WO2019214736A1 - Mutant de riz mir393am présentant une résistance à la delphacide brune du riz et une tolérance au sel élevées, et son application - Google Patents

Mutant de riz mir393am présentant une résistance à la delphacide brune du riz et une tolérance au sel élevées, et son application Download PDF

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WO2019214736A1
WO2019214736A1 PCT/CN2019/086476 CN2019086476W WO2019214736A1 WO 2019214736 A1 WO2019214736 A1 WO 2019214736A1 CN 2019086476 W CN2019086476 W CN 2019086476W WO 2019214736 A1 WO2019214736 A1 WO 2019214736A1
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plant
resistance
mir393a
brown planthopper
mir393am
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Chinese (zh)
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苗雪霞
马飞龙
时振英
李海超
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中国科学院上海生命科学研究院
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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
    • 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
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    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • 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)
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    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the invention relates to the field of crop genetics, and more particularly to a rice mutant miR393am which is highly resistant to brown planthopper and salt tolerant and its application.
  • miRNAs play an important role in plant growth and development and resistance, but have not been reported in rice against brown planthopper.
  • miRNAs are very diverse, their functions are different, and the organisms are complex. Therefore, the results of up-regulation and down-regulation of miRNAs are difficult to predict.
  • the object of the present invention is to provide a rice mutant miR393am which is highly resistant to brown planthopper and salt tolerant and its application.
  • an inhibitor of miR393a or an active fragment thereof for (i) resistance to brown planthopper, and/or (ii) enhancing plant stress tolerance, or preparing a formulation or composition
  • the formulation or composition is for (i) resistance to brown planthopper, and/or (ii) enhancing plant stress tolerance.
  • the stress resistance is selected from the group consisting of drought resistance, cold tolerance, salt tolerance, osmotic pressure resistance, heat resistance, or a combination thereof.
  • the formulation or composition is also used to upregulate expression of a synthetase gene of the SA signaling pathway.
  • the synthetase gene is selected from the group consisting of EDS1, NPR1, ICS1, PAD4, or a combination thereof.
  • the miR393a is derived from rice.
  • the miR393a inhibitor is selected from the group consisting of a small molecule compound, an antisense nucleic acid, a microRNA, an siRNA, an RNAi, a Crispr reagent, or a combination thereof.
  • the brown planthopper resistance comprises (a) reducing the amount of honeydew of the brown planthopper and/or (b) reducing the weight gain of the brown planthopper.
  • the composition is an agricultural composition.
  • the composition comprises (a) an inhibitor of miR393a or an active fragment thereof; and (b) an agronomically acceptable carrier.
  • the dosage form of the composition or formulation is selected from the group consisting of a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, an aerosol, or a combination thereof.
  • the plant comprises a crop, a forestry plant, a vegetable, a melon, a flower, a pasture (including turfgrass); preferably a grass, a leguminous, and a cruciferous plant, more preferably a rice, Corn, sorghum, wheat, soybean or Arabidopsis.
  • the plant comprises: rice, wheat, corn, sorghum, cruciferous (cabbage).
  • sequence of the miR393a is set forth in SEQ ID NO.: 1.
  • a second aspect of the invention provides a method of (i) resistance to brown planthopper, and/or (ii) enhancing plant stress resistance, comprising the steps of:
  • the reduction of expression or activity of a miR393a or miR393a active fragment in a plant is achieved by introducing into the plant an inhibitor of miR393a or an active fragment thereof.
  • the inhibitor of miR393a or an active fragment thereof is selected from the group consisting of a small molecule compound, an antisense nucleic acid, a microRNA, an siRNA, an RNAi, a Crispr reagent, or a combination thereof.
  • the "reduction" means that the expression or activity of the miR393a or miR393a active fragment is decreased to satisfy the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 60%, more preferably ⁇ 40%, optimally 0-30%; wherein A1 is the expression or activity of miR393a or miR393a active fragment; A0 is wild type Expression or activity of the same miR393a or miR393a active fragment in a plant of the same type.
  • the decrease means that the expression level E1 of the miR393a or miR393a active fragment in the plant is 0-80% of the wild type compared to the expression level E0 of the wild type miR393a or miR393a active fragment. Good land 0-60%, better 0-40%.
  • the expression or activity of the miR393a or miR393a active fragment in the reduced plant is achieved by a method selected from the group consisting of a gene mutation, a gene knockout, a gene disruption, an RNA interference technique, a Crispr technique, or combination.
  • the expression or activity of the miR393a or miR393a active fragment in the reduced plant is genetically engineered by miR393a or miR393a active fragments with one or more sgRNA-mediated Cas9 nucleases.
  • a third aspect of the invention provides a composition (i) resistant to brown planthopper, and/or (ii) enhancing plant stress resistance, the composition comprising: (a) an inhibitor of miR393a or an active fragment thereof; b) an agronomically acceptable carrier.
  • the composition comprises an agricultural composition.
  • the dosage form of the composition is selected from the group consisting of a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, an aerosol, or a combination thereof.
  • the composition contains 0.0001 to 99% by weight, preferably 0.1 to 90% by weight, of component (a), based on the total weight of the composition.
  • composition further comprises other substances which may (i) resist brown planthopper, and/or (ii) enhance plant resistance.
  • the other substance which may (i) resist brown planthopper, and/or (ii) enhance plant stress resistance is selected from the group consisting of cis-12-oxy-plant dienoic acid (OPDA), water. Salicylic acid (SA), or a combination thereof.
  • composition further comprises other genes which may (i) be resistant to brown planthopper, and/or (ii) enhance plant resistance or an enhancer thereof.
  • the gene is selected from the group consisting of Bph18, Bph14, Bph26, Bph29, Bph3, Bph9, Bph32, Bph6, or a combination thereof.
  • a fourth aspect of the invention provides the use of a composition according to the third aspect of the invention for (i) resistance to brown planthopper, and/or (ii) enhancing plant stress resistance.
  • a fifth aspect of the invention provides a method of preparing a genetically engineered plant tissue or plant cell, comprising the steps of:
  • the expression or activity of a miR393a or miR393a active fragment in a plant tissue or plant cell is reduced to obtain a genetically engineered plant tissue or plant cell.
  • a sixth aspect of the invention provides a method of preparing a transgenic plant, comprising the steps of:
  • the genetically engineered plant tissue or plant cell prepared by the method of the fifth aspect of the invention is regenerated into a plant body to obtain a transgenic plant.
  • a seventh aspect of the invention provides a transgenic plant prepared by the method of the sixth aspect of the invention.
  • Figure 1 shows the location of base deletions in miR393am plants. Among them, the sequence of A:miR393am mutant is different from that of wild type; B: the sequence of miR393a in wild type and mutant.
  • Figure 2 shows the expression of OsTIR1 and OsAFB2 genes in miR393am by qRT-PCR. Among them, the statistical data showed significant difference and extremely significant difference after t-test (** indicates P ⁇ 0.01).
  • Figure 3 shows the results of the single-plant method for determining the resistance of miR393am mutant plants to brown planthopper.
  • Figure 4 shows the change of honeydew and insect weight gain before and after feeding of brown planthopper by the single insect honeydew method.
  • A the change of honeydew amount before and after feeding of brown planthopper
  • B the change of insect weight gain before and after feeding of brown planthopper.
  • the statistical data showed a very significant difference after t-test (** means P ⁇ 0.01).
  • Figure 5 shows the changes in SA synthesis-related genes in miR393am plants.
  • Figure 6 shows the salt tolerance analysis of rice miR393am and wild type.
  • A wild type plants (Nip) and miR393am treatment, 200 mM Nacl treatment for 3 days, rehydration for 3 days phenotype
  • B survival analysis after 3 days of rehydration.
  • the statistical data showed a very significant difference after t-test (** means P ⁇ 0.01).
  • the present inventors have extensively and intensively studied, and for the first time, unexpectedly found that reducing the expression or activity of miR393a or miR393a active fragments in plants can significantly (i) resist brown planthopper (such as reducing honeydew amount and/or insect weight gain), and / or (ii) enhance plant stress resistance (such as salt tolerance), in addition, the present invention through a large number of screening, accidentally screened a strain (i) against brown planthopper, and / or (ii) enhanced plant stress resistance Excellent miR393am mutant plant. On the basis of this, the inventors completed the present invention.
  • brown planthopper such as reducing honeydew amount and/or insect weight gain
  • enhance plant stress resistance such as salt tolerance
  • the present invention provides a class of miRNAs involved in i) resistance to brown planthopper (such as reducing the amount of honeydew and/or gain of insects), and/or (ii) enhancing plant stress tolerance (e.g., salt tolerance).
  • miRNA refers to a class of RNA molecules that are processed from transcripts that form miRNA precursors. Mature miRNAs typically have 18-26 nucleotides (nt) (more specifically about 19-22 nt) and do not exclude miRNA molecules with other numbers of nucleotides. miRNAs are usually detected by Northern blotting.
  • Rice-derived miRNAs can be isolated from rice cells or tissues.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • the miRNA can be processed from a precursor miRNA (Pre-miRNA), which can be folded into a stable stem-loop (hairpin) structure, which is generally 50-100 bp in length. between.
  • the precursor miRNA can be folded into a stable stem-loop structure, and the stem portion of the stem-loop structure comprises two sequences that are substantially complementary.
  • the precursor miRNA can be natural or synthetic.
  • the precursor miRNA can be cleaved to generate a miRNA that is substantially complementary to at least a portion of the sequence encoding the mRNA of the gene.
  • substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (eg, a stem-loop structure).
  • at least 70% of the nucleotides of the two "substantially complementary" nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • mismatched nucleotides there may be up to 40 mismatched nucleotides between two sufficiently complementary molecules; preferably, up to 30 mismatched nucleotides; more preferably, up to 20 mismatched nucleosides Acid; further preferred, having up to 10 mismatched nucleotides, such as having 1, 2, 3, 4, 5, 8, 11 mismatched nucleotides.
  • a "stem loop” structure also referred to as a "hairpin” structure, refers to a nucleotide molecule that can form a secondary structure comprising a double-stranded region (stem), said double
  • the chain region is formed by two regions of the nucleotide molecule (on the same molecule), the two regions are flanked by two sides of the double-stranded portion; it also includes at least one "loop” structure, including non-complementary nucleotide molecules , that is, a single-chain area. Even if the two regions of the nucleotide molecule are not completely complementary, the double-stranded portion of the nucleotide can remain in a double-stranded state.
  • insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner to form a double-stranded region of the stem-loop structure.
  • Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.
  • the miRNA of the present invention is miR393a, and in rice, the sequence is: UCCAAAGGGAUCGCAUUGAUC (SEQ ID NO.: 1).
  • the invention also includes variants and derivatives of miR393a, which can be modified by one of ordinary skill in the art using general methods including, but not limited to, hydrocarbyl modification, glycosylation modification, nucleic acid modification, peptide Segment modification, lipid modification, halogen modification, and the like.
  • the glycosylated modifying group includes a 2-methoxy-glycosyl group, a hydrocarbyl-glycosyl group, a sugar ring group and the like.
  • at least one protective base such as "TT" or the like may be added to at least one end of the miRNA.
  • the present invention also provides an inhibitor of miR393 or an active fragment thereof against miR393 or an active fragment thereof, which inhibitor of miR393 or an active fragment thereof inhibits expression or activity of miR393 or an active fragment thereof.
  • the inhibitor of miR393 or an active fragment thereof is selected from the group consisting of a small molecule compound, an antisense nucleic acid, a microRNA, an siRNA, an RNAi, a Crispr reagent, or a combination thereof.
  • the invention also provides the use of inhibitors of miR393a or an active fragment thereof, which are used for (i) resistance to brown planthopper (such as reducing the amount of honeydew and/or gain of insects), and/or (ii) enhancing plant stress tolerance. (such as salt tolerance).
  • miR393a has the sequence set forth in SEQ ID NO.: 1 and is derived from rice.
  • the expression or activity of miR393a can be inhibited by techniques such as gene mutation, gene knockout, gene disruption, RNA interference technology, and Crispr technology.
  • the miR393a or miR393a active fragment can be genetically edited by Cas9 nuclease mediated by one or more sgRNAs.
  • the invention also provides a method of improving a plant, such as rice, the improvement comprising: (i) resistance to brown planthopper (such as reducing the amount of honeydew and/or gain of insects), and/or (ii) enhancing plant resistance Inverse (such as salt tolerance), including steps: reducing the expression or activity of a miR393a or miR393a active fragment in a plant, and adding an inhibitor of miR393a or an active fragment thereof.
  • brown planthopper such as reducing the amount of honeydew and/or gain of insects
  • enhancing plant resistance Inverse such as salt tolerance
  • the gene is selected from the group consisting of Bph18, Bph14, Bph26, Bph29, Bph3, Bph9, Bph32, Bph6, or a combination thereof.
  • the plant may be further treated with a substance which can (i) resist brown planthopper, and/or (ii) enhance plant stress resistance, thereby improving the trait of the corresponding plant.
  • the other substance which may (i) resist brown planthopper, and/or (ii) enhance plant stress resistance is selected from the group consisting of cis-12-oxy-plant dienoic acid (OPDA), water. Salicylic acid (SA), or a combination thereof.
  • the present invention is the first to discover that reducing the expression or activity of a miR393a or miR393a active fragment in a plant can significantly (i) resist brown planthopper (such as reducing the amount of honeydew and/or gain of insects), and/or (ii) enhance the plant. Resistance to stress (such as salt tolerance).
  • the present invention screens for the first time to screen a strain of (i) brown planthopper resistant to brown planthopper, and/or (ii) a miR393am mutant plant which enhances the plant's stress resistance.
  • the present invention targets miR393a as a target, designs a specific small guide RNA (sg-RNA), and designs specific primers according to the sequence of the sgRNA (Table 1), and after annealing, forms a double strand and is connected to a Crisper (UBI, OsU6) vector.
  • sg-RNA small guide RNA
  • UBI OsU6 vector
  • the present invention detects the expression of OsTIR1 and OsAFB2 in miR393am plants by qRT-PCR method, and found that the gene expression of OsTIR1 and OsAFB2 in miR393am plants was significantly up-regulated compared with wild-type Nipponbare (Fig. 2). This result proves on the one hand that OsTIR1 and OsAFB2 are indeed target genes of miR393a, and on the other hand, it is proved that miR393a is indeed knocked out.
  • Example 3 miR393am rice mutant showed high resistance to phenotype of brown planthopper
  • the present invention first detects the resistance of miR393am to brown planthopper by a single plant method.
  • the seeds of the wild type (Nippon Nip) and miR393am in the tillering stage were germinated and planted in small plastic pots, one seedling per pot.
  • the diameter was 8cm and the height was 40cm.
  • a transparent plastic cover covers the rice seedlings and is connected to 15 third-instar nymphs, each of which has 8 replicates to observe the degree of damage to the plants.
  • the wild type Nippon Nip
  • the plants of miR393am remained fresh green, showing significant resistance to brown planthopper (Fig. 3).
  • honeydew is the secretion of brown planthopper
  • the amount of secretion can reflect the feeding situation of brown planthopper to a large extent.
  • the weight gain of rice planthoppers before and after feeding on insect-resistant and susceptible varieties can more directly reflect whether rice has resistance.
  • the present invention detects the expression changes of several key synthetase genes EDS1, NPR1, ICS1 and PAD4 in the SA synthesis pathway by RT-PCR, and finds that the expression of these genes is significantly up-regulated in miR393am plants, therefore, It is speculated that the resistance of miR393am plants to brown planthopper is likely to be achieved by activating the SA signaling pathway.
  • the present invention tested the response of rice miR393am mutant to salt stress.
  • the wild-type seedlings, miR393am strains were grown in hydroponics in a 96-well plate with the bottom removed for 10 days, followed by treatment with 200 mM NaCl for 3 days, and then the salt solution was replaced with normal water nutrient solution for 3 days.
  • rice miR393am has a good growth rate and a high survival rate, showing significant salt tolerance (Fig. 6).
  • Example 6 The expression level of four stress-related genes in rice miR393am mutant was significantly increased.
  • the present invention also tested the expression levels of four stress-resistant genes EDS1, NPR1, ICS1 and PAD4 in the rice miR393am mutant. The results showed that compared with the wild type Nippon (Nip), the expression levels of these four genes were significantly increased, indicating that the increase of rice stress resistance after miR393am mutation is closely related to the increase of resistance-related gene expression (Fig. 5 ).
  • Hiei Y, Ohta S, Komari T, Kumashiro T Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

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Abstract

L'invention concerne un riz mutant miR393am ayant une résistance à la delphacide brune du riz et une tolérance au sel, un inhibiteur de miR393a, et son application. L'inhibiteur de miR393a et une préparation ou une composition préparée à partir de celui-ci peuvent être utilisés pour (i) résister à des delphacides brune du riz, et/ou (ii) améliorer la résistance au stress des plantes. La réduction de l'expression ou de l'activité de miR393a ou de fragments actifs de miR393a dans une plante peut (i) résister à des delphacides brunes de riz (par exemple, réduire la quantité de miel de miel et/ou la vitesse de croissance), et/ou (ii) améliorer la résistance au stress des plantes (telle que la tolérance au sel).
PCT/CN2019/086476 2018-05-10 2019-05-10 Mutant de riz mir393am présentant une résistance à la delphacide brune du riz et une tolérance au sel élevées, et son application WO2019214736A1 (fr)

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CN113564167B (zh) * 2021-07-30 2023-07-07 中山大学 一种水稻抗虫microRNA及其应用
CN113699179B (zh) * 2021-07-30 2022-04-05 广东省农业科学院植物保护研究所 osa-miR-162a在制备驱避褐飞虱绿色农药中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361346A (zh) * 2012-03-26 2013-10-23 北京林业大学 胡杨microRNAs前体的克隆与分析方法
CN105647962A (zh) * 2016-02-15 2016-06-08 浙江大学 运用CRISPR-Cas9系统敲除水稻MIRNA393b茎环序列的基因编辑方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533760A (zh) * 2011-11-29 2012-07-04 中国科学院华南植物园 提高水稻分蘖的小分子RNA Osa-miR393及用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361346A (zh) * 2012-03-26 2013-10-23 北京林业大学 胡杨microRNAs前体的克隆与分析方法
CN105647962A (zh) * 2016-02-15 2016-06-08 浙江大学 运用CRISPR-Cas9系统敲除水稻MIRNA393b茎环序列的基因编辑方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IGLESIAS, MARIA J. ET AL.: "MiR393 Regulation of Auxin Signaling and Redox-Related Components during Acclimation to Salinity in Arabidopsis", PLOS ONE, vol. 9, no. 9, 15 September 2014 (2014-09-15), XP055650701, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0107678 *
WINDELS, DAVID ET AL.: "miR393 is Required for Production of Proper Auxin Signalling Outputs", PLOS ONE, vol. 9, no. 4, 24 April 2014 (2014-04-24), XP055650696, ISSN: 1932-6203 *
ZHAO, JUNMING: "Functional Characterization of OsELF3-1 Gene for Heading Date in Rice (Oryza sativa L.) and Applications of Osa-miR393a in Perennial Turfgrass", WANFANG DISSERTATIONS, 27 February 2017 (2017-02-27) *

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