WO2023038075A1 - Genetically modified plant, method for producing plant, and growth stimulant - Google Patents

Genetically modified plant, method for producing plant, and growth stimulant Download PDF

Info

Publication number
WO2023038075A1
WO2023038075A1 PCT/JP2022/033654 JP2022033654W WO2023038075A1 WO 2023038075 A1 WO2023038075 A1 WO 2023038075A1 JP 2022033654 W JP2022033654 W JP 2022033654W WO 2023038075 A1 WO2023038075 A1 WO 2023038075A1
Authority
WO
WIPO (PCT)
Prior art keywords
oshho3
gene
nitrogen
plant
osamt1
Prior art date
Application number
PCT/JP2022/033654
Other languages
French (fr)
Japanese (ja)
Inventor
修一 柳澤
康仁 櫻庭
Original Assignee
国立大学法人東京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東京大学 filed Critical 国立大学法人東京大学
Priority to JP2023546978A priority Critical patent/JPWO2023038075A1/ja
Publication of WO2023038075A1 publication Critical patent/WO2023038075A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to genetically modified plants, methods for producing plants, and growth promoters.
  • the present invention relates to efficient use of nitrogen fertilizers in crops and improved crop production.
  • Plants acquire nitrogen, a building block of many molecules such as nucleic acids, amino acids and chlorophyll, from soil mainly as nitrate or ammonium ions.
  • Nitrate is the major nitrogen source for most terrestrial plants and ammonium ion is the major nitrogen source for plants growing in anaerobic conditions such as paddy fields.
  • a significant amount of the acquired nitrogen is utilized for the synthesis of photosynthetic proteins and the maintenance of photosynthesis, resulting in a positive correlation between leaf nitrogen content and plant photosynthetic rate and yield (e.g. See Patent Document 1.).
  • NUE nitrogen utilization efficiency
  • the present invention has been made in view of the above circumstances, and provides a genetically modified plant exhibiting high nitrogen utilization efficiency, a method for producing the plant, and a growth promoter.
  • the present invention includes the following aspects.
  • [1] A genetically modified plant in which the function of the OsHHO3 gene or its homologous gene is deleted or suppressed, and which exhibits growth promotion in a nitrogen-deficient environment.
  • [2] The genetically modified plant according to [1], wherein the function of the OsHHO3 gene or its homologous gene is deficient due to mutation in the OsHHO3 gene or its homologous gene.
  • the genetically modified plant of [1] in which ammonium ion uptake is promoted.
  • the genetically modified plant of [1] which is a gramineous plant.
  • [5] A method for producing a plant having improved growth in a low-nitrogen environment, wherein the function of the OsHHO3 gene or its homologous gene is deleted or suppressed in the plant.
  • [6] A growth promoter for plants in a nitrogen-deficient environment, the growth promoter comprising a genome-editing composition that disrupts the OsHHO3 gene or its homologue gene.
  • the present invention it is possible to provide a genetically modified plant exhibiting high nitrogen utilization efficiency, a method for producing a plant, and a growth promoter.
  • Sequence analysis was performed using T0 plant leaves of these oshho3-KO mutants (#1-#4).
  • black squares indicate coding regions
  • white squares and pentagons indicate 5'- and 3'-untranslated regions, respectively.
  • the protospacer adjacent motif (PAM) sequences are shown in ⁇ frames and the sequences used as gRNAs in WT are shown in upper case.
  • Predicted cleavage sites are indicated by black arrowheads.
  • Adenosine insertions and base deletions are indicated by ⁇ frames and "-", respectively.
  • Nucleotide and amino acid sequence abbreviations are shown in solid lines, amino acid sequences and amino acid number changes of products derived from mutant alleles are shown in gamma frames. Stop codons caused by genome editing are indicated by asterisks in the ⁇ frame. Editing at both alleles occurred in all four independent oshho3-KO lines (#1-#4). Photographs of wild type (WT), oshho3 knockout mutant (oshho3-KO), and OsHHO3 overexpressing (OsHHO3-OX) seedlings.
  • (b) Graph of root dry weight of WT, osho3-KO, and OsHHO3-OX seedlings (FIG. 3).
  • Dots indicate genes upregulated in roots only (725), shoots only (95), and both shoots and roots (39) (log 2 FC>1, p ⁇ 0.05); genes) and shoots only (79) downregulated (log 2 FC ⁇ 1, p ⁇ 0.05); genes upregulated in shoots but downregulated in roots (3) ; indicates genes that are neither upregulated nor downregulated in either roots or shoots.
  • Heatmaps represent changes in expression levels of AMT, NRT, and other nitrogen assimilation-related genes in shoots (S) and roots (R) by osho3 knockout mutations.
  • Results of a ChIP assay performed using OsHHO3-OX-#1 seedlings The OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoter regions analyzed by ChIP-qPCR are indicated using ⁇ horizontal lines.
  • the delta arrow indicates the transcription start site (TSS).
  • the region used as the promoter in FIG. 13 is indicated by a horizontal line in ⁇ , and the GATTC and GAATC sequence motifs are indicated by purple and blue vertical lines, respectively.
  • Nucleotide mutations (M1 and M2) introduced into the OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters are also indicated.
  • the OsUBQ5 gene served as a negative control. Co-transfection assay results.
  • Reporter plasmids contain the firefly luciferase gene (LUC) under the control of the WT or mutated versions of the OsAMT1;1, OsAMT1;2, or OsAMT1;3 promoters.
  • OsHHO3 expression vector or empty vector control was co-transfected with reporter plasmid and internal control plasmid. The LUC activity obtained with the empty vector control was set to 1. 1 is a graph showing the activity of OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters in WT, osho3-KO-#1, and OsHHO3-OX-#1 protoplasts. The 35S promoter served as a control.
  • the LUC activity of WT protoplasts was set to 1.
  • (a) Graph showing the nitrate uptake activity of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings grown in Yoshida nutrient solution.
  • (a)-(c) ND indicates no significant increase in nitrate uptake. Data are means ⁇ SD of four biological replicates. Asterisks above each bar represent significant differences compared to WT ( * p ⁇ 0.05, ** p ⁇ 0.01;Student's t-test).
  • (b) Graph showing nitrate ion content of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings grown in Yoshida nutrient solution.
  • OsIPT4 Os03g0810100
  • OsIPT5 Os07g0211700
  • OsCYP735A3 Os08g0429800
  • FIG. 4 is a graph showing repression of OsIPT4 and OsIPT5 promoters by OsHHO3.
  • Reporter plasmids containing the LUC reporter gene under the control of the OsIPT4 or OsIPT5 promoter were co-transfected into rice WT protoplasts along with the OsHHO3 expression vector or an empty control vector.
  • the 35S promoter served as a negative control.
  • Asterisks indicate significant differences between empty vector controls and OsHHO3-expressing vectors ( * p ⁇ 0.05, ** p ⁇ 0.01;Student's t-test). Results of a ChIP assay performed using OsHHO3-OX-#1 seedlings.
  • the regions amplified in the ChIP assay and used as promoters in (b) are indicated by ⁇ and ⁇ horizontal lines, respectively, and the GATTC and GAATC motifs are indicated by double-dash and single-dot vertical lines, respectively.
  • ⁇ arrow indicates TSS.
  • the OsUBQ5 gene served as a negative control. Asterisks indicate significant differences between OsUBQ5 control samples and other samples ( * p ⁇ 0.05, ** p ⁇ 0.01;Student's t-test).
  • FIG. 3 is a graph showing the cytokinin content of seedling shoots and roots. Data represent the mean ⁇ SD of four biological replicates. Asterisks indicate significant differences between WT and other genotypes ( * p ⁇ 0.05, ** p ⁇ 0.01;Student's t-test). Graph showing expression levels of cytokinin-related genes in WT, osho3-KO, and OsHHO3-OX seedlings.
  • OsIPT7 Os05g0551700
  • OsAHK3 Os01g0923700
  • OsPR6 Os12g0437800
  • the present invention provides genetically modified plants in which the function of the OsHHO3 gene or its homologous gene is deleted or repressed and which exhibit enhanced growth in nitrogen-deficient environments.
  • the OsHHO3 protein is a transcription factor of the NIGT1/HHO family.
  • the full-length amino acid sequence of OsHHO3 protein is shown in SEQ ID NO:1.
  • NIGT1s Arabidopsis thaliana nitrate-inducible NIGT1 genes
  • AtNIGT1 encodes GARP/G2-like transcription factors (TFs) that directly repress NRT2 (high-affinity nitrate transporter gene) and nitrogen starvation-induced genes. Under nitrogen starvation conditions, the gradual decline in AtNIGT1 transcription levels activates nitrogen starvation-inducible genes.
  • TFs GARP/G2-like transcription factors
  • ammonium ions are the main nitrogen source for rice.
  • no gene is known that promotes ammonium ion uptake in a low-nitrogen environment and improves growth in a low-nitrogen environment.
  • a method for producing rice plants with improved growth in a low-nitrogen environment is not known.
  • OsHHO3, a member of the NIGT1/HHO family, regulates the expression of three OsAMT1 (high-affinity ammonium transporter genes) responsible for over 95% of the ammonium ion uptake activity. was found to be a transcriptional repressor. Furthermore, CRISPR/Cas9-mediated inactivation of OsHHO3 enhanced ammonium ion uptake and plant growth under nitrogen-deficient conditions, improving nitrogen utilization efficiency.
  • the function of the OsHHO3 gene or its homologous gene is deleted or suppressed.
  • Defective function of the OsHHO3 gene or its homologous gene means loss of expression of the OsHHO3 gene product or its homologous gene product in the genetically modified plant of the present embodiment.
  • Loss of expression of the OsHHO3 gene product or its homologous gene product can be caused, for example, by introducing a mutation into the OsHHO3 gene or its homologous gene to disrupt the OsHHO3 gene or its homologous gene. Mutations can be caused by partial or whole deletion, substitution, insertion of arbitrary sequences, etc. in the OsHHO3 gene or its homologous gene, or the expression control region of these genes.
  • mutations can be introduced using methods such as treatment with mutagen, ultraviolet irradiation, gene targeting by homologous recombination techniques, gene knockout, conditional knockout by Cre-loxP system and the like. Genome editing technology may be used for gene targeting and gene knockout.
  • the suppression of the function of the OsHHO3 gene or its homologous gene means that the amount of the OsHHO3 gene product or its homologous gene product is suppressed in the genetically modified plant of the present embodiment compared to the wild-type control plant. It means that Suppression of expression of the OsHHO3 gene product or its homologous gene includes introducing into a cell or animal a nucleic acid sequence that causes the expression of an RNAi-inducing nucleic acid, an antisense nucleic acid, an aptamer or a ribozyme against the OsHHO3 gene product or its homologous gene, It can be generated by gene knockdown or the like.
  • Examples of genetically modified plants of this embodiment include gramineous plants and leguminous plants.
  • the gramineous plants include rice, wheat, barley, rye, corn, foxtail millet, sugar cane, and the like.
  • Examples of leguminous plants include soybean, adzuki bean, pea, and kidney bean.
  • Homologous genes of the OsHHO3 gene include, for example, genes encoding the following proteins.
  • Examples of gramineous plants include the homologous protein of Triticum aestivum (bread wheat) represented by SEQ ID NO: 2 (60.17% homology with OsHHO3 protein), the homologous protein of Hordeum vulgae (barley) represented by SEQ ID NO: 3 ( 62.50% homology with OsHHO3 protein), Zea mays (corn) homologous protein represented by SEQ ID NO: 4 (56.62% homology with OsHHO3 protein), and the like.
  • Examples of leguminous plants include the Glycine Max (soybean) homolog protein represented by SEQ ID NO: 5 (34.72% homology with OsHHO3 protein).
  • any one of the following gene functions (a) to (c) is deficient or suppressed.
  • (b) one to several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by (a)
  • (c) consisting of an amino acid sequence having 30% or more identity with the amino acid sequence represented in (a) above and having transcription repressing activity
  • the number of amino acids deleted, inserted, substituted or added is 1 to 200, preferably 1 to 100, more preferably 1 to 50, more preferably 1 to 30. , more preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.
  • the identity is preferably 50% or more, more preferably 55% or more, particularly preferably 85% or more, still more preferably 90% or more, and most preferably 95% or more.
  • transcriptional repressing activity means an activity that represses transcription of genes involved in ammonium ion uptake activity, such as high-affinity ammonium transporter genes.
  • High-affinity ammonium transporter genes include AMT1-3 genes, and in rice, OsAMT1;1, OsAMT1;2, and OsAMT1;3. Genes to be suppressed include NRT, other nitrogen assimilation-related genes; OsIPT4, OsIPT5, OsCYP735A3, and other cytokinin-related genes.
  • showing growth promotion in a nitrogen-deficient environment means that in a nitrogen-deficient environment, shoot dry weight, root dry weight, leaf Fv / Fm ratio, It means that at least one element selected from chlorophyll content, plant height, number of tillers, length of spike, number of spikelets per spike, and grain yield per plant is superior to wild-type plants. .
  • the genetically modified plant of this embodiment exhibits enhanced growth even in a nitrogen-deficient environment by promoting ammonium ion uptake and improving nitrogen utilization efficiency.
  • the present invention is a method for producing a plant that improves growth in a low-nitrogen environment, wherein the function of the OsHHO3 gene or its homologue gene is deleted or suppressed in the plant. I will provide a.
  • the OsHHO3 gene or its homologous gene is the same as explained in ⁇ genetically modified plant>>.
  • a plant in which the production of a functional gene product is deficient or suppressed is obtained.
  • method for example, there is a method of screening for plants in which the function of the OsHHO3 gene or its homologous gene is deleted or suppressed by treatment with a mutagen.
  • the production of functional gene products is deleted or suppressed. You can get plants.
  • a plant in which production of the gene product is deficient or suppressed may be obtained by introducing into the plant a nucleic acid that deficient or suppresses the expression of the OsHHO3 gene or its homologous gene.
  • the method of producing a plant of this embodiment may be a method of deleting or suppressing the function of the OsHHO3 gene or its homologous gene by modifying its promoter.
  • Modification methods include generating insertion-deficient (InDel) mutations by non-homologous end joining (NHEJ) after double-strand breaks in the target genomic DNA.
  • the InDel mutation induces a frameshift and a premature termination codon.
  • Another example is a method of inserting mutated foreign DNA into the target genome by homologous recombination repair after double-strand cleavage of the target genomic DNA.
  • the system used for double-strand cleavage of target genomic DNA is not particularly limited, and includes the CRISPR-Cas system, TALEN system, Zn finger nuclease system, and the like.
  • the method for introducing these systems into cells is not particularly limited, and the target genomic DNA-cleaving enzyme itself may be introduced into cells, or a target genomic DNA-cleaving enzyme expression vector may be introduced into cells.
  • Systems used for double-strand cleavage of target genomic DNA are introduced into cells simultaneously with, before, or after the exogenous DNA.
  • a Cas9 expression vector and an expression vector encoding a guide RNA that induces Cas9 to a site to be cleaved a method of introducing into a cell, and a recombinant Cas9 protein that is expressed and purified, and a guide RNA , into cells, and the like.
  • the system used for double-strand cleavage of target genomic DNA is preferably the CRISPR-Cas system.
  • genome editing may be performed in an in vitro culture system or in planta.
  • foreign genes, nucleic acids and proteins are introduced into cells by known methods such as the Agrobacterium method, the particle gun method and the whisker method for callus or tissue pieces.
  • introduction of foreign genes, nucleic acids and proteins into cells is carried out using known methods to exposed shoot tops of immature and mature embryos.
  • the plant produced in the present embodiment includes not only the plant body, but also the cell from which the plant body is derived, as long as the plant body is produced from the cell to improve the growth in a low-nitrogen environment.
  • used in A cell may be in a tissue or organ state.
  • Plants produced in this embodiment may be their progeny or clone organisms or cells.
  • the plant body of the clone includes a plant body obtained from cuttings or cuttings.
  • Plants produced by the method for producing a plant of the present embodiment include gramineous plants, leguminous plants and the like, with gramineous plants being preferred, and rice being more preferred.
  • the present invention provides a growth promoter for plants in nitrogen-deficient environments, the growth promoter comprising a genome editing composition that disrupts the OsHHO3 gene or its homologues.
  • a genome-editing composition contains a target genomic DNA-cleaving enzyme or DNA or mRNA encoding the enzyme.
  • Target genomic DNA cleaving enzymes include Cas9, TALENs, Zn finger nucleases and the like.
  • the DNA or mRNA encoding the enzyme includes DNA or mRNA encoding these proteins.
  • the genome editing composition preferably contains a guide RNA that induces Cas9.
  • the composition may also contain an expression vector encoding the guide RNA.
  • guide RNA include those having a protospacer represented by SEQ ID NO:6.
  • the genome editing composition preferably contains foreign DNA having a predetermined mutation with homologous arms at both ends.
  • disruption of the OsHHO3 gene or its homolog releases the suppression of expression of the three OsAMT1 involved in the ammonium ion uptake activity. promotes uptake of and promotes plant growth.
  • OsHHO3-OX OsHHO3 overexpressing lines
  • oshho3 -KO OsHHO3 gene-editing knockout mutant
  • the OsNIGT1 cDNA in the pCB-HYG-ZmUbi-OsNIGT1-MYC binary vector was ligated to the entire coding region of OsHHO3 using BamHI and StuI restriction enzymes. was replaced with a cDNA that Double-stranded DNA obtained by annealing two oligonucleotides (SEQ ID NOs: 7 and 8) corresponding to target sequences within the OsHHO3 locus to construct a binary vector for generating oshho3-KO strains.
  • the resulting binary plasmid was introduced into Agrobacterium tumefaciens strain EHA105, which was used for Agrobacterium-mediated transformation of Nipponbare (WT).
  • Transformed callus was selected on medium containing hygromycin B and regenerated to establish four independent OsHHO3-OX and oshho3-KO rice lines, respectively.
  • the nucleotide sequences of OsHHO3 genomic DNAs in four independent osho3-KO rice lines are shown in the sequence listing.
  • the nucleotide sequences of oshho3-KO#1 mutant OsHHO3 are shown in SEQ ID NO: 9 (#1 allele 1) and SEQ ID NO: 10 (#1 allele 2).
  • the nucleotide sequences of oshho3-KO#2 mutant OsHHO3 are shown in SEQ ID NO: 11 (#2 allele 1) and SEQ ID NO: 12 (#2 allele 2).
  • the nucleotide sequence of oshho3-KO#3 mutant OsHHO3 is shown in SEQ ID NO: 13 (#3 allele1, allele2).
  • the nucleotide sequences of oshho3-KO#4 mutant OsHHO3 are shown in SEQ ID NO: 14 (#4 allele 1) and SEQ ID NO: 15 (#4 allele 2).
  • the nucleotide sequence of OsHHO3 wild-type genomic DNA is shown in Sequence Listing 16.
  • T2 progeny Segregation of the hygromycin resistance marker gene in T2 progeny indicated that the transgene was introduced at a single locus in all four OsHHO3-OX lines. T2 seeds of T1 plants homozygous for the introduced gene were used for further experiments. DNA sequence analysis of T0 plants of all four independent osho3-KO lines revealed the presence of biallelic mutations at the target locus (see Figure 2).
  • the molecular basis of the superior oshho3-KO plant phenotype was investigated by examining changes in gene expression profiles given enhanced growth and improved yield of osho3-KO lines in a nitrogen-deficient environment.
  • the OsHHO3 knockout mutation affected the expression of far more genes in roots than in shoots, most of which were upregulated (see Figure 8, see Tables 1-10).
  • Heatmap analysis showed upregulation of a set of AMT and NRT genes and other nitrogen assimilation-related genes in the roots of osho3-KO seedlings (see Figure 9). Among these genes, OsAMT1, which encodes a high-affinity AMT, showed the most pronounced upregulation.
  • OsAMT1;1 is the most upregulated gene and OsAMT1;3 is the top 10 most upregulated genes. (see Table 1). OsAMT1;2 was also clearly upregulated in roots (see FIGS. 8 and 9).
  • More than 95% of the ammonium ion uptake activity is derived from rice OsAMT1;1, OsAMT1;2, and OsAMT1;3.
  • nitrogen starvation-induced expression was enhanced in oshho3-KO seedlings but attenuated in OsHHO3-OX seedlings. It was shown that there was a negative correlation between the expression level of AMT1 and the expression level of AMT1 (see FIG. 10).
  • ammonium ion uptake and content were higher in osho3-KO seedlings and lower in OsHHO3-OX seedlings than in WT seedlings, especially when the seedlings were exposed to nitrogen deficiency stress. (See FIG. 11).
  • nitrate uptake activity was slightly higher in osho3-KO seedlings than in WT seedlings when exposed to nitrogen depletion stress (see Figure 15). This is consistent with the weak upregulation of several NRT genes in oshho3-KO roots (see Figure 9).
  • OsHHO3 transcripts remained at low levels after nitrogen depletion treatment, whereas OsHHO3 expression remained low after nitrogen depletion treatment. It was found that it gradually decreased during the period (see FIG. 16). Therefore, since OsHHO3 directly represses AMT1, we hypothesized that reduction of OsHHO3 expression upon nitrogen depletion treatment induces the expression of these AMT1s. To test this hypothesis, chromatin immunoprecipitation (ChIP) and co-transfection assays were performed.
  • a ChIP assay revealed in vivo binding of OsHHO3 to regions a and d of OsAMT1;1, regions b and f of OsAMT1;2, and regions b and e of OsAMT1;3 (see Figure 12). Details of the ChIP assay are as follows.
  • OsAMT1;1 ( ⁇ 2,090 to +83 bp relative to the transcription start site [TSS, +1 bp]), OsAMT1;2 ( ⁇ 2,097 to +70 bp), OsAMT1;3 ( ⁇ 1, 922 to +93 bp), OsIPT4 (-1,859 to +158 bp), and OsIPT5 (-1,823 to +155 bp) were PCR amplified from genomic DNA of WT seedlings and cloned upstream of the LUC reporter gene in the pJD301 vector. Mutations in the OsAMT1 promoter were introduced using the Megaprimer PCR method.
  • the NLP7 cDNA of pHBT-NLP7-MYC6 was replaced with a cDNA corresponding to the entire coding region of OsHHO3.
  • Rice protoplasts were isolated from shoots of leaf sheaths and seedlings grown in the dark for 12 days and exposed to light for 16 hours.
  • the reporter plasmid (2 ⁇ g) and the effector plasmid (4 ⁇ g) were co-transfected into 1 ⁇ 10 5 rice protoplasts together with an internal control plasmid (pZmUbi-GUS, 1 ⁇ g) by polyethylene glycol (PEG)-mediated transfection.
  • Transfected protoplasts were incubated in protoplast culture medium (0.4 M mannitol, 15 mM MgCl 2 , and 4 mM MES-KOH [pH 5.8]) in the dark at room temperature for 16 hours.
  • the LUC and GUS activity levels of each cell lysate were then measured using the Luciferase Assay System Kit (Promega K.K., Tokyo, Japan) and 4-methylumbelliferyl ⁇ -D-glucuronide (a fluorescent substrate), respectively. determined and the relative LUC activity calculated.
  • OsHHO3 directly and synchronously represses the OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters by binding to sequences similar or identical to the NIGT1-binding motif.
  • disruption of OsHHO3 promotes ammonium ion uptake under nitrogen-deficient conditions, as OsHHO3 is the repressor responsible for the coordinated activation of three key AMT1s.
  • OsHHO3-mediated suppression of the three OsAMT1s could explain why oshho3-KO and OsHHO3-OX rice plants exhibited improved and decreased nitrogen utilization efficiency and growth, respectively.
  • OsHHO3 may also affect other nitrogen deficiency-related processes.
  • AtNIGT1 repressed AtIPT3 (adenylate isopentenyltransferase gene) and AtCYP735A2 (Cyt P450 monooxygenase gene) involved in cytokinin biosynthesis, resulting in increased tZ-type cytokinin content in the Arabidopsis nigt1 quadruple mutant.
  • cytokinin levels were higher in osho3-KO seedlings compared to WT, but lower in OsHHO3-OX seedlings (see Figure 20). Since active cytokinins are required for axillary bud growth (tillers) and the number of active tillers with panicles is an important factor in determining grain yield in rice, these results suggest that oshho3-KO This is consistent with the increased tiller number and yield of the plants and the decreased tiller number and yield of the OsHHO3-OX plants (see Figure 6(b)). Therefore, cytokinin-mediated regulation may also contribute to the agronomically superior phenotype of osho3-KO rice.
  • OsHHO3 was identified as a transcriptional repressor of three AMT1 genes that critically contribute to ammonium ion uptake in rice.
  • CRISPR/Cas9-mediated inactivation of OsHHO promotes ammonium ion uptake, improving nitrogen utilization efficiency, growth, and yield in low-nitrogen environments.
  • CRISPR/Cas9-mediated targeted mutagenesis of OsHHO3 could be a sustainable It may help develop rice cultivation that enables agriculture.
  • the coordinated activation of the three OsAMT1s is important for effectively promoting the acquisition of ammonium ions, emphasizing the importance of OsHHO3.
  • the distinct spatial localization patterns of OsAMT1;1, OsAMT1;2, and OsAMT1;3 in roots suggest distinct contributions to ammonium ion acquisition in roots, and knockout mutations of each of the three OsAMT1 All three triple knockout mutations of OsAMT1 reduced ammonium ion uptake to less than 5%, although individually or in pairs, they were insufficient to adequately reduce ammonium ion uptake.
  • the present invention can provide a strong foundation for improving the nitrogen utilization efficiency of various commercially cultivated rice cultivars.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Botany (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Environmental Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Biochemistry (AREA)
  • Physiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Developmental Biology & Embryology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Provided is a genetically modified plant in which the function of OsHHO3 gene or a homolog gene thereof is defected or suppressed and of which the growth is accelerated under a nitrogen-deficient environment.

Description

遺伝子改変植物、植物の作出方法、及び成長促進剤GENETICALLY MODIFIED PLANT, METHOD FOR PRODUCING PLANT, AND GROWTH PROMOTER
　本発明は、遺伝子改変植物、植物の作出方法、及び成長促進剤に関する。詳細には、本発明は、作物の窒素肥料の効率的利用と作物生産の向上に関する。 The present invention relates to genetically modified plants, methods for producing plants, and growth promoters. In particular, the present invention relates to efficient use of nitrogen fertilizers in crops and improved crop production.
　植物は、核酸、アミノ酸、クロロフィルなどの多数の分子の構成要素である窒素を、土壌から主に硝酸イオンまたはアンモニウムイオンとして獲得する。硝酸イオンは、ほとんどの陸上植物の主要な窒素源であり、アンモニウムイオンは、水田などの嫌気性条件で成長する植物の主要な窒素源である。
獲得した窒素のかなりの量が、光合成タンパク質の合成と光合成の維持に利用され、葉の窒素含有量と、植物の光合成速度及び収量との間に正の相関関係が生じている（例えば、非特許文献１参照。）。 Plants acquire nitrogen, a building block of many molecules such as nucleic acids, amino acids and chlorophyll, from soil mainly as nitrate or ammonium ions. Nitrate is the major nitrogen source for most terrestrial plants and ammonium ion is the major nitrogen source for plants growing in anaerobic conditions such as paddy fields.
A significant amount of the acquired nitrogen is utilized for the synthesis of photosynthetic proteins and the maintenance of photosynthesis, resulting in a positive correlation between leaf nitrogen content and plant photosynthetic rate and yield (e.g. See Patent Document 1.).
　そのため、世界の食糧需要を満たすべく高い収穫量を得るために、毎年大量の窒素肥料が畑に施用されている。 Therefore, large amounts of nitrogen fertilizer are applied to the fields every year in order to obtain high yields to meet the world's food demand.
　しかし、施用された窒素の５０％以上が畑から流出し、近隣の土地や水資源を汚染し、富栄養化やその他の環境汚染を引き起こし、水田からのアンモニアガス排出などの温室効果ガスの排出につながっている。
　したがって、特に窒素欠乏環境において、高い窒素利用効率（ＮＵＥ）を示す作物を開発する緊急の社会的必要性がある。 However, more than 50% of the applied nitrogen escapes from the fields, polluting neighboring land and water resources, causing eutrophication and other environmental pollution, and greenhouse gas emissions such as ammonia gas emissions from paddy fields. connected to
Therefore, there is an urgent social need to develop crops that exhibit high nitrogen utilization efficiency (NUE), especially in nitrogen-deficient environments.
　本発明は、上記事情を鑑みてなされたものであり、高い窒素利用効率を示す遺伝子改変植物、植物の作出方法、及び成長促進剤を提供する。 The present invention has been made in view of the above circumstances, and provides a genetically modified plant exhibiting high nitrogen utilization efficiency, a method for producing the plant, and a growth promoter.
　すなわち、本発明は、以下の態様を含む。
［１］ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損しており、又は抑制されており、窒素欠乏環境において、成長促進を示す、遺伝子改変植物。
［２］前記ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が、前記ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子に変異を有することによって、欠損している、［１］に記載の遺伝子改変植物。
［３］アンモニウムイオンの取り込みが促進された、［１］に記載の遺伝子改変植物。
［４］イネ科植物である［１］に記載の遺伝子改変植物。
［５］低窒素環境での生育が改善される植物の作出方法であって、前記植物内において、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能を欠損又は抑制させる、植物の作出方法。
［６］窒素欠乏環境における植物の成長促進剤であって、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子を破壊するゲノム編集組成物を含有する、成長促進剤。 That is, the present invention includes the following aspects.
[1] A genetically modified plant in which the function of the OsHHO3 gene or its homologous gene is deleted or suppressed, and which exhibits growth promotion in a nitrogen-deficient environment.
[2] The genetically modified plant according to [1], wherein the function of the OsHHO3 gene or its homologous gene is deficient due to mutation in the OsHHO3 gene or its homologous gene.
[3] The genetically modified plant of [1], in which ammonium ion uptake is promoted.
[4] The genetically modified plant of [1], which is a gramineous plant.
[5] A method for producing a plant having improved growth in a low-nitrogen environment, wherein the function of the OsHHO3 gene or its homologous gene is deleted or suppressed in the plant.
[6] A growth promoter for plants in a nitrogen-deficient environment, the growth promoter comprising a genome-editing composition that disrupts the OsHHO3 gene or its homologue gene.
　本発明によれば、高い窒素利用効率を示す遺伝子改変植物、植物の作出方法、及び成長促進剤を提供することができる。 According to the present invention, it is possible to provide a genetically modified plant exhibiting high nitrogen utilization efficiency, a method for producing a plant, and a growth promoter.
４つの独立したＯｓＨＨＯ３－ＯＸ系統における、ＯｓＨＨＯ３転写のレベルを示したグラフである。３週齢の土壌で成長したＷＴ（日本晴）植物と、４つの独立したＯｓＨＨＯ３－ＯＸ系統（＃１～＃４）のＴ２ホモ接合植物のシュートから単離されたＲＮＡを用いて、ＲＴ－ｑＰＣＲを行った。ＯｓＨＨＯ３の転写レベルは、最初にＯｓＵＢＱ５の転写レベルに対して規準化され、次にＷＴ実生で得られた値に対して規準化された。データは、３つの生物学的反復の平均±ＳＤを表し、各バーの上のアスタリスクは、各トランスジェニック系統とＷＴの間の有意差を表す（** p ＜０．０１;スチューデントｔ検定）。Graph showing levels of OsHHO3 transcription in four independent OsHHO3-OX lines. RT-qPCR using RNA isolated from shoots of 3-week-old soil-grown WT (Nipponbare) plants and T2 homozygous plants of four independent OsHHO3-OX lines (#1-#4) did Transcript levels of OsHHO3 were first normalized to those of OsUBQ5 and then to the values obtained in WT seedlings. Data represent the mean±SD of three biological replicates, asterisks above each bar represent significant differences between each transgenic line and WT (**p<0.01; Student's t-test) . ４つの独立したｏｓｈｈｏ３－ＫＯ系統における標的遺伝子座の配列分析結果である。これらｏｓｈｈｏ３－ＫＯ変異株（＃１～＃４）のＴ０植物の葉を使用して、配列分析を行った。遺伝子構造の概略図において、黒い四角形はコード領域を示し、白い四角形と五角形はそれぞれ５'－非翻訳領域と３'-非翻訳領域を示す。ＤＮＡ配列では、プロトスペーサー隣接モチーフ（ＰＡＭ）配列はα枠で示され、ＷＴでｇＲＮＡとして使用される配列は大文字で示される。予想される切断部位は黒い矢じりで示される。アデノシンの挿入と塩基の欠失は、それぞれβ枠と「－」で示される。ヌクレオチド配列とアミノ酸配列の省略は実線で示され、変異対立遺伝子に由来する産物のアミノ酸配列とアミノ酸数の変化はγ枠で示される。ゲノム編集によって引き起こされた終止コドンはγ枠中のアスタリスクで示される。両アレルでの編集は、４つの独立したｏｓｈｈｏ３－ＫＯ系統（＃１～＃４）すべてで発生した。Sequence analysis results of target loci in four independent osho3-KO lines. Sequence analysis was performed using T0 plant leaves of these oshho3-KO mutants (#1-#4). In schematic diagrams of gene structures, black squares indicate coding regions, white squares and pentagons indicate 5'- and 3'-untranslated regions, respectively. In the DNA sequence, the protospacer adjacent motif (PAM) sequences are shown in α frames and the sequences used as gRNAs in WT are shown in upper case. Predicted cleavage sites are indicated by black arrowheads. Adenosine insertions and base deletions are indicated by β frames and "-", respectively. Nucleotide and amino acid sequence abbreviations are shown in solid lines, amino acid sequences and amino acid number changes of products derived from mutant alleles are shown in gamma frames. Stop codons caused by genome editing are indicated by asterisks in the γ frame. Editing at both alleles occurred in all four independent oshho3-KO lines (#1-#4). 野生型 (ＷＴ)、ｏｓｈｈｏ３ノックアウト変異体 (ｏｓｈｈｏ３－ＫＯ)、および ＯｓＨＨＯ３ 過剰発現 (ＯｓＨＨＯ３－ＯＸ) の実生の写真である。実生は、栽培の最終段階で２１日間、唯一の窒素源として１．４２ｍＭ ＮＨ４ＮＯ３ (窒素十分なコントロール) または０．１４２ ｍＭ ＮＯ３ －（低ＮＯ３ －） またはＮＨ４ ＋ (低ＮＨ４ ＋) の存在下で生育した。スケールバー＝３ｃｍ。Photographs of wild type (WT), oshho3 knockout mutant (oshho3-KO), and OsHHO3 overexpressing (OsHHO3-OX) seedlings. Seedlings were treated with 1.42 mM NH 4 NO 3 (nitrogen-sufficient control) or 0.142 mM NO 3 − (low NO 3 − ) or NH 4 + (low NH 3 − ) as the sole nitrogen source for 21 days at the final stage of cultivation. 4 + ) were grown in the presence of Scale bar = 3 cm. （ａ）ＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸの実生（図３）のシュート乾燥重量のグラフである。（ｂ）ＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸの実生（図３）の根乾燥重量のグラフである。（ｃ）ＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸの実生（図３）の葉のＦｖ/Ｆｍ比のグラフである。（ｄ）ＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸの実生（図３）のシュートのクロロフィル含有量のグラフである。(a) Graph of shoot dry weight of WT, osho3-KO, and OsHHO3-OX seedlings (FIG. 3). (b) Graph of root dry weight of WT, osho3-KO, and OsHHO3-OX seedlings (FIG. 3). (c) Graph of Fv/Fm ratios in leaves of WT, osho3-KO, and OsHHO3-OX seedlings (FIG. 3). (d) Graph of chlorophyll content in shoots of WT, osho3-KO, and OsHHO3-OX seedlings (FIG. 3). 窒素欠乏土壌で生育した１２０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの写真である。スケールバー＝２０ ｃｍ。Photographs of 120-day-old WT, osho3-KO, and OsHHO3-OX rice plants grown in nitrogen-deficient soil. Scale bar = 20 cm. （ａ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの草丈のグラフである。（ｂ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの分げつ数のグラフである。（ｃ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの花穂の長さのグラフである。（ｄ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの穂あたりの小穂数のグラフである。（ｅ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの稔性のグラフである。（ｆ）土壌で生育した１５０日齢のＷＴ、ｏｓｈｈｏ３－ＫＯ、およびＯｓＨＨＯ３－ＯＸイネの植物あたりの穀物収量のグラフである。図３～図５では、植物は窒素十分な通常の土壌（コントロール）または窒素欠乏土壌（窒素欠乏）で栽培された。各バーは、５つの生物学的複製 （図４）または４つの生物学的複製（図６）の平均 ± 標準偏差（ＳＤ） を表す。ＷＴ、野生型日本晴； ＫＯ－♯１、ｏｓｈｈｏ３－ＫＯ ライン１; ＯＸ－♯１とＯＸ－♯２、それぞれＯｓＨＨＯ３－ＯＸライン１と２を示す。各バーの上のアスタリスクは、ＷＴと比較した有意差を表す (＊ｐ＜ ０．０５、＊＊ｐ＜ ０．０１; スチューデントの t 検定)。(a) Plant height graph of 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. (b) Graph of tiller number of 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. (c) Graph of spike length of 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. (d) Graph of number of spikelets per ear of 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. (e) Fertility graph of 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. (f) Grain yield per plant for 150-day-old WT, osho3-KO, and OsHHO3-OX rice grown in soil. In Figures 3-5, plants were grown in nitrogen-sufficient normal soil (control) or nitrogen-deficient soil (nitrogen-deficient). Each bar represents the mean±standard deviation (SD) of 5 biological replicates (FIG. 4) or 4 biological replicates (FIG. 6). WT, wild-type Nipponbare; KO-#1, osho3-KO line 1; OX-#1 and OX-#2, indicating OsHHO3- OX lines 1 and 2, respectively. Asterisks above each bar represent significant differences compared to WT ( * p<0.05, ** p<0.01;Student's t-test). 各遺伝子型の１５０ 日齢の植物から収集された充填および非充填の小穂の写真である。図６～図７では、植物は窒素十分な通常の土壌（コントロール）または窒素欠乏土壌（窒素欠乏）で栽培された。各バーは、５つの生物学的複製 （ｂ～ｅ）または４つの生物学的複製（ｇ～ｌ） の平均 ± 標準偏差（ＳＤ） を表す。ＷＴ、野生型日本晴； ＫＯ－♯１、ｏｓｈｈｏ３－ＫＯ ライン１; ＯＸ－♯１とＯＸ－♯２、それぞれＯｓＨＨＯ３－ＯＸライン１と２を示す。各バーの上のアスタリスクは、ＷＴと比較した有意差を表す (＊ｐ＜ ０．０５、＊＊ｐ＜ ０．０１; スチューデントの t 検定)。Photographs of filled and unfilled spikelets collected from 150 day old plants of each genotype. In Figures 6-7, plants were grown in nitrogen-sufficient normal soil (control) or nitrogen-deficient soil (nitrogen-deficient). Each bar represents the mean±standard deviation (SD) of 5 biological replicates (b-e) or 4 biological replicates (g-l). WT, wild-type Nipponbare; KO-#1, osho3-KO line 1; OX-#1 and OX-#2, indicating OsHHO3- OX lines 1 and 2, respectively. Asterisks above each bar represent significant differences compared to WT ( * p<0.05, ** p<0.01;Student's t-test). 散布図として表示される、ＷＴおよびｏｓｈｈｏ３－ＫＯ－♯１実生のトランスクリプトーム解析の結果である。ＯｓＨＨＯ３は、３ つのＡＭＴ１遺伝子を直接抑制し、アンモニウムイオンの取り込みを減少させる。散布図は、ｏｓｈｈｏ３－ＫＯ植物のシュートと根で上方制御された遺伝子と下方制御された遺伝子を示す。ドットは、根のみ（７２５個）、シュートのみ(９５個)、およびシュートと根の両方 (３９個) で上方制御された遺伝子 (ｌｏｇ２ＦＣ＞１、ｐ＜０．０５)；根（３５個）およびシュートのみ（７９個）下方制御された遺伝子（ｌｏｇ２ＦＣ＜－１、ｐ＜０．０５）；シュートでは上方制御されているが、根では下方制御されている遺伝子（３個）；根またはシュートのいずれでも上方制御も下方制御もされていない遺伝子を示す。Results of transcriptome analysis of WT and osho3-KO-#1 seedlings displayed as scatter plots. OsHHO3 directly represses the three AMT1 genes and reduces ammonium ion uptake. Scatter plots show up- and down-regulated genes in shoots and roots of osho3-KO plants. Dots indicate genes upregulated in roots only (725), shoots only (95), and both shoots and roots (39) (log 2 FC>1, p<0.05); genes) and shoots only (79) downregulated (log 2 FC<−1, p<0.05); genes upregulated in shoots but downregulated in roots (3) ; indicates genes that are neither upregulated nor downregulated in either roots or shoots. ヒートマップは、シュート（Ｓ）および根（Ｒ）におけるＡＭＴ、ＮＲＴ、およびその他の窒素同化関連遺伝子の発現レベルのｏｓｈｈｏ３ノックアウト変異による変化を表す。Heatmaps represent changes in expression levels of AMT, NRT, and other nitrogen assimilation-related genes in shoots (S) and roots (R) by osho3 knockout mutations. 窒素飢餓 (窒素フリー) または窒素飢餓無し (コントロール)で処理したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１実生の根におけるＡＭＴ１；１、ＡＭＴ１；２、およびＡＭＴ１；３のＲＴ－ｑＰＣＲ分析結果である。各値は、窒素飢餓の無いＷＴ実生の値と比較している。RT of AMT1;1, AMT1;2, and AMT1;3 in roots of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings treated with nitrogen starvation (nitrogen-free) or no nitrogen starvation (control) - qPCR analysis results. Each value is compared to that of WT seedlings without nitrogen starvation. （ａ）窒素十分な制御条件下で成長したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１実生のアンモニウムイオン取り込み活性を示すグラフである。（ｂ）窒素十分な制御条件下で成長したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１実生のアンモニウムイオン含有量を示すグラフである。(a) Ammonium ion uptake activity of WT, osho3-KO-#1 and OsHHO3-OX-#1 seedlings grown under nitrogen-sufficient control conditions. (b) Ammonium ion content of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings grown under nitrogen-sufficient control conditions. ＯｓＨＨＯ３－ＯＸ－♯１実生を使用して行われたＣｈＩＰアッセイの結果である。ＣｈＩＰ－ｑＰＣＲによって分析されたＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、および ＯｓＡＭＴ１;３プロモーター領域は、αの水平線を使用して示される。δの矢印は、転写開始部位（ＴＳＳ）を示す。図１３でプロモーターとして使用される領域は、εの水平線で示され、ＧＡＴＴＣおよびＧＡＡＴＣ配列モチーフは、それぞれ紫と青の垂直線で示される。ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、および ＯｓＡＭＴ１；３プロモーターに導入されたヌクレオチド変異 (Ｍ１およびＭ２)も示される。ＯｓＵＢＱ５遺伝子はネガティブコントロールとして機能した。Results of a ChIP assay performed using OsHHO3-OX-#1 seedlings. The OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoter regions analyzed by ChIP-qPCR are indicated using α horizontal lines. The delta arrow indicates the transcription start site (TSS). The region used as the promoter in FIG. 13 is indicated by a horizontal line in ε, and the GATTC and GAATC sequence motifs are indicated by purple and blue vertical lines, respectively. Nucleotide mutations (M1 and M2) introduced into the OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters are also indicated. The OsUBQ5 gene served as a negative control. コトランスフェクションアッセイの結果である。レポータープラスミドには、ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、またはＯｓＡＭＴ１；３プロモーターのＷＴまたは変異バージョンの制御下にホタルルシフェラーゼ遺伝子（ＬＵＣ）が含まれる。 ＯｓＨＨＯ３発現ベクターまたは空ベクターコントロールを、レポータープラスミドおよび内部コントロールプラスミドと共に同時トランスフェクトした。空のベクターコントロールで得られたＬＵＣ活性を１に設定した。Co-transfection assay results. Reporter plasmids contain the firefly luciferase gene (LUC) under the control of the WT or mutated versions of the OsAMT1;1, OsAMT1;2, or OsAMT1;3 promoters. OsHHO3 expression vector or empty vector control was co-transfected with reporter plasmid and internal control plasmid. The LUC activity obtained with the empty vector control was set to 1. ＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１プロトプラストにおけるＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、および ＯｓＡＭＴ１；３プロモーターの活性を示すグラフである。３５Ｓプロモーターはコントロールとして機能した。ＷＴプロトプラストのＬＵＣ活性を１に設定した。1 is a graph showing the activity of OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters in WT, osho3-KO-#1, and OsHHO3-OX-#1 protoplasts. The 35S promoter served as a control. The LUC activity of WT protoplasts was set to 1. （ａ）吉田養液で生育したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１実生の硝酸イオン取り込み活性を示すグラフである。（ａ）～（ｃ）において、Ｎ.Ｄ. は硝酸イオンの取り込みに有意な増加がないことを示す。データは、４つの生物学的複製の平均±ＳＤす。各バーの上のアスタリスクは、ＷＴと比較して有意差を表す (＊ｐ＜０．０５、＊＊ｐ＜０．０１; スチューデントの t 検定)。（ｂ）吉田養液で生育したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、およびＯｓＨＨＯ３－ＯＸ－♯１実生の硝酸イオン含有量を示すグラフである。（ｃ）窒素欠乏処理による硝酸イオン取り込み活性のｏｓｈｈｏ３ ＫＯ変異誘発性増加の増強を示す結果である。ＯｓＨＨＯ３－ＯＸ－♯１実生は、最初に吉田養液で生育させた後、吉田養液（コントロール）または窒素を含まない吉田養液条件 (窒素フリー) で５日間生育させた。(a) Graph showing the nitrate uptake activity of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings grown in Yoshida nutrient solution. In (a)-(c) ND indicates no significant increase in nitrate uptake. Data are means±SD of four biological replicates. Asterisks above each bar represent significant differences compared to WT ( * p<0.05, ** p<0.01;Student's t-test). (b) Graph showing nitrate ion content of WT, osho3-KO-#1, and OsHHO3-OX-#1 seedlings grown in Yoshida nutrient solution. (c) Results showing enhancement of osho3 KO mutagenic increase in nitrate uptake activity by nitrogen depletion treatment. OsHHO3-OX-#1 seedlings were first grown in Yoshida's nutrient solution and then grown in Yoshida's nutrient solution (control) or in nitrogen-free Yoshida's nutrient solution conditions (nitrogen-free) for 5 days. （ａ）窒素飢餓処理によるＯｓＨＨＯ３発現量の減少を示すグラフである。１/２吉田養液で４日間培養したＷＴ実生は、最初に吉田養液で１４日間生育させ、その後、窒素を含まない吉田養液でさらに指定された日数生育させた。（ｂ）１/２吉田養液で４日間培養したＷＴ実生は、最初に吉田養液で１４日間生育させ、その後、唯一の窒素源として２．８ｍＭ ＫＮＯ３を、添加窒素を含まない吉田養液でさらに指定された日数生育させた。（ａ）～（ｂ）において、ＲＴ－ｑＰＣＲは、シュートと根から分離されたＲＮＡを使用して実行された。ＯｓＨＨＯ３の転写レベルは、最初にＯｓＵＢＱ５の転写レベルに対して正規化され、次に０日時点で得られた対応する値に対して正規化された。データは、４ つの生物学的複製の平均±ＳＤを表す。(a) A graph showing the decrease in OsHHO3 expression level due to nitrogen starvation treatment. WT seedlings cultured for 4 days in 1/2 Yoshida broth were first grown in Yoshida broth for 14 days and then in nitrogen-free Yoshida broth for the indicated number of days. (b) WT seedlings cultured for 4 days in 1/2 Yoshida nutrient solution were first grown in Yoshida nutrient solution for 14 days, then 2.8 mM KNO3 as the sole nitrogen source and added nitrogen-free Yoshida nutrient solution. They were further grown in liquid for the indicated number of days. In (a)-(b) RT-qPCR was performed using RNA isolated from shoots and roots. Transcript levels of OsHHO3 were first normalized to those of OsUBQ5 and then to the corresponding values obtained at the day 0 time point. Data represent the mean±SD of four biological replicates. ＯｓＨＨＯ３は、サイトカイニン生合成を調節することを示すグラフである。窒素充分条件下で生育したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、並びにＯｓＨＨＯ３－ＯＸ－♯１および－♯２実生の根におけるＯｓＩＰＴ４（Ｏｓ０３ｇ０８１０１００）、ＯｓＩＰＴ５（Ｏｓ０７ｇ０２１１７００）、およびＯｓＣＹＰ７３５Ａ３ （Ｏｓ０８ｇ０４２９８００）の発現レベルを示すグラフである。各遺伝子の発現レベルは、ＷＴ実生で得られた値に対して正規化された。アスタリスクは、ＷＴと他の遺伝子型の間の有意差を示す (＊ｐ＜０．０５、＊＊ｐ＜０．０１； スチューデントのt検定)。OsHHO3 regulates cytokinin biosynthesis. Expression levels of OsIPT4 (Os03g0810100), OsIPT5 (Os07g0211700), and OsCYP735A3 (Os08g0429800) in the roots of WT, osho3-KO-#1, and OsHHO3-OX-#1 and -#2 seedlings grown under nitrogen-sufficient conditions were determined. It is a graph showing. Expression levels of each gene were normalized to the values obtained in WT seedlings. Asterisks indicate significant differences between WT and other genotypes ( * p<0.05, ** p<0.01;Student's t-test). ＯｓＨＨＯ３によるＯｓＩＰＴ４およびＯｓＩＰＴ５プロモーターを抑制することを示すグラフである。ＯｓＩＰＴ４またはＯｓＩＰＴ５プロモーターの制御下にあるＬＵＣレポーター遺伝子を含むレポータープラスミドを、ＯｓＨＨＯ３発現ベクターまたは空のコントロールベクターと共に、イネＷＴプロトプラストに同時トランスフェクトした。３５Ｓプロモーターは、ネガティブコントロールとして機能した。アスタリスクは、空のベクターコントロールとＯｓＨＨＯ３発現ベクターの間の有意差を示す (＊ｐ＜０．０５、＊＊ｐ＜０．０１； スチューデントのt検定)。FIG. 4 is a graph showing repression of OsIPT4 and OsIPT5 promoters by OsHHO3. Reporter plasmids containing the LUC reporter gene under the control of the OsIPT4 or OsIPT5 promoter were co-transfected into rice WT protoplasts along with the OsHHO3 expression vector or an empty control vector. The 35S promoter served as a negative control. Asterisks indicate significant differences between empty vector controls and OsHHO3-expressing vectors ( * p<0.05, ** p<0.01;Student's t-test). ＯｓＨＨＯ３－ＯＸ－♯１実生を使用して行われたＣｈＩＰアッセイの結果である。ＣｈＩＰアッセイで増幅された領域と（ｂ）でプロモーターとして使用された領域は、それぞれαとεの水平線で示され、ＧＡＴＴＣとＧＡＡＴＣモチーフはそれぞれ二点破線と一点破線の垂直線で示される。δ矢印はＴＳＳを示す。ＯｓＵＢＱ５遺伝子はネガティブコントロールとして機能した。アスタリスクは、ＯｓＵＢＱ５コントロールサンプルと他のサンプルの間の有意差を示す (＊ｐ＜０．０５、＊＊ｐ＜０．０１； スチューデントのt検定)。Results of a ChIP assay performed using OsHHO3-OX-#1 seedlings. The regions amplified in the ChIP assay and used as promoters in (b) are indicated by α and ε horizontal lines, respectively, and the GATTC and GAATC motifs are indicated by double-dash and single-dot vertical lines, respectively. δ arrow indicates TSS. The OsUBQ5 gene served as a negative control. Asterisks indicate significant differences between OsUBQ5 control samples and other samples ( * p<0.05, ** p<0.01;Student's t-test). 窒素十分な制御 (１．４２ｍＭ ＮＨ４ＮＯ３)および低窒素(０．０２８ｍＭ ＮＨ４ＮＯ３) 条件下で成長したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、並びにＯｓＨＨＯ３－ＯＸ－♯１および－♯２実生のシュートと根のサイトカイニン含有量を示すグラフである。データは、４つの生物学的複製の平均±ＳＤを表す。アスタリスクは、ＷＴと他の遺伝子型の間の有意差を示す (＊ｐ＜０．０５、＊＊ｐ＜０．０１； スチューデントのt検定)。WT, osho3-KO-#1 , and OsHHO3-OX-#1 and -#2 grown under nitrogen-sufficient (1.42 mM NH 4 NO 3 ) and low-nitrogen (0.028 mM NH 4 NO 3 ) conditions Fig. 3 is a graph showing the cytokinin content of seedling shoots and roots. Data represent the mean±SD of four biological replicates. Asterisks indicate significant differences between WT and other genotypes ( * p<0.05, ** p<0.01;Student's t-test). ＷＴ、ｏｓｈｈｏ３－ＫＯ、及びＯｓＨＨＯ３－ＯＸ実生におけるサイトカイニン関連遺伝子の発現レベルを示すグラフである。ＯｓＩＰＴ７の発現レベル (Ｏｓ０５ｇ０５５１７００)、ＯｓＡＨＫ３（Ｏｓ０１ｇ０９２３７００）、および ＯｓＰＲ６（Ｏｓ１２ｇ０４３７８００）は、窒素充分な条件下で成長したＷＴ、ｏｓｈｈｏ３－ＫＯ－♯１、並びにＯｓＨＨＯ３－ＯＸ－♯１および－♯２の実生の根で分析された。遺伝子発現レベルは、最初にＯｓＵＢＱ５の転写レベルに対して、次にＷＴ実生で得られた値に対して正規化された。データは、４つの生物学的複製の平均±ＳＤ を表す。アスタリスクは、ＷＴと他の遺伝子型の間の有意差を示す (＊ｐ＜０．０５、＊＊ｐ＜０．０１；スチューデントのt検定)。Graph showing expression levels of cytokinin-related genes in WT, osho3-KO, and OsHHO3-OX seedlings. The expression levels of OsIPT7 (Os05g0551700), OsAHK3 (Os01g0923700), and OsPR6 (Os12g0437800) were determined in WT, osho3-KO-#1, and OsHHO3-OX-#1 and -#2 seedlings grown under nitrogen-sufficient conditions. was analyzed in the roots of Gene expression levels were normalized first to the transcript levels of OsUBQ5 and then to the values obtained in WT seedlings. Data represent the mean ± SD of four biological replicates. Asterisks indicate significant differences between WT and other genotypes ( * p<0.05, ** p<0.01;Student's t-test).
≪遺伝子改変植物≫
　一実施形態において、本発明は、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損しており、又は抑制されており、窒素欠乏環境において、成長促進を示す、遺伝子改変植物を提供する。 ≪Genetically modified plant≫
In one embodiment, the present invention provides genetically modified plants in which the function of the OsHHO3 gene or its homologous gene is deleted or repressed and which exhibit enhanced growth in nitrogen-deficient environments.
　ＯｓＨＨＯ３タンパク質は、ＮＩＧＴ１／ＨＨＯファミリーの転写因子である。ＯｓＨＨＯ３タンパク質の全長アミノ酸配列を、配列番号１に示す。 The OsHHO3 protein is a transcription factor of the NIGT1/HHO family. The full-length amino acid sequence of OsHHO3 protein is shown in SEQ ID NO:1.
　頻繁に遭遇する窒素欠乏を克服するために、植物は根の構造を調節し、硝酸イオンおよびアンモニウムイオンの取り込みを強化する。窒素欠乏反応を制御する分子メカニズムはまだよく理解されていない。しかし、硝酸非誘導性ＨＨＯ遺伝子と共にＮＩＧＴ１／ＨＨＯ遺伝子ファミリーを形成するシロイヌナズナ硝酸誘導性ＮＩＧＴ１遺伝子（ＡｔＮＩＧＴ１ｓ）は、硝酸イオン応答の下方制御と窒素欠乏応答の誘導に関与することが示されている。 To overcome the frequently encountered nitrogen deficiency, plants modulate root structure and enhance nitrate and ammonium ion uptake. The molecular mechanisms controlling the nitrogen starvation reaction are still poorly understood. However, the Arabidopsis thaliana nitrate-inducible NIGT1 genes (AtNIGT1s), which together with the nitrate-inducible HHO genes form the NIGT1/HHO gene family, have been shown to be involved in the downregulation of nitrate responses and the induction of nitrogen starvation responses.
　ＡｔＮＩＧＴ１は、ＮＲＴ２（高親和性硝酸イオントランスポーター遺伝子）および窒素欠乏誘導遺伝子を直接抑制するＧＡＲＰ/Ｇ２様転写因子（ＴＦｓ）をコードする。窒素欠乏条件下で、ＡｔＮＩＧＴ１転写レベルが徐々に低下すると、窒素欠乏誘導性遺伝子が活性化される。 AtNIGT1 encodes GARP/G2-like transcription factors (TFs) that directly repress NRT2 (high-affinity nitrate transporter gene) and nitrogen starvation-induced genes. Under nitrogen starvation conditions, the gradual decline in AtNIGT1 transcription levels activates nitrogen starvation-inducible genes.
　作物の窒素利用効率の向上は、作物生産の向上と減肥農業の両方の鍵を握る。水田栽培では、アンモニウムイオンがイネの主たる窒素源となる。しかし、低窒素環境でアンモニウムイオンの吸収を促進し、低窒素環境での生育を改善する遺伝子は知られていない。また、低窒素環境での生育が改善されたイネの作出方法も知られていない。 Improving the nitrogen utilization efficiency of crops is the key to both improving crop production and reducing fertilizer. In paddy cultivation, ammonium ions are the main nitrogen source for rice. However, no gene is known that promotes ammonium ion uptake in a low-nitrogen environment and improves growth in a low-nitrogen environment. Moreover, a method for producing rice plants with improved growth in a low-nitrogen environment is not known.
　実施例で後述するように、本願発明において、ＮＩＧＴ１／ＨＨＯファミリーのメンバーであるＯｓＨＨＯ３が、９５％を超えるアンモニウムイオン取り込み活性に関与する３つのＯｓＡＭＴ１（高親和性アンモニウム輸送体遺伝子）の発現を調節する転写リプレッサーであることを明らかにした。更に、ＣＲＩＳＰＲ／Ｃａｓ９を介したＯｓＨＨＯ３の不活性化は、窒素欠乏条件下でアンモニウムイオンの取り込みと植物の成長を促進し、窒素利用効率を改善した。 As described later in the Examples, in the present invention, OsHHO3, a member of the NIGT1/HHO family, regulates the expression of three OsAMT1 (high-affinity ammonium transporter genes) responsible for over 95% of the ammonium ion uptake activity. was found to be a transcriptional repressor. Furthermore, CRISPR/Cas9-mediated inactivation of OsHHO3 enhanced ammonium ion uptake and plant growth under nitrogen-deficient conditions, improving nitrogen utilization efficiency.
　本実施形態の遺伝子改変植物において、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損している又は抑制されている。 In the genetically modified plant of this embodiment, the function of the OsHHO3 gene or its homologous gene is deleted or suppressed.
　ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損しているとは、本実施形態の遺伝子改変植物において、ＯｓＨＨＯ３遺伝子産物又はそのホモログ遺伝子産物の発現が喪失していることをいう。
　ＯｓＨＨＯ３遺伝子産物又はそのホモログ遺伝子産物の発現の喪失は、例えばＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子に変異を導入し、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子を破壊することにより生じさせることができる。
　変異は、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子、或いは、これら遺伝子の発現調節領域における一部又は全部の欠失、置換、任意の配列の挿入等により生じさせることができる。これらの変異の導入は、例えば、変異原性物質による処理、紫外線照射、相同組み換え技術等による遺伝子ターゲッティング、遺伝子ノックアウト、Ｃｒｅ-ｌｏｘＰ系等による条件的ノックアウト等の手法を用いて行うことができる。
　遺伝子ターゲティング、遺伝子ノックアウトについては、ゲノム編集技術を用いてもよい。 Defective function of the OsHHO3 gene or its homologous gene means loss of expression of the OsHHO3 gene product or its homologous gene product in the genetically modified plant of the present embodiment.
Loss of expression of the OsHHO3 gene product or its homologous gene product can be caused, for example, by introducing a mutation into the OsHHO3 gene or its homologous gene to disrupt the OsHHO3 gene or its homologous gene.
Mutations can be caused by partial or whole deletion, substitution, insertion of arbitrary sequences, etc. in the OsHHO3 gene or its homologous gene, or the expression control region of these genes. These mutations can be introduced using methods such as treatment with mutagen, ultraviolet irradiation, gene targeting by homologous recombination techniques, gene knockout, conditional knockout by Cre-loxP system and the like.
Genome editing technology may be used for gene targeting and gene knockout.
　ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が抑制されているとは、本実施形態の遺伝子改変植物において、コントロールとなる野生型の植物と比較して、ＯｓＨＨＯ３遺伝子産物又はそのホモログ遺伝子産物の量が抑制されていることをいう。
　ＯｓＨＨＯ３遺伝子産物又はそのホモログ遺伝子の発現の抑制は、ＯｓＨＨＯ３遺伝子産物又はそのホモログ遺伝子に対するＲＮＡｉ誘導性核酸、アンチセンス核酸、アプタマー若しくはリボザイムなどの発現を生じさせる核酸配列を、細胞又は動物に導入し、遺伝子ノックダウン等により生じさせることができる。 The suppression of the function of the OsHHO3 gene or its homologous gene means that the amount of the OsHHO3 gene product or its homologous gene product is suppressed in the genetically modified plant of the present embodiment compared to the wild-type control plant. It means that
Suppression of expression of the OsHHO3 gene product or its homologous gene includes introducing into a cell or animal a nucleic acid sequence that causes the expression of an RNAi-inducing nucleic acid, an antisense nucleic acid, an aptamer or a ribozyme against the OsHHO3 gene product or its homologous gene, It can be generated by gene knockdown or the like.
　本実施形態の遺伝子改変植物としては、イネ科植物、マメ科植物等が挙げられる。イネ科植物としては、イネ、コムギ、オオムギ、ライムギ、トウモロコシ、アワ、サトウキビなどが挙げられる。マメ科植物としては、ダイズ、アズキ、エンドウ、インゲンマメなどが挙げられる。 Examples of genetically modified plants of this embodiment include gramineous plants and leguminous plants. The gramineous plants include rice, wheat, barley, rye, corn, foxtail millet, sugar cane, and the like. Examples of leguminous plants include soybean, adzuki bean, pea, and kidney bean.
　ＯｓＨＨＯ３遺伝子のホモログ遺伝子としては、例えば、以下のタンパク質をコードする遺伝子が挙げられる。
　イネ科植物としては、配列番号２で表されるＴｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ（パンコムギ）のホモログタンパク質（ＯｓＨＨＯ３タンパク質との相同性６０．１７％）、配列番号３で表されるＨｏｒｄｅｕｍ ｖｕｌｇａｅ（オオムギ）のホモログタンパク質（ＯｓＨＨＯ３タンパク質との相同性６２．５０％）、配列番号４で表されるＺｅａ ｍａｙｓ（トウモロコシ）のホモログタンパク質（ＯｓＨＨＯ３タンパク質との相同性５６．６２％）等が挙げられる。
　マメ科植物としては、配列番号５で表されるＧｌｙｃｉｎｅ Ｍａｘ（ダイズ）のホモログタンパク質（ＯｓＨＨＯ３タンパク質との相同性３４．７２％）が挙げられる。 Homologous genes of the OsHHO3 gene include, for example, genes encoding the following proteins.
Examples of gramineous plants include the homologous protein of Triticum aestivum (bread wheat) represented by SEQ ID NO: 2 (60.17% homology with OsHHO3 protein), the homologous protein of Hordeum vulgae (barley) represented by SEQ ID NO: 3 ( 62.50% homology with OsHHO3 protein), Zea mays (corn) homologous protein represented by SEQ ID NO: 4 (56.62% homology with OsHHO3 protein), and the like.
Examples of leguminous plants include the Glycine Max (soybean) homolog protein represented by SEQ ID NO: 5 (34.72% homology with OsHHO3 protein).
　即ち、本実施形態の遺伝子改変植物は、以下の（ａ）～（ｃ）のいずれかの遺伝子の機能が欠損している又は抑制されていることが好ましい。
　（ａ）配列番号１で表されるアミノ酸配列からなるタンパク質をコードする遺伝子
　（ｂ）前記（ａ）で表されるアミノ酸配列において、１～数個のアミノ酸が欠失、挿入、置換若しくは付加されたアミノ酸配列からなり、且つ転写抑制活性を有するタンパク質をコードする遺伝子
　（ｃ）前記（ａ）で表されるアミノ酸配列と３０％以上の同一性を有するアミノ酸配列からなり、且つ転写抑制活性を有するタンパク質をコードする遺伝子 That is, in the genetically modified plant of the present embodiment, it is preferable that any one of the following gene functions (a) to (c) is deficient or suppressed.
(a) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 (b) one to several amino acids are deleted, inserted, substituted or added in the amino acid sequence represented by (a) (c) consisting of an amino acid sequence having 30% or more identity with the amino acid sequence represented in (a) above and having transcription repressing activity Genes that encode proteins
　（ｂ）において、欠失、挿入、置換若しくは付加されたアミノ酸の数としては、１～２００個であり、１～１００個が好ましく、１～５０個がより好ましく、１～３０個がより好ましく、１～２０個がより好ましく、１～１０個が更に好ましく、１～５個が最も好ましい。 In (b), the number of amino acids deleted, inserted, substituted or added is 1 to 200, preferably 1 to 100, more preferably 1 to 50, more preferably 1 to 30. , more preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.
　（ｃ）において、同一性としては、５０％以上が好ましく、５５％以上がより好ましく、８５％以上が特に好ましく、９０％以上が更に好ましく、９５％以上が最も好ましい。 In (c), the identity is preferably 50% or more, more preferably 55% or more, particularly preferably 85% or more, still more preferably 90% or more, and most preferably 95% or more.
　本発明において、「転写抑制活性」とは、高親和性アンモニウム輸送体遺伝子等のアンモニウムイオン取り込み活性に関与する遺伝子の転写を抑制する活性を意味する。
　高親和性アンモニウム輸送体遺伝子としては、ＡＭＴ１～３遺伝子が挙げられ、イネにおいては、ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、ＯｓＡＭＴ１；３が挙げられる。
　また、抑制対象の遺伝子としては、ＮＲＴ、その他の窒素同化関連遺伝子；ＯｓＩＰＴ４、ＯｓＩＰＴ５、ＯｓＣＹＰ７３５Ａ３、その他のサイトカイニン関連遺伝子も挙げられる。 In the present invention, the term "transcriptional repressing activity" means an activity that represses transcription of genes involved in ammonium ion uptake activity, such as high-affinity ammonium transporter genes.
High-affinity ammonium transporter genes include AMT1-3 genes, and in rice, OsAMT1;1, OsAMT1;2, and OsAMT1;3.
Genes to be suppressed include NRT, other nitrogen assimilation-related genes; OsIPT4, OsIPT5, OsCYP735A3, and other cytokinin-related genes.
　本実施形態の遺伝子改変植物において、窒素欠乏環境において、成長促進を示すとは、実施例にて後述するように、窒素欠乏環境において、シュート乾燥重量、根乾燥重量、葉のＦｖ/Ｆｍ比、クロロフィル含有量、草丈、分げつ数、花穂の長さ、穂あたりの小穂数、及び植物あたりの穀物収量から選ばれる少なくとも一要素について野生型植物と比較して優れていることを意味する。
　本実施形態の遺伝子改変植物は、アンモニウムイオンの取り込みを促進し、窒素利用効率を改善することにより、窒素欠乏環境においても、成長促進を示す。 In the genetically modified plant of the present embodiment, showing growth promotion in a nitrogen-deficient environment means that in a nitrogen-deficient environment, shoot dry weight, root dry weight, leaf Fv / Fm ratio, It means that at least one element selected from chlorophyll content, plant height, number of tillers, length of spike, number of spikelets per spike, and grain yield per plant is superior to wild-type plants. .
The genetically modified plant of this embodiment exhibits enhanced growth even in a nitrogen-deficient environment by promoting ammonium ion uptake and improving nitrogen utilization efficiency.
≪植物の作出方法≫
　一実施形態において、本発明は、低窒素環境での生育が改善される植物の作出方法であって、前記植物内において、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能を欠損又は抑制させる、植物の作出方法を提供する。 ≪Method of producing plants≫
In one embodiment, the present invention is a method for producing a plant that improves growth in a low-nitrogen environment, wherein the function of the OsHHO3 gene or its homologue gene is deleted or suppressed in the plant. I will provide a.
　ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子は、≪遺伝子改変植物≫において説明したものと同様である。 The OsHHO3 gene or its homologous gene is the same as explained in <<genetically modified plant>>.
　遺伝子の機能を欠損又は抑制させる方法としては、例えば、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子のゲノムに人為的に改変又は変異を加えることにより、機能的な遺伝子産物の生成が欠損又は抑制された植物を得る方法が挙げられる。
　例えば、突然変異誘発剤の処理により、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損又は抑制された植物をスクリーニングする方法が挙げられる。
　また例えば、遺伝子ターゲッティング法や、ゲノム編集技術、トランスジェニック等によって、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子のゲノムに人為的に改変又は変異を加えることにより、機能的な遺伝子産物の生成が欠損又は抑制された植物を得てもよい。
 または、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の発現を欠損又は抑制させる核酸を植物に導入することによって、遺伝子産物の生成が欠損又は抑制された植物を得てもよい。 As a method for deficient or suppressed gene function, for example, by artificially modifying or mutating the genome of the OsHHO3 gene or its homologous gene, a plant in which the production of a functional gene product is deficient or suppressed is obtained. method.
For example, there is a method of screening for plants in which the function of the OsHHO3 gene or its homologous gene is deleted or suppressed by treatment with a mutagen.
Also, for example, by artificially modifying or mutating the genome of the OsHHO3 gene or its homologous gene by gene targeting method, genome editing technology, transgenic, etc., the production of functional gene products is deleted or suppressed. You can get plants.
Alternatively, a plant in which production of the gene product is deficient or suppressed may be obtained by introducing into the plant a nucleic acid that deficient or suppresses the expression of the OsHHO3 gene or its homologous gene.
 本実施形態の植物の作出方法は、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能を、そのプロモーターを改変することによって、欠損又は抑制する方法でもよい。 The method of producing a plant of this embodiment may be a method of deleting or suppressing the function of the OsHHO3 gene or its homologous gene by modifying its promoter.
　ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子及び/又はそのプロモーターを改変する方法としては、効率の観点からゲノム編集技術を用いることが好ましい。
　改変する方法としては、標的ゲノムＤＮＡの二重鎖切断後の非相同末端結合（ＮＨＥＪ）による挿入欠損（ＩｎＤｅｌ）変異を生じさせる方法が挙げられる。ＩｎＤｅｌ変異により、フレームシフトや未成熟終始コドンが誘発される。
　また、標的ゲノムＤＮＡの二重鎖切断後、相同組換え型修復により、変異を導入した外来ＤＮＡを標的ゲノム中に挿入する方法も挙げられる。 As a method for modifying the OsHHO3 gene or its homologous gene and/or its promoter, it is preferable to use genome editing technology from the viewpoint of efficiency.
Modification methods include generating insertion-deficient (InDel) mutations by non-homologous end joining (NHEJ) after double-strand breaks in the target genomic DNA. The InDel mutation induces a frameshift and a premature termination codon.
Another example is a method of inserting mutated foreign DNA into the target genome by homologous recombination repair after double-strand cleavage of the target genomic DNA.
　標的ゲノムＤＮＡの二重鎖切断に用いるシステムとしては、特に限定されず、ＣＲＩＳＰＲ－Ｃａｓシステム、ＴＡＬＥＮシステム、Ｚｎフィンガーヌクレアーゼシステム等が挙げられる。これらのシステムの細胞への導入方法としては、特に限定されず、標的ゲノムＤＮＡ切断酵素自体を細胞に導入してもよく、標的ゲノムＤＮＡ切断酵素発現ベクターを細胞に導入してもよい。標的ゲノムＤＮＡの二重鎖切断に用いるシステムは、外来ＤＮＡと同時又は外来ＤＮＡの前若しくは後に細胞に導入される。 The system used for double-strand cleavage of target genomic DNA is not particularly limited, and includes the CRISPR-Cas system, TALEN system, Zn finger nuclease system, and the like. The method for introducing these systems into cells is not particularly limited, and the target genomic DNA-cleaving enzyme itself may be introduced into cells, or a target genomic DNA-cleaving enzyme expression vector may be introduced into cells. Systems used for double-strand cleavage of target genomic DNA are introduced into cells simultaneously with, before, or after the exogenous DNA.
　例えば、ＣＲＩＳＰＲ－Ｃａｓシステムにおいては、Ｃａｓ９発現ベクターと、切断したい箇所にＣａｓ９を誘導するガイドＲＮＡをコードする発現ベクターと、を細胞に導入する方法や、発現精製した組み換えＣａｓ９タンパク質と、ガイドＲＮＡと、を細胞に導入する方法等が挙げられる。
　 本実施形態において、標的ゲノムＤＮＡの二重鎖切断に用いるシステムは、ＣＲＩＳＰＲ－Ｃａｓシステムが好ましい。 For example, in the CRISPR-Cas system, a Cas9 expression vector and an expression vector encoding a guide RNA that induces Cas9 to a site to be cleaved, a method of introducing into a cell, and a recombinant Cas9 protein that is expressed and purified, and a guide RNA , into cells, and the like.
In this embodiment, the system used for double-strand cleavage of target genomic DNA is preferably the CRISPR-Cas system.
　本実施形態において、ゲノム編集は、ｉｎ　ｖｉｔｒｏ培養系で行われても、ｉｎ　ｐｌａｎｔａで行われてもよい。ｉｎ　ｖｉｔｒｏ培養系でのゲノム編集方法においては、外来遺伝子、核酸及びタンパク質の細胞への導入は、カルス又は組織片に対して、アグロバクテリウム法、パーティクルガン法及びウィスカー法等の公知の方法を用いて行われる。ｉｎ　ｐｌａｎｔａにおけるゲノム編集方法においては、外来遺伝子、核酸及びタンパク質の細胞への導入は、露出させた未熟胚や完熟胚のシュート頂に対して、公知の方法を用いて行われる。 In this embodiment, genome editing may be performed in an in vitro culture system or in planta. In the genome editing method in an in vitro culture system, foreign genes, nucleic acids and proteins are introduced into cells by known methods such as the Agrobacterium method, the particle gun method and the whisker method for callus or tissue pieces. performed using In the method of genome editing in planta, introduction of foreign genes, nucleic acids and proteins into cells is carried out using known methods to exposed shoot tops of immature and mature embryos.
　本実施形態において作出される植物は、植物体のみならず、細胞から植物体を作出した場合に、低窒素環境での生育が改善されるものであれば、その由来となる細胞を包含する意味で用いている。細胞は、組織又は器官の状態であってもよい。本実施形態において作出される植物とは、それらの後代若しくはクローンの生物又は細胞であってもよい。例えば、前記クローンの植物体としては挿し木または挿し芽等により得られた植物体が挙げられる。 The plant produced in the present embodiment includes not only the plant body, but also the cell from which the plant body is derived, as long as the plant body is produced from the cell to improve the growth in a low-nitrogen environment. used in A cell may be in a tissue or organ state. Plants produced in this embodiment may be their progeny or clone organisms or cells. For example, the plant body of the clone includes a plant body obtained from cuttings or cuttings.
　本実施形態の植物の作出方法で作出される植物は、イネ科植物、マメ科植物等が挙げられ、イネ科植物が好ましく、イネがより好ましい。 Plants produced by the method for producing a plant of the present embodiment include gramineous plants, leguminous plants and the like, with gramineous plants being preferred, and rice being more preferred.
≪成長促進剤≫
　一実施形態において、本発明は、窒素欠乏環境における植物の成長促進剤であって、ＯｓＨＨＯ３遺伝子又はそのホモログを破壊するゲノム編集組成物を含有する、成長促進剤を提供する。 ≪Growth Promoter≫
In one embodiment, the present invention provides a growth promoter for plants in nitrogen-deficient environments, the growth promoter comprising a genome editing composition that disrupts the OsHHO3 gene or its homologues.
　ゲノム編集組成物は、標的ゲノムＤＮＡ切断酵素又は前記酵素をコードするＤＮＡ若しくはｍＲＮＡを含む。標的ゲノムＤＮＡ切断酵素としては、Ｃａｓ９、ＴＡＬＥＮ、Ｚｎフィンガーヌクレアーゼ等が挙げられる。また、前記酵素をコードするＤＮＡ又はｍＲＮＡとしては、これらのタンパク質をコードするＤＮＡ又はｍＲＮＡが挙げられる。 A genome-editing composition contains a target genomic DNA-cleaving enzyme or DNA or mRNA encoding the enzyme. Target genomic DNA cleaving enzymes include Cas9, TALENs, Zn finger nucleases and the like. In addition, the DNA or mRNA encoding the enzyme includes DNA or mRNA encoding these proteins.
　標的ゲノムＤＮＡ切断酵素として、Ｃａｓ９を用いる場合には、ゲノム編集組成物は、Ｃａｓ９を誘導するガイドＲＮＡを含有することが好ましい。また、組成物は、ガイドＲＮＡをコードする発現ベクターを含有してもよい。ガイドＲＮＡとしては、例えば、配列番号６で表されるプロトスペーサーを有するものが挙げられる。 When Cas9 is used as the target genomic DNA cleaving enzyme, the genome editing composition preferably contains a guide RNA that induces Cas9. The composition may also contain an expression vector encoding the guide RNA. Examples of guide RNA include those having a protospacer represented by SEQ ID NO:6.
　相同組換により、ＯｓＨＨＯ３遺伝子又はそのホモログを破壊する場合には、ゲノム編集組成物は、相同性アームを両端に備えた所定の変異を有する外来ＤＮＡを含むことが好ましい。 When the OsHHO3 gene or its homolog is disrupted by homologous recombination, the genome editing composition preferably contains foreign DNA having a predetermined mutation with homologous arms at both ends.
　本実施形態の成長促進剤によれば、ＯｓＨＨＯ３遺伝子又はそのホモログが破壊されることにより、アンモニウムイオン取り込み活性に関与する３つのＯｓＡＭＴ１の発現抑制が解除されるため、、窒素欠乏条件下でアンモニウムイオンの取り込みを促進し、植物の成長を促進する。 According to the growth promoter of the present embodiment, disruption of the OsHHO3 gene or its homolog releases the suppression of expression of the three OsAMT1 involved in the ammonium ion uptake activity. promotes uptake of and promotes plant growth.
　以下に実施例を挙げて本発明を更に詳述するが、本発明はこれらの実施例に限定されるものではない。 Although the present invention will be described in more detail with examples below, the present invention is not limited to these examples.
　イネの窒素欠乏応答におけるＯｓＨＨＯ３の役割を調べるために、ジャポニカ米品種日本晴を用いて、４つの独立したＯｓＨＨＯ３過剰発現（ＯｓＨＨＯ３－ＯＸ）系統（図１参照。）とＯｓＨＨＯ３遺伝子編集ノックアウト変異体（ｏｓｈｈｏ３－ＫＯ）系統（図２参照。）を作製した。 To investigate the role of OsHHO3 in the nitrogen starvation response of rice, four independent OsHHO3 overexpressing (OsHHO3-OX) lines (see Fig. 1) and an OsHHO3 gene-editing knockout mutant (oshho3 -KO) lines (see FIG. 2) were produced.
　ＯｓＨＨＯ３－ＯＸ系統の生成のためのバイナリーベクターを構築するために、ｐＣＢ－ＨＹＧ－ＺｍＵｂｉ－ＯｓＮＩＧＴ１－ＭＹＣ バイナリーベクターのＯｓＮＩＧＴ１ ｃＤＮＡを、ＢａｍＨＩおよびＳｔｕＩ制限酵素を使用して、ＯｓＨＨＯ３のコード領域全体に対応するｃＤＮＡに置き換えた。
ｏｓｈｈｏ３－ＫＯ系統を生成するためのバイナリーベクターを構築するために、ＯｓＨＨＯ３遺伝子座内の標的配列に対応する２つのオリゴヌクレオチド(配列番号７及び８)をアニーリングすることによって得られた二本鎖ＤＮＡを、ｐＵ６ｇＲＮＡに挿入した後、そこからＵ６プロモーター、標的配列、ｓｇＲＮＡ ｓｃａｆｆｏｌｄ、ｐｏｌｙ Ｔを含むＤＮＡ断片をＣＲＩＳＰＲ/Ｃａｓ９ベクターｐＺＨ＿ＯｓＵ６ｇＲＮＡ＿ＰｕｂｉＭＭＣａｓ９にクローニングした。 To construct a binary vector for the generation of OsHHO3-OX strains, the OsNIGT1 cDNA in the pCB-HYG-ZmUbi-OsNIGT1-MYC binary vector was ligated to the entire coding region of OsHHO3 using BamHI and StuI restriction enzymes. was replaced with a cDNA that
Double-stranded DNA obtained by annealing two oligonucleotides (SEQ ID NOs: 7 and 8) corresponding to target sequences within the OsHHO3 locus to construct a binary vector for generating oshho3-KO strains. was inserted into pU6gRNA, from which a DNA fragment containing the U6 promoter, target sequence, sgRNA scaffold, poly T was cloned into the CRISPR/Cas9 vector pZH_OsU6gRNA_PubiMMCas9.
　得られたバイナリープラスミドを、アグロバクテリウム・ツメファシエンス株ＥＨＡ１０５に導入し、これを日本晴(ＷＴ)のアグロバクテリウム媒介形質転換に使用した。形質転換されたカルスは、ハイグロマイシンＢを含む培地で選択され、再生され、４つの独立したＯｓＨＨＯ３－ＯＸおよびｏｓｈｈｏ３－ＫＯイネ系統がそれぞれ確立された。４つの独立したｏｓｈｈｏ３－ＫＯイネ系統におけるＯｓＨＨＯ３のｇｅｎｏｍｉｃＤＮＡの塩基配列を配列表に示す。ｏｓｈｈｏ３－ＫＯ♯１の変異ＯｓＨＨＯ３の塩基配列を配列番号９（♯１ ａｌｌｅｌｅ１）、及び配列番号１０（♯１ ａｌｌｅｌｅ２）に示す。ｏｓｈｈｏ３－ＫＯ♯２の変異ＯｓＨＨＯ３の塩基配列を配列番号１１（♯２ ａｌｌｅｌｅ１）、及び配列番号１２（♯２ ａｌｌｅｌｅ２）に示す。ｏｓｈｈｏ３－ＫＯ♯３の変異ＯｓＨＨＯ３の塩基配列を配列番号１３（♯３ ａｌｌｅｌｅ１、 ａｌｌｅｌｅ２）に示す。ｏｓｈｈｏ３－ＫＯ♯４の変異ＯｓＨＨＯ３の塩基配列を配列番号１４（♯４ ａｌｌｅｌｅ１）、及び配列番号１５（♯４ ａｌｌｅｌｅ２）に示す。ＯｓＨＨＯ３野生型のｇｅｎｏｍｉｃＤＮＡの塩基配列を配列表１６に示す。 The resulting binary plasmid was introduced into Agrobacterium tumefaciens strain EHA105, which was used for Agrobacterium-mediated transformation of Nipponbare (WT). Transformed callus was selected on medium containing hygromycin B and regenerated to establish four independent OsHHO3-OX and oshho3-KO rice lines, respectively. The nucleotide sequences of OsHHO3 genomic DNAs in four independent osho3-KO rice lines are shown in the sequence listing. The nucleotide sequences of oshho3-KO#1 mutant OsHHO3 are shown in SEQ ID NO: 9 (#1 allele 1) and SEQ ID NO: 10 (#1 allele 2). The nucleotide sequences of oshho3-KO#2 mutant OsHHO3 are shown in SEQ ID NO: 11 (#2 allele 1) and SEQ ID NO: 12 (#2 allele 2). The nucleotide sequence of oshho3-KO#3 mutant OsHHO3 is shown in SEQ ID NO: 13 (#3 allele1, allele2). The nucleotide sequences of oshho3-KO#4 mutant OsHHO3 are shown in SEQ ID NO: 14 (#4 allele 1) and SEQ ID NO: 15 (#4 allele 2). The nucleotide sequence of OsHHO3 wild-type genomic DNA is shown in Sequence Listing 16.
　Ｔ２子孫におけるハイグロマイシン耐性マーカー遺伝子の分離は、導入遺伝子が４つのＯｓＨＨＯ３－ＯＸ系統全てにおいて単一の遺伝子座に導入されたことを示した。導入された遺伝子についてホモ接合であるＴ１植物のＴ２種子を、更なる実験のために使用した。４つの独立したｏｓｈｈｏ３－ＫＯ系統全てのＴ０植物のＤＮＡ配列分析により、標的遺伝子座に両対立遺伝子変異が存在することが明らかになった（図２参照。）。 Segregation of the hygromycin resistance marker gene in T2 progeny indicated that the transgene was introduced at a single locus in all four OsHHO3-OX lines. T2 seeds of T1 plants homozygous for the introduced gene were used for further experiments. DNA sequence analysis of T0 plants of all four independent osho3-KO lines revealed the presence of biallelic mutations at the target locus (see Figure 2).
　ＯｓＨＨＯ３のノックアウト変異とＯｓＨＨＯ３の過剰発現が、植物の成長に及ぼす影響を調べるために、野生型（ＷＴ）、ＯｓＨＨＯ３－ＯＸ、及びｏｓｈｈｏ３－ＫＯの実生を、さまざまな窒素栄養条件下で２１日間成長させた。ＯｓＨＨＯ３－ＯＸとｏｓｈｈｏ３－ＫＯの実生は、それぞれ成長障害と成長促進を示し、それぞれシュートと根の乾燥重量の減少と増加を伴った（図３、図４（ａ）～（ｂ）参照。）。
　さらに、ＷＴと比較して、ｏｓｈｈｏ３－ＫＯの葉の光化学系ＩＩの最大量子収率（Ｆｖ/Ｆｍ）とクロロフィル含有量は、窒素欠乏条件下（低アンモニウムイオンおよび低硝酸イオン条件下）で高かったのに対し、ＯｓＨＨＯ３－ＯＸの葉のそれらは低かった（図４（ｃ）、（ｄ）参照。）。 To investigate the effect of knockout mutation of OsHHO3 and overexpression of OsHHO3 on plant growth, seedlings of wild-type (WT), OsHHO3-OX, and oshho3-KO were grown for 21 days under different nitrogen nutrient conditions. let me Seedlings of OsHHO3-OX and oshho3-KO exhibited stunting and accelerated growth, respectively, with decreased and increased shoot and root dry weights, respectively (see Figures 3 and 4(a)-(b)). .
Furthermore, compared to WT, the maximum quantum yield (Fv/Fm) and chlorophyll content of photosystem II in osho3-KO leaves were higher under nitrogen-deficient conditions (low ammonium and nitrate conditions). In contrast, those in OsHHO3-OX leaves were low (see FIGS. 4(c) and (d)).
　ＯｓＨＨＯ３ノックアウト変異と過剰発現における、正と負の効果は、窒素欠乏土壌で育てられた１２０日齢の植物で、顕著であり（図５参照。）、１５０日齢のｏｓｈｈｏ３－ＫＯ植物とＯｓＨＨＯ３－ＯＸ植物は、ＷＴと比較して、それぞれ優れた農業特性と劣った農業特性を示した（図６～図７参照。）。 重要なことに、ｏｓｈｈｏ３－ＫＯおよびＯｓＨＨＯ３－ＯＸ植物におけるＯｓＨＨＯ３発現レベルの変化は、分げつ数に影響を与えたが、稔性には影響を与えなかったため、ＷＴと比較して、それぞれ植物当たり、ｏｓｈｈｏ３－ＫＯ植物は、高い収量を示し、ＯｓＨＨＯ３－ＯＸ植物は低い収量を示した（図６（ｂ）～（ｆ）、図７参照。）。 The positive and negative effects of OsHHO3 knockout mutation and overexpression were pronounced in 120-day-old plants grown in nitrogen-deficient soil (see FIG. 5), and 150-day-old oshho3-KO plants and OsHHO3- OX plants exhibited superior and inferior agronomic performance, respectively, compared to WT (see Figures 6-7). Importantly, changes in OsHHO3 expression levels in oshho3-KO and OsHHO3-OX plants affected tiller number, but not fertility, thus compared to WT, each plant On the other hand, osho3-KO plants showed high yields and OsHHO3-OX plants showed low yields (Fig. 6(b)-(f), see Fig. 7).
　窒素欠乏環境でのｏｓｈｈｏ３－ＫＯ系統の成長の促進と収量の向上を考慮して遺伝子発現プロファイルの変化を調べることにより、ｏｓｈｈｏ３－ＫＯ植物の優れた表現型の分子基盤を調べた。ＯｓＨＨＯ３のノックアウト変異は、シュートよりも根ではるかに多くの遺伝子の発現に影響を及ぼし、そのほとんどは上方制御されていた（図８参照、表１～表１０参照。）。ヒートマップ分析は、ｏｓｈｈｏ３－ＫＯ実生の根におけるＡＭＴおよびＮＲＴ遺伝子のセットと、他の窒素同化関連遺伝子の上方制御を示した（図９参照。）。
　これらの遺伝子の中で、高親和性ＡＭＴをコードするＯｓＡＭＴ１は、最も顕著な上方制御を示した。４つの未知の遺伝子と１つのＨＳＰ（Ｏｓ０３ｇ０２６６９００）を除くすべての上方制御された遺伝子の中で、ＯｓＡＭＴ１；１が最も上方制御された遺伝子であり、ＯｓＡＭＴ１；３が最も上方制御された遺伝子トップ１０の１つだった（表１参照。）。ＯｓＡＭＴ１；２も明らかに根で上方制御されていた（図８、図９参照。）。 The molecular basis of the superior oshho3-KO plant phenotype was investigated by examining changes in gene expression profiles given enhanced growth and improved yield of osho3-KO lines in a nitrogen-deficient environment. The OsHHO3 knockout mutation affected the expression of far more genes in roots than in shoots, most of which were upregulated (see Figure 8, see Tables 1-10). Heatmap analysis showed upregulation of a set of AMT and NRT genes and other nitrogen assimilation-related genes in the roots of osho3-KO seedlings (see Figure 9).
Among these genes, OsAMT1, which encodes a high-affinity AMT, showed the most pronounced upregulation. Among all the upregulated genes except 4 unknown genes and 1 HSP (Os03g0266900), OsAMT1;1 is the most upregulated gene and OsAMT1;3 is the top 10 most upregulated genes. (see Table 1). OsAMT1;2 was also clearly upregulated in roots (see FIGS. 8 and 9).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
 　
Figure JPOXMLDOC01-appb-T000004
 　
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　アンモニウムイオン取り込み活性の９５％以上は、イネのＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、およびＯｓＡＭＴ１；３に由来する。根におけるこれらＡＭＴ１の窒素欠乏誘発性発現を精査したところ、窒素欠乏誘発性発現が、ｏｓｈｈｏ３－ＫＯ実生で増強されるが、ＯｓＨＨＯ３－ＯＸ実生では軽減されることを見出し、ＯｓＨＨＯ３の発現レベルと３つのＡＭＴ１の発現レベルとの間に負の相関関係があることが示された（図１０参照。）。 More than 95% of the ammonium ion uptake activity is derived from rice OsAMT1;1, OsAMT1;2, and OsAMT1;3. Upon probing the nitrogen starvation-induced expression of these AMT1s in roots, we found that nitrogen starvation-induced expression was enhanced in oshho3-KO seedlings but attenuated in OsHHO3-OX seedlings. It was shown that there was a negative correlation between the expression level of AMT1 and the expression level of AMT1 (see FIG. 10).
　これらＡＭＴ１の同調抑制と一致して、アンモニウムイオンの取り込みと含有量は、特に実生が窒素欠乏ストレスに曝された場合、ＷＴ実生よりもｏｓｈｈｏ３－ＫＯ実生では高くなり、ＯｓＨＨＯ３－ＯＸ実生では低くなった（図１１参照。）。さらに、硝酸イオン取り込み活性は、窒素欠乏ストレスに曝された場合（図１５参照。）、ＷＴ実生よりもｏｓｈｈｏ３－ＫＯ実生でわずかに高かった。これは、ｏｓｈｈｏ３－ＫＯの根におけるいくつかのＮＲＴ遺伝子の弱い上方制御と一致している（図９参照。）。 Consistent with these synchronous suppression of AMT1, ammonium ion uptake and content were higher in osho3-KO seedlings and lower in OsHHO3-OX seedlings than in WT seedlings, especially when the seedlings were exposed to nitrogen deficiency stress. (See FIG. 11). In addition, nitrate uptake activity was slightly higher in osho3-KO seedlings than in WT seedlings when exposed to nitrogen depletion stress (see Figure 15). This is consistent with the weak upregulation of several NRT genes in oshho3-KO roots (see Figure 9).
　ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、およびＯｓＡＭＴ１；３のアンモニウムイオン欠乏誘導性発現とは対照的に、ＯｓＨＨＯ３転写産物は、窒素枯渇処理後も低レベルのままだったが、ＯｓＨＨＯ３の発現は窒素枯渇処理中に徐々に減少することが分かった（図１６参照。）。
　したがって、ＯｓＨＨＯ３はＡＭＴ１を直接抑制するため、窒素枯渇処理時のＯｓＨＨＯ３発現の低下がこれらＡＭＴ１の発現を誘導すると仮定した。この仮説を検証するために、クロマチン免疫沈降（ＣｈＩＰ）およびコトランスフェクションアッセイを実施した。 In contrast to the ammonium ion deprivation-induced expression of OsAMT1;1, OsAMT1;2, and OsAMT1;3, OsHHO3 transcripts remained at low levels after nitrogen depletion treatment, whereas OsHHO3 expression remained low after nitrogen depletion treatment. It was found that it gradually decreased during the period (see FIG. 16).
Therefore, since OsHHO3 directly represses AMT1, we hypothesized that reduction of OsHHO3 expression upon nitrogen depletion treatment induces the expression of these AMT1s. To test this hypothesis, chromatin immunoprecipitation (ChIP) and co-transfection assays were performed.
　ＣｈＩＰアッセイは、ＯｓＡＭＴ１；１の領域ａとｄ、ＯｓＡＭＴ１；２の領域ｂとｆ、およびＯｓＡＭＴ１；３の領域ｂとｅへのＯｓＨＨＯ３のインビボ結合を明らかにした（図１２参照。）。ＣｈＩＰアッセイの詳細は、以下の通りである。 A ChIP assay revealed in vivo binding of OsHHO3 to regions a and d of OsAMT1;1, regions b and f of OsAMT1;2, and regions b and e of OsAMT1;3 (see Figure 12). Details of the ChIP assay are as follows.
　各ＯｓＨＨＯ３－ＯＸ系統とＷＴの５つの実生を収穫し、１５ｍｌの架橋バッファー (０．４Ｍ スクロース、１０ｍＭ Ｔｒｉｓ－ＨＣｌ［ｐＨ８．０］、１ｍＭ フェニルメチルスルホニルフルオリド、１ｍＭ ＥＤＴＡ、および１％ ホルムアルデヒド) 中で、２０分間室温真空下でインキュベートした。次に、各サンプルに２ｍｌ の２Ｍ グリシンを添加して、架橋反応を停止させた。 Ｂｉｏｒｕｐｔｏｒ ＩＩ（Ｓｏｎｉｃｂｉｏ Ｃｏ．、神奈川、日本）を用いて、核の単離および溶解並びに核溶解物の超音波処理を行った。次いで、抗ＭＹＣポリクローナル抗体（クローン９Ｅ１０、Ｍｉｌｌｉｐｏｒｅ ＃０５－４１９）およびプロテインＧアガロースビーズ（Ｒｏｃｈｅ）による免疫沈降、回収ＤＮＡの精製、およびｑＰＣＲを行った。 Five seedlings of each OsHHO3-OX line and WT were harvested and added to 15 ml of cross-linking buffer (0.4 M sucrose, 10 mM Tris-HCl [pH 8.0], 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, and 1% formaldehyde). incubate for 20 minutes at room temperature under vacuum. 2 ml of 2M glycine was then added to each sample to stop the cross-linking reaction. Isolation and lysis of nuclei and sonication of nuclear lysates were performed using a Bioruptor II (Sonicbio Co., Kanagawa, Japan). Immunoprecipitation with anti-MYC polyclonal antibody (clone 9E10, Millipore #05-419) and protein G agarose beads (Roche), purification of recovered DNA, and qPCR were then performed.
　さらに、ＷＴ、並びに、変異型ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、およびＯｓＡＭＴ１；３プロモーターを使用したコトランスフェクションアッセイ（図１３参照。）により、ＯｓＨＨＯ３は、３つすべてのＷＴ ＡＭＴ１プロモーターの活性を抑制するが、３５Ｓプロモーター（コントロール）の活性は抑制しないことが明らかとなり（図１３参照。）、インビボでＯｓＨＨＯ３によって結合された領域内のＮＩＧＴ１結合モチーフ（５'－ＧＡＴＴＣ－３'およびその逆相補体５'－ＧＡＡＴＣ－３'）の変異（図１２参照。）は、ＯｓＨＨＯ３による抑制を弱めることが明らかとなった（図１３参照。）。コトランスフェクションアッセイの詳細は、以下の通りである。 Furthermore, co-transfection assays using the WT and mutant OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters (see FIG. 13) showed that OsHHO3 repressed the activity of all three WT AMT1 promoters. However, it was found not to repress the activity of the 35S promoter (control) (see Figure 13), indicating that the NIGT1 binding motifs (5'-GATTC-3' and its reverse complement) within the region bound by OsHHO3 in vivo 5′-GAATC-3′) mutation (see FIG. 12) was found to weaken the suppression by OsHHO3 (see FIG. 13). Details of the cotransfection assay are as follows.
　レポーター プラスミドを構築するために、ＯｓＡＭＴ１；１ (転写開始部位 [ＴＳＳ、＋１ｂｐ] に対して－２，０９０～ ＋８３ｂｐ)、ＯｓＡＭＴ１；２（－２，０９７～＋７０ｂｐ）、ＯｓＡＭＴ１；３（－１，９２２～＋９３ｂｐ）、ＯｓＩＰＴ４ (－１，８５９～＋１５８ｂｐ)、および ＯｓＩＰＴ５ (－１，８２３～＋１５５ｂｐ) を、ＷＴ 実生のゲノムＤＮＡからＰＣＲ増幅し、ｐＪＤ３０１ ｖｅｃｔｏｒにおいてＬＵＣレポーター遺伝子の上流にクローニングした。ＯｓＡＭＴ１プロモーターの変異は、Ｍｅｇａｐｒｉｍｅｒ ＰＣＲ法を使用して導入した。
　ＯｓＨＨＯ３発現ベクター (エフェクター プラスミド) を構築するために、ｐＨＢＴ－ＮＬＰ７－ＭＹＣ６のＮＬＰ７ ｃＤＮＡをＯｓＨＨＯ３のコード領域全体に対応するｃＤＮＡに置き換えた。
　イネのプロトプラストは、暗所で１２日間栽培された葉鞘と実生のシュートから分離し、１６時間光照射した。レポータープラスミド (２μｇ) とエフェクタープラスミド (４μｇ) を、内部コントロール プラスミド（ｐＺｍＵｂｉ－ＧＵＳ、１μｇ）と共に、ポリエチレングリコール（ＰＥＧ）を介したトランスフェクション法によって１×１０５個のイネプロトプラストに同時に導入した。トランスフェクトされたプロトプラストを、プロトプラスト培養液 (０．４Ｍ マンニトール、１５ｍＭ ＭｇＣｌ２、および４ｍＭ ＭＥＳ－ＫＯＨ［ｐＨ ５．８］) 中、暗所、室温で１６ 時間インキュベートした。次に、各細胞ライセートのＬＵＣおよびＧＵＳ活性レベルを、それぞれＬｕｃｉｆｅｒａｓｅ Ａｓｓａｙ Ｓｙｓｔｅｍ Ｋｉｔ（Ｐｒｏｍｅｇａ Ｋ.Ｋ.、東京、日本）および４－メチルウンベリフェリルβ－Ｄ－グルクロニド（蛍光基質）を使用して決定し、相対ＬＵＣ活性を計算した。 OsAMT1;1 (−2,090 to +83 bp relative to the transcription start site [TSS, +1 bp]), OsAMT1;2 (−2,097 to +70 bp), OsAMT1;3 (−1, 922 to +93 bp), OsIPT4 (-1,859 to +158 bp), and OsIPT5 (-1,823 to +155 bp) were PCR amplified from genomic DNA of WT seedlings and cloned upstream of the LUC reporter gene in the pJD301 vector. Mutations in the OsAMT1 promoter were introduced using the Megaprimer PCR method.
To construct the OsHHO3 expression vector (effector plasmid), the NLP7 cDNA of pHBT-NLP7-MYC6 was replaced with a cDNA corresponding to the entire coding region of OsHHO3.
Rice protoplasts were isolated from shoots of leaf sheaths and seedlings grown in the dark for 12 days and exposed to light for 16 hours. The reporter plasmid (2 μg) and the effector plasmid (4 μg) were co-transfected into 1×10 5 rice protoplasts together with an internal control plasmid (pZmUbi-GUS, 1 μg) by polyethylene glycol (PEG)-mediated transfection. Transfected protoplasts were incubated in protoplast culture medium (0.4 M mannitol, 15 mM MgCl 2 , and 4 mM MES-KOH [pH 5.8]) in the dark at room temperature for 16 hours. The LUC and GUS activity levels of each cell lysate were then measured using the Luciferase Assay System Kit (Promega K.K., Tokyo, Japan) and 4-methylumbelliferyl β-D-glucuronide (a fluorescent substrate), respectively. determined and the relative LUC activity calculated.
　これらの結果は、ＯｓＨＨＯ３が、ＮＩＧＴ１結合モチーフと類似または同一の配列に結合することにより、ＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、およびＯｓＡＭＴ１；３プロモーターを直接かつ同調的に抑制することを示す。したがって、ＯｓＨＨＯ３は、３つの重要なＡＭＴ１の協調的活性化の責任リプレッサーであるため、ＯｓＨＨＯ３の破壊は、窒素欠乏条件下でアンモニウムイオンの取り込みを促進すると結論付けた。 These results indicate that OsHHO3 directly and synchronously represses the OsAMT1;1, OsAMT1;2, and OsAMT1;3 promoters by binding to sequences similar or identical to the NIGT1-binding motif. We therefore conclude that disruption of OsHHO3 promotes ammonium ion uptake under nitrogen-deficient conditions, as OsHHO3 is the repressor responsible for the coordinated activation of three key AMT1s.
　結論と一致して、これらのＯｓＡＭＴ１プロモーターの活性は、ＷＴプロトプラストと比較してｏｓｈｈｏ３－ＫＯプロトプラストでは高かったが、ＯｓＨＨＯ３－ＯＸプロトプラストでは低く（図１４参照。）、ＯｓＨＨＯ３とＯｓＡＭＴ１との発現レベル間の逆相関関係をさらに支持している。 Consistent with our conclusion, the activity of these OsAMT1 promoters was higher in oshho3-KO protoplasts than in WT protoplasts, but lower in OsHHO3-OX protoplasts (see FIG. 14), indicating that the expression levels between OsHHO3 and OsAMT1 were lower. further support the inverse correlation of
　３つのＯｓＡＭＴ１のＯｓＨＨＯ３を介した抑制は、ｏｓｈｈｏ３－ＫＯおよびＯｓＨＨＯ３－ＯＸイネが、それぞれ改善および減少した窒素利用効率と成長を示した理由を説明できる。
　しかし、ＡｔＮＩＧＴ１と同様に、ＯｓＨＨＯ３も他の窒素欠乏関連プロセスに影響を与える可能性がある。シロイヌナズナでは、ＡｔＮＩＧＴ１がサイトカイニン生合成に関与するＡｔＩＰＴ３（アデニル酸イソペンテニルトランスフェラーゼ遺伝子）とＡｔＣＹＰ７３５Ａ２（Ｃｙｔ Ｐ４５０モノオキシゲナーゼ遺伝子）を抑制したため、シロイヌナズナのｎｉｇｔ１四重変異体では、ｔＺ型サイトカイニン含有量が増加した。
　ｏｓｈｈｏ３－ＫＯイネのトランスクリプトーム解析でも、ＡｔＩＰＴ３のイネホモログであるＯｓＩＰＴ４のＯｓＨＨＯ３を介した抑制が示唆されたが、ＯｓＨＨＯ３がＡｔＩＰＴ３の別のホモログであるＯｓＩＰＴ５とＯｓＣＹＰ７３５Ａを抑制するかどうかは不明であった。 OsHHO3-mediated suppression of the three OsAMT1s could explain why oshho3-KO and OsHHO3-OX rice plants exhibited improved and decreased nitrogen utilization efficiency and growth, respectively.
However, similar to AtNIGT1, OsHHO3 may also affect other nitrogen deficiency-related processes. In Arabidopsis thaliana, AtNIGT1 repressed AtIPT3 (adenylate isopentenyltransferase gene) and AtCYP735A2 (Cyt P450 monooxygenase gene) involved in cytokinin biosynthesis, resulting in increased tZ-type cytokinin content in the Arabidopsis nigt1 quadruple mutant. .
Transcriptome analysis of oshho3-KO rice also suggested OsHHO3-mediated repression of OsIPT4, the rice homolog of AtIPT3, but it was unclear whether OsHHO3 represses other homologs of AtIPT3, OsIPT5 and OsCYP735A. rice field.
　したがって、本発明者らは、ｏｓｈｈｏ３－ＫＯ、及びＯｓＨＨＯ３－ＯＸ実生におけるＯｓＩＰＴ４、ＯｓＩＰＴ５、ＯｓＣＹＰ７３５Ａ３、および他のいくつかのサイトカイニン関連遺伝子の発現レベルを調べた。結果は、ＯｓＨＨＯ３がこれらの遺伝子を抑制することを示した（図１７、図２１参照。）。さらに、コトランスフェクションアッセイにより、ＯｓＨＨＯ３によるＯｓＩＰＴ４およびＯｓＩＰＴ５プロモーターの抑制が明らかになった（図１８参照。）。ＣｈＩＰアッセイは、ＯｓＨＨＯ３が少なくともインビボでＯｓＩＰＴ５プロモーター領域に結合することも明らかにした（図１９参照。）。 Therefore, we examined the expression levels of OsIPT4, OsIPT5, OsCYP735A3, and several other cytokinin-related genes in oshho3-KO and OsHHO3-OX seedlings. The results showed that OsHHO3 repressed these genes (see Figures 17 and 21). Furthermore, co-transfection assays revealed repression of OsIPT4 and OsIPT5 promoters by OsHHO3 (see Figure 18). ChIP assays also revealed that OsHHO3 binds to the OsIPT5 promoter region, at least in vivo (see Figure 19).
　さらに、サイトカイニンレベルは、ＷＴと比較してｏｓｈｈｏ３－ＫＯ実生では高かったが、ＯｓＨＨＯ３－ＯＸ実生では低かった（図２０参照。）。活性サイトカイニンは、腋芽の成長（分げつ）に必要であり、穂を伴う活性分げつの数は、イネの穀粒収量を決定する重要な要因であるため、これらの結果は、ｏｓｈｈｏ３－ＫＯ植物の分げつ数と収量の増加および、ＯｓＨＨＯ３－ＯＸ植物の分げつ数と収量の減少と一致する（図６（ｂ）参照。）。したがって、サイトカイニンを介した調節は、ｏｓｈｈｏ３－ＫＯイネの農業的に優れた表現型にも寄与する可能性がある。 Furthermore, cytokinin levels were higher in osho3-KO seedlings compared to WT, but lower in OsHHO3-OX seedlings (see Figure 20). Since active cytokinins are required for axillary bud growth (tillers) and the number of active tillers with panicles is an important factor in determining grain yield in rice, these results suggest that oshho3-KO This is consistent with the increased tiller number and yield of the plants and the decreased tiller number and yield of the OsHHO3-OX plants (see Figure 6(b)). Therefore, cytokinin-mediated regulation may also contribute to the agronomically superior phenotype of osho3-KO rice.
　本発明では、ＯｓＨＨＯ３を、イネのアンモニウムイオン取り込みに決定的に寄与する３つのＡＭＴ１遺伝子の転写抑制因子として同定した。また、ＣＲＩＳＰＲ／Ｃａｓ９を介したＯｓＨＨＯ３の不活性化は、アンモニウムイオンの取り込みを促進し、低窒素環境での窒素利用効率、成長、および収量を改善することも示した。 In the present invention, OsHHO3 was identified as a transcriptional repressor of three AMT1 genes that critically contribute to ammonium ion uptake in rice. We also showed that CRISPR/Cas9-mediated inactivation of OsHHO promotes ammonium ion uptake, improving nitrogen utilization efficiency, growth, and yield in low-nitrogen environments.
　低窒素条件下での作物の成長の改善は、少量の窒素肥料を使用する持続可能な農業にとって非常に必要な特性であるため、ＣＲＩＳＰＲ／Ｃａｓ９を介したＯｓＨＨＯ３の標的変異誘発は、持続可能な農業を可能にするイネ栽培の開発に役立つ可能性がある。 CRISPR/Cas9-mediated targeted mutagenesis of OsHHO3 could be a sustainable It may help develop rice cultivation that enables agriculture.
　３つのＯｓＡＭＴ１の協調的な活性化は、アンモニウムイオンの獲得を効果的に促進するために重要であり、ＯｓＨＨＯ３の重要性を強調する。実際、根におけるＯｓＡＭＴ１；１、ＯｓＡＭＴ１；２、およびＯｓＡＭＴ１；３の明確な空間的局在パターンは、根におけるアンモニウムイオン獲得への明確な寄与を示唆し、３つのＯｓＡＭＴ１のそれぞれのノックアウト変異は、個別にまたはペアで、アンモニウムイオンの取り込みを適切に減少させるには不十分だったが、３つすべてのＯｓＡＭＴ１のトリプルノックアウト変異は、アンモニウムイオン取り込みを５％未満に減少させた。 The coordinated activation of the three OsAMT1s is important for effectively promoting the acquisition of ammonium ions, emphasizing the importance of OsHHO3. Indeed, the distinct spatial localization patterns of OsAMT1;1, OsAMT1;2, and OsAMT1;3 in roots suggest distinct contributions to ammonium ion acquisition in roots, and knockout mutations of each of the three OsAMT1 All three triple knockout mutations of OsAMT1 reduced ammonium ion uptake to less than 5%, although individually or in pairs, they were insufficient to adequately reduce ammonium ion uptake.
　本発明によれば、さまざまな商業的に栽培されたイネ品種の窒素利用効率を改善するための強力な基盤を提供することができる。 The present invention can provide a strong foundation for improving the nitrogen utilization efficiency of various commercially cultivated rice cultivars.

Claims (6)

  1. 　ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が欠損しており、又は抑制されており、窒素欠乏環境において、成長促進を示す、遺伝子改変植物。 A genetically modified plant in which the function of the OsHHO3 gene or its homologous gene is deleted or suppressed and which exhibits enhanced growth in a nitrogen-deficient environment.
  2. 　前記ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能が、前記ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子に変異を有することによって、欠損している、請求項１に記載の遺伝子改変植物。 The genetically modified plant according to claim 1, wherein the function of the OsHHO3 gene or its homologous gene is deficient due to mutation in the OsHHO3 gene or its homologous gene.
  3. 　アンモニウムイオンの取り込みが促進された、請求項１に記載の遺伝子改変植物。 The genetically modified plant according to claim 1, wherein uptake of ammonium ions is promoted.
  4. 　イネ科植物である請求項１に記載の遺伝子改変植物。 The genetically modified plant according to claim 1, which is a gramineous plant.
  5. 　低窒素環境での生育が改善される植物の作出方法であって、前記植物内において、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子の機能を欠損又は抑制させる、植物の作出方法。 A method for producing a plant with improved growth in a low-nitrogen environment, wherein the function of the OsHHO3 gene or its homologous gene is deleted or suppressed in the plant.
  6. 　窒素欠乏環境における植物の成長促進剤であって、ＯｓＨＨＯ３遺伝子又はそのホモログ遺伝子を破壊するゲノム編集組成物を含有する、成長促進剤。 A growth promoter for plants in a nitrogen-deficient environment, the growth promoter containing a genome-editing composition that disrupts the OsHHO3 gene or its homolog gene.
PCT/JP2022/033654 2021-09-11 2022-09-08 Genetically modified plant, method for producing plant, and growth stimulant WO2023038075A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023546978A JPWO2023038075A1 (en) 2021-09-11 2022-09-08

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163243110P 2021-09-11 2021-09-11
US63/243,110 2021-09-11

Publications (1)

Publication Number Publication Date
WO2023038075A1 true WO2023038075A1 (en) 2023-03-16

Family

ID=85506426

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/033654 WO2023038075A1 (en) 2021-09-11 2022-09-08 Genetically modified plant, method for producing plant, and growth stimulant

Country Status (2)

Country Link
JP (1) JPWO2023038075A1 (en)
WO (1) WO2023038075A1 (en)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI QIAN, ZHOU LUYAN, LI YUHONG, ZHANG DONGPING, GAO YONG: "Plant NIGT1/HRS1/HHO Transcription Factors: Key Regulators with Multiple Roles in Plant Growth, Development, and Stress Responses", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 22, no. 16, 1 January 2021 (2021-01-01), pages 8685, XP093044070, DOI: 10.3390/ijms22168685 *
LIU KEXIN, YOSHIAKI UEDA , NAMIE OHTSUKI , YASUHITO SAKURABA , SHUICHI YANAGISAWA: "Functional analysis of GARP-type transcription factors involved in nitrogen deficiency response in rice", JAPANESE SOCIETY OF SOIL AND FERTILIZERS LECTURE ABSTRACTS, 1 September 2021 (2021-09-01), pages 48 - 48, XP093044024, Retrieved from the Internet <URL:https://www.jstage.jst.go.jp/article/dohikouen/67/0/67_48_2/_pdf/-char/ja> [retrieved on 20230503], DOI: 10.20710/dohikouen.67.0_48_2 *
SAFI ALAEDDINE, MEDICI ANNA, SZPONARSKI WOJCIECH, MARTIN FLORENCE, CLÉMENT-VIDAL ANNE, MARSHALL-COLON AMY, RUFFEL SANDRINE, GAYMAR: "GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways", JOURNAL OF EXPERIMENTAL BOTANY, OXFORD UNIVERSITY PRESS, GB, vol. 72, no. 10, 4 May 2021 (2021-05-04), GB , pages 3881 - 3901, XP093044073, ISSN: 0022-0957, DOI: 10.1093/jxb/erab114 *
UEDA YOSHIAKI, OHTSUKI NAMIE, KADOTA KOJI, TEZUKA AYUMI, NAGANO ATSUSHI J., KADOWAKI TARO, KIM YONGHYUN, MIYAO MITSUE, YANAGISAWA : "Gene regulatory network and its constituent transcription factors that control nitrogen‐deficiency responses in rice", NEW PHYTOLOGIST, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 227, no. 5, 1 September 2020 (2020-09-01), GB , pages 1434 - 1452, XP093044071, ISSN: 0028-646X, DOI: 10.1111/nph.16627 *

Also Published As

Publication number Publication date
JPWO2023038075A1 (en) 2023-03-16

Similar Documents

Publication Publication Date Title
US11873499B2 (en) Methods of increasing nutrient use efficiency
US10988775B2 (en) Wheat plants resistant to powdery mildew
US20210198682A1 (en) Application of sdg40 gene or encoded protein thereof
KR20110020307A (en) Genetically modified plant capable of biosynthesizing capsinoid
CN109748960B (en) Gene for regulating and controlling anti-aluminum virus transcription factor STOP1 protein and application thereof
CN114717241B (en) Rice salt tolerance related gene OsMSRFP, encoding protein and application thereof
CN111087457A (en) Protein NGR5 for improving nitrogen utilization rate and crop yield, and coding gene and application thereof
WO2023038075A1 (en) Genetically modified plant, method for producing plant, and growth stimulant
KR20200070357A (en) Plant resistance
CN111154767A (en) Root length regulatory gene LOGL5, corresponding construct and application thereof
CN114573669A (en) Application of protein Ghd7 in regulation and control of low nitrogen resistance of plants
US20220259611A1 (en) Method for increasing yield in plants
CN104450739B (en) A kind of paddy rice source anti insect related gene OsHR1 and coded product thereof and application
CN114934063B (en) Application of rice DT1 protein in regulation of drought resistance of rice
US20230365985A1 (en) PROTEINS AND BIOLOGICAL MATERIALS RELATED TO RICE (Oryza sativa L.) YIELD, AND USE THEREOF IN RICE YIELD INCREASE
CN111635896B (en) Application of USB1 protein in regulation and control of plant salt tolerance
CN115044592B (en) Gene ZmADT2 for regulating and controlling maize plant type and resistance to tumor smut, and encoding protein and application thereof
KR102629157B1 (en) Recombinant vector for gene editing of Solanum lycopersicum using Potato Virus X vector and uses thereof
US20230392160A1 (en) Compositions and methods for increasing genome editing efficiency
WO2023008076A1 (en) Genetically modified potato with reduced glycoalkaloid and method for producing same
WO2021070549A1 (en) Method for genome editing in wheat and use thereof
WO2004092372A1 (en) Gene capable of imparting salt stress resistance
KR20240023384A (en) Application of Philosophyll DoObgC and its splicing variants in promoting hypocotyl elongation.
CN117683809A (en) Yield-related protein OsSOE, biological material and application thereof
US20050188435A1 (en) Method of increasing the GGT activity in plants, plants with increased GGT activity, and a method of producing such plants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22867396

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023546978

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE