WO2021004269A1 - 一种miR396或其编码基因的突变体在调控植物农艺性状的应用 - Google Patents

一种miR396或其编码基因的突变体在调控植物农艺性状的应用 Download PDF

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WO2021004269A1
WO2021004269A1 PCT/CN2020/097783 CN2020097783W WO2021004269A1 WO 2021004269 A1 WO2021004269 A1 WO 2021004269A1 CN 2020097783 W CN2020097783 W CN 2020097783W WO 2021004269 A1 WO2021004269 A1 WO 2021004269A1
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plant
gene
mir396
grain
sequence
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PCT/CN2020/097783
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English (en)
French (fr)
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张金山
李峰
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山东舜丰生物科技有限公司
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Priority to CN202080003128.7A priority Critical patent/CN112513275B/zh
Publication of WO2021004269A1 publication Critical patent/WO2021004269A1/zh

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    • 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
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • 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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
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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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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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
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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
    • C—CHEMISTRY; METALLURGY
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of crop genetics, in particular to the application of miR396 or a mutant of its coding gene in regulating plant agronomic traits.
  • Rice is one of the most important food crops in the world and the largest food crop in my country.
  • the dwarf breeding from the late 1950s to the early 1960s and the successful application of three-line hybrid indica rice in the 1970s enabled my country to achieve two major leaps in rice yield per unit area, and made great contributions to satisfying my country's food self-sufficiency.
  • Over the past decade or so with the continuous increase in the population of our country, the requirements for the total rice output have been increasing.
  • the use of chemical fertilizers, especially the application of nitrogen fertilizers has played a pivotal role in promoting the development of agricultural production.
  • people use a large amount of nitrogen fertilizer it also brings many problems and impacts.
  • MicroRNA is a class of non-coding single-stranded small RNA molecules with a length of 20-24 nucleotides. It binds to the mRNA of the target gene through base pairing, thereby causing mRNA degradation or translation inhibition. In plants, miRNA controls the growth, development, and stress tolerance of plants by regulating the accumulation of protein-encoding genes in in situ cells.
  • the purpose of the present invention is to provide a method that can significantly improve plant agronomic traits.
  • nucleic acid construct has a 5'-3' structure of formula I:
  • X1 is none or selected from positions 1-12 of SEQ ID NO.:1 or positions 1-9 of SEQ ID NO.:2;
  • X2 is selected from the mature sequence/conserved sequence of miR396;
  • X3 is none or selected from positions 44-184 of SEQ ID NO.: 1 or positions 41-176 of SEQ ID NO.: 2;
  • each "-" is a bond or a nucleotide connection sequence.
  • the agronomic traits of the regulated plant include:
  • the mature sequence/conserved sequence of miR396 includes the mature sequence/conserved sequence of miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g, and/or miR396h.
  • the mature sequence/conserved sequence of miR396 includes the mature sequence/conserved sequence of miR396e and/or miR396f.
  • the mature sequence of miR396 is the mature sequence of miR396e and/or the mature sequence of miR396f.
  • the conservative sequence of miR396 is selected from positions 13-43 of SEQ ID NO.:1 or positions 10-40 of SEQ ID NO.:2.
  • the low-nitrogen condition refers to the ratio of the nitrogen content N L of the medium or field growth conditions to the nitrogen content N0 (N L /N0) of the vegan medium or field growth conditions.
  • N L nitrogen content of the medium or field growth conditions
  • N0 nitrogen content of the vegan medium or field growth conditions.
  • -1 preferably, 0.01-0.9, preferably 0.1-0.9, preferably 0.3-0.8, more preferably 0.5-0.8, more preferably 0.6-0.8.
  • the low-nitrogen condition is that the amount of nitrogen fertilizer is lower than the amount of conventional fertilizer applied during crop growth, or the low-nitrogen condition is that no additional nitrogen fertilizer is applied to the soil, and the amount of conventional fertilizer is Those skilled in the art or farmers know that the amount of fertilizer used to ensure stable yield and high quality of crops, such as rice, wheat, and corn is 150-350 kg per hectare. The amount varies for different crops and regions. The conventional amount can be determined by the field The technician or farmer's planting experience determines that in the present invention, the application amount of nitrogen fertilizer is preferably 210-270 kg per hectare.
  • the low-nitrogen condition is that no additional nitrogen fertilizer is applied to the growth environment of the plant, and the total nitrogen content in the soil is about 0.5-3.0 g/kg of soil when no additional nitrogen fertilizer is applied; In different embodiments, without applying additional nitrogen fertilizer, the total nitrogen content in the soil is 1-2.5 g/kg of soil, preferably, 1.2-2.2 g/kg of soil.
  • nucleic acid construct is selected from the following group:
  • sequence of the nucleic acid construct is shown in SEQ ID NO.: 1 or 2.
  • the coding gene encodes the aforementioned nucleic acid construct.
  • the coding genes include MIR396a, MIR396b, MIR396c, MIR396d, MIR396e, MIR396f, MIR396g and/or MIR396h.
  • the coding gene includes MIR396e and/or MIR396f.
  • the nucleic acid construct or its encoding gene is derived from one or more plants selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, Rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, lettuce.
  • the nucleic acid construct or its encoding gene is derived from rice.
  • the nucleic acid construct or its encoding gene mutant is derived from one or more plants selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat , Sorghum, rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, lettuce.
  • nucleic acid construct or the mutant of the encoding gene is derived from rice.
  • the nucleic acid construct or the mutant of the encoding gene includes miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and/or miR396h mutants.
  • the mutant of the nucleic acid construct or its encoding gene includes a mutant of miR396e and/or miR396f.
  • the composition is an agricultural composition.
  • the composition comprises (a) a miR396 inhibitor; and (b) an agronomically acceptable carrier.
  • the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperm is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cunninghamaceae, Cupressaceae, Tricuspidae Cunninghamaceae, Taxaceae, Ephedraceae, Maizeaceae, Monotypicaceae, Centipedeaceae, or combinations thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, legumes, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, Lettuce or a combination thereof.
  • the rice is selected from the group consisting of indica rice, japonica rice or a combination thereof.
  • nucleic acid construct or the mutant of the encoding gene is natural or artificially synthesized.
  • the nucleic acid construct or the mutant of its encoding gene includes substitution, insertion, and/or deletion of the base of the nucleic acid construct or its encoding gene, preferably large fragment deletion.
  • the expression or activity of the nucleic acid construct or its encoding gene mutant is reduced by ⁇ 50%, more preferably, ⁇ 70% compared to the expression or activity of the wild-type nucleic acid construct or its encoding gene. %, more preferably, ⁇ 90% or 100%.
  • the ratio of the activity E1 of the nucleic acid construct or its encoding gene mutant to the background activity E0 of the wild-type nucleic acid construct or its encoding gene mutant is ⁇ 1/2 , Preferably ⁇ 1/5, more preferably ⁇ 1/10, and more preferably 0.
  • nucleic acid construct or the mutant of the gene encoding it reduces, silences or loses its regulatory effect on the target gene.
  • the target gene includes GRF gene.
  • the GRF gene is selected from the group consisting of GRF4, GRF6, GRF8, or a combination thereof.
  • the GRF gene includes GRF8.
  • the mutation site of the nucleic acid construct or its encoding gene includes at least a part of a mature sequence region or a conserved sequence region.
  • the mutation site of the nucleic acid construct or its encoding gene is in the mature sequence region or the conservative sequence region.
  • the second aspect of the present invention provides the use of a GRF gene or its encoded protein promoter for regulating plant agronomic traits under low nitrogen conditions or preparing a composition or preparation for regulating plant agronomic traits under low nitrogen conditions, wherein,
  • the agronomic traits of the plant are selected from one or more of the following group:
  • the GRF gene includes GRF4, GRF6 and/or GRF8.
  • the GRF gene includes a GRF protein encoding gene and a GRF gene conserved region sequence.
  • the GRF gene includes wild-type GRF gene and mutant GRF gene.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein).
  • polypeptide encoded by the mutant GRF gene is the same or substantially the same as the polypeptide encoded by the wild-type GRF gene.
  • the mutant GRF gene includes a polynucleotide with a homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%) compared with the wild-type GRF gene.
  • the mutant GRF gene is included in the 5'end and/or 3'end of the wild-type GRF gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • the GRF gene is selected from the following group: cDNA sequence, genomic sequence, or a combination thereof.
  • nucleotide sequence of the GRF4 gene is selected from the following group:
  • nucleotide sequence of the GRF4 gene is shown in SEQ ID NO.:5.
  • nucleotide sequence of the GRF6 gene is selected from the following group:
  • nucleotide sequence of the GRF6 gene is shown in SEQ ID NO.:7.
  • nucleotide sequence of the GRF8 gene is selected from the following group:
  • nucleotide sequence of the GRF8 gene is shown in SEQ ID NO.:9.
  • amino acid sequence of the GRF4 protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 3 or 4 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and a polypeptide derived from (i) ;or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 3 or 4 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), having the GRF4 activity Of peptides.
  • amino acid sequence of the GRF4 protein is shown in SEQ ID NO.: 3 or 4.
  • amino acid sequence of the GRF6 protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 6 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and is a polypeptide derived from (i); or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 6 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), a polypeptide having the GRF6 activity .
  • amino acid sequence of the GRF6 protein is shown in SEQ ID NO.:6.
  • amino acid sequence of the GRF8 protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 8 is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and is a polypeptide derived from (i); or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 8 is ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95% or ⁇ 98%), a polypeptide having the GRF8 activity .
  • amino acid sequence of the GRF8 protein is shown in SEQ ID NO.: 8.
  • the GRF gene or its encoded protein promoter includes a substance that promotes the expression or activity of the GRF gene or its encoded protein.
  • the expression or activity of the GRF gene or its encoded protein refers to increasing the expression or activity of the GRF gene or its encoded protein by ⁇ 10%, preferably, ⁇ 20%, preferably, ⁇ 50% , More preferably, ⁇ 70%.
  • the GRF gene or its encoded protein promoter is selected from the following group: small molecule compounds, nucleic acid molecules, enzymes, or combinations thereof.
  • the GRF gene or its encoded protein is derived from one or more plants selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape , Spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, lettuce.
  • the GRF gene or its encoded protein is derived from rice.
  • the composition is an agricultural composition.
  • the composition comprises (a) a promoter of GRF gene or its encoded protein; and (b) an agronomically acceptable carrier.
  • the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperm is selected from the group consisting of Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cunninghamaceae, Cupressaceae, Tricuspidae Cunninghamaceae, Taxaceae, Ephedraceae, Maizeaceae, Monotypicaceae, Centipedeaceae, or combinations thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of Solanaceae, Gramineae, Leguminous plants, or a combination thereof.
  • the woody plant is selected from the group consisting of Actinidiaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of cruciferous plants, gramineous plants, legumes, Solanaceae, Actinidiaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or combinations thereof .
  • the plant is selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, wheat, sorghum, rape, spinach, lettuce, cucumber, chrysanthemum, water spinach, celery, Lettuce or a combination thereof.
  • the rice is selected from the group consisting of indica rice, japonica rice or a combination thereof.
  • the third aspect of the present invention provides a method for improving agronomic traits of plants, including:
  • the reduction of the expression or activity of miR396 can be achieved in any of the following ways:
  • the miR396 is miR396e and/or miR396f.
  • the GRF gene is selected from the group consisting of GRF4, GRF6, GRF8, or a combination thereof.
  • the GRF gene includes GRF8.
  • the reduction of the expression or activity of miR396 in plants is achieved in the following manner:
  • the method includes the steps:
  • the miR396 inhibitor includes miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and/or miR396h inhibitor.
  • the miR396 inhibitor includes miR396e and/or miR396f inhibitor.
  • the miR396 inhibitor is selected from the group consisting of small molecule compounds, antisense nucleic acids, microRNA, siRNA, RNAi, Crispr reagent, or a combination thereof.
  • the "decrease” means that the expression or activity of miR396 is decreased to meet the following conditions:
  • the ratio of A1/A0 is ⁇ 80%, preferably ⁇ 60%, more preferably ⁇ 40%, and most preferably 0-30%; where A1 is the expression or activity of miR396 in the plant; A0 is the wild type The expression or activity of the same miR396 in plants of the same type.
  • the reduction means that the expression level E1 of MIR396 in the plant is 0-80% of that of the wild type, preferably 0-60%, compared with the expression level E0 of wild-type MIR396. Preferably 0-40%, more preferably 0-30%.
  • the reduction of the expression or activity of miR396 in a plant is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology, or a combination thereof.
  • the reduction of the expression or activity of miR396 in the plant is performed by gene editing of miR396 with one or more sgRNA-mediated Cas9 nucleases.
  • the method includes administering a plant GRF gene or a promoter of the polypeptide encoded by it.
  • the method includes introducing exogenous GRF gene or its encoded protein into the plant.
  • the method includes the steps:
  • the method includes the steps:
  • step (b) Contacting the plant cell or tissue or organ with the Agrobacterium in step (a), so that the GRF gene sequence is transferred into the plant cell and integrated into the chromosome of the plant cell;
  • step (d) Regenerating the plant cells or tissues or organs in step (c) into plants.
  • the improved plant agronomic traits include:
  • the fourth aspect of the present invention provides a composition for improving agronomic traits of plants under low nitrogen conditions, including:
  • the composition includes an agricultural composition.
  • the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the composition contains 0.0001-99wt%, preferably 0.1-90wt% of component (a), based on the total weight of the composition.
  • the improved plant agronomic traits include:
  • the fifth aspect of the present invention provides a use of the composition according to the fourth aspect of the present invention for improving agronomic traits of plants under low nitrogen conditions.
  • the sixth aspect of the present invention provides a method for preparing gene-edited plant tissues or plant cells, including the steps:
  • the seventh aspect of the present invention provides a method for preparing a gene-edited plant, including the steps:
  • the gene-edited plant tissue or plant cell prepared by the method of the sixth aspect of the present invention is regenerated into a plant body, thereby obtaining a gene-edited plant.
  • the eighth aspect of the present invention provides a gene-edited plant prepared by the method described in the seventh aspect of the present invention.
  • Figure 1 shows the effect of mir396e mutation type on seed grain type.
  • Figure 2 shows the difference between wild-type and mir396e mutant seeds.
  • Figure 3 shows the growth of plants under different nitrogen conditions.
  • Figure 4 shows the effect of miR396ef mutation on seed grain shape and thousand-grain weight under low nitrogen conditions.
  • Figure 5 shows the slice observation of the seed.
  • Figure 6 shows the effect of mir396ef mutation on ear length, main branches, and grain number per ear.
  • Figure 7 shows the effect of the mir396ef mutation on rice yield.
  • Figure 8 shows the effect of miR396ef mutation on rice leaf length.
  • Figure 9 shows the effect of mir396ef mutation on rice plant height.
  • Figure 10 shows the effect of the mir396ef mutation on rice biomass.
  • Figure 11 shows that mir396ef regulates grain and panicle development through the miR396-GRF4/6/8-GIF1/2/3 pathway.
  • nucleic acid construct of the present invention or the mutant of its encoding gene can significantly improve plant agronomic traits under low nitrogen conditions, including: (a) increase yield and/or biomass; (b) promote ear type and/or grain type development ; (C) size, weight and/or quantity of fruit and/or seeds; (d) grain length; (e) grain width; (f) ear length; (g) thousand-grain weight; (h) leaf length; (i) Leaf width; (j) effective tiller number; (k) fruit pod number, etc.
  • GRF GFR4, 6, and/or 8 genes or their encoded proteins can significantly improve plant agronomic traits under low nitrogen conditions, including: (a) increase yield And/or biomass; (b) promote ear type and/or grain type development; (c) fruit and/or seed size, weight and/or quantity; (d) grain length; (e) grain width; (f) ) Ear length; (g) Thousand-grain weight; (h) Leaf length; (i) Leaf width; (j) Effective tiller number; (k) Fruit pod number.
  • GRF such as GFR4, 6, and/or 8
  • the present invention finds for the first time that the nucleic acid construct of the present invention or its code Gene mutants under low nitrogen conditions affect plant yield and biomass through GRF (such as GRF4/6/8); ear type and/or grain type development; fruit and/or seed size, weight and/or number; Length; grain width; ear length; thousand-grain weight; leaf length; leaf width; effective tiller number; fruit pod number and other traits.
  • GRF such as GRF4/6/8
  • ear type and/or grain type development fruit and/or seed size, weight and/or number
  • Length; grain width; ear length; thousand-grain weight; leaf length; leaf width; effective tiller number; fruit pod number and other traits On this basis, the inventor completed the present invention.
  • GRF gene and “gene of the present invention” can be used interchangeably, and both refer to the gene of the present invention that regulates plant agronomic traits.
  • the genes of the present invention include GRF4, GRF6, and GRF8.
  • the genes of the present invention include GRF8. More preferably, the GRF gene of the present invention is derived from rice.
  • GRF the full name of growth-regulating factors
  • GIF GIF-interaction factor
  • the GRF gene of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • the genomic DNA may be the same as the sequence shown in SEQ ID NO.: 5, 7, 9 or a degenerate variant.
  • the DNA of the present invention may be single-stranded or double-stranded, and the DNA may be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 5, 7, 9 or a degenerate variant.
  • degenerate variant in the present invention refers to a protein that encodes a protein with SEQ ID NO.: 3, 4, 6 or 8, but differs from the code shown in SEQ ID NO.: 5, 7, 9 Nucleic acid sequences that differ in region sequence.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO.: 3, 4, 6 or 8 includes: the coding sequence of only the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and any Optional additional coding sequence) and non-coding sequence.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides having the same amino acid sequence as the present invention or polypeptide fragments, analogs and derivatives.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 2.
  • the present invention also relates to nucleic acid fragments that hybridize with the aforementioned sequences.
  • the "nucleic acid fragment” has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to determine and/or isolate polynucleotides encoding polypeptides related to heat resistance.
  • polypeptide of the present invention As used herein, the terms "polypeptide of the present invention”, “polypeptide encoded by the GRF gene”, “protein encoded by the GRF gene” and “GRF polypeptide” are used interchangeably, and all refer to the plant of the present invention that can regulate and control plants under low nitrogen conditions. Agronomic traits of peptides.
  • the polypeptide of the present invention includes GRF4, GRF6 and/or GRF8. More preferably, the polypeptide of the present invention is derived from rice.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insect and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of GRF polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity as the natural GRF polypeptide of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with substitution groups in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as leader sequence or secretory sequence, or the sequence used to purify the polypeptide or proprotein sequence, or fusion protein). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.
  • the polypeptide of the present invention refers to a polypeptide having the sequence of SEQ ID NO.: 3, 4, 6 or 8 that regulates plant agronomic traits under low nitrogen conditions. It also includes variant forms of the sequence of SEQ ID NO.: 3, 4, 6 or 8 that have the same function as the GRF polypeptide. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and the addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of GRF polypeptides.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA encoded by DNA that can hybridize with the DNA of the SPL polypeptide under high or low stringency conditions Proteins, and polypeptides or proteins obtained using anti-GRF polypeptide antisera.
  • the present invention also provides other polypeptides, such as fusion proteins comprising GRF polypeptides or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes soluble fragments of GRF polypeptides.
  • the fragment has at least about 10 consecutive amino acids of the GRF polypeptide sequence, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids. Consecutive amino acids.
  • the present invention also provides GRF polypeptides or analogs thereof.
  • the difference between these analogs and the natural GRF polypeptide may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis through radiation or exposure to mutagens, site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually without changing the primary structure) forms include: chemically derived forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation. Modified forms also include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). It also includes polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • GRF polypeptide conservative variant polypeptide refers to the amino acid sequence shown in SEQ ID NO.: 3, 4, 6 or 8, at most 10, preferably at most 8, and more preferably At most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein, and adding one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the promoter of the GRF gene or its encoded protein includes a substance that can increase the expression and/or activity of the GRF gene or its encoded protein.
  • the promotion of the GRF gene or its encoded protein is not particularly limited, as long as it can promote the expression of GRF or enhance the activity of the GRF protein is within the protection scope of the present invention.
  • the promoter of the GRF gene or its encoded protein includes small molecule compounds, nucleic acids, enzymes and the like.
  • miR396 is a non-coding single-stranded small RNA molecule with a length of 20-24 nucleotides, which is mainly located at the 5'end of the coding sequence, and its 5'end and 3'end are matched with each other to form a neck loop structure. , which contains 8 members miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g, miR396h. It binds to the mRNA of the target gene through base pairing, thereby causing mRNA degradation or translation inhibition, and plays an important regulatory role in the growth and development of plants.
  • the precursor sequence of miR396e is shown in SEQ ID NO.:1
  • the precursor sequence of miR396f is shown in SEQ ID NO.:2.
  • the mature sequence/conserved sequence refers to the truly functional sequence formed after the RNA precursor sequence is sheared.
  • miR396 represents the RNA precursor sequence; the mature sequence is the RNA sequence formed after the RNA precursor sequence is sheared; the conservative sequence refers to the RNA fragments with the same sequence among different species, and the mature sequence can be equal Regarding conserved sequences, mature and conserved sequences can cross, and conserved sequences can be longer than mature sequences.
  • MIR396 represents the DNA sequence encoding miR396.
  • the similarity of mature sequence/conserved sequence among subtypes a, b, c, d, e, f, g, and h of the miR396 family is very high.
  • the mature sequence of the miR396 family is well-known in the art.
  • the mature sequence of miR396e is uuccacaggcuuucuugaacug (SEQ ID NO.:1 12-33)
  • the mature sequence of miR396f is cuccacaggcuuucuugaacug (SEQ ID NO.: 2nd). 9-30).
  • nucleic acid construct of the present invention or the mutant of its coding gene
  • the present invention provides a nucleic acid construct or a mutant of its coding gene, which is used to regulate agronomic traits of plants under low nitrogen conditions.
  • nucleic acid construct of the present invention has a 5'-3' formula I structure:
  • X1 is none or selected from positions 1-12 of SEQ ID NO.:1 or positions 1-9 of SEQ ID NO.:2;
  • X2 is selected from the mature sequence/conserved sequence of miR396;
  • X3 is none or selected from positions 44-184 of SEQ ID NO.: 1 or positions 41-176 of SEQ ID NO.: 2;
  • each "-" is a bond or a nucleotide connection sequence.
  • the nucleic acid construct of the present invention or the mutant of its encoding gene includes miR396a, miR396b, miR396c, miR396d, miR396e, miR396f, miR396g and/or miR396h mutants.
  • the nucleic acid construct of the present invention or the mutant of its encoding gene may be a single mutation, a double mutation, or a multiple mutation, preferably a single mutation or a double mutation, and more preferably a miR396e and/or miR396f mutation.
  • Inserting the mutant or GRF gene of the coding gene of the construct of the present invention into an exogenous vector constitutes the vector of the present invention.
  • the vector of the present invention is transformed into a plant cell to mediate the integration of the vector of the present invention into the chromosome of the plant cell to produce a transgenic plant cell.
  • the transgenic plant cell of the present invention is regenerated into a plant body, thereby obtaining a transgenic plant.
  • mutants of the coding genes of the nucleic acid constructs constructed by the present invention can be introduced into plant cells through conventional genetic transformation techniques (for example, Agrobacterium transformation techniques) to obtain the coding gene mutants carrying the nucleic acid constructs ( Or a plant cell with a vector encoding a gene mutant of the nucleic acid construct), or a plant cell in which the gene encoding a mutant of the nucleic acid construct is integrated in the genome.
  • conventional genetic transformation techniques for example, Agrobacterium transformation techniques
  • the present invention also provides an inhibitor for miR396, which can inhibit the expression or activity of miR396.
  • the miR396 inhibitor is selected from the following group: small molecule compounds, antisense nucleic acids, microRNA, siRNA, RNAi, Crispr reagent, or a combination thereof.
  • the present invention also provides the use of the nucleic acid construct represented by formula I or its encoding gene mutant, miR396 inhibitor, and/or GRF gene or its encoding protein promoter of the present invention, which are used for regulation under low nitrogen conditions Agronomic traits of plants.
  • the nucleic acid construct represented by formula I of the present invention or a mutant of its encoding gene is derived from rice.
  • the expression or activity of miR396 can be inhibited by techniques such as gene mutation, gene knockout, gene interruption, RNA interference technology, Crispr technology and the like.
  • miR396 can be gene-edited by Cas9 nuclease mediated by one or more sgRNAs.
  • the present invention also provides a method for improving plants (such as rice).
  • the improvement includes: (a) increasing yield and/or biomass; (b) promoting ear type and/or grain type development; (c) increasing Grain length; (d) increase grain width; (e) increase ear length; (f) increase thousand-grain weight; (g) increase leaf length; (h) increase leaf width, including steps: reduce miR396 in plants under low nitrogen conditions.
  • the same trait in different types of crops can be characterized by different terms, or the same trait can be characterized by multiple terms with different other traits.
  • crops such as wheat and rice
  • it can be characterized by ear length, ear grain
  • the number, grain type and other shapes reflect its yield; in soybeans, the number of pods and grain types can be used to reflect the yield; in tomatoes, the size and quantity of the fruit can reflect its yield; therefore, the present invention has an impact on crop traits.
  • the improvement is not limited to the traits listed in the present invention, but also includes other traits that have the same concept as the traits described in the present invention in other crops not listed in the present invention.
  • the present invention finds for the first time a new type of nucleic acid construct represented by formula I of miR396 and its family members or mutants of its coding genes, especially miR396e and/or miR396f subtype mutants, which can be used under low nitrogen conditions
  • a new type of nucleic acid construct represented by formula I of miR396 and its family members or mutants of its coding genes, especially miR396e and/or miR396f subtype mutants which can be used under low nitrogen conditions
  • Significantly improve plant traits under the following conditions including (a) increase yield and/or biomass; (b) promote ear type and/or grain type development; (c) increase grain length; (d) increase grain width; (e) increase ears Length; (f) Increase thousand-grain weight; (g) Increase leaf length; (h) Increase leaf width.
  • the present invention finds for the first time that the nucleic acid construct represented by formula I of the present invention or a mutant of its coding gene can up-regulate the expression of GRF8 gene.
  • the present invention finds for the first time that the nucleic acid construct represented by the formula I of the present invention or the mutant of its encoding gene regulates the development of grain and ear shape through miR396-GRF4/6/8-GIF1/2/3 pathway.
  • the present invention finds for the first time that the nucleic acid construct represented by formula I of the present invention or a mutant of its coding gene can reduce the amount of nitrogen fertilizer used, increase the utilization rate of nitrogen fertilizer, increase the amount of rice biomass energy accumulation, and increase the amount of rice Yield and biomass, while reducing the environmental pollution of nitrogen fertilizer.
  • GRF GRF4, 6, and/or 8, especially GRF8 genes or their encoded proteins in plants can significantly improve plant traits under low nitrogen conditions, including ( a) Increase yield and/or biomass; (b) Promote ear type and/or grain type development; (c) Increase grain length; (d) Increase grain width; (e) Increase ear length; (f) Increase thousand-grain weight; (g) Increase leaf length; (h) Increase leaf width.
  • the reagents or materials used in the embodiments of the present invention are all commercially available products.
  • the medium used in the present invention is a commercially available Kimura B medium
  • the vegan condition is the commercially available Kimura B medium of the present invention, containing various components required for plant growth, and the nitrogen content is (0.034 g/L);
  • the nitrogen-free condition is based on the commercially available Kimura B medium used in the present invention, which does not contain nitrogen but contains all other components.
  • the low-nitrogen condition of the present invention is that the amount of nitrogen fertilizer is lower than the amount of conventional fertilizer applied during crop growth or no additional nitrogen fertilizer is applied to the land.
  • the amount of conventional fertilizer is known to those skilled in the art or farmers to ensure crops The amount of fertilizer used for stable yield and high quality.
  • the low-nitrogen condition is the case where no additional nitrogen fertilizer is applied in the plant growth environment, and the total nitrogen content in the field is 0.5-3.0 g/kg without additional application of nitrogen fertilizer. Soil; In different embodiments, without additional nitrogen fertilizer, the total nitrogen content in the field can be 1-2.5g/kg soil, 1.2-2.2g/kg soil.
  • the conventional amount of nitrogen fertilizer such as rice, wheat, and corn, is 150-350 kg per hectare.
  • the amount varies in different crops and regions.
  • the conventional amount can be determined by those skilled in the art or farmers’ planting experience.
  • nitrogen fertilizer is preferred.
  • the application rate is 210-270 kg per hectare.
  • Example 1 Under low nitrogen conditions, a single miR396e mutation can increase rice yield
  • the target site is selected from the stem-loop sequence of the miR396 precursor, and the sgRNA sequence is designed for the target site: GCUCAUGUUGGGAUUGUGGU (SEQ ID NO.: 10).
  • the hulled seeds are soaked and disinfected with NaClO, washed with sterile water and then inoculated into NB induction medium, and cultured in an incubator for 10-15 days.
  • Agrobacterium infiltration and resistant callus screening the Agrobacterium EH105 strain transferred into the target vector is expanded, and then the callus in a better state is soaked.
  • the callus was sucked up and the liquid was inoculated on a selection medium containing antibiotics and cultured under light for two weeks.
  • Callus differentiation culture select the vigorously growing callus (resistant callus) and transfer it to the differentiation medium containing antibiotics. Within a week, most of the callus grew rapidly, and green spots appeared in the callus, and the green callus would quickly differentiate into seedlings.
  • the single miR396e mutation can change the grain type of rice seeds, specifically increasing its grain length, grain width and/or grain thickness, and ultimately increase the yield of rice by changing the grain type.
  • mutants such as miR396ef and mir396abcef were obtained using the method described in Experimental Example 1.
  • Different gRNAs were designed to obtain miR396ef double mutants and other different types of mutants such as mir396abcef.
  • miR396ef mutation can increase the number of rice grains per panicle
  • mir396ef Compared with the wild type, mir396ef has ear length (20.55 ⁇ 1.83cm vs 17.93 ⁇ 1.64cm) and more main branches (8.8 ⁇ 1.34 vs 7.33 ⁇ 1.56) under field conditions and under low nitrogen conditions (no nitrogen fertilizer applied). Longer panicle length and more panicle branches are usually associated with more grains. The number of panicles in the mir396ef mutant is higher than that of the wild type (77.82 ⁇ 8.89 vs 72.35 ⁇ 10.96). ( Figure 6, a-c)
  • the mir396ef double mutation has a more significant impact on rice grain type, grain number per panicle, plant height and other phenotypes than under normal cultivation conditions. Through the impact on the phenotype, it can finally increase rice yield. .
  • Example 3 The miR396ef mutant can increase rice biomass under low nitrogen conditions
  • miR396ef mutation can increase rice leaf length
  • each individual plant selects three main tillers, a total of 20 individual plants are selected; cut the leaf sheath at the flag leaf sheath with the three tillers of each individual plant selected above; The ruler measures the distance from the tip of the leaf to the sheath. Compared with the wild-type flag leaf, the mir396ef double mutant flag leaf is 32.8% longer (Figure 8).
  • the mutant plant height increased; the flag leaf area increased, the total photosynthetic rate increased, and more photosynthetic products could be accumulated; the nitrogen absorption and utilization rate increased, these factors became the favorable factors for the increase of the dry matter quality of the mir396ef mutant plant .
  • Example 4 mir396ef regulates grain and panicle development through miR396-GRF4/6/8-GIF1/2/3 pathway
  • GRF has been shown to interact with GIFs, a transcriptional co-activator.
  • GIF4 and GRF6 we performed a yeast two-hybrid screen. It was found that OsGIF1, OsGIF2 and OsGIF3 interacted with OsGRF4, and OsGIF3 also interacted with OsGRF6 ( Figure 11d).
  • the GIF1 mutant was generated using CRISPR/Cas9 technology, and the expected short leaf phenotype was observed. Compared with the wild type, the gif1 mutant also showed a smaller plant and aborted seed phenotype (Figure 11e).

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Abstract

提供了一种miR396或其编码基因的突变体在调控植物农艺性状中的应用。还提供了一种GRF基因或其编码蛋白促进剂的用途，一种改良植物农艺性状的方法，一种在低氮条件下改良植物农艺性状的组合物及其用途，一种制备经基因编辑的植物组织或植物细胞的方法以及一种制备经基因编辑的植物的方法。

Description

一种miR396或其编码基因的突变体在调控植物农艺性状的应用 技术领域
本发明涉及作物遗传学领域，具体地涉及一种miR396或其编码基因的突变体在调控植物农艺性状的应用。
背景技术
水稻是世界上最重要的粮食作物之一，也是我国第一大粮食作物。20世纪50年代末至60年代初的矮化育种和70年代三系杂交籼稻的成功应用，使我国水稻单产实现了两次大的飞跃，为满足我国粮食自给自足的作出了巨大贡献。进十几年来，随着我国人口数量的不断增加，对水稻总产的要求日渐提高。在现代农业生产过程中，人们对化学肥料的使用，尤其是氮肥的施用，对促进农业生产的发展起到了举足轻重的作用。但是，在人们大量施用氮肥的同时，也带来了诸多问题和影响，不仅对经济和资源造成巨大的浪费，还导致生态环境受到严重威胁，过量的使用氮肥导致土壤酸化、次生盐渍化，还会导致温室效应和臭氧层空洞，水体的富营养化等。因此，培育在低氮条件下，能够维持或提高现有水稻单产的新品种，是遗传育种家的重要课题。
microRNA(miRNA)是一类20–24个核苷酸长度的非编码单链小RNA分子，它通过碱基配对结合靶基因的mRNA，从而引起mRNA的降解或翻译抑制。miRNA在植物中通过调控编码蛋白基因在原位细胞中的积累来控制植物的生长、发育、耐逆性等。
而目前的miRNA靶标在改良植物农艺性状方面的能力有限。
因此本领域迫切需要开发一种能够显著改良植物农艺性状的方法。
发明内容
本发明的目的是提供一种能够显著改良植物农艺性状的方法。
在本发明的第一方面，提供了一种核酸构建物或其编码基因的突变体的用 途，用于低氮条件下调控植物农艺性状或制备用于低氮条件下调控植物农艺性状的组合物或制剂，其中，所述植物的农艺性状选自下组的一种或多种：
(a)产量和/或生物量；
(b)穗型和/或粒型发育；
(c)果实和/或种子的大小、重量和/或数量；
(d)粒长；
(e)粒宽；
(f)穗长；
(g)千粒重；
(h)叶长；
(i)叶宽；
(j)有效分蘖数；
(k)果荚数；
其中，所述核酸构建物具有5’-3’的式I结构：
X1-X2-X3  (I)
式中，X1为无或选自SEQ ID NO.:1的1-12位或SEQ ID NO.:2的1-9位；
X2选自miR396的成熟序列/保守序列；
X3为无或选自SEQ ID NO.:1的44-184位或SEQ ID NO.:2的41-176位；
并且，各“-”为键或核苷酸连接序列。
在另一优选例中，所述调控植物的农艺性状包括：
(a)提高产量和/或生物量；
(b)促进穗型和/或粒型发育；
(c)增加果实和/或种子的大小、重量和/或数量；
(d)增加粒长；
(e)增加粒宽；
(f)增加穗长；
(g)增加千粒重；
(h)增加叶长。
在另一优选例中，所述miR396的成熟序列/保守序列包括miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g、和/或miR396h的成熟序列/保守序列。
在另一优选例中，所述miR396的成熟序列/保守序列包括miR396e、和/或miR396f的成熟序列/保守序列。
在另一优选例中，所述miR396的成熟序列为miR396e的成熟序列和/或miR396f的成熟序列。
在另一优选例中，所述miR396的保守序列选自SEQ ID NO.:1的13-43位或SEQ ID NO.:2的10-40位。
在另一优选例中，所述低氮条件指所述培养基或大田生长条件的氮含量N L与全素培养基或大田生长条件下中氮含量N0之比(N L/N0)为0-1，较佳地，0.01-0.9，较佳地0.1-0.9，较佳地0.3-0.8，更佳地0.5-0.8，更佳地0.6-0.8。
在另一优选例中，所述低氮条件为氮肥用量低于作物生长过程中所施用的常规肥量，或者所述低氮条件为土壤中不施用额外的氮肥，所述的常规肥量为本领域技术人员或农民所知的为保证农作物稳产、高质所施用的肥量，比如水稻，小麦，玉米为150-350千克每公顷，不同作物不同地区用量有所不同，常规量可以由本领域技术人员或农民种植经验进行确定，在本发明中，优选氮肥的施用量为210-270千克每公顷。
在另一优选例中，所述的低氮条件为植物的生长环境中未施加额外的氮肥，未施加额外的氮肥的情况下，土壤中全氮的含量约为0.5-3.0g/kg土壤；在不同的实施方式中，未施加额外的氮肥的情况下，土壤中全氮的含量为1-2.5g/kg土壤，优选，1.2-2.2g/kg土壤。
在另一优选例中，所述核酸构建物选自下组：
(a)具有SEQ ID NO:1或2所示的核苷酸序列；
(b)核苷酸序列与SEQ ID NO.:1或2所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；
(c)在SEQ ID NO.:1或2所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸。
在另一优选例中，所述核酸构建物的序列如SEQ ID NO.:1或2所示。
在另一优选例中，所述编码基因编码上述核酸构建物。
在另一优选例中，所述编码基因包括MIR396a、MIR396b、MIR396c、MIR396d、MIR396e、MIR396f、MIR396g和/或MIR396h。
在另一优选例中，所述编码基因包括MIR396e和/或MIR396f。
在另一优选例中，所述核酸构建物或其编码基因来源于选自下组的一种或多种植物：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜。
在另一优选例中，所述核酸构建物或其编码基因来源于水稻。
在另一优选例中，所述核酸构建物或其编码基因的突变体来源于选自下组的一种或多种植物：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜。
在另一优选例中，所述核酸构建物或其编码基因的突变体来源于水稻。
在另一优选例中，所述核酸构建物或其编码基因的突变体包括miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g和/或miR396h的突变体。
在另一优选例中，所述核酸构建物或其编码基因的突变体包括miR396e和/或miR396f的突变体。
在另一优选例中，所述组合物为农用组合物。
在另一优选例中，所述组合物包含(a)miR396抑制剂；和(b)农学上可接受的载体。
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜或其组合。
在另一优选例中，所述的水稻选自下组：籼稻、粳稻或其组合。
在另一优选例中，所述核酸构建物或其编码基因的突变体是天然的或人工合成的。
在另一优选例中，所述核酸构建物或其编码基因的突变体包括对上述核酸构建物或其编码基因的碱基的替换、插入、和/或缺失，优选大片段缺失。
在另一优选例中，所述核酸构建物或其编码基因的突变体的表达或活性比野生型的核酸构建物或其编码基因的表达或活性降低了≥50％，更佳地，≥70％，更佳地，≥90％或100％。
在另一优选例中，当所述核酸构建物或其编码基因的突变体的活性E1与所述野生型的核酸构建物或其编码基因的突变体的本底活性E0之比≤1/2，较佳地≤1/5，更佳地≤1/10，更佳地，为0。
在另一优选例中，所述核酸构建物或其编码基因的突变体降低、沉默或丧失对靶标基因的调控作用。
在另一优选例中，所述靶标基因包括GRF基因。
在另一优选例中，所述GRF基因选自下组：GRF4、GRF6、GRF8、或其组合。
在另一优选例中，所述GRF基因包括GRF8。
在另一优选例中，所述核酸构建物或其编码基因的突变位点至少包括部分成熟序列区或保守序列区。
在另一优选例中，所述核酸构建物或其编码基因的突变位点在成熟序列区或保守序列区。
本发明第二方面提供了一种GRF基因或其编码蛋白促进剂的用途，用于低氮条件下调控植物农艺性状或制备用于低氮条件下调控植物农艺性状的组合物或制剂，其中，所述植物的农艺性状选自下组的一种或多种：
(a)产量和/或生物量；
(b)穗型和/或粒型发育；
(c)果实和/或种子的大小、重量和/或数量；
(d)粒长；
(e)粒宽；
(f)穗长；
(g)千粒重；
(h)叶长；
(i)叶宽；
(j)有效分蘖数；
(k)果荚数。
在另一优选例中，所述GRF基因包括GRF4、GRF6和/或GRF8。
在另一优选例中，所述GRF基因包括GRF蛋白编码基因和GRF基因保守区序列。
在另一优选例中，所述GRF基因包括野生型GRF基因和突变型GRF基因。
在另一优选例中，所述的突变型包括突变后编码蛋白的功能未发生改变的突变形式(即功能与野生型编码蛋白相同或基本相同)。
在另一优选例中，所述的突变型GRF基因编码的多肽与野生型GRF基因所编码的多肽相同或基本相同。
在另一优选例中，所述的突变型GRF基因包括与野生型GRF基因相比，同源性≥80％(较佳地≥90％，更佳地≥95％)的多核苷酸。
在另一优选例中，所述的突变型GRF基因包括在野生型GRF基因的5＇端和/或3＇端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸。
在另一优选例中，所述的GRF基因选自下组：cDNA序列、基因组序列、或其组合。
在另一优选例中，所述GRF4基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:3或4所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:5所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:5所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；
(d)在SEQ ID NO.:5所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述GRF4基因的核苷酸序列如SEQ ID NO.:5所示。
在另一优选例中，所述GRF6基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:6所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:7所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:7所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；
(d)在SEQ ID NO.:7所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述GRF6基因的核苷酸序列如SEQ ID NO.:7所示。
在另一优选例中，所述GRF8基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:8所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:9所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:9所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；
(d)在SEQ ID NO.:9所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述GRF8基因的核苷酸序列如SEQ ID NO.:9所示。
在另一优选例中，所述GRF4蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:3或4所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:3或4所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO.:3或4所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述GRF4活性的多肽。
在另一优选例中，所述GRF4蛋白的氨基酸序列如SEQ ID NO.:3或4所示。
在另一优选例中，所述GRF6蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:6所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:6所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO.:6所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述GRF6活性的多肽。
在另一优选例中，所述GRF6蛋白的氨基酸序列如SEQ ID NO.:6所示。
在另一优选例中，所述GRF8蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:8所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:8所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO.:8所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述GRF8活性的多肽。
在另一优选例中，所述GRF8蛋白的氨基酸序列如SEQ ID NO.:8所示。
在另一优选例中，所述GRF基因或其编码蛋白促进剂包括促进GRF基因或其编码蛋白表达或活性的物质。
在另一优选例中，所述GRF基因或其编码蛋白表达或活性指将GRF基因或其编码蛋白的表达或活性提高≥10％，较佳地，≥20％，较佳地，≥50％，更佳地，≥70％。
在另一优选例中，所述GRF基因或其编码蛋白促进剂选自下组：小分子化合 物、核酸分子、酶、或其组合。
在另一优选例中，所述GRF基因或其编码蛋白来源于选自下组的一种或多种植物：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜。
在另一优选例中，所述GRF基因或其编码蛋白来源于水稻。
在另一优选例中，所述组合物为农用组合物。
在另一优选例中，所述组合物包含(a)GRF基因或其编码蛋白的促进剂；和(b)农学上可接受的载体。
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、小麦、高粱、油菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜或其组合。
在另一优选例中，所述的水稻选自下组：籼稻、粳稻或其组合。
本发明第三方面提供了一种改良植物农艺性状的方法，包括：
在低氮条件下，降低植物中miR396的表达或活性或提高植物中GRF基因或 其编码蛋白的表达或活性。
在另一优选例中，所述降低miR396的表达或活性可以通过以下任一方式实现：
I、对miR396的前体序列进行突变从而降低miR396成熟序列的表达或活性；
II、对miR396的成熟序列进行突变从而降低其表达或活性。
在另一优选例中，所述miR396为miR396e和/或miR396f。
在另一优选例中，所述GRF基因选自下组：GRF4、GRF6、GRF8、或其组合。
在另一优选例中，所述GRF基因包括GRF8。
在另一优选例中，所述的降低植物中miR396的表达或活性是通过以下方式实现：
(1)使植物体内miR396突变获得上述的核酸构建物突变体，和/或
(2)在所述植物中导入miR396的抑制剂。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将miR396的抑制剂导入所述植物或植物细胞，从而获得miR396表达下调的植物或植物细胞。
在另一优选例中，所述miR396的抑制剂包括miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g和/或miR396h的抑制剂。
在另一优选例中，所述miR396的抑制剂包括miR396e和/或miR396f的抑制剂。
在另一优选例中，所述的miR396的抑制剂选自下组：小分子化合物、反义核酸、microRNA、siRNA、RNAi、Crispr试剂、或其组合。
在另一优选例中，所述“降低”是指miR396的表达或活性降低满足以下条件：
A1/A0的比值≤80％，较佳地≤60％，更佳地≤40％，最佳地为0-30％；其中，A1为所述植株中miR396的表达或活性；A0为野生型同种类型植物植株中相同miR396的表达或活性。
在另一优选例中，所述的降低指与野生型MIR396的表达水平E0相比，所述植株中MIR396的表达水平E1为野生型的0-80％，较佳地0-60％，更佳地0-40％， 更佳地，0-30％。
在另一优选例中，所述的降低植株中miR396的表达或活性通过选自下组的方式实现：基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术、或其组合。
在另一优选例中，所述的降低植株中miR396的表达或活性通过用1个或多个sgRNA介导的Cas9核酸酶对miR396进行基因编辑。
在另一优选例中，所述方法包括给予植物GRF基因或其编码的多肽的促进剂。
在另一优选例中，所述方法包括向植物中导入外源的GRF基因或其编码蛋白。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将GRF基因序列导入所述植物或植物细胞，从而获得GRF基因表达上调的植物或植物细胞。
在另一优选例中，所述方法包括步骤：
(a)提供携带GRF基因序列的表达载体的农杆菌；
(b)将植物细胞或组织或器官与步骤(a)中的农杆菌接触，从而使GRF的基因序列转入植物细胞，并且整合到植物细胞的染色体上；
(c)选择已转入GRF基因序列的植物细胞或组织或器官；和
(d)将步骤(c)中的植物细胞或组织或器官再生为植株。
在另一优选例中，所述改良植物农艺性状包括：
(a)提高产量和/或生物量；
(b)促进穗型和/或粒型发育；
(c)增加果实和/或种子的大小、重量和/或数量；
(d)增加粒长；
(e)增加粒宽；
(f)增加穗长；
(g)增加千粒重；
(h)增加叶长；
(i)增加有效分蘖数。
本发明第四方面提供了一种在低氮条件下改良植物农艺性状的组合物，包括：
(i)miR396抑制剂、和/或GRF基因或其编码蛋白的促进剂；和
(ii)农学上可接受的载体。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述组合物的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述组合物中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(a)，以所述组合物的总重量计。
在另一优选例中，所述改良植物农艺性状包括：
(a)提高产量和/或生物量；
(b)促进穗型和/或粒型发育；
(c)增加果实和/或种子的大小、重量和/或数量；
(d)增加粒长；
(e)增加粒宽；
(f)增加穗长；
(g)增加千粒重；
(h)增加叶长。
本发明第五方面提供了一种本发明第四方面所述的组合物的用途，用于低氮条件下改良植物的农艺性状。
本发明第六方面提供了一种制备经基因编辑的植物组织或植物细胞的方法，包括步骤：
在低氮条件下，降低植物组织或植物细胞中的miR396的表达或活性、和/或提高植物组织或植物细胞中GRF基因或其编码蛋白的表达或活性，从而获得经基因编辑的植物组织或植物细胞。
本发明第七方面提供了一种制备经基因编辑的植物的方法，包括步骤：
将本发明第六方面所述的方法制备的经基因编辑的植物组织或植物细胞再生为植物体，从而获得经基因编辑的植物。
本发明第八方面提供了一种经基因编辑的植物，所述的植物是用本发明第七方面所述的方法制备的。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了mir396e突变类型对种子粒型的影响。
图2显示了野生型和mir396e突变型种子粒型差异。
图3显示了不同氮素条件下，植株的生长态势。
图4显示了低氮条件下，miR396ef突变对种子粒型、千粒重的影响。
其中，(a)miR396ef突变对粒长、粒宽的影响；
(b)miR396ef突变对粒厚的影响；
(c)miR396ef突变对千粒重的影响。
图5显示了种子的切片观察。
其中，(a)miR396ef突变对颖壳细胞长的影响；
(b)miR396ef突变对内稃和外稃细胞数量的影响。
图6显示了mir396ef突变对穗长、主枝、穗粒数的影响。
其中，(a)miR396ef突变对穗长的影响；
(b)miR396ef突变对主分枝数的影响；
(c)miR396ef突变对穗粒数的影响。
图7显示了mir396ef突变对水稻产量的影响。
其中，(a)非低氮条件下，miR396ef突变体的产量；
(b)低氮条件下，miR396ef突变体的产量。
图8显示了miR396ef突变对水稻叶长的影响。
图9显示了mir396ef突变对水稻株高的影响。
图10显示了mir396ef突变对水稻生物量的影响。
图11显示了mir396ef通过miR396-GRF4/6/8-GIF1/2/3途径调控子粒和穗型发育。
其中，(a)mir396靶向抗性基因序列；
(b)np:rGRF4、np:rGRF6和np:rGRF8对籽粒粒型的影响；
(c)np:rGRF6和np:rGRF8对穗长的影响；
(d)GRE-GIF的相互作用；
(e)gif1突变体的株型和种子育型。
具体实施方式
本发明人经过广泛而深入的研究，首次发现或合成一类新的miR396及其家族成员的式I所示的核酸构建物或其编码基因的突变体。本发明的核酸构建物或其编码基因的突变体可在低氮条件下显著改善植物农艺性状，包括：(a)提高产量和/或生物量；(b)促进穗型和/或粒型发育；(c)果实和/或种子的大小、重量和/或数量；(d)粒长；(e)粒宽；(f)穗长；(g)千粒重；(h)叶长；(i)叶宽；(j)有效分蘖数；(k)果荚数等。此外，发明人还意外发现，提高GRF(比如GFR4、6、和/或8)基因或其编码蛋白的表达或活性，可在低氮条件下显著改善植物农艺性状，包括：(a)提高产量和/或生物量；(b)促进穗型和/或粒型发育；(c)果实和/或种子的大小、重量和/或数量；(d)粒长；(e)粒宽；(f)穗长；(g)千粒重；(h)叶长；(i)叶宽；(j)有效分蘖数；(k)果荚数，此外本发明首次发现，本发明的核酸构建物或其编码基因的突变体在低氮条件下通过GRF(如GRF4/6/8)影响植物产量、生物量；穗型和/或粒型发育；果实和/或种子的大小、重量和/或数量；粒长；粒宽；穗长；千粒重；叶长；叶宽；有效分蘖数；果荚数等性状。在此基础上，本发明人完成了本发明。
GRF基因
如本文所用，术语“GRF基因”、“本发明基因”可以互换使用，都是指本发 明的具有调控植物农艺性状的基因。
在一优选例中，本发明基因包括GRF4、GRF6、GRF8，优选的，本发明的基因包括GRF8。更佳地，本发明GRF基因来源于水稻。
GRF，全称为growth-regulating factors,是植物特有的一类转录因子。它可以通过N-末端QLQ结构域与GIF(GRF-interaction factor)蛋白中的SNH结构域互作,形成功能复合体,共同参与下游基因的表达调控。不同物种间其成员个数略有不同，拟南芥中含有9个，水稻中含有12个，玉米含有14个。
本发明的GRF基因可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。基因组DNA可以是与SEQ ID NO.:5、7、9所示的序列相同或者是简并的变异体。本发明的DNA可以是单链的或是双链的，DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:5、7、9所示的编码区序列相同或者是简并的变异体。
如本文所用，“简并的变异体”在本发明中是指编码具有SEQ ID NO.:3、4、6或8的蛋白质，但与SEQ ID NO.:5、7、9所示的编码区序列有差别的核酸序列。
编码SEQ ID NO.:3、4、6或8的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指:(1)在 较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO.:2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码耐热性能相关多肽的多聚核苷酸。
GRF基因编码的多肽
如本文所用，术语“本发明多肽”、“GRF基因编码的多肽”、“GRF基因编码的蛋白”、“GRF多肽”可以互换使用，都是指本发明的具有在低氮条件下调控植物农艺性状的多肽。
在一优选例中，本发明多肽包括GRF4、GRF6和/或GRF8。更佳地，本发明多肽来源于水稻。
本发明的多肽可以是重组多肽、天然多肽、合成多肽，优选重组多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括GRF多肽的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明的天然GRF多肽相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或 (iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在优选例中，本发明多肽指具有在低氮条件下调控植物农艺性状的SEQ ID NO.:3、4、6或8的序列的多肽。还包括具有与GRF多肽相同功能的、SEQ ID NO.:3、4、6或8的序列的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括GRF多肽的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与SPL多肽的DNA杂交的DNA所编码的蛋白、以及利用抗GRF多肽的抗血清获得的多肽或蛋白。本发明还提供了其他多肽，如包含GRF多肽或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了GRF多肽的可溶性片段。通常，该片段具有GRF多肽序列的至少约10个连续氨基酸，通常至少约30个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
本发明还提供GRF多肽或其类似物。这些类似物与天然GRF多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化。修饰形式还包括具有磷酸化氨基酸残 基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，“GRF多肽保守性变异多肽”指与SEQ ID NO.:3、4、6或8所示的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保守性变异多肽最好根据下表进行氨基酸替换而产生。
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
GRF基因或其编码蛋白的促进剂
在本发明中，GRF基因或其编码蛋白的促进剂包括能够提高GRF基因或其编码蛋白的表达和/或活性的物质。
在本发明中，所述GRF基因或其编码蛋白的促进没有特别限制，只要能够促进GRF的表达或者增强GRF蛋白活性都在本发明的保护范围内。
在一优选实施方式中，所述GRF基因或其编码蛋白的促进剂包括小分子化合物、核酸、酶等。
miR396
miR396是含有20–24个核苷酸长度的非编码单链小RNA分子，主要位于编码序列的5＇端，其5＇端与3＇端配部分碱基相互配对，使其形成颈环结构，其包含8个成员miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g、miR396h。通过碱基配对结合靶基因的mRNA，从而引起mRNA的降解或翻译抑制，在植物生长发育过程中发挥着重要的调节作用。
在一优选实施方式中，miR396e的前体序列如SEQ ID NO.:1所示，miR396f的前体序列如SEQ ID NO.:2所示。
miR396的成熟序列/保守序列
在本发明中，成熟序列/保守序列指RNA前体序列经过剪切加工后形成的真正发挥功能的序列。
具体地，本发明中miR396代表RNA前体序列；所述的成熟序列是RNA前体序列经过剪切加工后形成的RNA序列；保守序列是指不同物种间序列一致的RNA片段，成熟序列可以等同于保守序列，成熟序列与保守序列可以存在交叉，保守序列可以比成熟序列长。MIR396代表编码miR396的DNA序列。
在miR396家族中，miR396家族的亚型a、b、c、d、e、f、g、h之间的成熟序列/保守序列的相似度非常高。所述miR396家族的成熟序列都是本领域公知的，例如，miR396e的成熟序列为uuccacaggcuuucuugaacug(SEQ ID NO.:1第12-33位)，miR396f的成熟序列为cuccacaggcuuucuugaacug(SEQ ID NO.:2第9-30位)。
本发明的核酸构建物或其编码基因的突变体
本发明提供了一种核酸构建物或其编码基因的突变体，用于低氮条件下调控植物的农艺性状。
在本发明中，本发明的核酸构建物具有5’-3’的式I结构：
X1-X2-X3  (I)
式中，X1为无或选自SEQ ID NO.:1的1-12位或SEQ ID NO.:2的1-9位；
X2选自miR396的成熟序列/保守序列；
X3为无或选自SEQ ID NO.:1的44-184位或SEQ ID NO.:2的41-176位；
并且，各“-”为键或核苷酸连接序列。
在本发明中，本发明的核酸构建物或其编码基因的突变体包括miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g和/或miR396h突变体。
在本发明中，本发明的核酸构建物或其编码基因的突变体可以是单一突变，可以是双突变，可以是多突变，优选单突变或双突变，更优选miR396e和/或miR396f的突变。
本发明的构建物中所用的各种元件或者是本领域中已知的，或者可用本领域技术人员已知的方法制备。
将本发明的构建物的编码基因的突变体或GRF基因插入外源载体(尤其是适合转基因植物操作的载体)，就构成了本发明的载体。
将本发明的载体转化植物细胞从而介导本发明的载体对植物细胞染色体进行整合，制得转基因的植物细胞。
将本发明的转基因的植物细胞再生为植物体，从而获得转基因的植物。
将本发明构建好的上述核酸构建物的编码基因的突变体，通过常规的遗传转化技术(例如农杆菌转化技术)，可以导入植物细胞，从而获得携带所述核酸构建物的编码基因突变体(或带有所述核酸构建物的编码基因突变体的载体)的植物细胞，或获得基因组中整合有所述核酸构建物的编码基因突变体的植物细胞。
miR396的抑制剂
本发明还提供了一种针对miR396的抑制剂，所述miR396的抑制剂可抑制miR396的表达或活性。在本发明中，所述miR396的抑制剂选自下组：小分子化合物、反义核酸、microRNA、siRNA、RNAi、Crispr试剂、或其组合。
用途
本发明还提供了本发明的式I所示核酸构建物或其编码基因的突变体、miR396抑制剂、和/或GRF基因或其编码蛋白促进剂的用途，它们被用于低氮条件下调控植物的农艺性状。在本发明中，本发明的式I所示核酸构建物或其编码基因的突变体来源于水稻。
在本发明中，可通过基因突变、基因敲除、基因中断、RNA干扰技术、Crispr技术等技术抑制miR396的表达或活性。
在一优选实施方式中，可通过用1个或多个sgRNA介导的Cas9核酸酶对miR396进行基因编辑。
植物(如水稻)改良
本发明还提供了一种改良植物(如水稻)的方法，所述的改良包括：(a)提高产量和/或生物量；(b)促进穗型和/或粒型发育；(c)增加粒长；(d)增加粒宽；(e)增加穗长；(f)增加千粒重；(g)增加叶长；(h)增加叶宽，包括步骤：在低氮条件下，降低植物中miR396的表达或活性、施用miR396的抑制剂、本发明第一方面所定义的核酸构建物或其编码基因的突变体、和/或提高植物中GRF基因或其编码蛋白的表达或活性。
本领域技术人员公知，不同种类作物中同一性状可以采用不同的术语表征，或者同一性状通过多种术语不同的其他性状进行表征，如在小麦、水稻等作物中，其可以通过穗长、穗粒数、粒型等形状反应其产量；在大豆中，可通过果荚数、粒型来反映其产量；在番茄中，可以通过果实大小、数量来反映其产量；因此，本发明对农作物性状的改良不局限于本发明所列举的性状，还包括本发明未列举的在其他农作物中与本发明所述性状相同概念的其他性状。
在本发明中，还可进一步用常规方法将其他可以调控植物性状的物质处理植物或植物种子，从而改良对应植物的性状。
本发明的主要优点包括：
(1)本发明首次发现一类新的miR396及其家族成员的式I所示的核酸构建物或其编码基因的突变体，尤其是miR396e和/或miR396f亚型突变体，可在低氮条件下显著改良植物性状，包括(a)提高产量和/或生物量；(b)促进穗型和/ 或粒型发育；(c)增加粒长；(d)增加粒宽；(e)增加穗长；(f)增加千粒重；(g)增加叶长；(h)增加叶宽。
(2)本发明首次发现，本发明的式I所示的核酸构建物或其编码基因的突变体可上调GRF8基因表达。
(3)本发明首次发现，本发明的式I所示的核酸构建物或其编码基因的突变体通过miR396-GRF4/6/8-GIF1/2/3途径调控子粒和穗型发育。
(4)本发明首次发现，本发明的式I所示的核酸构建物或其编码基因的突变体可降低氮肥的使用量，提高氮肥的利用率，提高水稻生物质能源积累量，提高水稻的产量和生物量，同时降低氮肥对环境污染。
(5)本发明首次发现，提高植物中GRF(包括GRF4、6、和/或8，尤其是GRF8)基因或其编码蛋白的表达或活性，可在低氮条件下显著改良植物性状，包括(a)提高产量和/或生物量；(b)促进穗型和/或粒型发育；(c)增加粒长；(d)增加粒宽；(e)增加穗长；(f)增加千粒重；(g)增加叶长；(h)增加叶宽。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
如无特别说明，本发明实施例中所用的试剂或材料均为市售产品。
在本发明中，本发明所用培养基为市售木村B培养基，全素条件为本发明的市售木村B培养基，含有植物生长中所需的各种成分，其中氮素含量为(0.034g/L)；无氮条件为在本发明所用市售木村B培养基的基础上，不含有氮素但含有其他全部分成分。
本发明所述的低氮条件为氮肥用量低于作物生长过程中所施用的常规肥量或土地中不施用额外氮肥，所述的常规肥量为本领域技术人员或农民所知的为保证农作物稳产、高质所施用的肥量。本发明实施例所述的大田操作中，所 述的低氮条件为植物生长环境中未额外施加氮肥的情况，未额外施加氮肥的情况下，大田中全氮的含量为0.5-3.0g/kg土壤；在不同的实施方式中，未额外施加氮肥的情况下，大田中全氮的含量为可以为1-2.5g/kg土壤、1.2-2.2g/kg土壤。
氮肥的常规用量，比如水稻，小麦，玉米为150-350千克每公顷，不同作物不同地区用量有所不同，常规量可以由本领域技术人员或农民种植经验进行确定，在本发明中，优选氮肥的施用量为210-270千克每公顷。
实施例1在低氮条件下，miR396e单突变可以增加水稻产量
1、基因编辑位点设计
为设计针对miR396e的基因编辑载体，避免同时靶向miR396的其它家族成员。特选取在miR396前体的stem-loop序列上选取靶向位点，针对该靶标位点设计sgRNA序列：GCUCAUGUUGGGAUUGUGGU(SEQ ID NO.:10)。
2、CRISPR-Cas9基因编辑工具的构建
A)ddH 2O溶解引物至10μM，正/反向引物各1μl加入8μl退火溶液(anneal buffer：TE buffer加50mM NaCl)，混匀；
B)将混匀的引物运行annealling program，PCR仪升温至95℃保持5min，然后每1s降0.1℃，降至16℃；
C)用Bsa I酶切CRISPR-Cas9载体，回收载体片段，备用；
D)连接gRNA和Crispr-Cas9载体，
Figure PCTCN2020097783-appb-000001
加水至10μl,16℃连接2hr。
E)转化大肠杆菌，挑单克隆M13F测序验证片段成功连入载体。
3、载体遗传转化
A)上述构建质粒直接转化农杆菌EHA105：
1.农杆菌感受态细胞中加入质粒DNA，之后冰浴30min,放入液氮中1min，然后立即放入37℃水浴锅中水浴2min。
2.取出离心管，加入LB培养基，振荡培养3～5hr。
3.取出菌液与含相应抗生素的LB培养基平板上涂板，在培养箱中倒置培养。2天左右菌落可见。
B)水稻转基因：
1.愈伤组织诱导，去壳种子用NaClO浸泡消毒，无菌水冲洗后接种于NB诱导培养基中，在培养箱中培养10–15天。
2.愈伤组织继代，将诱导出的愈伤组织用单面刀切下，放入继代培养基，相同条件下培养。
3.农杆菌浸染与抗性愈伤组织筛选，将转入目的载体的农杆菌EH105菌株扩繁，后浸泡状态较好的愈伤组织。
4.吸出或倒掉菌液，将愈伤组织放在箱暗培养48–72h。
5.共培养结束后的愈伤组织用Carbenicillin抗性的无菌水浸泡冲洗，以除去农杆菌。
6.愈伤组织吸干液体，将其接种于含抗生素的筛选培养基上，光照培养两周。
7.愈伤组织分化培养，挑选生长旺盛的愈伤组织(抗性愈伤组织)转移到含抗生素的分化培养基上。一周时间内大部分愈伤组织快速生长，并在愈伤组织表明出现绿点，变绿的愈伤组织会快速分化出幼苗。
4、植株培养及突变体筛选
A)将分化出的健壮幼苗转移至含抗生素的生根培养基中生根培养一周，室温练苗2-3天后，温室基质栽培15-20天后移栽入大田。
B)取每株植物的叶片，提取基因组DNA，在靶向位点两侧设计引物。扩增得到的片段进行Sanger测序，确定每株植物的基因型。
C)检测miR396e基因突变类型，在T0代筛选到在miR396e成熟区一系列的突变类型，并经过4-5代的不断扩繁，不断增加突变类型群体。
D)以miR396e作为检测探针，用Northern blot杂交检测mir396e/f突变 体中成熟体miR396e的积累水平。
5、实验结果
检测到针对miR396e单突变的多种突变类型，观察分析发现，mir396e突变体表型表现在可以影响水稻粒型(粒长，粒宽和/或粒重)(图1)。
进一步分析，mir396e突变体植株相比野生型植株，种子粒长增加4.77％，粒宽增加5.58％(图2)。
6、实验结论
miR396e单突变，可以改变水稻种子粒型，具体的可以增加其粒长、粒宽和/或粒厚，通过粒型的改变，最终提高水稻的产量。
实施例2在低氮条件下，miR396ef双突变体可以提高水稻的产量
采用实验例1中所述方法获得miR396ef、mir396abcef等不同类型突变体；设计不同的gRNA，获得了miR396ef双突变体以及其他的如mir396abcef等不同类型突变体。
(1)在无氮条件下，miR396ef突变体(miR396ef双突变体)生长态势的影响
对在不同氮素条件下生长的突变体和野生型幼苗进行观察，发现在全素(氮含量0.034g/L)培养条件下，mir396ef突变体与野生型几乎没有任何差异；但在无氮素(氮含量0g/L)的培养基中，三个mir396e/f突变体生长势明显强于野生型(图3)。本实验结果初步表明，miR396e/f可以参与响应外界的缺氮营养胁迫过程，并且对水稻的生长势有着重要的影响。
(1)在低氮条件下，miR396ef对粒型的影响
大田环境下，低氮条件下(未施用氮肥，土壤全氮含量约为1.5g/kg土壤)，miR396ef不同突变类型可以增加水稻的粒长8.46％、粒宽7.95％、粒厚8.16％和千粒重26.51％(图4，a－c)。mir396abcef突变体粒型与mir396ef突变体粒型一致，无明显统计学差异。
对野生型和突变型种子进行切片观察，发现粒型的改变主要表现在种子颖壳细胞数目的增多和体积的增大。(图5，a-b)
(2)在低氮条件下，miR396ef突变可以增加水稻的穗粒数
大田条件下，低氮条件下(未施用氮肥)，与野生型相比，mir396ef穗长(20.55±1.83cm VS 17.93±1.64cm)，主枝多(8.8±1.34VS 7.33±1.56)。较长的穗长和更多的穗分枝通常与更多的谷粒相关，mir396ef突变体穗粒数高于野生型(77.82±8.89VS 72.35±10.96)。(图6，a-c)
(3)在低氮条件下，miR396ef突变可以增加水稻的产量
我们的大田产量试验表明，正常栽培条件下施用270kg ha-1氮肥的田块(2m×2m)，mir396ef突变体谷物产量相对野生型增加了约4％。
在低氮条件下(未施用氮肥)，mir396ef突变体水稻的谷物产量较野生型显著增加约15％。(图7，a-b)
综上，mir396ef双突变在低氮条件下，相比正常栽培条件下，对水稻粒型、穗粒数、株高等表型的影响更加显著，通过对表型的影响，最后可提高水稻的产量。
实施例3在低氮条件下miR396ef突变体可以提高水稻的生物量
(1)miR396ef突变可以增加水稻叶长
在大田条件下，灌浆中期，每个单株选取主效的三个分蘖，共选取20个单株；将以上选取的每个单株的三个分蘖，在旗叶叶鞘处剪断叶鞘；用刻度尺测量叶片顶端到叶鞘的距离。与野生型旗叶相比，mir396ef双突变旗叶长32.8％(图8)。
(2)miR396ef突变可以增加水稻株高
在大田条件下，灌浆后期，共选取20个单株；用刻度尺测量每个单株由地表到穗的距离。结果显示，mir396ef突变株(74.13±3.54cm)高于野生型(69.33±3.20cm)，但分蘖数与野生型相似。(图9)
(3)miR396ef突变可以增加水稻的地上部生物量积累
在大田条件下，水稻成熟后，选取20个单株；将以上单株由地平面剪断；将剪断的地上部所有植株组织放入60℃烘箱，烘烤两周；称量统计每个单株的干物质重量。与野生植物相比，在低氮栽培条件下，mir396ef突变株每株植物 地上干生物量增加了25％(图10)。检测分析发现旗叶单位叶面积的光合速率与野生型相比无显著差异。因此，突变体株高增加；旗叶叶面积增大，总的光合速率增加，可以积累跟多的光合产物；氮素吸收和利用速率增加，这些因素都成为mir396ef突变植株干物质量增加的有利因素。
实施例4 mir396ef通过miR396-GRF4/6/8-GIF1/2/3途径调控子粒和穗型发育
水稻中的12个GRF转录因子基因都携带着miR396靶位点。我们发现，与野生型相比，mir396ef突变植物中只有OsGRF4、OsGRF6和OsGRF8上调。RLM-race(5’RNA连接酶介导的cDNA末端的快速扩增)分析表明，miR396在体内可以在miR396配对区的特定位置直接切割OsGRF4和OsGRF6的mRNA。
为了研究mir396介导的OsGRF4、OsGRF6和OsGRF8调控种子和穗的发育，我们构建了OsGRF4、OsGRF6和OsGRF8(分别命名为np:rGRF4、np:rGRF6和np:rGRF8)的mir396靶向抗性基因序列(图11a)，并在它们的本身启动子的调控下转入水稻。我们发现：(1)np:rGRF4、np:rGRF6和np:rGRF8的籽粒比野生型植物大，np:rGRF4的粒型增长最大，np:rGRF6的粒型增长最小(图11b)。(2)np:rGRF6和np:rGRF8的穗长均有所增加(图11c)，np:rGRF4的穗长和分枝数与野生型相似。这些结果表明，mir396ef分子通过调控其靶基因OsGRF4、OsGRF6和OsGRF8来调控种子和穗的发育。
GRF已被证明与转录辅助活化因子GIFs相互作用。为了发现与GRF4和GRF6相互作用的GIF，我们进行了酵母双杂交筛选。发现OsGIF1、OsGIF2和OsGIF3与OsGRF4相互作用，OsGIF3也与OsGRF6相互作用(图11d)。用CRISPR/Cas9技术产生了GIF1突变体，并观察了预期的短叶表型。与野生型相比，gif1突变体也表现出较小的植株和败育种子表型(图11e)。这些结果表明，水稻株型和粒型的发育受miR396EF-GRF4/6/8-GIF1/2/3模块的调控。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种核酸构建物或其编码基因的突变体的用途，其特征在于，用于低氮条件下调控植物农艺性状或制备用于低氮条件下调控植物农艺性状的组合物或制剂，其中，所述植物的农艺性状选自下组的一种或多种：
    (a)产量和/或生物量；
    (b)穗型和/或粒型发育；
    (c)果实和/或种子的大小、重量和/或数量；
    (d)粒长；
    (e)粒宽；
    (f)穗长；
    (g)千粒重；
    (h)叶长；
    (i)叶宽；
    (j)有效分蘖数；
    (k)果荚数；
    其中，所述核酸构建物具有5’-3’的式I结构：
    X1-X2-X3  (I)
    式中，X1为无或选自SEQ ID NO.:1的1-12位或SEQ ID NO.:2的1-9位；
    X2选自miR396的成熟序列或miR396的保守序列；
    X3为无或选自SEQ ID NO.:1的44-184位或SEQ ID NO.:2的41-176位；
    并且，各“-”为键或核苷酸连接序列。
  2. 如权利要求1所述的用途，其特征在于，所述调控植物的农艺性状包括：
    (a)提高产量和/或生物量；和/或
    (b)促进穗型和/或粒型发育；和/或
    (c)增加果实和/或种子的大小、重量和/或数量；和/或
    (d)增加粒长；和/或
    (e)增加粒宽；和/或
    (f)增加穗长；和/或
    (g)增加千粒重；和/或
    (h)增加叶长。
  3. 如权利要求1所述的用途，其特征在于，所述miR396的成熟序列或miR396的保守序列包括miR396a、miR396b、miR396c、miR396d、miR396e、miR396f、miR396g、和/或miR396h的成熟序列或其保守序列。
  4. 如权利要求1所述的用途，其特征在于，所述miR396的成熟序列为miR396e的成熟序列和/或miR396f的成熟序列。
  5. 如权利要求1所述的用途，其特征在于，所述miR396的保守序列选自SEQ ID NO.:1的13-43位或SEQ ID NO.:2的10-40位。
  6. 一种GRF基因或其编码蛋白促进剂的用途，其特征在于，用于低氮条件下调控植物农艺性状或制备用于低氮条件下调控植物农艺性状的组合物或制剂，其中，所述植物的农艺性状选自下组的一种或多种：
    (a)产量和/或生物量；
    (b)穗型和/或粒型发育；
    (c)果实和/或种子的大小、重量和/或数量；
    (d)粒长；
    (e)粒宽；
    (f)穗长；
    (g)千粒重；
    (h)叶长；
    (i)叶宽；
    (j)有效分蘖数；
    (k)果荚数。
  7. 一种改良植物农艺性状的方法，其特征在于，包括：
    在低氮条件下，降低植物中miR396的表达或活性或提高植物中GRF基因或其编码蛋白的表达或活性。
  8. 一种在低氮条件下改良植物农艺性状的组合物，其特征在于，包括：
    (i)miR396抑制剂、和/或GRF基因或其编码蛋白的促进剂；和
    (ii)农学上可接受的载体。
  9. 一种权利要求8所述的组合物的用途，其特征在于，用于低氮条件下改良植物的农艺性状。
  10. 一种制备经基因编辑的植物组织或植物细胞的方法，其特征在于，包括步骤：
    在低氮条件下，降低植物组织或植物细胞中的miR396的表达或活性、和/或提高植物组织或植物细胞中GRF基因或其编码蛋白的表达或活性，从而获得经基因编辑的植物组织或植物细胞。
  11. 一种制备经基因编辑的植物的方法，其特征在于，包括步骤：
    将权利要求10所述的方法制备的经基因编辑的植物组织或植物细胞再生为植物体，从而获得经基因编辑的植物。
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