株高和千粒重均无显著性差异(图2)。
3.敲除Flotillin1基因对RSV在水稻中扩散率的影响
通过植物免疫荧光技术，探究了敲除Flotillin1对水稻中RSV扩散率的影响，结果 表明(图3)，当RSV侵染接种叶12h后，与WT相比，Flotillin1突变体中，RSV在维管束鞘细胞，筛管及伴胞的扩散率分别显著降低55％，36％及34％；当RSV侵染系统叶1d后，与WT相比，Flotillin1突变体中，RSV在筛管及伴胞的扩散率分别显著降低64％及57％。
4.敲除Flotillin1基因对RSV NP在水稻中表达量的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Flotillin1基因对RSV NP在水稻中表达量的影响。结果表明(图4)，在Flotillin1突变体中，与WT相比，当RSV侵染系统叶1d后，RSV NP的表达量无显著性差异，当RSV侵染系统叶4d和7d后，RSV NP的表达量分别显著降低87％及53％。
5.敲除Flotillin1基因对水稻的发病率的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Flotillin1基因对水稻发病率的影响。结果表明(图5)，在Flotillin1突变体中，与WT相比，当虫口密度为10头时，RSV的发病率显著降低20％，当虫口密度为2头时，RSV的发病率显著降低30％。
实施例5.Importinα4突变体的研究
1.敲除Importinα4对水稻株高及千粒重的影响
与WT相比，Importinα4突变体中，水稻的株高及千粒重均显著性下降(图6)。
2.敲除-Importinα4对RSV NP在水稻中表达量的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Flotillin1-Importinα4对RSV NP在水稻中表达量的影响。结果表明(图7)，在Importinα4突变体中，与WT相比，Importinα4突变体中，当RSV侵染系统叶1d和4d后，RSV NP的表达量无显著性差异，当RSV侵染系统叶7d后，RSV NP的表达量显著降低84％。
3.敲除Importinα4对水稻的发病率的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Importinα4对水稻发病率的影响。结果表明(图8)，在Importinα4突变体中，与WT相比，当虫口密度为10头时，Importinα4突变体中，RSV的发病率显著降低24％，当虫口密度为2头时，RSV的发病率显著降低27％。
实施例6.Flotillin1-Importinα4突变体的研究
1.敲除Flotillin1-Importinα4对水稻株高及千粒重的影响
与WT相比，Flotillin1-Importinα4突变体中，水稻的株高及千粒重均无显著性差异(图9)。
2.敲除Flotillin1-Importinα4对RSV NP在水稻中表达量的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Flotillin1-Importinα4对RSV NP在水稻中表达量的影响。结果表明(图10)，在Flotillin1-Importinα4突变体中，与WT相比，Flotillin1-Importinα4突变体中，当RSV侵染系统叶1d，4d和7d后，RSV NP的表达量分别显著降低82％，97％和70％。
3.敲除Flotillin1-Importinα4对水稻的发病率的影响
通过给水稻接种RSV高带毒灰飞虱，探究了敲除Flotillin1-Importinα4对水稻发病率的影响。结果表明(图11)，在Flotillin1-Importinα4突变体中，与WT相比，当虫口密度为10头时，Flotillin1-Importinα4突变体中，RSV的发病率显著降低37％，当虫口密度为2头时，RSV的发病率显著降低40％。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种获得能够抵抗病原体的植株的方法，所述方法包括：减少或抑制所述植株中Flotillin1基因和Importinα4基因的转录或翻译，或者减少或抑制所述植株中Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平。
  2. 如权利要求1所述的方法，其中，所述方法具有选自下列的一项或多项特征：
    (1)所述植物是禾本科植物；优选地，所述植物选自小麦，大麦，玉米，水稻，高粱；
    (2)所述病原体是病毒；优选地，所述病原体是水稻条纹病毒；
    (3)所述Importinα4基因是水稻的Importinα4基因；优选地，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示；
    (4)所述Flotillin1基因是水稻的Flotillin1基因；优选地，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
  3. 如权利要求1或2所述的方法，减少或抑制所述植物Flotillin1基因和Importinα4基因的表达是通过下列的任意一项方法：一个或几个核苷酸的置换、缺失或添加(例如1个，2个或3个核苷酸的置换、缺失或添加)，位点特异性诱变，甲磺酸乙酯诱变，定向诱导基因组局部突变，或基因编辑(例如，由sgRNA引导的基因敲除)。
  4. 如权利要求1-3任一项所述的方法，当所述植物细胞中存在Flotillin1基因和Importinα4基因的几个(例如，1个，2个，3个，4个)拷贝时，使Flotillin1基因和Importinα4基因的每个拷贝均变成有缺陷的。
  5. 如权利要求1-4任一项所述的方法，所述方法通过以下步骤(a)至(f)实现：
    (a)构建含有sgRNA1和sgRNA2的载体，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段；
    (b)将所述载体转化农杆菌；
    (c)将所述农杆菌侵染植物细胞；
    (d)任选地，选择具有缺陷的Flotillin1基因和Importinα4基因的植物细胞；
    (e)将步骤(c)或(d)的植物细胞生成植物；
    (f)任选地，筛选具有缺陷的Flotillin1基因和Importinα4基因的植物，以获得能够抵抗病原体的植物。
  6. 如权利要求5所述的方法，所述方法具有选自下列的一项或多项特征：
    (1)所述sgRNA1和sgRNA2构建于同一载体或不同的载体中；优选地，所述sgRNA1和sgRNA2构建于pYLCRISPR/Cas9Pμbi-H载体中；
    (2)所述农杆菌是EHA105农杆菌；
    (3)在步骤(f)中，提取所述植物的RNA，将所述RNA逆转录为cDNA，通过引物扩增所述cDNA中包含sgRNA1和sgRNA2靶向的核苷酸片段，以进行筛选；或者，提取所述植物的基因组DNA，通过引物扩增所述DNA中包含sgRNA1和sgRNA2靶向的核苷酸片段，以进行筛选。
  7. 如权利要求5或6所述的方法，所述方法具有选自下列的一项或多项特征：
    (1)所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6；
    (2)所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
  8. 特异性抑制Flotillin1基因和Importinα4基因的转录或翻译或特异性抑制Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平的分子在制备试剂盒中的用途，所述试剂盒用于获得能够抵抗病原体的植株，或用于提高植株抵抗病原体的能力；
    优选地，所述分子是核酸分子；
    优选地，所述核酸分子选自反义寡核苷酸，dsRNA，siRNA，shRNA，或sgRNA；
    优选地，所述核酸分子包含sgRNA1和sgRNA2，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段；
    优选地，所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6；
    优选地，所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8。
  9. 如权利要求8所述的用途，其中，所述试剂盒还包含：载体，农杆菌，用于培养植物细胞或组织的培养基和/或试剂，用于提取植物DNA或RNA的试剂，扩增包含 sgRNA1和sgRNA2分别靶向的片段的引物组，或其任何组合；
    优选地，所述载体是pYLCRISPR/Cas9Pμbi-H；
    优选地，所述sgRNA1和sgRNA2包含或不包含于所述载体中；
    优选地，所述农杆菌是EHA105农杆菌。
  10. 如权利要求8或9所述的用途，其中，所述方法具有选自下列的一项或多项特征：
    (1)所述植物是禾本科植物；优选地，所述植物选自小麦，大麦，玉米，水稻，高粱；
    (2)所述病原体是病毒；优选地，所述病原体是水稻条纹病毒；
    (3)所述Importinα4基因是水稻的Importinα4基因；优选地，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示；
    (4)所述Flotillin1基因是水稻的Importinα4基因；优选地，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示。
  11. 一种试剂盒，其包含以下(1)至(6)的任意一项或多项：
    (1)特异性抑制Flotillin1基因和Importinα4基因的转录或翻译或特异性抑制Flotillin1基因和Importinα4基因所分别编码的蛋白质的表达水平的核酸分子；
    (2)包含所述核酸分子的载体；
    (3)包含所述载体的农杆菌；
    (4)用于培养植物细胞或组织的培养基和/或试剂；
    (5)用于提取植物DNA或RNA的试剂；
    (6)用于扩增包含Flotillin1基因和Importinα4基因的核苷酸片段的引物组；
    优选地，所述核酸分子选自反义寡核苷酸，dsRNA，siRNA，shRNA，或sgRNA；
    优选地，所述核酸分子包含sgRNA1和sgRNA2，所述sgRNA1能够靶向所述Flotillin1基因或其片段，所述sgRNA2能够靶向所述Importinα4基因或其片段。
  12. 如权利要求11所述的试剂盒，其中，所述试剂盒用于获得能够抵抗病原体的植株，或用于提高植株抵抗病原体的能力；
    优选地，所述试剂盒具有选自下列的一项或多项特征：
    (1)所述植物是禾本科植物；优选地，所述植物选自小麦，大麦，玉米，水稻，高粱；
    (2)所述病原体是病毒；优选地，所述病原体是水稻条纹病毒；
    (3)所述Importinα4基因是水稻的Importinα4基因；优选地，所述Importinα4基因编码的蛋白质的氨基酸序列如SEQ ID NO:3所示；
    (4)所述Flotillin1基因是水稻的Importinα4基因；优选地，所述Flotillin1基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示；
    (5)所述sgRNA1的核苷酸序列为SEQ ID NO:5或SEQ ID NO:6；
    (6)所述sgRNA2的核苷酸序列为SEQ ID NO:7或SEQ ID NO:8；
    (7)所述载体是pYLCRISPR/Cas9Pμbi-H；
    (8)所述农杆菌是EHA105农杆菌。
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