WO2023203988A1 - Plant with improved deep-rootedness - Google Patents

Plant with improved deep-rootedness Download PDF

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WO2023203988A1
WO2023203988A1 PCT/JP2023/013277 JP2023013277W WO2023203988A1 WO 2023203988 A1 WO2023203988 A1 WO 2023203988A1 JP 2023013277 W JP2023013277 W JP 2023013277W WO 2023203988 A1 WO2023203988 A1 WO 2023203988A1
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amino acid
seq
qsor1
protein
acid sequence
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Japanese (ja)
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優作 宇賀
和彦 杉本
悠花 桂田
凌 黒田
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国立研究開発法人農業・食品産業技術総合研究機構
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Publication of WO2023203988A1 publication Critical patent/WO2023203988A1/en

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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/06Roots
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a plant with improved deep rooting ability that has a gene encoding a mutant qSOR1 protein.
  • Roots are the only organ for plants to acquire and absorb nutrients and water from the soil, so root system morphology is extremely important for crop growth, but the root system morphology suitable for crop production differs depending on the soil environment.
  • the property that roots grow deeply (deep roots) is advantageous for drought tolerance, nitrogen absorption, avoidance of heavy metals, etc., while the property that roots grow shallowly (shallow roots) is resistant to phosphorus deficiency and oxygen deficiency in flooded conditions. It is advantageous for avoidance, phytoremediation of heavy metals, etc. Therefore, improving root system morphology to suit the soil environment is important for developing crops that are resistant to environmental stress.
  • Cross breeding is commonly used as a method for developing crops.
  • cross breeding requires phenotypic selection, and when evaluating the morphology of the root system in the soil, it is usually necessary to dig up and investigate the roots, and root sampling requires a great deal of time and effort.
  • roots cannot be investigated until after the above-ground parts have been harvested, so there has been little progress in improving crop roots through cross-breeding.
  • Non-Patent Document 1 a plurality of gravitropism genes (Non-Patent Document 1) and hydrotropism genes (Non-Patent Document 2) have been identified. Furthermore, in monocotyledonous plants including rice and maize, many genes involved in roots have been identified through mutant analysis.
  • Non-Patent Document 7 It is known that homologous genes of the rice DRO1 gene and the rice qSOR1 gene exist widely in angiosperms and form a large gene group called the DRO1 family (Non-Patent Document 7). Furthermore, it is known that DRO1 family proteins have five domains that are highly conserved among plants (Non-patent Document 8), and research using Arabidopsis and rice revealed that the fifth domain (CCL domain) is known to be involved in the function of gravitropism (Non-patent Documents 6, 7, and 9).
  • qSOR1 gene Since the qSOR1 gene is widely present in both monocots and dicots, useful alleles of the qSOR1 gene are found not only in monocot crops such as wheat and maize, but also in the root systems of dicot crops such as soybean and rapeseed. It is thought that it can be widely used for improvements. However, the number of qSOR1 gene alleles useful as breeding material is limited, and at present, technology for improving deep rooting ability of plants has not been sufficiently developed.
  • the present inventors searched for lines with non-synonymous substitutions in the qSOR1 gene from rice mutant lines into which random mutations were introduced, and examined the root phenotypes of the obtained mutant lines.
  • deep rooting was found in the mutant line containing the gene encoding the mutant qSOR1 protein containing an amino acid substitution in the third domain (domain III), the function of which was unknown until now. It was found that it was improved compared to When these mutant lines were backcrossed to the original variety and the root morphology of the resulting lines was examined, it was proven that the improvement in deep rooting ability was due to the above amino acid substitution that occurred in the qSOR1 protein.
  • the present inventors have completed the present invention based on the above findings. That is, the present invention includes the following.
  • a deep-rooted protein that has a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein that contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • Plants with improved [2] The amino acid substitution is an amino acid selected from the group consisting of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 and leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2.
  • the plant according to [1] which is a substitution.
  • the amino acid substitution is a substitution of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 to serine, or a substitution of leucine to phenylalanine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2.
  • the mutant qSOR1 protein is (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16, (ii) An amino acid that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12 and contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. a protein consisting of a sequence and exhibiting an activity of improving deep rooting of plants, or (iii) Has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 12, and corresponds to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • a base sequence that encodes a protein that contains and exhibits an activity of improving deep rooting of plants or (vi) Has an insertion, deletion, substitution, and/or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1 or 11, and is in the 140th to 145th sequence of the amino acid sequence shown in SEQ ID NO: 2.
  • a nucleotide sequence that contains a nucleotide mutation that causes an amino acid substitution and that encodes a protein that exhibits an activity to improve deep rooting ability of plants The plant according to any one of [1] to [4], comprising: [6] The plant according to any one of [1] to [5], which is a monocot or a dicot.
  • a method for producing plants with improved deep rooting [8] Plant a vector containing a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • a method for producing plants with improved deep rooting including the step of introducing the plant into a plant.
  • [9] A step of cross-breeding plants using the plants according to any one of [1] to [6] as breeding parents to obtain progeny plants, and a step in which the gene encoding the mutant qSOR1 protein is introduced.
  • a method for selecting plants with improved deep rooting ability comprising the step of identifying a plant having a gene encoding a mutant qSOR1 protein containing an amino acid substitution in the sequence.
  • Figure 1A shows the qSOR1 proteins of monocotyledonous plants (rice qSOR1 protein, maize qSOR1 (ZmqSOR1) protein, sorghum qSOR1 (SbqSOR1) protein, wheat qSOR1 (TaqSOR1) protein, Minato chinensis qSOR1 (BdqSOR1) protein) and dicot.
  • FIG. 3 is a diagram showing the results of comparison. Five domains (domains I to V) that are highly conserved among multiple plant species are each indicated by a square. This is a continuation of FIG. 1A.
  • FIG. 2 is a diagram showing the amino acid sequence of Koshihikari qSOR1 protein.
  • FIG. 3 is a diagram showing the locations of mutations in the qSOR1 protein amino acid sequences of four rice qSOR1 mutant lines from the original variety.
  • FIG. 4 is a diagram showing the mutation locations of the qSOR1 gene CDS of four rice qSOR1 mutant lines from the original variety.
  • FIG. 5 is a diagram showing the results of comparing the amino acid sequences of rice qSOR1 protein, Arabidopsis LZY2 protein, Arabidopsis LZY3 protein, and Medicago NGR protein. Five domains (domains I to V) that are highly conserved among multiple plant species are each indicated by a square.
  • FIG. 1 Five domains (domains I to V) that are highly conserved among multiple plant species are each indicated by a square.
  • FIG. 6 is a diagram showing the root phenotypes of the original variety (Koshihikari) and the rice qSOR1 mutant line evaluated by the cup method.
  • A is a representative photograph showing the roots of each line grown by the cup method. Scale bar indicates 1 cm. ⁇ rga indicates the root elongation angle.
  • B is a graph showing the root elongation angle of each line grown by the cup method. Data are expressed as mean ⁇ standard deviation. * (asterisk) indicates that there is a significant difference at the 0.1% level by Dunnett's test using the original variety (Koshihikari) as the control group.
  • FIG. 7 is a diagram showing the root phenotypes of the original variety (Koshihikari) and the rice qSOR1 mutant line evaluated by the basket method.
  • A is a representative photograph showing the roots of each line grown by the basket method. Scale bar indicates 1 cm.
  • B is a graph showing the deep root rate of each line cultivated by the basket method. Data are expressed as mean ⁇ standard deviation. * indicates a significant difference at the 0.1% level by Dunnett's test using the original variety (Koshihikari) as the control group.
  • FIG. 8 is a diagram showing root bending angles of the original variety (Koshihikari) and the rice qSOR1 mutant line.
  • A is a representative photograph showing the gravitropic response of each strain 4 hours after the square plate was rotated 90°.
  • Scale bar indicates 5 mm.
  • ⁇ rac indicates the root bending angle.
  • g indicates the direction of gravity.
  • B is a graph showing the root bending angle of each line. Data are expressed as mean ⁇ standard deviation. * indicates that there is a significant difference at the 0.1% level compared to the original variety (Koshihikari) by Student's t-test. n indicates the number of individuals measured.
  • FIG. 9 is a diagram showing a comparison of the yields of the original variety (Koshihikari) and the rice qSOR1 mutant line (strain name 2792M). Data are shown as the average of three replicates of the dry weight of chaff (total of 24 plants in one plot) ⁇ standard deviation.
  • FIG. 10 is a diagram showing the amino acid sequence of Arabidopsis LZY3 protein.
  • FIG. 11 is a diagram showing the locations of mutations from the wild type in the amino acid sequence of Arabidopsis LZY3 mutant proteins (dLZY3(P130S), dLZY3(L131F)).
  • FIG. 12 is a diagram showing the locations of mutations from the wild type in the base sequence encoding Arabidopsis LZY3 mutant proteins (dLZY3(P130S), dLZY3(L131F)).
  • FIG. 13 is a photograph showing the root phenotype of Arabidopsis wild type, lzy2 single mutant, lzy2lzy3 double mutant, dLZY3(P130S)/lzy2lzy3 mutant, and dLZY3(L131F)/lzy2lzy3 mutant.
  • the present invention provides a deep rooting plant with a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein (mutant qSOR1 gene) containing an amino acid substitution that improves deep rooting ability.
  • the present invention relates to a plant with improved root strength (also referred to as "plant of the present invention").
  • Deep rooting refers to the property of roots extending in the direction of gravity (typically underground) at a deep angle with respect to the horizontal plane (typically the ground surface). Deep rootability can be evaluated using, for example, root elongation angle, deep root ratio, or degree of gravitropic response of roots as an index.
  • root extension angle refers to the angle at which roots extend toward the direction of gravity with respect to the horizontal plane.
  • deep root ratio means the ratio of the number of deep roots to the total number of roots.
  • deep root refers to a certain angle (e.g., an appropriate angle within the range of 20 degrees to 70 degrees, such as 20 degrees, 30 degrees, 40 degrees, 50 degrees, 60 degrees, or 70 degrees) with respect to the horizontal plane. ) means a root extending in the direction of gravity at an angle greater than As used herein, “gravitropic response” means that plants sense the direction of gravity and change the growth direction of roots to the direction of gravity, and change the growth direction of above-ground parts to the direction opposite to gravity. do.
  • deep rootability is improved means that the deep rootability is improved compared to the original variety or original line of the plant (for example, a wild type plant).
  • “Deep rooting has improved” means that the root elongation angle has increased (for example, the average value of the root elongation angle has increased by 10 degrees or more, or the root elongation angle has increased statistically significantly).
  • the deep root ratio has increased (e.g., the average value of the deep root ratio has increased by 10% or more, or the deep root ratio has increased statistically significantly)
  • Included is an enhanced gravitropic response of the roots.
  • original variety and “original line” refer to the variety and line from which the plant of the present invention is derived.
  • the mutant qSOR1 gene possessed by the plant of the present invention is one in which a nucleotide mutation that improves deep rooting has been introduced into the qSOR1 gene on the genome, it is considered to be the "original variety” or “original strain”. ” may mean a variety or line before the above-mentioned nucleotide mutation that improves deep rooting is generated or introduced.
  • the mutant qSOR1 gene possessed by the plant of the present invention is a foreign gene
  • the "original variety” and “original line” may mean the variety or line before the mutant qSOR1 gene is introduced.
  • Whether or not a test plant has improved deep rooting can be determined using, for example, the cup method (Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86). This can be determined by evaluating the root elongation angle. Specifically, for example, seeds of the test plant and its original variety or line are sown in a cup-shaped container filled with soil, and cultivated for a predetermined period (for example, 3 weeks). Thereafter, the plant is removed from the cup, the roots are washed, and the root elongation angle of each root is measured using a protractor or the like. If the root elongation angle of the test plant is increased as described above compared to its original variety or original line, it can be determined that the test plant has improved deep rooting ability.
  • the cup method Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86. This can be determined by evaluating the root elongation angle.
  • Whether or not the deep rooting ability of the test plants has improved can also be determined by evaluating the deep rooting ratio using the basket method (Patent No. 5791049; Kitomi Y. et al., Rice, 2015, 8: 16). Can be judged. Specifically, for example, seeds of the test plant and its original variety or strain are sown in a stainless steel mesh colander filled with soil, and cultivated in a hydroponic solution for a predetermined period of time (for example, about 1.5 months). . After that, the number of deep roots and the total number of roots are measured, and the deep root ratio is calculated. If the deep root rate of the test plant is increased as described above compared to its original variety or original line, it can be determined that the test plant has improved deep root ability.
  • the basket method Patent No. 5791049; Kitomi Y. et al., Rice, 2015, 8: 16. Can be judged. Specifically, for example, seeds of the test plant and its original variety or strain are sown in
  • Whether or not the deep rooting ability of the test plant has improved can also be determined by evaluating the gravitropic response of the roots. Specifically, for example, seeds of germinated test plants and their original varieties or original lines are sown on a plate containing agarose gel, and kept at a predetermined temperature for several days (for example, 2 days) with the sides of the plate facing down. Grow under dark conditions. Then, rotate the plate 90 degrees vertically, and measure the root bending angle (the angle between the direction of root elongation before bending and the direction of root elongation after bending) after several hours (for example, 4 hours). .
  • the root bending angle the angle between the direction of root elongation before bending and the direction of root elongation after bending
  • the root flexion angle of the test plant is increased compared to its original variety or line (for example, the average value of the root flexion angle is increased by 5 degrees or more, preferably 10 degrees or more, or the root flexion angle is statistically (significantly increased), it can be determined that the test plant has enhanced root gravitropism response and has improved deep rooting ability.
  • roots In plants, roots first differentiate as radicles during embryonic development, and the radicles develop to become primary roots. In dicotyledonous plants, the primary root develops to become a taproot, from which lateral roots arise, forming a root system called the taproot system. On the other hand, in monocotyledonous plants, primary roots do not develop much, and many nodal roots arise from the nodes of the stem, forming a root system called a bearded root (mainly consisting of seed roots and nodal roots). The radicle and the roots derived from it are called fixed roots (seed roots), and the roots produced from parts other than the radicle (such as stems) are called adventitious roots. Adventitious roots that grow from the stems of rice, corn, etc.
  • the "root” may be any type of root, including, but not limited to, the above-mentioned fixed roots, adventitious roots, dicot roots (e.g., tap roots and lateral roots), monocot roots ( Examples include seed roots and nodal roots), crown roots, and the like.
  • Dro1-NIL quasi-isogenic line
  • IR64 and Dro1-NIL have improved deep rooting ability. It has been reported that the yield was significantly higher with Dro1-NIL than with IR64 when grown under the following conditions (Patent No. 5791049; Uga Y. et al., Nature Genetics, 2013, 45(9): 1097-1102 ).
  • the plant of the present invention which has improved deep rooting compared to the original variety or line, has higher drought tolerance than the original variety or line, and is particularly useful for cultivation under drought conditions. Conceivable.
  • mutant qSOR1 protein refers to a protein that has an amino acid mutation (for example, an amino acid substitution that improves deep rootability) that alters protein function in the amino acid sequence of the wild-type qSOR1 protein.
  • amino acid mutation for example, an amino acid substitution that improves deep rootability
  • wild-type qSOR1 protein means a qSOR1 protein that does not have the above-mentioned amino acid substitution that improves deep rooting ability.
  • amino acid mutation includes insertions, deletions, substitutions, additions, and the like.
  • qSOR1 protein means a protein encoded by the qSOR1 gene.
  • qSOR1 protein is a protein involved in gravitropism, especially root gravitropism, and corresponds to positions 1 to 12, 58 to 64, 140 to 145, and 224 to 241 of the amino acid sequence shown in SEQ ID NO: 2. It has highly conserved sequences (domains IV, respectively) at the same positions.
  • the qSOR1 protein has the amino acid sequence PLDRFL in domain III in the wild type.
  • qSOR1 protein examples include, but are not limited to, rice qSOR1, maize qSOR1 (ZmqSOR1) protein, sorghum qSOR1 (SbqSOR1) protein, wheat qSOR1 (TaqSOR1) protein, Minato chinensis qSOR1 (BdqSOR1) protein, Arabidopsis LZY2 (AtLZY2) protein, These include Arabidopsis LZY3 (AtLZY3) protein, soybean NGR2 (GmNGR2) protein, Lotus japonicus NGR (LjNGR) protein, Alfalfa NGR (MtNGR) protein, poplar NGR (PtNGR) protein, and peach NGR (PpeNGR) protein ( Figures 1 and Figure 5).
  • the "qSOR1 gene” (gene encoding the qSOR1 protein) includes the rice qSOR1 gene (also referred to as the DRL1 gene or OsNGR2 gene) and its homologous genes. Homologues of the rice qSOR1 gene can be found in a wide range of plant species, including sorghum, maize, barley, wheat, monocots such as Albatross, Arabidopsis, Alfalfa, cucumber, lotus, tomato, poplar, soybean, Lotus japonicus, It is present in dicotyledonous plants such as peaches.
  • Homologous genes of rice qSOR1 gene include, but are not limited to, sorghum qSOR1 gene (SbqSOR1; SORBI_3002G373700), maize qSOR1 gene (ZmqSOR1; Zm00001d022133), barley qSOR1 gene (HvqSOR1), wheat qSOR1 gene (TaAqSOR1, TaBqS OR1, TaDqSOR1) , qSOR1 genes such as B.
  • sorghum qSOR1 gene SbqSOR1; SORBI_3002G373700
  • maize qSOR1 gene ZmqSOR1; Zm00001d022133
  • barley qSOR1 gene HvqSOR1 gene
  • wheat qSOR1 gene TaAqSOR1, TaBqS OR1, TaDqSOR1 gene
  • qSOR1 genes such as B.
  • thaliana qSOR1 gene BdqSOR1 gene
  • LZY genes such as Arabidopsis LZY2 gene (AtLZY2), Arabidopsis LZY3 gene (AtLZY3), Arabidopsis LZY4 gene (AtLZY4), Alfalfa NGR (NEGATIVE GRAVITROPIC RESPONSE OF RO) OTS) gene (MtNGR ) NGR genes such as soybean NGR2 (GmNGR2), Lotus japonicum NGR (LjNGR), poplar NGR (PtNGR), and peach NGR (PpeNGR), DRL1 gene, etc. are included.
  • the rice qSOR1 quantitative trait locus for SOIL SURFACE ROOTING 1 gene is present in paddy rice Gemdjah Beton, which forms surface roots (a phenotype in which some crown roots become shallow and extend to the soil surface), and paddy rice Sasanishiki, which does not form surface roots.
  • paddy rice Gemdjah Beton which forms surface roots (a phenotype in which some crown roots become shallow and extend to the soil surface)
  • paddy rice Sasanishiki which does not form surface roots.
  • the amino acid sequence of the natural rice (Koshihikari) qSOR1 protein is shown in SEQ ID NO: 2, for example. Furthermore, a CDS encoding the natural rice (Koshihikari) qSOR1 protein is shown in SEQ ID NO: 1, for example.
  • Arabidopsis LZY4 protein is involved only in root gravitropism, and LZY2 and LZY3 proteins are involved in both shoot and root gravitropism (Taniguchi M. et al., The Plant Cell, 2017 , 29: 1984-1999).
  • the amino acid sequences of the natural Arabidopsis LZY2 protein and LZY3 protein are shown, for example, in SEQ ID NOs: 17 and 12, respectively.
  • the CDS encoding the natural Arabidopsis LZY3 protein is shown in SEQ ID NO: 11, for example.
  • Alfalfa NGR protein is also involved in root gravitropism (Ge L. and Chen R., Nature Plants, 2016, 2(11): 16155).
  • the amino acid sequence of the natural Alfalfa NGR protein is shown, for example, in SEQ ID NO: 18.
  • Figures 1A and B show rice qSOR1 protein (SEQ ID NO: 2), maize qSOR1 (ZmqSOR1) protein (SEQ ID NO: 24), sorghum qSOR1 (SbqSOR1) protein (SEQ ID NO: 25), and wheat qSOR1 (TaqSOR1) protein (SEQ ID NO: 26).
  • Minato thaliana qSOR1 (BdqSOR1) protein (SEQ ID NO: 27), Arabidopsis LZY3 (AtLZY3) protein (SEQ ID NO: 12), soybean NGR2 (GmNGR2) protein (SEQ ID NO: 28), Lotus japonicum NGR (LjNGR) protein (SEQ ID NO: 29),
  • MtNGR alfalfa NGR
  • PtNGR poplar NGR
  • peach NGR PpeNGR
  • FIG. 5 shows the results of comparing the amino acid sequences of rice qSOR1 protein (SEQ ID NO: 2), Arabidopsis LZY2 protein (SEQ ID NO: 17), Arabidopsis LZY3 protein (SEQ ID NO: 12), and Alfalfa NGR protein (SEQ ID NO: 18).
  • the amino acid sequences of these qSOR1 proteins especially the amino acid sequences of domains IV, have very high similarity.
  • these qSOR1 proteins all have the amino acid sequence PLDRFL in domain III.
  • Domain III is the 140th to 145th amino acid sequence shown in SEQ ID NO: 2, the 130th to 135th amino acid sequence shown in SEQ ID NO: 12, the 128th to 133rd amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence shown in SEQ ID NO: 18.
  • gene includes protein coding sequences (CDS).
  • a gene may include an untranslated region (UTR), an exon and an intron, a promoter, an enhancer, an insulator, a terminator, and/or a polyA sequence, etc., depending on the case.
  • a gene includes not only a double-stranded nucleic acid but also its constituent single strands such as the positive strand (sense strand) or complementary strand (antisense strand), and unless otherwise specified, it includes genomic DNA, DNA, Includes RNA, mRNA, cDNA, etc.
  • a gene can be, for example, a polynucleotide encoding a protein.
  • polynucleotide includes both DNA and RNA, and in the case of DNA, it may be single-stranded or double-stranded.
  • amino acid substitutions that improve deep-rootedness contained in the mutant qSOR1 protein include amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2 refers to the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. refers to the substitution of at least one amino acid (eg, 1, 2, 3, 4, 5, or 6 amino acids) in.
  • the above amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions.
  • the above amino acid substitutions include, for example, proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2, leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2, and leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2.
  • substitutions for proline at position 140 include polar uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), and acidic amino acids (glutamic acid or aspartic acid). , or a basic amino acid (lysine, arginine, or histidine), preferably substitution with serine, threonine, glutamine, asparagine, or cysteine, more preferably substitution with serine or threonine, most preferably substitution with serine, threonine, glutamine, asparagine, or cysteine. Preferred is substitution with serine.
  • Substitutions for leucine at position 141 include, for example, polar uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), and acidic amino acids (glutamic acid or aspartic acid). or a basic amino acid (lysine, arginine, or histidine), preferably substitution with phenylalanine or tryptophan, more preferably substitution with phenylalanine.
  • the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 means any amino acid sequence (amino acid sequence of any qSOR1 protein) aligned with the amino acid sequence shown in SEQ ID NO: 2. refers to the position of the amino acid aligned with proline located at position 140 of the amino acid sequence shown in SEQ ID NO:2.
  • similar expressions such as “the position corresponding to the "x"th position of the amino acid sequence shown in SEQ ID NO: 2” and "the position corresponding to the 140th to 145th positions of the amino acid sequence shown in SEQ ID NO: 2" are also interpreted in the same way. .
  • the present invention provides a plant with improved deep rooting ability that expresses a mutant qSOR1 protein comprising the above-described amino acid substitutions that improve deep rooting ability.
  • a gene encoding a mutant qSOR1 protein is one in which a nucleotide mutation that improves deep rootability has occurred in the endogenous qSOR1 gene in the genome, or has been introduced (for example, artificially) into the endogenous qSOR1 gene. It may be something like that.
  • a nucleotide mutation that improves deep rootability refers to a nucleotide mutation that causes an amino acid substitution that improves deep rootability.
  • nucleotide mutation contained in the qSOR1 gene refers to a mutation in the base sequence of the wild-type qSOR1 gene, and includes nucleotide insertions, deletions, substitutions, additions, and the like.
  • the mutant qSOR1 gene may also be a foreign gene.
  • exogenous gene refers to a gene that is artificially introduced into a host plant through genetic manipulation such as transformation.
  • the amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 4 is an amino acid sequence in which proline at position 140 of the amino acid sequence shown in SEQ ID NO: 2 (the amino acid sequence of the original qSOR1 protein) is replaced with serine.
  • the amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 6 is the amino acid sequence in which leucine at position 141 of the amino acid sequence shown in SEQ ID NO: 2 is replaced with phenylalanine.
  • the amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 14 is such that proline at position 130 (corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2) of the amino acid sequence of SEQ ID NO: 12 (the amino acid sequence of wild-type LZY3 protein) is This is an amino acid sequence substituted with serine.
  • the amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 16 is an amino acid sequence in which leucine at position 131 in the amino acid sequence shown in SEQ ID NO: 12 (corresponding to position 141 in the amino acid sequence shown in SEQ ID NO: 2) is replaced with phenylalanine.
  • the mutant qSOR1 protein may be, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14, or 16.
  • the mutant qSOR1 protein also has at least 40%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24-31. , a sequence having sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, 99%, or 99.5% or more and corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • It may also be a protein that consists of an amino acid sequence that includes amino acid substitutions and that exhibits an activity for improving deep rooting of plants.
  • the mutant qSOR1 protein also has 1 to 50, 1 to 25, 1 to 10, 1 to 5, In the sequence having an insertion, deletion, substitution, and/or addition of 1 to 3, 1 to 2, or 1 amino acid and corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. It may also be a protein that consists of an amino acid sequence that includes an amino acid substitution, and that exhibits an activity for improving deep rooting of plants.
  • the base sequences shown in SEQ ID NOs: 3, 5, 13, and 15 are CDSs encoding the mutant qSOR1 proteins shown in SEQ ID NOs: 4, 6, 14, and 16 above, respectively.
  • the base sequence shown in SEQ ID NO: 3 is a base sequence in which the 418th to 420th codon CCG of the base sequence shown in SEQ ID NO: 1 (CDS encoding the original variety qSOR1 protein) is replaced with TCG.
  • the base sequence shown in SEQ ID NO: 5 is a base sequence in which the 421st to 423rd codons CTC of the base sequence shown in SEQ ID NO: 1 are replaced with TTC.
  • the base sequence shown in SEQ ID NO: 13 is a base sequence in which the 388th to 390th codons CCT of the base sequence shown in SEQ ID NO: 11 (CDS encoding wild-type LZY3 protein) are replaced with TCT.
  • the base sequence shown in SEQ ID NO: 15 is a base sequence in which the 391st to 393rd codons TTG of the base sequence shown in SEQ ID NO: 11 are replaced with TTC.
  • the gene encoding the mutant qSOR1 protein may include the base sequence shown in SEQ ID NO: 3, 5, 13, or 15.
  • the gene encoding the mutant qSOR1 protein also has 40% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more of the base sequence shown in SEQ ID NO: 1 or 11. % or more, 98% or more, 99%, or 99.5% or more, and contains a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.
  • the gene encoding the mutant qSOR1 protein also has 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, and 1 to 3 in the base sequence shown in SEQ ID NO: 1 or 11. , 1 to 2, or 1 base insertion, deletion, substitution, and/or addition, and an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. It may also contain a nucleotide mutation that causes a nucleotide mutation and a base sequence that encodes a protein that exhibits the activity of improving deep rooting ability of plants.
  • the plants used in the present invention are typically, but not limited to, angiosperms.
  • the plants may be either annual plants or perennial plants, and may be monocotyledonous plants or dicotyledonous plants.
  • Examples of monocotyledonous plants include, but are not limited to, plants of the Poaceae, Liliaceae, Pineapple family, Arocaceae, Araceae, Zingiberaceae, Orchidaceae, and the like.
  • Dicotyledonous plants include, but are not limited to, Cucurbitaceae, Brassicaceae, Fabaceae, Asteraceae, Lamiaceae, Solanaceae, Rosaceae, Apiaceae, Convolvulaceae, Lotus family, and Salicaceae.
  • the plants may be agricultural crops such as ornamental flower plants and edible vegetables and fruits.
  • ornamental flower plants include morning glory, sunflower, cosmos, sweet pea, marigold, pansy, viola, daisy, snapdragon, gerbera, bellflower, clematis, canna, cyclamen, chrysanthemum, tulip, rose, carnation, petunia, and gypsophila. , lily, orchid, etc.
  • Examples of agricultural crops include rice, wheat, barley, rye, oats, adlay, corn, millet, millet, millet, sorghum, finger millet, pearl millet, teff, sugar cane, Arabidopsis, rapeseed, cabbage, komatsuna, radish, Chinese cabbage, broccoli, Examples include soybeans, kidney beans, broad beans, green onions, rapeseed, cabbage, lettuce, tobacco, tomatoes, strawberries, eggplants, carrots, potatoes, cotton, onions, garlic, potatoes, taro, yams, sweet potatoes, cucumbers, lotus, peaches, and the like.
  • the plants may also be plants used as street trees, such as poplars, plane trees, weeping willows, locusts, cherry blossoms, and sycamores.
  • the plant used in the present invention is preferably a plant of the Poaceae family, Brassicaceae family, or Fabaceae family.
  • Plants of the Poaceae family include, but are not limited to, rice, wheat, barley, rye, oat, adlay, corn, millet, millet, millet, sorghum, finger millet, pearl millet, teff, sugarcane, timothy, Kentucky bluegrass, and orchard.
  • Grass Italian ryegrass, perennial ryegrass, tall fescue, bahiagrass, and minato ryegrass.
  • Examples of plants belonging to the Cruciferae family include, but are not limited to, Arabidopsis, rapeseed, cabbage, Japanese radish, Japanese radish, Chinese cabbage, broccoli, and the like.
  • Examples of plants belonging to the leguminous family include, but are not limited to, soybean, kidney bean, fava bean, adzuki bean, pea, alfalfa, Lotus japonicus, and the like.
  • the plant used in the present invention is more preferably rice.
  • "rice” means any plant belonging to the genus Poaceae of the family Poaceae.
  • Rice includes cultivated rice and wild rice.
  • Examples of cultivated rice include Asian rice (Oryza sativa) and African rice (Oryza glaberrima), and examples of Asian rice include japonica (Oryza sativa subsp. japonica) and indica (Oryza sativa subsp. indica).
  • Japonica varieties include Koshihikari, Toyomeki, Momiroman, Hokuriku 193, Yamadawara, and Sasanishiki, and examples of Indica varieties include IR64.
  • a plant includes the whole plant or a part thereof (leaves, stems, roots, shoot tips, anthers, pollen, embryos, callus, cells, etc.), seeds, and the like.
  • Plants may have multiple genomes.
  • wild species of rice include allotetraploids, which have two types of genomes, genome B and genome C.
  • the plant of the present invention may have the mutant qSOR1 gene described above on at least one genome.
  • the plant of the present invention may also have the above-mentioned mutant qSOR1 gene homozygous or heterozygous on at least one genome.
  • the plant of the present invention may have an unchanged grass type.
  • the plant type has not changed means that the plant type has not changed compared to the original variety or original line.
  • grass type refers to the general shape of the above-ground part of a plant, which is defined by characteristics such as stem and branching.
  • the grass type includes the height of the rice, the number of panicles, the length of the panicle, the length of the cob, the number of primary branches per panicle, and the number of secondary branches per panicle. It can be evaluated by the number of stalks, the number of seeds per panicle, etc.
  • the plant of the present invention may have a maintained or increased yield, especially when it is a cultivated plant (crop).
  • yield is maintained means that the yield is maintained compared to the original variety or line (for example, the yield is increased or decreased by less than 5%).
  • yield is increased means that the yield is increased compared to the original variety or original line (for example, the yield is increased by 5% or more).
  • the plant of the present invention is rice, the amount of refined rice dry matter can be measured as the yield.
  • the plants of the present invention can be produced, for example, by introducing the above-mentioned nucleotide mutations that improve deep rooting into the qSOR1 gene on the genome of the plant. Therefore, the present invention provides a method for producing a plant with improved deep rooting ability (plant of the present invention), which includes the step of introducing a nucleotide mutation that improves deep rooting ability into the qSOR1 gene of the plant (hereinafter referred to as ⁇ the mutation of the present invention''). (also referred to as "method for producing introduced plants”).
  • the target plant to which the mutation is introduced may be a plant (for example, a wild-type plant) that does not have a nucleotide mutation that improves deep rooting in the qSOR1 gene, or It may also be a plant that has a nucleotide mutation that improves deep rooting ability.
  • the plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice.
  • the nucleotide mutation that improves deep rooting ability is, for example, a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, for example, A nucleotide mutation that causes the substitution of proline with serine at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2, or the substitution of leucine with phenylalanine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO: 2.
  • the mutant qSOR1 gene produced by introducing a nucleotide mutation that improves deep rooting ability is, for example, (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16, (ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or (iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It may consist of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No. 2, and may encode a protein that exhibits an activity for improving deep rooting ability of plants.
  • the introduction of nucleotide mutations that improve deep rooting ability can be carried out by, for example, introducing random mutations into the genome of the plant, and selecting nucleotide mutations that improve deep rooting ability from among the resulting mutants. This can be carried out by selecting mutants that have introduced or accumulated .
  • Random mutations can be caused by, for example, irradiation with radiation such as x-rays or gamma rays, mutagenic chemicals (nitroso compounds (e.g., nitrosoguanidine), base-like compounds (e.g., bromodeoxyuridine), alkylating agents (e.g., ethyl It can be introduced by treatment with nitrosourea (ENU), ethyl methanesulfonate (EMS), etc.).
  • mutagenic chemicals nitroso compounds (e.g., nitrosoguanidine), base-like compounds (e.g., bromodeoxyuridine), alkylating agents (e.g., ethyl It can be introduced by treatment with nitrosourea (ENU), ethyl methanesulfonate (EMS), etc.
  • Site-directed mutagenesis can be performed using, for example, site-directed mutagenesis based on homologous recombination such as the Gateway (R) method, site-directed mutagenesis based on PCR, or transcription activator-like effector nuclease (TALEN) (specifically Table 2012-514976 Publication, Special Table 2013-513389 Publication), Zinc Finger Nuclease (Patent No. 4350907, Patent No. 4555292), CRISPR/Cas9 (Jinek et al. A programmable dual-RNA-guided DNA endonuclease) This can be done by various site-directed mutagenesis techniques such as genome editing techniques using in adaptive bacterial immunity. Science 337, 816-821. (2012)).
  • the above-mentioned method for producing a mutated plant of the present invention may include the step of selecting a mutant in which the above-mentioned nucleotide mutation that improves deep rooting ability has been introduced or accumulated. Selection of mutants can be carried out, for example, by determining the base sequence of the qSOR1 gene and selecting mutants in which the determined base sequence contains the above-mentioned nucleotide mutation that improves deep-rootedness.
  • the plants of the present invention can also be produced by transformation. Therefore, the present invention involves the step of introducing into the plant a vector containing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability, to create a plant with improved deep rooting ability (plant of the present invention).
  • a method hereinafter also referred to as "method for producing a transformed plant of the present invention" is provided.
  • the present invention also provides the above genes and vectors used in such methods for producing transformed plants.
  • the target plant into which the vector is introduced may be a plant into which the vector has not been introduced (for example, a wild-type plant).
  • the plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice.
  • amino acid substitutions that improve deep rooting ability include, for example, amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; It may be a substitution of proline at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2 with serine, or a substitution of leucine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO: 2 with phenylalanine.
  • the mutant qSOR1 protein is (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16, (ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or (iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It can be a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No.
  • a selection marker gene can be appropriately incorporated into the vector in addition to the above-mentioned mutant qSOR1 gene.
  • the vector may be introduced into plants using any technique commonly used in the art, such as the Agrobacterium method, particle gun method, electroporation method, etc.
  • the plant used for vector introduction may be a plant body, a plant organ, or a piece of plant tissue, or a callus or protoplast may be prepared and used.
  • the above method for producing a transformed plant of the present invention may include a step of selecting a transformed plant into which the above vector has been introduced.
  • Plants into which the vector has been introduced can be selected, for example, based on the presence or absence of expression of the selectable marker gene incorporated into the vector.
  • the selection marker gene is not particularly limited, but for example, antibiotic resistance genes commonly used in the art can be suitably used. Examples of antibiotic resistance genes that can be suitably used include, but are not limited to, kanamycin resistance genes, neomycin resistance genes, ampicillin resistance genes, hygromycin resistance genes, and the like.
  • the present invention also includes a step of hybridizing plants using the plants of the present invention as breeding parents to obtain progeny plants, and a step encoding the mutant qSOR1 protein.
  • a method for producing a plant (plant of the present invention) with improved deep rooting ability also referred to as "breeding method of the present invention”
  • breeding method of the present invention includes the step of selecting progeny plants into which a gene has been introduced.
  • breeding plants using the plants of the present invention as breeding parents refers to breeding the plants of the present invention with each other, or the plants of the present invention with plants of the same or closely related species. Mating may be carried out once or repeatedly. For example, a plant of the present invention may be crossed with a plant of the same or closely related species (recurrent parent), the progeny plant may be crossed with the recurrent parent (backcrossing), and the progeny plant may be further crossed with the recurrent parent, which are repeated. Good (continuous backcrossing). Alternatively, the plants of the present invention may be crossed with plants of the same or related species, and the progeny plants may be crossed with other plants of the same or related species.
  • Selection of progeny plants into which the gene encoding the mutant qSOR1 protein has been introduced can be carried out by the method described for the method for producing a mutated plant and the method for producing a transformed plant of the present invention.
  • Method for selecting plants with improved deep rooting ability Provided is a method for selecting plants with improved deep rooting ability (also referred to as the "selection method of the present invention"), which includes the step of identifying a plant that has a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves.
  • test plant means a plant that is subjected to the selection method of the present invention.
  • the "plant” is as defined in the above description of the plant of the present invention.
  • the plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice.
  • test plants include, for example, mutants obtained by introducing random mutations into plants, progeny plants obtained by crossbreeding plants using the plants of the present invention as breeding parents, etc. It can be.
  • amino acid substitutions that improve deep rooting ability include, for example, amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; This may be a substitution of proline with serine at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2, or a substitution of phenylalanine with leucine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO:2.
  • the mutant qSOR1 protein is (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16, (ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or (iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It can be a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No. 2, and exhibiting an activity for improving deep rooting ability of plants.
  • nucleic acid amplification techniques include polymerase chain reaction (PCR) method, LAMP (Loop-Mediated Isothermal Amplification) method, TMA (Transcription Mediated Amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR Any method such as the (Ligase Chain Reaction) method can be used.
  • PCR polymerase chain reaction
  • LAMP Loop-Mediated Isothermal Amplification
  • TMA Transcription Mediated Amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • LCR Any method such as the (Ligase Chain Reaction) method can be used.
  • the template for nucleic acid amplification may be genomic DNA or cDNA derived from a test plant.
  • the primer used for nucleic acid amplification may have a length of 15 bases or more, or 20 bases or more, and may have a length of 50 bases or less or 30 bases or less.
  • the primers may also be, for example, 15-50 bases long, 20-50 bases long, or 20-30 bases long.
  • part of the qSOR1 gene is a region containing the nucleotide sequence encoding domain III of the qSOR1 protein (sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2). Good to have.
  • the selection method of the present invention includes, for example, direct sequencing using the Sanger method, HRM (High Resolution Melting) method, KASP TM (Kompetitive Allele Specific PCR) genotyping assay (LGC Biosearch Technologies), dCAPS (derived amplified polymorphic sequence) method, etc. This can be done using
  • the selection method of the present invention may further include the step of evaluating the root elongation angle, deep root ratio, or gravitropism of the test plant.
  • the methods for evaluating root elongation angle, deep root ratio, or gravitropism are as described for the plants of the present invention.
  • nucleic acid amplification is performed using genomic DNA derived from a test plant as a template.
  • a primer set including a reverse primer containing at least 15 consecutive bases of the base sequence shown in SEQ ID NO: 20 or 21 may be used.
  • the above primer set is, for example, (i) A forward primer containing the base sequence shown in SEQ ID NO: 19, and (ii) It may contain a reverse primer containing the base sequence shown in SEQ ID NO: 20 or 21.
  • the nucleotide sequence shown in SEQ ID NO: 19 corresponds to the nucleotide sequence on the second intron of the rice qSOR1 gene (positions 629 to 652 of the nucleotide sequence in SEQ ID NO: 23), and the nucleotide sequences shown in SEQ ID NO: 20 and 21 correspond to the nucleotide sequence on the second intron of the rice qSOR1 gene.
  • Corresponds to the nucleotide sequence on the third intron of the qSOR1 gene corresponds to the nucleotide sequence on the third intron of the qSOR1 gene (respectively, positions 1453 to 1478 and 1301 to 1327 of the base sequence of SEQ ID NO: 23). Therefore, by performing nucleic acid amplification using the above primer set, the region including the third exon (including the region encoding domain III of the qSOR1 protein) is amplified.
  • the nucleotide sequence of the amplified product is determined by the Sanger method or the like to determine whether or not the qSOR1 gene has a nucleotide mutation that improves deep rootability.
  • a plant determined to have a nucleotide mutation that improves deep rooting ability in the qSOR1 gene can be identified as a plant that has a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability.
  • the primer used for nucleic acid amplification in the selection method of the present invention preferably contains the base sequence defined above at its 3' end.
  • the selection method of the present invention may include a step of preparing a test plant before the step of performing nucleic acid amplification.
  • the step of preparing a test plant includes, for example, a step of introducing a nucleotide mutation that improves deep rooting ability into the qSOR1 gene of the plant to make it a test plant, and a step of introducing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability.
  • This may be a step of introducing a vector containing the vector into a plant to obtain a test plant, or a step of cross-breeding the plants using the plant of the present invention as a breeding parent to obtain a progeny plant as a test plant.
  • the selection method of the present invention can be used, for example, in the method of producing a mutated plant, the method of producing a transformed plant, or the breeding method of the present invention.
  • the obtained PCR product was heated to 98°C and cooled to dissociate and reassociate the double-stranded DNA, and then treated with Cel-I nuclease extracted from celery and subjected to agarose electrophoresis. Since Cel-I nuclease cleaves double-stranded DNA at mismatch sites, strains in which cleavage fragments were detected in the agarose electrophoresis described above can be determined to be strains with substitution mutations in the qSOR1 gene.
  • Analysis of the determined nucleotide sequences revealed four strains with non-synonymous substitutions in the qSOR1 gene.
  • the CDS of the qSOR1 gene determined for these four qSOR1 mutant lines are shown in SEQ ID NOs: 3, 5, 7, and 9, respectively. Furthermore, the amino acid sequences encoded by these are shown in SEQ ID NOs: 4, 6, 8, and 10, respectively.
  • SEQ ID NO: 2 As a result of comparing the CDS of the qSOR1 gene of these four qSOR1 mutant lines with the CDS of the qSOR1 gene of the original variety (Koshihikari) (SEQ ID NO: 2), it was revealed that they had the following mutations ( Figure 3 and (see 4).
  • Strain name 0951M A mutation (missense mutation) resulting in the substitution of proline at position 140 with serine (P140S) has occurred.
  • Strain name 2792M A mutation resulting in the substitution of leucine at position 141 with phenylalanine (L141F) has occurred.
  • Strain name 0909M A mutation has occurred that results in the substitution of cysteine for arginine at position 204 (R204C).
  • the positions of the above amino acid substitutions are numbered according to the amino acid sequence of SEQ ID NO: 2. The same applies to Examples 1 to 5 below.
  • the arginine at position 204 forms a salt bridge with aspartic acid at position 17 and glutamic acid at position 226, which contributes to stabilizing the three-dimensional structure of the qSOR1 protein. It was expected that there might be a possibility that In particular, strain name 0909M, in which the basic amino acid arginine was replaced with the neutral amino acid cysteine at position 204, was expected to show a change in root phenotype compared to the original variety. On the other hand, the amino acid substitutions of P140S and L141F did not change the structure of the qSOR1 protein and were not expected to affect the root phenotype.
  • amino acid sequences (SEQ ID NOs: 2, 17, 12, 18, respectively) encoded by the rice (Koshihikari) qSOR1 gene and the isolated homologous genes, the Arabidopsis LZY2 gene, the Arabidopsis LZY3 gene, and the Taruuma alfalfa NGR gene.
  • proline at position 140 and leucine at position 141 were found to be present in domain III, whose amino acid sequence is highly conserved among different species.
  • arginine at position 204 was conserved in rice and alfalfa, but it was a different amino acid in Arabidopsis (Figure 5).
  • Root phenotype of qSOR1 mutant line In order to investigate whether the root elongation angle of the qSOR1 mutant line obtained in Example 1 had changed compared to the original variety (Koshihikari), the root elongation angle was measured by the cup method.
  • the qSOR1 mutant lines (strain names 0909M, 0951M, and 2792M) with changes in root elongation angle in Example 2 were each backcrossed to the original variety three times and selfed. It was confirmed by direct sequencing that the three BC3F3 lines obtained (quasi-isogenic lines with Koshihikari background) maintained the above-mentioned mutation in the qSOR1 gene.
  • the root phenotypes of these three BC3F3 lines were investigated using a modified basket method using a stainless steel mesh colander.
  • a custom-made stainless steel colander with a diameter of 7.5 cm was filled with fertilizer-free culture soil, and sterilized seeds of the original variety or BC3F3 strain were sown and cultivated in hydroponic solution for about a month and a half. After the cultivation period ended, roots that extended downward at an angle exceeding 30 degrees to the ground surface were considered deep roots, and the number of deep roots and the total number of roots were measured.
  • the number of deep roots divided by the total number of roots was calculated as the deep root ratio (RDR30; Ratio of Deeper Root than 30 degrees). The larger the value of the deep root ratio, the deeper the root.
  • the deep root rate was investigated for 20 individuals each of the original variety and three BC3F3 lines.
  • the change in the root phenotype of the qSOR1 mutant line is due to the mutation that occurred in the qSOR1 gene, that is, the R204C substitution in the amino acid sequence of the rice qSOR1 protein has the effect of shallowing the roots of rice. Contrary to predictions from the original amino acid sequence, the P140S and L141F substitutions (substitutions in domain III) were shown to have the effect of deepening the roots of rice.
  • the rice husks were removed from 30 seeds to obtain brown rice. After washing the brown rice three times with 10 ml of sterilized water, it was placed in a petri dish containing 10 ml of a medium containing 1% PPM (PLANT PRESERVATIVE MIXTURE TM , Plant Cell Technology) as a disinfectant, and allowed to stand at 30°C for 1 day. Germinated seeds were sown on square plates containing 0.4% agarose gel and left standing in the dark at 28°C for 2 days. Thereafter, the square plate was rotated 90 degrees, and after 4 hours, the roots were photographed and the bending angle of the roots was measured.
  • PPM PANT PRESERVATIVE MIXTURE TM , Plant Cell Technology
  • rice that has the gene encoding the qSOR1 protein containing the R204C substitution has shallower roots due to a weaker gravitropic response than the original variety, and the P140S and L141F substitutions (substitutions in domain III).
  • Rice that has the gene encoding the qSOR1 protein has been shown to have deeper roots due to a stronger gravitropic response than the original variety.
  • the heading dates of the original variety and the 2792M-derived BC3F3 line in 2020 were August 6th to 7th in all three plots, and the heading dates of both lines were almost simultaneous. Ta.
  • the heading date of the original variety in 2021 was July 30th to 31st in all three plots, and the heading date of the 2792M-derived BC3F3 line in 2021 was July 28th in all three plots. Because the summer temperature in 2021 was higher than in 2020, it is thought that the heading date in 2021 was about one week earlier than the heading date in 2020 for both lines. The difference between lines in heading date was smaller than this difference between years.
  • amino acid sequences of the dLZY3(P130S) and dLZY3(L131F) mutant proteins are shown in SEQ ID NOs: 14 and 16, respectively, and the nucleotide sequences encoding them are shown in SEQ ID NOs: 13 and 15, respectively.
  • LZY3p:LZY3-mCherry a vector that expresses a fusion protein of LZY3 protein and mCherry (red fluorescent protein) under the LZY3 promoter (Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999, University Joint Use
  • the nucleotide sequence of the construct contained in the vector which was provided by the National Institute for Basic Biology of the National Institutes of Natural Sciences, is shown in SEQ ID NO: 22), by substituting C at position 388 of the LZY3 gene with T
  • a vector (LZY3p:dLZY3(P130S)-mCherry) expressing a fusion protein of the (P130S) mutant protein and mCherry under the LZY3 promoter was created.
  • LZY3p:dLZY3(P130S)-mCherry and LZY3p:dLZY3(L131F)-mCherry were isolated from Arabidopsis lzy2lzy3 double mutant (Arabidopsis Columbia strain in which LZY2 and LZY3 genes are deleted). mutant). Seeds of the T1 generation of the obtained transformed plants were sown on a 1/2MS agar medium containing 20 ⁇ g/ml of hygromycin, and plants showing resistance to hygromycin were selected. The selected plants were newly transplanted onto a 1/2 MS agar medium, grown on the agar medium vertically, and the morphology of the root system was observed.
  • Arabidopsis Columbia strain wild type
  • Arabidopsis Columbia strain mutant in which the LZY2 gene has been deleted
  • Arabidopsis Columbia strain in which the LZY2 and LZY3 genes have been deleted (lzy2lzy3 double mutant)
  • the agar medium was grown vertically and the morphology of the root system was observed.
  • the lzy2lzy3 double mutant (dLZY3(P130S)/lzy2lzy3 mutant) into which LZY3p:dLZY3(P130S)-mCherry was introduced and the lzy2lzy3 double mutant into which LZY3p:dLZY3(L131F)-mCherry was introduced ( dLZY3(L131F)/lzy2lzy3 mutant) all had roots elongated downward compared to the original line, the lzy2lzy3 double mutant, and complemented the phenotype of the lzy2lzy3 double mutant.
  • the roots of the dLZY3(P130S)/lzy2lzy3 mutant and the dLZY3(L131F)/lzy2lzy3 mutant significantly elongate downward, even compared to the wild type and lzy2 single mutant that express the wild-type LZY3 protein.
  • the roots of the dLZY3(P130S)/lzy2lzy3 mutant and dLZY3(L131F)/lzy2lzy3 mutant appear to be slightly shorter than those of the wild type, lzy2 single mutant, and lzy2lzy3 double mutant; This is considered to be an effect of hygromycin in the medium.
  • the grass types of the above-mentioned dLZY3(P130S)/lzy2lzy3 mutant and dLZY3(L131F)/lzy2lzy3 mutant were not significantly changed compared to the lzy2lzy3 double mutant.

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Abstract

The problem addressed by the present invention is to provide technology for improving the deep-rootedness of a plant. The present invention provides a plant with improved deep-rootedness which has a gene encoding a mutant qSOR1 protein including an amino acid substitution that improves deep-rootedness.

Description

深根性が向上した植物Plants with improved deep roots
 本発明は、変異型qSOR1タンパク質をコードする遺伝子を有する深根性が向上した植物に関する。 The present invention relates to a plant with improved deep rooting ability that has a gene encoding a mutant qSOR1 protein.
 現在、世界の人口は発展途上国を中心に増加しており、これらの人口を養うための食料の増産は必須である。一方、近年の地球規模の気候変化に伴い、干ばつや豪雨などの降雨の極端化現象が世界各地で発生している。さらに、長年の過耕作などによる土壌の荒廃や塩類集積なども問題となっている。このような不安定な環境下で安定的、かつ、持続的な食料生産を行うためには、環境ストレスに強い作物の開発が必要である。 Currently, the world's population is increasing, mainly in developing countries, and it is essential to increase food production to feed this population. On the other hand, with recent global climate changes, extreme rainfall events such as droughts and heavy rains are occurring in many parts of the world. Furthermore, soil deterioration and salt accumulation due to years of over-cultivation have also become problems. In order to achieve stable and sustainable food production in such an unstable environment, it is necessary to develop crops that are resistant to environmental stress.
 根は植物が土中の養水分を獲得・吸収するための唯一の器官であるため、根系形態は作物の生長にとって非常に重要であるが、作物生産に適した根系形態は土壌環境によって異なる。例えば、根が深く伸びる性質(深根性)は、干ばつ耐性、窒素吸収、重金属回避などに有利に働く一方、根が浅く伸びる性質(浅根性)は、リン欠耐性、湛水状態での酸欠回避、重金属のファイトレメディエーションなどに有利に働く。したがって、土壌環境に合わせた根系形態の改良は、環境ストレスに強い作物の開発にとって重要である。 Roots are the only organ for plants to acquire and absorb nutrients and water from the soil, so root system morphology is extremely important for crop growth, but the root system morphology suitable for crop production differs depending on the soil environment. For example, the property that roots grow deeply (deep roots) is advantageous for drought tolerance, nitrogen absorption, avoidance of heavy metals, etc., while the property that roots grow shallowly (shallow roots) is resistant to phosphorus deficiency and oxygen deficiency in flooded conditions. It is advantageous for avoidance, phytoremediation of heavy metals, etc. Therefore, improving root system morphology to suit the soil environment is important for developing crops that are resistant to environmental stress.
 作物の開発方法としては、交雑育種が一般的によく用いられている。しかしながら、交雑育種には表現型選抜が必要であり、土中にある根系形態を評価する際、通常は根を掘り起こして調査する必要があること、根のサンプリングに多大な時間や労力を必要とすること、穀類などでは地上部の収穫の後でなければ根を調査できないことから、交雑育種による作物根の改良はほとんど進んでいない。 Cross breeding is commonly used as a method for developing crops. However, cross breeding requires phenotypic selection, and when evaluating the morphology of the root system in the soil, it is usually necessary to dig up and investigate the roots, and root sampling requires a great deal of time and effort. In the case of grains, roots cannot be investigated until after the above-ground parts have been harvested, so there has been little progress in improving crop roots through cross-breeding.
 一方、モデル植物であるシロイヌナズナでは、突然変異体の解析によって、根の形態発生に関与する遺伝子や環境ストレス下における根の形態形成に関与する遺伝子が多数同定され、根の形態制御のメカニズムの解明が進んでいる。例えば、根系形態に大きく影響する屈性に関しては、複数の重力屈性遺伝子(非特許文献1)や水分屈性遺伝子(非特許文献2)が同定されている。また、イネやトウモロコシを含む単子葉植物でも、突然変異体の解析によって、多くの根に関与する遺伝子が同定されており、例えば、イネでは、DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1(DOCS1)や、OsPIN2をコードするLARGE ROOT ANGLE1の突然変異体は浅根となること(非特許文献3及び4)、アクチン結合タンパク質をコードするRice Morphology Determinant(RMD)の突然変異体は、浅根が有利な低リン酸条件下で深根化することが報告されている(非特許文献5)。しかし、突然変異体は、原品種(野生型)と比較し、形態や生理機能の異常が伴うことが多く、突然変異体から同定した遺伝子の多くはそのまま育種素材として利用することが困難である。 On the other hand, in the model plant Arabidopsis thaliana, many genes involved in root morphogenesis and those involved in root morphogenesis under environmental stress have been identified through the analysis of mutants, and the mechanism of controlling root morphology has been elucidated. is progressing. For example, regarding tropism, which greatly affects root system morphology, a plurality of gravitropism genes (Non-Patent Document 1) and hydrotropism genes (Non-Patent Document 2) have been identified. Furthermore, in monocotyledonous plants including rice and maize, many genes involved in roots have been identified through mutant analysis. For example, in rice, DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) and OsPIN2 Mutants of LARGE ROOT ANGLE1, which encodes ANGLE1, develop shallow roots (Non-Patent Documents 3 and 4), and mutants of Rice Morphology Determinant (RMD), which encodes an actin-binding protein, develop shallow roots under low phosphate conditions. It has been reported that the roots become deep at the bottom (Non-patent Document 5). However, mutants often have abnormalities in morphology and physiological function compared to the original variety (wild type), and many of the genes identified from mutants are difficult to use directly as breeding materials. .
 このような状況の中、近年、イネを用いた量的形質遺伝子座(QTL: quantitative trait locus)解析によって、根伸長角度に関与するQTLとして、DRO1(DEEPER ROOTING 1)遺伝子及びその相同遺伝子であるqSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)遺伝子が単離され、これらの遺伝子は重力屈性に関与することが報告された(特許文献1、非特許文献6及び7)。 Under these circumstances, in recent years, quantitative trait locus (QTL) analysis using rice has revealed that the DRO1 (DEEPER ROOTING 1) gene and its homologous genes are QTL involved in root elongation angle. The qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene was isolated, and it was reported that these genes are involved in gravitropism (Patent Document 1, Non-Patent Documents 6 and 7).
 イネDRO1遺伝子及びイネqSOR1遺伝子の相同遺伝子は被子植物に広く存在し、DRO1ファミリーと称される大きな遺伝子群を形成することが知られている(非特許文献7)。また、DRO1ファミリータンパク質には、植物間で高く保存されている5つのドメインが存在することが知られており(非特許文献8)、シロイヌナズナやイネを用いた研究から5つ目のドメイン(CCLドメイン)が重力屈性の機能に関与することが知られている(非特許文献6、7及び9)。 It is known that homologous genes of the rice DRO1 gene and the rice qSOR1 gene exist widely in angiosperms and form a large gene group called the DRO1 family (Non-Patent Document 7). Furthermore, it is known that DRO1 family proteins have five domains that are highly conserved among plants (Non-patent Document 8), and research using Arabidopsis and rice revealed that the fifth domain (CCL domain) is known to be involved in the function of gravitropism (Non-patent Documents 6, 7, and 9).
特許第5791049号公報Patent No. 5791049
 qSOR1遺伝子は単子葉植物と双子葉植物の両方に広く存在していることから、qSOR1遺伝子の有用アリルは、コムギやトウモロコシなどの単子葉作物だけでなく、ダイズやナタネなどの双子葉作物の根系改良にも広く利用できると考えられる。
 しかしながら、育種素材として有用なqSOR1遺伝子アリルは限られており、現状では植物の深根性を向上させる技術は十分に開発されていない。
Since the qSOR1 gene is widely present in both monocots and dicots, useful alleles of the qSOR1 gene are found not only in monocot crops such as wheat and maize, but also in the root systems of dicot crops such as soybean and rapeseed. It is thought that it can be widely used for improvements.
However, the number of qSOR1 gene alleles useful as breeding material is limited, and at present, technology for improving deep rooting ability of plants has not been sufficiently developed.
 本発明者らは、ランダム変異が導入されたイネ突然変異系統から、qSOR1遺伝子中に非同義置換を有する系統を探索し、得られた突然変異系統の根の表現型を調べた。その結果、驚くべきことに、これまで機能が未知であった3つ目のドメイン(ドメインIII)にアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を有する突然変異系統において、深根性が原品種に比べて向上していることが判明した。これらの突然変異系統を原品種に戻し交配し、得られた系統の根の形態を調べたところ、上記深根性の向上はqSOR1タンパク質内に生じた上記アミノ酸置換に起因することが証明された。さらに、同様のアミノ酸置換を有するシロイヌナズナLZY3タンパク質(イネqSOR1タンパク質に対応。本明細書では「qSOR1タンパク質」と称する場合もある。)を発現するシロイヌナズナ形質転換体の根の形態を調べたところ、深根性が顕著に向上していることが判明した。 The present inventors searched for lines with non-synonymous substitutions in the qSOR1 gene from rice mutant lines into which random mutations were introduced, and examined the root phenotypes of the obtained mutant lines. As a result, surprisingly, deep rooting was found in the mutant line containing the gene encoding the mutant qSOR1 protein containing an amino acid substitution in the third domain (domain III), the function of which was unknown until now. It was found that it was improved compared to When these mutant lines were backcrossed to the original variety and the root morphology of the resulting lines was examined, it was proven that the improvement in deep rooting ability was due to the above amino acid substitution that occurred in the qSOR1 protein. Furthermore, we investigated the morphology of the roots of Arabidopsis transformants expressing Arabidopsis LZY3 protein (corresponding to rice qSOR1 protein, sometimes referred to as "qSOR1 protein" in this specification), which has similar amino acid substitutions. It was found that his perseverance was significantly improved.
 本発明者らは、上記の知見に基づき、本発明を完成するに至った。すなわち、本発明は以下を包含する。
[1]配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)タンパク質をコードする遺伝子を有する、深根性が向上した植物。
[2]前記アミノ酸置換が、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリン及び配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンからなる群から選択されるアミノ酸の置換である、[1]に記載の植物。
[3]前記アミノ酸置換が、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリンのセリンへの置換、又は配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンのフェニルアラニンへの置換である、[1]又は[2]に記載の植物。
[4]前記変異型qSOR1タンパク質が、
(i) 配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質、
(ii) 配列番号2若しくは12に示すアミノ酸配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質、又は
(iii) 配列番号2若しくは12に示すアミノ酸配列において1~10個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質
である、[1]~[3]のいずれかに記載の植物。
[5]前記変異型qSOR1タンパク質をコードする遺伝子が、
(iv) 配列番号3、5、13若しくは15に示す塩基配列、
(v) 配列番号1若しくは11に示す塩基配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列、又は
(vi) 配列番号1若しくは11に示す塩基配列において1~10個の塩基の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列、
を含む、[1]~[4]のいずれかに記載の植物。
[6]単子葉植物又は双子葉植物である、[1]~[5]のいずれかに記載の植物。
[7]植物のqSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)遺伝子に、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を導入する工程を含む、深根性が向上した植物を作出する方法。
[8]配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)タンパク質をコードする遺伝子を含むベクターを植物に導入する工程を含む、深根性が向上した植物を作出する方法。
[9][1]~[6]のいずれかに記載の植物を育種親として用いて植物の交配を行い、子孫植物を取得する工程、及び前記変異型qSOR1タンパク質をコードする遺伝子が導入された子孫植物を選抜する工程を含む、深根性が向上した植物を作出する方法。
[10]被験植物由来のDNAを鋳型としてqSOR1遺伝子の全体又はその一部について核酸増幅を行う工程、核酸増幅の結果に基づいて、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を有する植物を同定する工程を含む、深根性が向上した植物を選抜する方法。
 本明細書は本願の優先権の基礎となる日本国特許出願番号2022-068983号の開示内容を包含する。
The present inventors have completed the present invention based on the above findings. That is, the present invention includes the following.
[1] A deep-rooted protein that has a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein that contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. Plants with improved
[2] The amino acid substitution is an amino acid selected from the group consisting of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 and leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2. The plant according to [1], which is a substitution.
[3] The amino acid substitution is a substitution of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 to serine, or a substitution of leucine to phenylalanine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2. The plant according to [1] or [2], which is a substitution of.
[4] The mutant qSOR1 protein is
(i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16,
(ii) An amino acid that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12 and contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. a protein consisting of a sequence and exhibiting an activity of improving deep rooting of plants, or
(iii) Has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 12, and corresponds to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. The plant according to any one of [1] to [3], which is a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence at a position and exhibiting an activity of improving deep rooting of plants.
[5] The gene encoding the mutant qSOR1 protein,
(iv) the base sequence shown in SEQ ID NO: 3, 5, 13 or 15,
(v) A nucleotide mutation that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 or 11 and causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. a base sequence that encodes a protein that contains and exhibits an activity of improving deep rooting of plants, or
(vi) Has an insertion, deletion, substitution, and/or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1 or 11, and is in the 140th to 145th sequence of the amino acid sequence shown in SEQ ID NO: 2. a nucleotide sequence that contains a nucleotide mutation that causes an amino acid substitution and that encodes a protein that exhibits an activity to improve deep rooting ability of plants;
The plant according to any one of [1] to [4], comprising:
[6] The plant according to any one of [1] to [5], which is a monocot or a dicot.
[7] A step of introducing into the plant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. A method for producing plants with improved deep rooting.
[8] Plant a vector containing a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. A method for producing plants with improved deep rooting, including the step of introducing the plant into a plant.
[9] A step of cross-breeding plants using the plants according to any one of [1] to [6] as breeding parents to obtain progeny plants, and a step in which the gene encoding the mutant qSOR1 protein is introduced. A method for producing plants with improved deep rooting, including a step of selecting descendant plants.
[10] Nucleic acid amplification of the whole or part of the qSOR1 gene using DNA from the test plant as a template, based on the results of the nucleic acid amplification, the position corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. A method for selecting plants with improved deep rooting ability, the method comprising the step of identifying a plant having a gene encoding a mutant qSOR1 protein containing an amino acid substitution in the sequence.
This specification includes the disclosure content of Japanese Patent Application No. 2022-068983, which is the basis of the priority of this application.
 本発明によれば、深根性が向上している植物及びその作出方法を提供することができる。 According to the present invention, it is possible to provide a plant with improved deep rooting ability and a method for producing the same.
図1Aは、単子葉植物のqSOR1タンパク質(イネqSOR1タンパク質、トウモロコシqSOR1(ZmqSOR1)タンパク質、ソルガムqSOR1(SbqSOR1)タンパク質、コムギqSOR1(TaqSOR1)タンパク質、ミナトカモジグサqSOR1(BdqSOR1)タンパク質)と、双子葉植物のqSOR1タンパク質(シロイヌナズナLZY3(AtLZY3)タンパク質、ダイズNGR2(GmNGR2)タンパク質、ミヤコグサNGR(LjNGR)タンパク質、タルウマゴヤシNGR(MtNGR)タンパク質、ポプラNGR(PtNGR)タンパク質、モモNGR(PpeNGR)タンパク質)のアミノ酸配列を比較した結果を示す図である。複数植物種間で保存性の高い5つのドメイン(ドメインI~V)をそれぞれ四角で示す。Figure 1A shows the qSOR1 proteins of monocotyledonous plants (rice qSOR1 protein, maize qSOR1 (ZmqSOR1) protein, sorghum qSOR1 (SbqSOR1) protein, wheat qSOR1 (TaqSOR1) protein, Minato chinensis qSOR1 (BdqSOR1) protein) and dicot. Amino acid sequence of qSOR1 protein (Arabidopsis LZY3 (AtLZY3) protein, soybean NGR2 (GmNGR2) protein, Lotus japonicum NGR (LjNGR) protein, Alfalfa NGR (MtNGR) protein, poplar NGR (PtNGR) protein, peach NGR (PpeNGR) protein) FIG. 3 is a diagram showing the results of comparison. Five domains (domains I to V) that are highly conserved among multiple plant species are each indicated by a square. 図1Aの続きである。This is a continuation of FIG. 1A. 図2は、コシヒカリqSOR1タンパク質のアミノ酸配列を示す図である。FIG. 2 is a diagram showing the amino acid sequence of Koshihikari qSOR1 protein. 図3は、4つのイネqSOR1突然変異系統のqSOR1タンパク質アミノ酸配列の原品種からの変異箇所を示す図である。FIG. 3 is a diagram showing the locations of mutations in the qSOR1 protein amino acid sequences of four rice qSOR1 mutant lines from the original variety. 図4は、4つのイネqSOR1突然変異系統のqSOR1遺伝子CDSの原品種からの変異箇所を示す図である。FIG. 4 is a diagram showing the mutation locations of the qSOR1 gene CDS of four rice qSOR1 mutant lines from the original variety. 図5は、イネ(Rice)qSOR1タンパク質、シロイヌナズナ(Arabidopsis)LZY2タンパク質、シロイヌナズナLZY3タンパク質、タルウマゴヤシ(Medicago)NGRタンパク質のアミノ酸配列を比較した結果を示す図である。複数植物種間で保存性の高い5つのドメイン(ドメインI~V)をそれぞれ四角で示す。FIG. 5 is a diagram showing the results of comparing the amino acid sequences of rice qSOR1 protein, Arabidopsis LZY2 protein, Arabidopsis LZY3 protein, and Medicago NGR protein. Five domains (domains I to V) that are highly conserved among multiple plant species are each indicated by a square. 図6は、カップ法により評価した原品種(コシヒカリ)及びイネqSOR1突然変異系統の根の表現型を示す図である。Aは、カップ法で栽培した各系統の根を示す代表的な写真である。スケールバーは1 cmを示す。θrgaは、根伸長角度を示す。Bは、カップ法で栽培した各系統の根伸長角度を示すグラフである。データは、平均値±標準偏差として表す。*(アステリスク)は、原品種(コシヒカリ)をコントロール群とするDunnett検定により0.1%水準で有意差があることを示す。FIG. 6 is a diagram showing the root phenotypes of the original variety (Koshihikari) and the rice qSOR1 mutant line evaluated by the cup method. A is a representative photograph showing the roots of each line grown by the cup method. Scale bar indicates 1 cm. θrga indicates the root elongation angle. B is a graph showing the root elongation angle of each line grown by the cup method. Data are expressed as mean ± standard deviation. * (asterisk) indicates that there is a significant difference at the 0.1% level by Dunnett's test using the original variety (Koshihikari) as the control group. 図7は、バスケット法により評価した原品種(コシヒカリ)及びイネqSOR1突然変異系統の根の表現型を示す図である。Aは、バスケット法で栽培した各系統の根を示す代表的な写真である。スケールバーは1 cmを示す。Bは、バスケット法で栽培した各系統の深根率を示すグラフである。データは、平均値±標準偏差として表す。*は原品種(コシヒカリ)をコントロール群とするDunnett検定により0.1%水準で有意差があることを示す。FIG. 7 is a diagram showing the root phenotypes of the original variety (Koshihikari) and the rice qSOR1 mutant line evaluated by the basket method. A is a representative photograph showing the roots of each line grown by the basket method. Scale bar indicates 1 cm. B is a graph showing the deep root rate of each line cultivated by the basket method. Data are expressed as mean ± standard deviation. * indicates a significant difference at the 0.1% level by Dunnett's test using the original variety (Koshihikari) as the control group. 図8は、原品種(コシヒカリ)及びイネqSOR1突然変異系統の根屈曲角度を示す図である。Aは、角形プレートを90°回転してから4時間後の各系統の重力屈性応答を示す代表的な写真である。スケールバーは5 mmを示す。θracは根屈曲角度を示す。gは重力方向を示す。Bは、各系統の根屈曲角度を示すグラフである。データは、平均値±標準偏差として表す。*は原品種(コシヒカリ)に対しStudentのt検定により0.1%水準で有意な差があることを示す。nは測定した個体数を示す。FIG. 8 is a diagram showing root bending angles of the original variety (Koshihikari) and the rice qSOR1 mutant line. A is a representative photograph showing the gravitropic response of each strain 4 hours after the square plate was rotated 90°. Scale bar indicates 5 mm. θrac indicates the root bending angle. g indicates the direction of gravity. B is a graph showing the root bending angle of each line. Data are expressed as mean ± standard deviation. * indicates that there is a significant difference at the 0.1% level compared to the original variety (Koshihikari) by Student's t-test. n indicates the number of individuals measured. 図9は、原品種(コシヒカリ)及びイネqSOR1突然変異系統(系統名2792M)の収量の比較を示す図である。データは、精籾乾物重(1区画24株の合計)の3反復平均±標準偏差として示す。FIG. 9 is a diagram showing a comparison of the yields of the original variety (Koshihikari) and the rice qSOR1 mutant line (strain name 2792M). Data are shown as the average of three replicates of the dry weight of chaff (total of 24 plants in one plot)±standard deviation. 図10は、シロイヌナズナLZY3タンパク質のアミノ酸配列を示す図である。FIG. 10 is a diagram showing the amino acid sequence of Arabidopsis LZY3 protein. 図11は、シロイヌナズナLZY3変異体タンパク質(dLZY3(P130S)、dLZY3(L131F))のアミノ酸配列の野生型からの変異箇所を示す図である。FIG. 11 is a diagram showing the locations of mutations from the wild type in the amino acid sequence of Arabidopsis LZY3 mutant proteins (dLZY3(P130S), dLZY3(L131F)). 図12は、シロイヌナズナLZY3変異体タンパク質(dLZY3(P130S)、dLZY3(L131F))をコードする塩基配列の野生型からの変異箇所を示す図である。FIG. 12 is a diagram showing the locations of mutations from the wild type in the base sequence encoding Arabidopsis LZY3 mutant proteins (dLZY3(P130S), dLZY3(L131F)). 図13は、シロイヌナズナ野生型、lzy2単一変異体、lzy2lzy3二重変異体、dLZY3(P130S)/lzy2lzy3変異体、及びdLZY3(L131F)/lzy2lzy3変異体の根の表現型を示す写真である。野生型、lzy2単一変異体、lzy2lzy3二重変異体については播種後14日目の根の表現型を、dLZY3(P130S)/lzy2lzy3変異体及びdLZY3(L131F)/lzy2lzy3変異体については播種後19日目の根の表現型を示す。スケールバーは5 mmを示す。gは重力方向を示す。FIG. 13 is a photograph showing the root phenotype of Arabidopsis wild type, lzy2 single mutant, lzy2lzy3 double mutant, dLZY3(P130S)/lzy2lzy3 mutant, and dLZY3(L131F)/lzy2lzy3 mutant. The root phenotype on day 14 after sowing for the wild type, lzy2 single mutant, and lzy2lzy3 double mutant, and 19 days after sowing for the dLZY3(P130S)/lzy2lzy3 mutant and dLZY3(L131F)/lzy2lzy3 mutant. Showing the root phenotype on day one. Scale bar indicates 5 mm. g indicates the direction of gravity.
 以下、本発明を詳細に説明する。 Hereinafter, the present invention will be explained in detail.
(1)深根性が向上した植物
 本発明は、深根性を向上させるアミノ酸置換を含む変異型qSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)タンパク質をコードする遺伝子(変異型qSOR1遺伝子)を有する、深根性が向上した植物(「本発明の植物」とも称する。)に関する。
(1) Plants with improved deep rooting ability The present invention provides a deep rooting plant with a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein (mutant qSOR1 gene) containing an amino acid substitution that improves deep rooting ability. The present invention relates to a plant with improved root strength (also referred to as "plant of the present invention").
 本明細書において「深根性」とは、根が水平面(典型的には地表)に対して深い角度で重力方向(典型的には地中)に伸長する性質を意味する。深根性は、例えば、根伸長角度、深根率、又は根の重力屈性応答の程度を指標として評価することができる。本明細書において「根伸長角度」とは、水平面に対する、根が重力方向に向かって伸長する角度を意味する。本明細書において「深根率」とは、根の総数に対する深根の数の割合を意味する。本明細書において「深根」とは、水平面に対して一定の角度(例えば20度~70度の範囲内の適切な角度、例えば20度、30度、40度50度、60度又は70度)よりも大きい角度で重力方向に伸長している根を意味する。本明細書において「重力屈性応答」とは、植物が重力の方向を感知して根の成長方向を重力方向に変更し、地上部の成長方向を重力と反対の方向に変更することを意味する。 As used herein, "deep rooting" refers to the property of roots extending in the direction of gravity (typically underground) at a deep angle with respect to the horizontal plane (typically the ground surface). Deep rootability can be evaluated using, for example, root elongation angle, deep root ratio, or degree of gravitropic response of roots as an index. As used herein, the term "root extension angle" refers to the angle at which roots extend toward the direction of gravity with respect to the horizontal plane. As used herein, "deep root ratio" means the ratio of the number of deep roots to the total number of roots. As used herein, "deep root" refers to a certain angle (e.g., an appropriate angle within the range of 20 degrees to 70 degrees, such as 20 degrees, 30 degrees, 40 degrees, 50 degrees, 60 degrees, or 70 degrees) with respect to the horizontal plane. ) means a root extending in the direction of gravity at an angle greater than As used herein, "gravitropic response" means that plants sense the direction of gravity and change the growth direction of roots to the direction of gravity, and change the growth direction of above-ground parts to the direction opposite to gravity. do.
 本明細書において、「深根性が向上した」とは、原品種又は原系統の植物(例えば野生型植物)に比べ、深根性が向上していることを意味する。「深根性が向上した」には、根伸長角度が増大している(例えば、根伸長角度の平均値が10度以上増大しているか、又は、根伸長角度が統計学的に有意に増大している)こと、深根率が増大している(例えば、深根率の平均値が10%以上増大しているか、又は深根率が統計学的に有意に増大している)こと、及び根の重力屈性応答が増強されていることが包含される。本明細書において、「原品種」、「原系統」とは、本発明の植物が由来する品種及び系統を意味する。例えば、本発明の植物が有する変異型qSOR1遺伝子が、ゲノム上のqSOR1遺伝子に深根性を向上させるヌクレオチド変異が生じたか、又は導入されたものである場合には、「原品種」、「原系統」は、上記の深根性を向上させるヌクレオチド変異が生じるか導入される前の品種、系統を意味し得る。本発明の植物が有する変異型qSOR1遺伝子が外来性遺伝子である場合には、「原品種」、「原系統」は、変異型qSOR1遺伝子が導入される前の品種、系統を意味し得る。 As used herein, "deep rootability is improved" means that the deep rootability is improved compared to the original variety or original line of the plant (for example, a wild type plant). "Deep rooting has improved" means that the root elongation angle has increased (for example, the average value of the root elongation angle has increased by 10 degrees or more, or the root elongation angle has increased statistically significantly). ), the deep root ratio has increased (e.g., the average value of the deep root ratio has increased by 10% or more, or the deep root ratio has increased statistically significantly), and Included is an enhanced gravitropic response of the roots. As used herein, "original variety" and "original line" refer to the variety and line from which the plant of the present invention is derived. For example, if the mutant qSOR1 gene possessed by the plant of the present invention is one in which a nucleotide mutation that improves deep rooting has been introduced into the qSOR1 gene on the genome, it is considered to be the "original variety" or "original strain". ” may mean a variety or line before the above-mentioned nucleotide mutation that improves deep rooting is generated or introduced. When the mutant qSOR1 gene possessed by the plant of the present invention is a foreign gene, the "original variety" and "original line" may mean the variety or line before the mutant qSOR1 gene is introduced.
 被験植物(例えば、本発明の植物)の深根性が向上しているか否かは、例えば、カップ法(Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86)を用いて根伸長角度を評価することによって判定できる。具体的には、例えば、培土を充填したカップ状の容器に被験植物及びその原品種若しくは原系統の種子を播種し、所定期間(例えば3週間)栽培する。その後、カップから植物を取り出し、根を洗浄し、各根の根伸長角度を分度器等で測定する。被験植物の根伸長角度がその原品種又は原系統に比べ上記のように増大している場合には、被験植物は、深根性が向上していると判断することができる。 Whether or not a test plant (for example, a plant of the present invention) has improved deep rooting can be determined using, for example, the cup method (Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86). This can be determined by evaluating the root elongation angle. Specifically, for example, seeds of the test plant and its original variety or line are sown in a cup-shaped container filled with soil, and cultivated for a predetermined period (for example, 3 weeks). Thereafter, the plant is removed from the cup, the roots are washed, and the root elongation angle of each root is measured using a protractor or the like. If the root elongation angle of the test plant is increased as described above compared to its original variety or original line, it can be determined that the test plant has improved deep rooting ability.
 被験植物の深根性が向上しているか否かはまた、バスケット法(特許第5791049号; Kitomi Y. et al., Rice, 2015, 8: 16)を用いて深根率を評価することによっても判定できる。具体的には、例えば、培土を充填したステンレス製のメッシュ状のザルに被験植物及びその原品種若しくは原系統の種子を播種し、水耕液中で所定期間(例えば1.5か月程度)栽培する。その後、深根の数と総根数を測定し、深根率を算出する。被験植物の深根率がその原品種又は原系統に比べ上記のように増大している場合には、被験植物は深根性が向上していると判断することができる。 Whether or not the deep rooting ability of the test plants has improved can also be determined by evaluating the deep rooting ratio using the basket method (Patent No. 5791049; Kitomi Y. et al., Rice, 2015, 8: 16). Can be judged. Specifically, for example, seeds of the test plant and its original variety or strain are sown in a stainless steel mesh colander filled with soil, and cultivated in a hydroponic solution for a predetermined period of time (for example, about 1.5 months). . After that, the number of deep roots and the total number of roots are measured, and the deep root ratio is calculated. If the deep root rate of the test plant is increased as described above compared to its original variety or original line, it can be determined that the test plant has improved deep root ability.
 被験植物の深根性が向上しているか否かはまた、根の重力屈性応答を評価することによって判定できる。具体的には、例えば、発芽させた被験植物及びその原品種若しくは原系統の種子をアガロースゲルが入ったプレートに播種し、所定の温度で数日間(例えば2日間)、プレートの側面を下にした状態で暗黒条件下にて生育させる。その後、プレートを垂直方向に90度回転させ、数時間後(例えば4時間後)に根屈曲角度(屈曲前の根の伸長方向と屈曲後の根の伸長方向との間の角度)を計測する。被験植物の根屈曲角度がその原品種又は原系統に比べ増大している(例えば、根屈曲角度の平均値が5度以上、好ましくは10度以上増大しているか、又は根屈曲角度が統計学的に有意に増大している)場合には、被験植物は根の重力屈性応答が増強されており、深根性が向上していると判断することができる。 Whether or not the deep rooting ability of the test plant has improved can also be determined by evaluating the gravitropic response of the roots. Specifically, for example, seeds of germinated test plants and their original varieties or original lines are sown on a plate containing agarose gel, and kept at a predetermined temperature for several days (for example, 2 days) with the sides of the plate facing down. Grow under dark conditions. Then, rotate the plate 90 degrees vertically, and measure the root bending angle (the angle between the direction of root elongation before bending and the direction of root elongation after bending) after several hours (for example, 4 hours). . The root flexion angle of the test plant is increased compared to its original variety or line (for example, the average value of the root flexion angle is increased by 5 degrees or more, preferably 10 degrees or more, or the root flexion angle is statistically (significantly increased), it can be determined that the test plant has enhanced root gravitropism response and has improved deep rooting ability.
 植物において、根は最初胚発生中に幼根として分化し、幼根は発達して初生根となる。双子葉植物等では、初生根が発達して主根となり、そこから側根が生じ、主根系と称される根系が形成される。一方、単子葉植物等では、初生根はあまり発達せず、茎の節から節根が多数生じ、ひげ根(主に種子根及び節根からなる)と称される根系が形成される。幼根及びそれに由来する根は定根(種子根)と称され、幼根以外の部位(茎等)から生じた根は不定根と称される。イネやトウモロコシ等の茎から生える不定根は、冠根と称される。本発明において、「根」は任意の種類の根であってよく、限定されないが、例えば、上記の定根、不定根、双子葉植物の根(例えば、主根及び側根)、単子葉植物の根(例えば種子根及び節根)、冠根等を包含する。 In plants, roots first differentiate as radicles during embryonic development, and the radicles develop to become primary roots. In dicotyledonous plants, the primary root develops to become a taproot, from which lateral roots arise, forming a root system called the taproot system. On the other hand, in monocotyledonous plants, primary roots do not develop much, and many nodal roots arise from the nodes of the stem, forming a root system called a bearded root (mainly consisting of seed roots and nodal roots). The radicle and the roots derived from it are called fixed roots (seed roots), and the roots produced from parts other than the radicle (such as stems) are called adventitious roots. Adventitious roots that grow from the stems of rice, corn, etc. are called crown roots. In the present invention, the "root" may be any type of root, including, but not limited to, the above-mentioned fixed roots, adventitious roots, dicot roots (e.g., tap roots and lateral roots), monocot roots ( Examples include seed roots and nodal roots), crown roots, and the like.
 一般に、深根性が高いほど干ばつ耐性に有利となることが知られている。例えば、浅根の水稲IR64と比較し、深根の陸稲Kinandang Patong由来のDRO1遺伝子を導入したIR64背景の準同質遺伝子系統(Dro1-NIL)は深根性が向上すること、IR64とDro1-NILを干ばつ下で栽培したところ、IR64よりDro1-NILで有意に収量が増大したことが報告されている(特許第5791049号; Uga Y. et al., Nature Genetics, 2013, 45(9): 1097-1102)。 In general, it is known that the deeper the roots, the better the drought tolerance. For example, compared to shallow-rooted paddy rice IR64, a quasi-isogenic line (Dro1-NIL) on the IR64 background into which the DRO1 gene derived from deep-rooted upland rice Kinandang Patong has been introduced has improved deep rooting, and IR64 and Dro1-NIL have improved deep rooting ability. It has been reported that the yield was significantly higher with Dro1-NIL than with IR64 when grown under the following conditions (Patent No. 5791049; Uga Y. et al., Nature Genetics, 2013, 45(9): 1097-1102 ).
 したがって、原品種又は原系統に比べて深根性が向上している本発明の植物は、原品種又は原系統よりも高い干ばつ耐性を有しており、干ばつ下での栽培に特に有用であると考えられる。 Therefore, the plant of the present invention, which has improved deep rooting compared to the original variety or line, has higher drought tolerance than the original variety or line, and is particularly useful for cultivation under drought conditions. Conceivable.
 本明細書において、「変異型qSOR1タンパク質」とは、野生型qSOR1タンパク質のアミノ酸配列に対し、タンパク質機能の改変をもたらすアミノ酸変異(例えば、深根性を向上させるアミノ酸置換)を有するタンパク質を意味する。本明細書において、「野生型qSOR1タンパク質」とは、上記の深根性を向上させるアミノ酸置換を有しないqSOR1タンパク質を意味する。本明細書において、「アミノ酸変異」には、挿入、欠失、置換、付加等が包含される。 As used herein, the term "mutant qSOR1 protein" refers to a protein that has an amino acid mutation (for example, an amino acid substitution that improves deep rootability) that alters protein function in the amino acid sequence of the wild-type qSOR1 protein. As used herein, "wild-type qSOR1 protein" means a qSOR1 protein that does not have the above-mentioned amino acid substitution that improves deep rooting ability. As used herein, "amino acid mutation" includes insertions, deletions, substitutions, additions, and the like.
 本明細書において、「qSOR1タンパク質」とは、qSOR1遺伝子によりコードされるタンパク質を意味する。qSOR1タンパク質は、重力屈性、特に根の重力屈性に関与するタンパク質であり、配列番号2に示すアミノ酸配列の1~12番目、58~64番目、140~145番目、224~241番目に対応する位置に、高度に保存された配列(それぞれ、ドメインI~V)を有する。qSOR1タンパク質は、野生型ではドメインIIIにアミノ酸配列PLDRFLを有する。qSOR1タンパク質としては、限定されないが、例えば、イネqSOR1、トウモロコシqSOR1(ZmqSOR1)タンパク質、ソルガムqSO1(SbqSOR1)タンパク質、コムギqSOR1(TaqSOR1)タンパク質、ミナトカモジグサqSOR1(BdqSOR1)タンパク質、シロイヌナズナLZY2(AtLZY2)タンパク質、シロイヌナズナLZY3(AtLZY3)タンパク質、ダイズNGR2(GmNGR2)タンパク質、ミヤコグサNGR(LjNGR)タンパク質、タルウマゴヤシNGR(MtNGR)タンパク質、ポプラNGR(PtNGR)タンパク質、モモNGR(PpeNGR)タンパク質が包含される(図1及び図5)。 As used herein, "qSOR1 protein" means a protein encoded by the qSOR1 gene. qSOR1 protein is a protein involved in gravitropism, especially root gravitropism, and corresponds to positions 1 to 12, 58 to 64, 140 to 145, and 224 to 241 of the amino acid sequence shown in SEQ ID NO: 2. It has highly conserved sequences (domains IV, respectively) at the same positions. The qSOR1 protein has the amino acid sequence PLDRFL in domain III in the wild type. Examples of the qSOR1 protein include, but are not limited to, rice qSOR1, maize qSOR1 (ZmqSOR1) protein, sorghum qSOR1 (SbqSOR1) protein, wheat qSOR1 (TaqSOR1) protein, Minato chinensis qSOR1 (BdqSOR1) protein, Arabidopsis LZY2 (AtLZY2) protein, These include Arabidopsis LZY3 (AtLZY3) protein, soybean NGR2 (GmNGR2) protein, Lotus japonicus NGR (LjNGR) protein, Alfalfa NGR (MtNGR) protein, poplar NGR (PtNGR) protein, and peach NGR (PpeNGR) protein (Figures 1 and Figure 5).
 本明細書において、「qSOR1遺伝子」(qSOR1タンパク質をコードする遺伝子)には、イネqSOR1遺伝子(DRL1遺伝子又はOsNGR2遺伝子とも称される。)及びその相同遺伝子が包含される。イネqSOR1遺伝子の相同遺伝子(ホモログ)は、広範な植物種、例えば、ソルガム、トウモロコシ、オオムギ、コムギ、ミナトカモジクサ等の単子葉植物、シロイヌナズナ、タルウマゴヤシ、キュウリ、ハス、トマト、ポプラ、ダイズ、ミヤコグサ、モモ等の双子葉植物に存在する。イネqSOR1遺伝子の相同遺伝子としては、限定されないが、例えば、ソルガムqSOR1遺伝子(SbqSOR1; SORBI_3002G373700)、トウモロコシqSOR1遺伝子(ZmqSOR1; Zm00001d022133)、オオムギqSOR1遺伝子(HvqSOR1)、コムギqSOR1遺伝子(TaAqSOR1、TaBqSOR1、TaDqSOR1)、ミナトカモジクサqSOR1遺伝子(BdqSOR1)等のqSOR1遺伝子、シロイヌナズナLZY2遺伝子(AtLZY2)、シロイヌナズナLZY3遺伝子(AtLZY3)、シロイヌナズナLZY4遺伝子(AtLZY4)等のLZY遺伝子、タルウマゴヤシNGR(NEGATIVE GRAVITROPIC RESPONSE OF ROOTS)遺伝子(MtNGR)ダイズNGR2(GmNGR2)、ミヤコグサNGR(LjNGR)、ポプラNGR(PtNGR)、モモNGR(PpeNGR)等のNGR遺伝子、DRL1遺伝子等が包含される。 As used herein, the "qSOR1 gene" (gene encoding the qSOR1 protein) includes the rice qSOR1 gene (also referred to as the DRL1 gene or OsNGR2 gene) and its homologous genes. Homologues of the rice qSOR1 gene can be found in a wide range of plant species, including sorghum, maize, barley, wheat, monocots such as Albatross, Arabidopsis, Alfalfa, cucumber, lotus, tomato, poplar, soybean, Lotus japonicus, It is present in dicotyledonous plants such as peaches. Homologous genes of rice qSOR1 gene include, but are not limited to, sorghum qSOR1 gene (SbqSOR1; SORBI_3002G373700), maize qSOR1 gene (ZmqSOR1; Zm00001d022133), barley qSOR1 gene (HvqSOR1), wheat qSOR1 gene (TaAqSOR1, TaBqS OR1, TaDqSOR1) , qSOR1 genes such as B. thaliana qSOR1 gene (BdqSOR1), LZY genes such as Arabidopsis LZY2 gene (AtLZY2), Arabidopsis LZY3 gene (AtLZY3), Arabidopsis LZY4 gene (AtLZY4), Alfalfa NGR (NEGATIVE GRAVITROPIC RESPONSE OF RO) OTS) gene (MtNGR ) NGR genes such as soybean NGR2 (GmNGR2), Lotus japonicum NGR (LjNGR), poplar NGR (PtNGR), and peach NGR (PpeNGR), DRL1 gene, etc. are included.
 イネqSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)遺伝子は、地表根(一部の冠根が浅根化し、土壌表面に伸長する表現型)を形成する水稲Gemdjah Betonと地表根を形成しない水稲ササニシキの交雑集団を用いたQTL解析により、根伸長角度に関与するQTLとしてイネ第7染色体上に発見された(Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86)。イネqSOR1タンパク質は重力屈性に関与し、根伸長角度を制御することが報告されている。天然のイネ(コシヒカリ)qSOR1タンパク質のアミノ酸配列は、例えば配列番号2に示される。さらに、天然のイネ(コシヒカリ)qSOR1タンパク質をコードするCDSは、例えば配列番号1に示される。 The rice qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene is present in paddy rice Gemdjah Beton, which forms surface roots (a phenotype in which some crown roots become shallow and extend to the soil surface), and paddy rice Sasanishiki, which does not form surface roots. Through QTL analysis using a hybrid population, it was discovered on rice chromosome 7 as a QTL involved in root elongation angle (Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86). It has been reported that rice qSOR1 protein is involved in gravitropism and controls root elongation angle. The amino acid sequence of the natural rice (Koshihikari) qSOR1 protein is shown in SEQ ID NO: 2, for example. Furthermore, a CDS encoding the natural rice (Koshihikari) qSOR1 protein is shown in SEQ ID NO: 1, for example.
 シロイヌナズナLZY4タンパク質は根の重力屈性のみに、LZY2及びLZY3タンパク質はシュートと根の重力屈性の双方に関与していることが報告されている(Taniguchi M. et al., The Plant Cell, 2017, 29: 1984-1999)。天然のシロイヌナズナLZY2タンパク質及びLZY3タンパク質のアミノ酸配列は、例えば、それぞれ配列番号17、12に示される。さらに、天然のシロイヌナズナLZY3タンパク質をコードするCDSは、例えば、配列番号11に示される。 It has been reported that Arabidopsis LZY4 protein is involved only in root gravitropism, and LZY2 and LZY3 proteins are involved in both shoot and root gravitropism (Taniguchi M. et al., The Plant Cell, 2017 , 29: 1984-1999). The amino acid sequences of the natural Arabidopsis LZY2 protein and LZY3 protein are shown, for example, in SEQ ID NOs: 17 and 12, respectively. Furthermore, the CDS encoding the natural Arabidopsis LZY3 protein is shown in SEQ ID NO: 11, for example.
 タルウマゴヤシNGRタンパク質も根の重力屈性に関与していることが報告されている(Ge L. and Chen R., Nature Plants, 2016, 2(11): 16155)。天然のタルウマゴヤシNGRタンパク質のアミノ酸配列は、例えば、配列番号18に示される。 It has been reported that Alfalfa NGR protein is also involved in root gravitropism (Ge L. and Chen R., Nature Plants, 2016, 2(11): 16155). The amino acid sequence of the natural Alfalfa NGR protein is shown, for example, in SEQ ID NO: 18.
 その他、天然のトウモロコシqSOR1(ZmqSOR1)、ソルガムqSOR1(SbqSOR1)、コムギqSOR1(TaqSOR1)、ミナトカモジグサqSOR1(BdqSOR1)、ダイズNGR2(GmNGR2)、ミヤコグサNGR(LjNGR)、ポプラNGR(PtNGR)、モモNGR(PpeNGR)のアミノ酸配列は、例えば、それぞれ配列番号24~31に示される。 In addition, natural maize qSOR1 (ZmqSOR1), sorghum qSOR1 (SbqSOR1), wheat qSOR1 (TaqSOR1), Minato chinensis qSOR1 (BdqSOR1), soybean NGR2 (GmNGR2), Lotus japonica NGR (LjNGR), poplar NGR (PtNGR), peach NGR ( The amino acid sequences of PpeNGR) are shown, for example, in SEQ ID NOs: 24 to 31, respectively.
 図1A及びBは、イネqSOR1タンパク質(配列番号2)、トウモロコシqSOR1(ZmqSOR1)タンパク質(配列番号24)、ソルガムqSOR1(SbqSOR1)タンパク質(配列番号25)、コムギqSOR1(TaqSOR1)タンパク質(配列番号26)、ミナトカモジグサqSOR1(BdqSOR1)タンパク質(配列番号27)、シロイヌナズナLZY3(AtLZY3)タンパク質(配列番号12)、ダイズNGR2(GmNGR2)タンパク質(配列番号28)、ミヤコグサNGR(LjNGR)タンパク質(配列番号29)、タルウマゴヤシNGR(MtNGR)タンパク質(配列番号18)、ポプラNGR(PtNGR)タンパク質(配列番号30)、モモNGR(PpeNGR)タンパク質(配列番号31)のアミノ酸配列を比較した結果を示す。図5は、イネqSOR1タンパク質(配列番号2)、シロイヌナズナLZY2タンパク質(配列番号17)、シロイヌナズナLZY3タンパク質(配列番号12)、タルウマゴヤシNGRタンパク質(配列番号18)のアミノ酸配列を比較した結果を示す。図1及び5に示されるように、これらのqSOR1タンパク質のアミノ酸配列、特にドメインI~Vのアミノ酸配列は非常に高い類似性を有する。特に、これらのqSOR1タンパク質はすべてドメインIIIにアミノ酸配列PLDRFLを有する。ドメインIIIは、配列番号2に示すアミノ酸配列の140~145番目、配列番号12に示すアミノ酸配列の130~135番目、配列番号17に示すアミノ酸配列の128~133番目、配列番号18に示すアミノ酸配列の109~114番目、配列番号24に示すアミノ酸配列の127~132番目、配列番号25に示すアミノ酸配列の131~136番目、配列番号26に示すアミノ酸配列の121~126番目、配列番号27に示すアミノ酸配列の122~127番目、配列番号28に示すアミノ酸配列の111~116番目、配列番号29に示すアミノ酸配列の105~110番目、配列番号30に示すアミノ酸配列の112~117番目、配列番号31に示すアミノ酸配列の110~115番目に存在する。 Figures 1A and B show rice qSOR1 protein (SEQ ID NO: 2), maize qSOR1 (ZmqSOR1) protein (SEQ ID NO: 24), sorghum qSOR1 (SbqSOR1) protein (SEQ ID NO: 25), and wheat qSOR1 (TaqSOR1) protein (SEQ ID NO: 26). , Minato thaliana qSOR1 (BdqSOR1) protein (SEQ ID NO: 27), Arabidopsis LZY3 (AtLZY3) protein (SEQ ID NO: 12), soybean NGR2 (GmNGR2) protein (SEQ ID NO: 28), Lotus japonicum NGR (LjNGR) protein (SEQ ID NO: 29), The results of comparing the amino acid sequences of alfalfa NGR (MtNGR) protein (SEQ ID NO: 18), poplar NGR (PtNGR) protein (SEQ ID NO: 30), and peach NGR (PpeNGR) protein (SEQ ID NO: 31) are shown. FIG. 5 shows the results of comparing the amino acid sequences of rice qSOR1 protein (SEQ ID NO: 2), Arabidopsis LZY2 protein (SEQ ID NO: 17), Arabidopsis LZY3 protein (SEQ ID NO: 12), and Alfalfa NGR protein (SEQ ID NO: 18). As shown in Figures 1 and 5, the amino acid sequences of these qSOR1 proteins, especially the amino acid sequences of domains IV, have very high similarity. In particular, these qSOR1 proteins all have the amino acid sequence PLDRFL in domain III. Domain III is the 140th to 145th amino acid sequence shown in SEQ ID NO: 2, the 130th to 135th amino acid sequence shown in SEQ ID NO: 12, the 128th to 133rd amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence shown in SEQ ID NO: 18. 109th to 114th of the amino acid sequence shown in SEQ ID NO: 24, 127th to 132nd of the amino acid sequence shown in SEQ ID NO: 24, 131st to 136th of the amino acid sequence shown in SEQ ID NO: 25, 121st to 126th of the amino acid sequence shown in SEQ ID NO: 26, shown in SEQ ID NO: 27 122nd to 127th amino acid sequence, 111th to 116th amino acid sequence shown in SEQ ID NO: 28, 105th to 110th amino acid sequence shown in SEQ ID NO: 29, 112nd to 117th amino acid sequence shown in SEQ ID NO: 30, SEQ ID NO: 31 It is present at positions 110 to 115 of the amino acid sequence shown in .
 本明細書において、「遺伝子」は、タンパク質コード配列(CDS)を含む。遺伝子は、場合により、非翻訳領域(UTR)を含んでもよいし、エクソン及びイントロンを含んでもよいし、プロモーター、エンハンサー、インシュレーター、ターミネーター、及び/又はポリA配列等を含んでもよい。遺伝子は、二本鎖の核酸だけでなく、それを構成する正鎖(センス鎖)又は相補鎖(アンチセンス鎖)等の一本鎖を包含し、さらに特に言及しない限り、ゲノムDNA、DNA、RNA、mRNA、cDNA等を包含する。遺伝子は、例えば、タンパク質をコードするポリヌクレオチドであり得る。本明細書において、「ポリヌクレオチド」にはDNAおよびRNAの双方が含まれ、DNAである場合には、一本鎖であっても二本鎖であってもよい。 As used herein, "gene" includes protein coding sequences (CDS). A gene may include an untranslated region (UTR), an exon and an intron, a promoter, an enhancer, an insulator, a terminator, and/or a polyA sequence, etc., depending on the case. A gene includes not only a double-stranded nucleic acid but also its constituent single strands such as the positive strand (sense strand) or complementary strand (antisense strand), and unless otherwise specified, it includes genomic DNA, DNA, Includes RNA, mRNA, cDNA, etc. A gene can be, for example, a polynucleotide encoding a protein. As used herein, "polynucleotide" includes both DNA and RNA, and in the case of DNA, it may be single-stranded or double-stranded.
 本発明において、変異型qSOR1タンパク質に含まれる深根性を向上させるアミノ酸置換としては、例えば、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中のアミノ酸置換が挙げられる。本明細書において、「配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中のアミノ酸置換」とは、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中の少なくとも1個のアミノ酸(例えば、1個、2個、3個、4個、5個、又は6個のアミノ酸)の置換を意味する。 In the present invention, examples of amino acid substitutions that improve deep-rootedness contained in the mutant qSOR1 protein include amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. As used herein, "amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2" refers to the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. refers to the substitution of at least one amino acid (eg, 1, 2, 3, 4, 5, or 6 amino acids) in.
 上記のアミノ酸置換は、保存的アミノ酸置換であってもよく、非保存的アミノ酸置換であってもよい。上記のアミノ酸置換は、例えば、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリン、配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシン、配列番号2に示すアミノ酸配列の142番目に対応する位置のアスパラギン酸、配列番号2に示すアミノ酸配列の143番目に対応する位置のアルギニン、配列番号2に示すアミノ酸配列の144番目に対応する位置のフェニルアラニン、及び配列番号2に示すアミノ酸配列の145番目に対応する位置のロイシンからなる群から選択されるアミノ酸の置換(例えば、非保存的アミノ酸置換)であり得る。上記140番目のプロリンの置換としては、例えば、極性非電荷アミノ酸(セリン、トレオニン、グルタミン、アスパラギン、若しくはシステイン)、芳香族アミノ酸(フェニルアラニン、チロシン、若しくはトリプトファン)、酸性アミノ酸(グルタミン酸、若しくはアスパラギン酸)、又は塩基性アミノ酸(リジン、アルギニン、若しくはヒスチジン)への置換が挙げられるが、好ましくは、セリン、トレオニン、グルタミン、アスパラギン、又はシステインへの置換、より好ましくは、セリン又はトレオニンへの置換、最も好ましくはセリンへの置換である。上記141番目のロイシンの置換としては、例えば、極性非電荷アミノ酸(セリン、トレオニン、グルタミン、アスパラギン、若しくはシステイン)、芳香族アミノ酸(フェニルアラニン、チロシン、若しくはトリプトファン)、酸性アミノ酸(グルタミン酸、若しくはアスパラギン酸)、又は塩基性アミノ酸(リジン、アルギニン、若しくはヒスチジン)への置換が挙げられるが、好ましくは、フェニルアラニン又はトリプトファンへの置換、より好ましくは、フェニルアラニンへの置換である。 The above amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. The above amino acid substitutions include, for example, proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2, leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2, and leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2. Aspartic acid at the position corresponding to the 142nd position, arginine at the position corresponding to the 143rd position in the amino acid sequence shown in SEQ ID NO: 2, phenylalanine at the position corresponding to the 144th position in the amino acid sequence shown in SEQ ID NO: 2, and as shown in SEQ ID NO: 2. It may be a substitution of an amino acid selected from the group consisting of leucine at a position corresponding to position 145 of the amino acid sequence (for example, a non-conservative amino acid substitution). Examples of substitutions for proline at position 140 include polar uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), and acidic amino acids (glutamic acid or aspartic acid). , or a basic amino acid (lysine, arginine, or histidine), preferably substitution with serine, threonine, glutamine, asparagine, or cysteine, more preferably substitution with serine or threonine, most preferably substitution with serine, threonine, glutamine, asparagine, or cysteine. Preferred is substitution with serine. Substitutions for leucine at position 141 include, for example, polar uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), and acidic amino acids (glutamic acid or aspartic acid). or a basic amino acid (lysine, arginine, or histidine), preferably substitution with phenylalanine or tryptophan, more preferably substitution with phenylalanine.
 本明細書において、「配列番号2に示すアミノ酸配列の140番目に対応する位置」とは、配列番号2で示されるアミノ酸配列とアラインメントされた任意のアミノ酸配列(任意のqSOR1タンパク質のアミノ酸配列)中で、配列番号2に示すアミノ酸配列の140番目に位置するプロリンに対してアラインメントされるアミノ酸の位置を指す。なお、「配列番号2に示すアミノ酸配列の"x"番目に対応する位置」及び「配列番号2に示すアミノ酸配列の140~145番目に対応する位置」等の類似の表現も同様に解される。
 一実施形態では、本発明は、上記の深根性を向上させるアミノ酸置換を含む変異型qSOR1タンパク質を発現する、深根性が向上した植物を提供する。
As used herein, "the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2" means any amino acid sequence (amino acid sequence of any qSOR1 protein) aligned with the amino acid sequence shown in SEQ ID NO: 2. refers to the position of the amino acid aligned with proline located at position 140 of the amino acid sequence shown in SEQ ID NO:2. In addition, similar expressions such as "the position corresponding to the "x"th position of the amino acid sequence shown in SEQ ID NO: 2" and "the position corresponding to the 140th to 145th positions of the amino acid sequence shown in SEQ ID NO: 2" are also interpreted in the same way. .
In one embodiment, the present invention provides a plant with improved deep rooting ability that expresses a mutant qSOR1 protein comprising the above-described amino acid substitutions that improve deep rooting ability.
 本発明において、変異型qSOR1タンパク質をコードする遺伝子(変異型qSOR1遺伝子)は、ゲノム上の内在性のqSOR1遺伝子に深根性を向上させるヌクレオチド変異が生じたか、又は(例えば、人為的に)導入されたものであってもよい。本明細書において、「深根性を向上させるヌクレオチド変異」とは、深根性を向上させるアミノ酸置換を引き起こすヌクレオチド変異を意味する。本明細書において、qSOR1遺伝子に含まれる「ヌクレオチド変異」とは、野生型qSOR1遺伝子の塩基配列に対する変異を意味し、ヌクレオチドの挿入、欠失、置換、付加等が包含される。 In the present invention, a gene encoding a mutant qSOR1 protein (mutant qSOR1 gene) is one in which a nucleotide mutation that improves deep rootability has occurred in the endogenous qSOR1 gene in the genome, or has been introduced (for example, artificially) into the endogenous qSOR1 gene. It may be something like that. As used herein, "a nucleotide mutation that improves deep rootability" refers to a nucleotide mutation that causes an amino acid substitution that improves deep rootability. As used herein, the term "nucleotide mutation" contained in the qSOR1 gene refers to a mutation in the base sequence of the wild-type qSOR1 gene, and includes nucleotide insertions, deletions, substitutions, additions, and the like.
 本発明において、変異型qSOR1遺伝子はまた、外来性遺伝子であり得る。本明細書において、「外来性」遺伝子とは、形質転換などの遺伝子操作により、人為的に宿主植物に導入された遺伝子を意味する。 In the present invention, the mutant qSOR1 gene may also be a foreign gene. As used herein, the term "exogenous" gene refers to a gene that is artificially introduced into a host plant through genetic manipulation such as transformation.
 配列番号4に示す変異型qSOR1タンパク質のアミノ酸配列は、配列番号2に示すアミノ酸配列(原品種qSOR1タンパク質のアミノ酸配列)の140番目のプロリンがセリンに置換されたアミノ酸配列である。配列番号6に示す変異型qSOR1タンパク質のアミノ酸配列は、配列番号2に示すアミノ酸配列の141番目のロイシンがフェニルアラニンに置換されたアミノ酸配列である。配列番号14に示す変異型LZY3タンパク質のアミノ酸配列は、配列番号12のアミノ酸配列(野生型LZY3タンパク質のアミノ酸配列)の130番目(配列番号2に示すアミノ酸配列の140番目に対応する)のプロリンがセリンに置換されたアミノ酸配列である。配列番号16に示す変異型LZY3タンパク質のアミノ酸配列は、配列番号12のアミノ酸配列の131番目(配列番号2に示すアミノ酸配列の141番目に対応)のロイシンがフェニルアラニンに置換されたアミノ酸配列である。 The amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 4 is an amino acid sequence in which proline at position 140 of the amino acid sequence shown in SEQ ID NO: 2 (the amino acid sequence of the original qSOR1 protein) is replaced with serine. The amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 6 is the amino acid sequence in which leucine at position 141 of the amino acid sequence shown in SEQ ID NO: 2 is replaced with phenylalanine. The amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 14 is such that proline at position 130 (corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2) of the amino acid sequence of SEQ ID NO: 12 (the amino acid sequence of wild-type LZY3 protein) is This is an amino acid sequence substituted with serine. The amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 16 is an amino acid sequence in which leucine at position 131 in the amino acid sequence shown in SEQ ID NO: 12 (corresponding to position 141 in the amino acid sequence shown in SEQ ID NO: 2) is replaced with phenylalanine.
 本発明において、変異型qSOR1タンパク質は、例えば、配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質であってもよい。変異型qSOR1タンパク質はまた、配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列と40%以上、60%以上、70%以上、80%以上、90%以上、95%以上、96%以上、97%以上、98%以上、99%、又は99.5%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質であってもよい。変異型qSOR1タンパク質はまた、配列番号2、12、17、18、24~31のいずれか1つに示すアミノ酸配列において1~50個、1~25個、1~10個、1~5個、1~3個、1~2個、又は1個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質であってもよい。 In the present invention, the mutant qSOR1 protein may be, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14, or 16. The mutant qSOR1 protein also has at least 40%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24-31. , a sequence having sequence identity of 95% or more, 96% or more, 97% or more, 98% or more, 99%, or 99.5% or more and corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. It may also be a protein that consists of an amino acid sequence that includes amino acid substitutions and that exhibits an activity for improving deep rooting of plants. The mutant qSOR1 protein also has 1 to 50, 1 to 25, 1 to 10, 1 to 5, In the sequence having an insertion, deletion, substitution, and/or addition of 1 to 3, 1 to 2, or 1 amino acid and corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. It may also be a protein that consists of an amino acid sequence that includes an amino acid substitution, and that exhibits an activity for improving deep rooting of plants.
 配列番号3、5、13、及び15に示す塩基配列は、それぞれ、上記の配列番号4、6、14、16に示す変異型qSOR1タンパク質をコードするCDSである。具体的には、配列番号3に示す塩基配列は、配列番号1に示す塩基配列(原品種qSOR1タンパク質をコードするCDS)の418~420番目のコドンCCGがTCGに置換された塩基配列である。配列番号5に示す塩基配列は、配列番号1に示す塩基配列の421~423番目のコドンCTCがTTCに置換された塩基配列である。配列番号13に示す塩基配列は、配列番号11に示す塩基配列(野生型LZY3タンパク質をコードするCDS)の388~390番目のコドンCCTがTCTに置換された塩基配列である。配列番号15に示す塩基配列は、配列番号11に示す塩基配列の391~393番目のコドンTTGがTTCに置換された塩基配列である。 The base sequences shown in SEQ ID NOs: 3, 5, 13, and 15 are CDSs encoding the mutant qSOR1 proteins shown in SEQ ID NOs: 4, 6, 14, and 16 above, respectively. Specifically, the base sequence shown in SEQ ID NO: 3 is a base sequence in which the 418th to 420th codon CCG of the base sequence shown in SEQ ID NO: 1 (CDS encoding the original variety qSOR1 protein) is replaced with TCG. The base sequence shown in SEQ ID NO: 5 is a base sequence in which the 421st to 423rd codons CTC of the base sequence shown in SEQ ID NO: 1 are replaced with TTC. The base sequence shown in SEQ ID NO: 13 is a base sequence in which the 388th to 390th codons CCT of the base sequence shown in SEQ ID NO: 11 (CDS encoding wild-type LZY3 protein) are replaced with TCT. The base sequence shown in SEQ ID NO: 15 is a base sequence in which the 391st to 393rd codons TTG of the base sequence shown in SEQ ID NO: 11 are replaced with TTC.
 本発明において、変異型qSOR1タンパク質をコードする遺伝子は、配列番号3、5、13又は15に示す塩基配列を含むものであってもよい。変異型qSOR1タンパク質をコードする遺伝子はまた、配列番号1若しくは11に示す塩基配列と40%以上、60%以上、70%以上、80%以上、90%以上、95%以上、96%以上、97%以上、98%以上、99%、又は99.5%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列を含むものであってもよい。変異型qSOR1タンパク質をコードする遺伝子はまた、配列番号1若しくは11に示す塩基配列において1~100個、1~50個、1~25個、1~10個、1~5個、1~3個、1~2個、又は1個の塩基の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列を含むものであってもよい。 In the present invention, the gene encoding the mutant qSOR1 protein may include the base sequence shown in SEQ ID NO: 3, 5, 13, or 15. The gene encoding the mutant qSOR1 protein also has 40% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more of the base sequence shown in SEQ ID NO: 1 or 11. % or more, 98% or more, 99%, or 99.5% or more, and contains a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. , and may contain a base sequence encoding a protein that exhibits an activity of improving deep rooting ability of plants. The gene encoding the mutant qSOR1 protein also has 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, and 1 to 3 in the base sequence shown in SEQ ID NO: 1 or 11. , 1 to 2, or 1 base insertion, deletion, substitution, and/or addition, and an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. It may also contain a nucleotide mutation that causes a nucleotide mutation and a base sequence that encodes a protein that exhibits the activity of improving deep rooting ability of plants.
 本発明において用いる植物は、限定されないが、典型的には被子植物である。植物は、一年草及び多年草のいずれであっても良く、また、単子葉植物及び双子葉植物のいずれであっても良い。単子葉植物としては、限定されないが、例えば、イネ科、ユリ科、パイナップル科、ヤシ科、サトイモ科、ショウガ科、ラン科等の植物が挙げられる。双子葉植物としては、限定されないが、ウリ科、アブラナ科、マメ科、キク科、シソ科、ナス科、バラ科、セリ科、ヒルガオ科、ハス科、ヤナギ科が挙げられる。 The plants used in the present invention are typically, but not limited to, angiosperms. The plants may be either annual plants or perennial plants, and may be monocotyledonous plants or dicotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, plants of the Poaceae, Liliaceae, Pineapple family, Arocaceae, Araceae, Zingiberaceae, Orchidaceae, and the like. Dicotyledonous plants include, but are not limited to, Cucurbitaceae, Brassicaceae, Fabaceae, Asteraceae, Lamiaceae, Solanaceae, Rosaceae, Apiaceae, Convolvulaceae, Lotus family, and Salicaceae.
 植物は、観賞用の花卉植物、食用の野菜・果物等の農作物であっても良い。観賞用の花卉植物としては、例えば、アサガオ、ヒマワリ、コスモス、スイートピー、マリーゴールド、パンジー、ビオラ、デイジー、キンギョソウ、ガーベラ、キキョウ、クレマチス、カンナ、シクラメン、キク、チューリップ、バラ、カーネーション、ペチュニア、カスミソウ、ユリ、ラン等が挙げられる。農作物としては、例えば、イネ、コムギ、オオムギ、ライムギ、エンバク、ハトムギ、トウモロコシ、キビ、アワ、ヒエ、ソルガム、シコクビエ、トウジンビ、テフ、サトウキビ、シロイヌナズナ、ナタネ、キャベツ、コマツナ、ダイコン、ハクサイ、ブロッコリー、ダイズ、インゲンマメ、ソラマメ、ネギ、ナタネ、キャベツ、レタス、タバコ、トマト、イチゴ、ナス、ニンジン、バレイショ、ワタ、タマネギ、ニンニク、ジャガイモ、サトイモ、ヤマイモ、サツマイモ、キュウリ、ハス、モモ等が挙げられる。植物はまた、ポプラ、プラタナス、シダレヤナギ、ニセアカシア、サクラ、アオギリ等の街路樹等として利用される植物であってもよい。 The plants may be agricultural crops such as ornamental flower plants and edible vegetables and fruits. Examples of ornamental flower plants include morning glory, sunflower, cosmos, sweet pea, marigold, pansy, viola, daisy, snapdragon, gerbera, bellflower, clematis, canna, cyclamen, chrysanthemum, tulip, rose, carnation, petunia, and gypsophila. , lily, orchid, etc. Examples of agricultural crops include rice, wheat, barley, rye, oats, adlay, corn, millet, millet, millet, millet, sorghum, finger millet, pearl millet, teff, sugar cane, Arabidopsis, rapeseed, cabbage, komatsuna, radish, Chinese cabbage, broccoli, Examples include soybeans, kidney beans, broad beans, green onions, rapeseed, cabbage, lettuce, tobacco, tomatoes, strawberries, eggplants, carrots, potatoes, cotton, onions, garlic, potatoes, taro, yams, sweet potatoes, cucumbers, lotus, peaches, and the like. The plants may also be plants used as street trees, such as poplars, plane trees, weeping willows, locusts, cherry blossoms, and sycamores.
 本発明において用いる植物は、好ましくは、イネ科、アブラナ科、又はマメ科の植物である。イネ科の植物としては、限定されないが、例えば、イネ、コムギ、オオムギ、ライムギ、エンバク、ハトムギ、トウモロコシ、キビ、アワ、ヒエ、ソルガム、シコクビエ、トウジンビ、テフ、サトウキビ、チモシー、ケンタッキーブルーグラス、オーチャードグラス、イタリアンライグラス、ペレニアルライグラス、トールフェスク、バヒアグラス、ミナトカモジグサなどが挙げられる。アブラナ科の植物としては、限定されないが、例えば、シロイヌナズナ、ナタネ、キャベツ、コマツナ、ダイコン、ハクサイ、ブロッコリー等が挙げられる。マメ科の植物としては、限定されないが、例えば、ダイズ、インゲンマメ、ソラマメ、アズキ、エンドウ、タルウマゴヤシ、ミヤコグサ等が挙げられる。 The plant used in the present invention is preferably a plant of the Poaceae family, Brassicaceae family, or Fabaceae family. Plants of the Poaceae family include, but are not limited to, rice, wheat, barley, rye, oat, adlay, corn, millet, millet, millet, sorghum, finger millet, pearl millet, teff, sugarcane, timothy, Kentucky bluegrass, and orchard. Grass, Italian ryegrass, perennial ryegrass, tall fescue, bahiagrass, and minato ryegrass. Examples of plants belonging to the Cruciferae family include, but are not limited to, Arabidopsis, rapeseed, cabbage, Japanese radish, Japanese radish, Chinese cabbage, broccoli, and the like. Examples of plants belonging to the leguminous family include, but are not limited to, soybean, kidney bean, fava bean, adzuki bean, pea, alfalfa, Lotus japonicus, and the like.
 本発明において用いる植物は、より好ましくはイネである。本明細書において、「イネ」とは、イネ科イネ属に属する任意の植物を意味する。イネには、栽培イネ及び野生イネが包含される。栽培イネとしては、アジアイネ(Oryza sativa)及びアフリカイネ(Oryza glaberrima)が挙げられ、アジアイネとしては、ジャポニカ種(Oryza sativa subsp. japonica)及びインディカ種(Oryza sativa subsp. indica)が挙げられる。ジャポニカ種の品種としては、例えば、コシヒカリ、とよめき、モミロマン、北陸193号、やまだわら、ササニシキが挙げられ、インディカ種の品種としては、例えばIR64が挙げられる。
 本明細書において、植物は、植物体の全体又はその一部(葉、茎、根、茎頂、葯、花粉、胚、カルス、細胞等)、種子等を包含する。
The plant used in the present invention is more preferably rice. As used herein, "rice" means any plant belonging to the genus Poaceae of the family Poaceae. Rice includes cultivated rice and wild rice. Examples of cultivated rice include Asian rice (Oryza sativa) and African rice (Oryza glaberrima), and examples of Asian rice include japonica (Oryza sativa subsp. japonica) and indica (Oryza sativa subsp. indica). Examples of Japonica varieties include Koshihikari, Toyomeki, Momiroman, Hokuriku 193, Yamadawara, and Sasanishiki, and examples of Indica varieties include IR64.
In the present specification, a plant includes the whole plant or a part thereof (leaves, stems, roots, shoot tips, anthers, pollen, embryos, callus, cells, etc.), seeds, and the like.
 植物は複数のゲノムを有する場合がある。例えば、イネの野生種には、ゲノムBとゲノムCの2種類のゲノムを有する異質4倍体等が存在する。本発明の植物は、少なくとも1つのゲノム上に上記変異型qSOR1遺伝子を有するものであってもよい。本発明の植物はまた、少なくとも1つのゲノム上に上記変異型qSOR1遺伝子をホモ接合性又はヘテロ接合性で有するものであってよい。 Plants may have multiple genomes. For example, wild species of rice include allotetraploids, which have two types of genomes, genome B and genome C. The plant of the present invention may have the mutant qSOR1 gene described above on at least one genome. The plant of the present invention may also have the above-mentioned mutant qSOR1 gene homozygous or heterozygous on at least one genome.
 一般に、植物のストレス耐性(干ばつ耐性等を含む)を向上させるために、植物の遺伝子に変異を導入した場合、収量や草型等に悪影響を及ぼす場合があるが、本発明者らは、植物のqSOR1遺伝子に上述の深根性を向上させるヌクレオチド変異を導入しても原品種に比べて収量が低下せず、むしろ増加する傾向があること、草型は変化しないことを見い出した。 Generally, when mutations are introduced into plant genes to improve stress tolerance (including drought tolerance, etc.) of plants, this may have an adverse effect on yield, plant type, etc. We found that even if we introduced the above-mentioned nucleotide mutation that improves deep rooting ability into the qSOR1 gene of the plant, the yield did not decrease compared to the original variety, but rather tended to increase, and the plant type did not change.
 したがって、本発明の植物は、草型が変化していないものであってもよい。本明細書において「草型が変化していない」とは、原品種又は原系統に比べて草型が変化していないことを意味する。本明細書において「草型」とは、茎や枝張りなどの特性によって規定される植物の地上部の概形を意味する。例えば、本発明の植物がイネである場合、草型は、イネの丈、穂の数、穂の長さ、穂軸の長さ、一穂当たりの一次枝梗の数、一穂当たりの二次枝梗の数、一穂当たりの種子の数等によって評価され得る。 Therefore, the plant of the present invention may have an unchanged grass type. As used herein, "the plant type has not changed" means that the plant type has not changed compared to the original variety or original line. As used herein, "grass type" refers to the general shape of the above-ground part of a plant, which is defined by characteristics such as stem and branching. For example, when the plant of the present invention is rice, the grass type includes the height of the rice, the number of panicles, the length of the panicle, the length of the cob, the number of primary branches per panicle, and the number of secondary branches per panicle. It can be evaluated by the number of stalks, the number of seeds per panicle, etc.
 本発明の植物は、特に栽培植物(作物)である場合には、収量が維持されているか又は増大しているものであってもよい。本明細書において「収量が維持されている」とは、原品種又は原系統に比べて収量が維持されている(例えば、収量が5%未満しか増大又は低下していない)ことを意味する。本明細書において「収量が増大している」とは、原品種又は原系統に比べて収量が増大している(例えば、収量が5%以上増大している)ことを意味する。例えば、本発明の植物がイネである場合、精籾乾物量を収量として測定することができる。 The plant of the present invention may have a maintained or increased yield, especially when it is a cultivated plant (crop). As used herein, "yield is maintained" means that the yield is maintained compared to the original variety or line (for example, the yield is increased or decreased by less than 5%). As used herein, "yield is increased" means that the yield is increased compared to the original variety or original line (for example, the yield is increased by 5% or more). For example, when the plant of the present invention is rice, the amount of refined rice dry matter can be measured as the yield.
(2)変異導入植物の作出方法
 本発明の植物は、例えば、植物のゲノム上のqSOR1遺伝子に上述の深根性を向上させるヌクレオチド変異を導入することによって作出することができる。したがって、本発明は、植物のqSOR1遺伝子に、深根性を向上させるヌクレオチド変異を導入する工程を含む、深根性が向上した植物(本発明の植物)を作出する方法(以下、「本発明の変異導入植物作出方法」とも称する)を提供する。
(2) Method for producing mutated plants The plants of the present invention can be produced, for example, by introducing the above-mentioned nucleotide mutations that improve deep rooting into the qSOR1 gene on the genome of the plant. Therefore, the present invention provides a method for producing a plant with improved deep rooting ability (plant of the present invention), which includes the step of introducing a nucleotide mutation that improves deep rooting ability into the qSOR1 gene of the plant (hereinafter referred to as ``the mutation of the present invention''). (also referred to as "method for producing introduced plants").
 上記の本発明の変異導入植物作出方法において、「植物」、「qSOR1遺伝子」、「深根性を向上させるヌクレオチド変異」、「深根性が向上した」等の用語は、本発明の植物についての説明において定義した通りである。上記の本発明の変異導入植物作出方法において、変異を導入する対象の植物は、qSOR1遺伝子に深根性を向上させるヌクレオチド変異を有しない植物(例えば、野生型植物)であってもよく、qSOR1遺伝子に深根性を向上させるヌクレオチド変異を有する植物であってもよい。植物は、例えば単子葉植物又は双子葉植物、例えばイネ科又はアブラナ科、例えばイネであり得る。本発明の変異導入植物作出方法において、深根性を向上させるヌクレオチド変異は、例えば、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異、例えば、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリンのセリンへの置換、又は配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンのフェニルアラニンへの置換を引き起こすヌクレオチド変異であり得る。本発明の変異導入植物作出方法において、深根性を向上させるヌクレオチド変異を導入することにより生成される変異型qSOR1遺伝子は、例えば、
(i) 配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質、
(ii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質、又は
(iii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列において1~10個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質
をコードしていてもよい。
In the above-mentioned method for producing a mutated plant of the present invention, terms such as "plant,""qSOR1gene,""nucleotide mutation that improves deep rooting ability," and "improved deep rooting ability" are used to describe the plant of the present invention. As defined in . In the above-mentioned method for producing a mutated plant of the present invention, the target plant to which the mutation is introduced may be a plant (for example, a wild-type plant) that does not have a nucleotide mutation that improves deep rooting in the qSOR1 gene, or It may also be a plant that has a nucleotide mutation that improves deep rooting ability. The plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice. In the method for producing mutagenized plants of the present invention, the nucleotide mutation that improves deep rooting ability is, for example, a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, for example, A nucleotide mutation that causes the substitution of proline with serine at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2, or the substitution of leucine with phenylalanine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO: 2. obtain. In the method for producing mutagenized plants of the present invention, the mutant qSOR1 gene produced by introducing a nucleotide mutation that improves deep rooting ability is, for example,
(i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16,
(ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or
(iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It may consist of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No. 2, and may encode a protein that exhibits an activity for improving deep rooting ability of plants.
 上記の本発明の変異導入植物作出方法において、深根性を向上させるヌクレオチド変異の導入は、例えば、植物のゲノムにランダム変異を導入し、得られた変異体の中から深根性を向上させるヌクレオチド変異が導入され又は集積した変異体を選抜することにより行うことができる。ランダム変異は、例えば、x線、γ線等の放射線の照射、変異原性化学物質(ニトロソ化合物(例えば、ニトロソグアニジン)、塩基類似化合物(例えば、ブロモデオキシウリジン)、アルキル化剤(例えば、エチルニトロソウレア(ENU)、メタンスルホン酸エチル(EMS))等)による処理等によって導入することができる。 In the above-mentioned method for producing a mutated plant of the present invention, the introduction of nucleotide mutations that improve deep rooting ability can be carried out by, for example, introducing random mutations into the genome of the plant, and selecting nucleotide mutations that improve deep rooting ability from among the resulting mutants. This can be carried out by selecting mutants that have introduced or accumulated . Random mutations can be caused by, for example, irradiation with radiation such as x-rays or gamma rays, mutagenic chemicals (nitroso compounds (e.g., nitrosoguanidine), base-like compounds (e.g., bromodeoxyuridine), alkylating agents (e.g., ethyl It can be introduced by treatment with nitrosourea (ENU), ethyl methanesulfonate (EMS), etc.).
 深根性を向上させるヌクレオチド変異の導入はまた、植物のゲノムに部位特異的変異を導入することにより行うこともできる。部位特異的変異は、例えば、Gateway(R)法などの相同組換えに基づく部位特異的変異誘発、PCRをベースにした部位特異的変異導入法、又はTranscription activator-like effector nuclease(TALEN)(特表2012-514976号公報、特表2013-513389号公報)、ジンクフィンガーヌクレアーゼ(特許第4350907号公報、特許第4555292号公報)、CRISPR/Cas9(Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337, 816-821.(2012))等を用いたゲノム編集技術などの様々な部位特異的変異誘発技術によって行うことができる。 Introduction of nucleotide mutations that improve deep rooting ability can also be carried out by introducing site-specific mutations into the plant genome. Site-directed mutagenesis can be performed using, for example, site-directed mutagenesis based on homologous recombination such as the Gateway (R) method, site-directed mutagenesis based on PCR, or transcription activator-like effector nuclease (TALEN) (specifically Table 2012-514976 Publication, Special Table 2013-513389 Publication), Zinc Finger Nuclease (Patent No. 4350907, Patent No. 4555292), CRISPR/Cas9 (Jinek et al. A programmable dual-RNA-guided DNA endonuclease) This can be done by various site-directed mutagenesis techniques such as genome editing techniques using in adaptive bacterial immunity. Science 337, 816-821. (2012)).
 上記の本発明の変異導入植物作出方法は、上記の深根性を向上させるヌクレオチド変異が導入され又は集積した変異体の選抜を行う工程を含んでもよい。変異体の選抜は、例えば、qSOR1遺伝子の塩基配列を決定し、決定した塩基配列中に上記の深根性を向上させるヌクレオチド変異が存在する変異体を選抜することによって行うことができる。 The above-mentioned method for producing a mutated plant of the present invention may include the step of selecting a mutant in which the above-mentioned nucleotide mutation that improves deep rooting ability has been introduced or accumulated. Selection of mutants can be carried out, for example, by determining the base sequence of the qSOR1 gene and selecting mutants in which the determined base sequence contains the above-mentioned nucleotide mutation that improves deep-rootedness.
(3)形質転換植物の作出方法
 本発明の植物はまた、形質転換によって作出することもできる。したがって、本発明は、深根性を向上させるアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を含むベクターを植物に導入する工程を含む、深根性が向上した植物(本発明の植物)を作出する方法(以下、「本発明の形質転換植物作出方法」とも称する)を提供する。本発明はまた、そのような形質転換植物作出方法に使用される上記遺伝子及びベクターも提供する。
 上記の本発明の形質転換植物作出方法において、「深根性を向上させるアミノ酸置換」、「変異型qSOR1タンパク質」、「遺伝子」、「深根性が向上した」、「植物」等の用語は、本発明の植物についての説明において定義した通りである。上記の本発明の形質転換植物作出方法において、ベクターを導入する対象の植物は、ベクターが導入されていない植物(例えば、野生型植物)であってもよい。植物は、例えば単子葉植物又は双子葉植物、例えばイネ科又はアブラナ科、例えばイネであり得る。本発明の形質転換植物作出方法において、深根性を向上させるアミノ酸置換は、例えば、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中のアミノ酸置換、例えば、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリンのセリンへの置換、又は配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンのフェニルアラニンへの置換であり得る。
 上記の本発明の形質転換植物作出方法において、変異型qSOR1タンパク質は、
(i) 配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質、
(ii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質、又は
(iii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列において1~10個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質
であり得る。
 上記の本発明の形質転換植物作出方法において、ベクターには、上記の変異型qSOR1遺伝子に加えて選択マーカー遺伝子を適宜組み込むことができる。
(3) Method for producing transformed plants The plants of the present invention can also be produced by transformation. Therefore, the present invention involves the step of introducing into the plant a vector containing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability, to create a plant with improved deep rooting ability (plant of the present invention). A method (hereinafter also referred to as "method for producing a transformed plant of the present invention") is provided. The present invention also provides the above genes and vectors used in such methods for producing transformed plants.
In the above-mentioned method for producing a transformed plant of the present invention, terms such as "amino acid substitution that improves deep rooting", "mutant qSOR1 protein", "gene", "improved deep rooting", and "plant" are used herein. As defined in the description of the plants of the invention. In the above-described method for producing a transformed plant of the present invention, the target plant into which the vector is introduced may be a plant into which the vector has not been introduced (for example, a wild-type plant). The plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice. In the method for producing a transformed plant of the present invention, amino acid substitutions that improve deep rooting ability include, for example, amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; It may be a substitution of proline at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2 with serine, or a substitution of leucine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO: 2 with phenylalanine.
In the above method for producing a transformed plant of the present invention, the mutant qSOR1 protein is
(i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16,
(ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or
(iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It can be a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No. 2, and exhibiting an activity for improving deep rooting ability of plants.
In the above-mentioned method for producing a transformed plant of the present invention, a selection marker gene can be appropriately incorporated into the vector in addition to the above-mentioned mutant qSOR1 gene.
 植物へのベクターの導入は、当分野において通常行われている技術のいずれを用いても良く、例えばアグロバクテリウム法、パーティクルガン法、エレクトロポレーション法等を好適に使用することができる。ベクターの導入のために用いる植物は、植物体、植物器官、植物組織片を使用しても良く、またカルスやプロトプラストを調製して使用しても良い。 The vector may be introduced into plants using any technique commonly used in the art, such as the Agrobacterium method, particle gun method, electroporation method, etc. The plant used for vector introduction may be a plant body, a plant organ, or a piece of plant tissue, or a callus or protoplast may be prepared and used.
 上記の本発明の形質転換植物作出方法は、上記のベクターが導入された形質転換植物の選抜を行う工程を含んでもよい。 The above method for producing a transformed plant of the present invention may include a step of selecting a transformed plant into which the above vector has been introduced.
 ベクターが導入された植物は、例えばベクター中に組み込まれた選択マーカー遺伝子の発現の有無を利用して選抜することができる。選択マーカー遺伝子としては、特に限定するものではないが、例えば当分野において通常使用される抗生物質耐性遺伝子を好適に使用することができる。好適に使用され得る抗生物質耐性遺伝子としては、限定するものではないが、例えばカナマイシン耐性遺伝子、ネオマイシン耐性遺伝子、アンピシリン耐性遺伝子、ハイグロマイシン耐性遺伝子等が挙げられる。 Plants into which the vector has been introduced can be selected, for example, based on the presence or absence of expression of the selectable marker gene incorporated into the vector. The selection marker gene is not particularly limited, but for example, antibiotic resistance genes commonly used in the art can be suitably used. Examples of antibiotic resistance genes that can be suitably used include, but are not limited to, kanamycin resistance genes, neomycin resistance genes, ampicillin resistance genes, hygromycin resistance genes, and the like.
 植物へのベクターの導入はまた、PCR法、サザンハイブリダイゼーション法、ノーザンハイブリダイゼーション法、ウェスタンブロッティング法等によって確認することができる。 Introduction of the vector into plants can also be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, etc.
(4)交配による深根性が向上した植物の作出方法
 本発明はまた、本発明の植物を育種親として用いて植物の交配を行い、子孫植物を取得する工程、及び上記変異型qSOR1タンパク質をコードする遺伝子が導入された子孫植物を選抜する工程を含む、深根性が向上した植物(本発明の植物)を作出する方法(「本発明の育種方法」とも称する。)を提供する。
(4) Method for producing plants with improved deep rooting ability by hybridization The present invention also includes a step of hybridizing plants using the plants of the present invention as breeding parents to obtain progeny plants, and a step encoding the mutant qSOR1 protein. To provide a method for producing a plant (plant of the present invention) with improved deep rooting ability (also referred to as "breeding method of the present invention"), which includes the step of selecting progeny plants into which a gene has been introduced.
 本発明の植物を「育種親として用いて植物の交配を行う」とは、本発明の植物同士、又は、本発明の植物と同種又は近縁種の植物とを交配することを指す。交配は1回でもよいし、繰り返し行ってもよい。例えば、本発明の植物を同種又は近縁種の植物(反復親)と交配し、その子孫植物を反復親と交配し(戻し交配)、その子孫植物をさらに反復親と交配することを繰り返してもよい(連続戻し交配)。あるいは、本発明の植物を同種又は近縁種の植物と交配し、その子孫植物を別の同種又は近縁種の植物と交配してもよい。 "Mating plants using the plants of the present invention as breeding parents" refers to breeding the plants of the present invention with each other, or the plants of the present invention with plants of the same or closely related species. Mating may be carried out once or repeatedly. For example, a plant of the present invention may be crossed with a plant of the same or closely related species (recurrent parent), the progeny plant may be crossed with the recurrent parent (backcrossing), and the progeny plant may be further crossed with the recurrent parent, which are repeated. Good (continuous backcrossing). Alternatively, the plants of the present invention may be crossed with plants of the same or related species, and the progeny plants may be crossed with other plants of the same or related species.
 上記変異型qSOR1タンパク質をコードする遺伝子が導入された子孫植物の選抜は、本発明の変異導入植物作出方法及び形質転換植物作出方法に関して記載した方法により行うことができる。 Selection of progeny plants into which the gene encoding the mutant qSOR1 protein has been introduced can be carried out by the method described for the method for producing a mutated plant and the method for producing a transformed plant of the present invention.
(5)深根性が向上した植物の選抜方法
 本発明はまた、被験植物由来のDNAを鋳型としてqSOR1遺伝子の全体又はその一部について核酸増幅を行う工程、核酸増幅の結果に基づいて、深根性を向上させるアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を有する植物を同定する工程を含む、深根性が向上した植物を選抜する方法(「本発明の選抜方法」とも称する)を提供する。
(5) Method for selecting plants with improved deep rooting ability Provided is a method for selecting plants with improved deep rooting ability (also referred to as the "selection method of the present invention"), which includes the step of identifying a plant that has a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves.
 本発明の選抜方法において、「被験植物」とは、本発明の選抜方法に供される植物を意味する。本発明の選抜方法において、「植物」とは、上記の本発明の植物についての説明において定義した通りである。植物は、例えば単子葉植物又は双子葉植物、例えばイネ科又はアブラナ科、例えばイネであり得る。本発明の選抜方法において、被験植物は、例えば、植物にランダム変異を導入して得られた変異体、本発明の植物を育種親として用いて植物の交配を行うことにより得られた子孫植物等であり得る。本発明の選抜方法において、「DNA」、「qSOR1遺伝子」、「深根性を向上させるアミノ酸置換」、「変異型qSOR1タンパク質」、「遺伝子」、「深根性が向上した」等の用語も、上記の本発明の植物についての説明において定義した通りである。本発明の選抜方法において、深根性を向上させるアミノ酸置換は、例えば、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中のアミノ酸置換、例えば、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリンのセリンへの置換、又は配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンのフェニルアラニンへの置換であり得る。
 上記の本発明の形質転換植物作出方法において、変異型qSOR1タンパク質は、
(i) 配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質、
(ii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質、又は
(iii) 配列番号2、12、17、18、及び24~31のいずれか1つに示すアミノ酸配列において1~10個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質
であり得る。
In the selection method of the present invention, the "test plant" means a plant that is subjected to the selection method of the present invention. In the selection method of the present invention, the "plant" is as defined in the above description of the plant of the present invention. The plant may be, for example, a monocot or a dicotyledon, such as a member of the Poaceae or Brassicaceae family, such as rice. In the selection method of the present invention, test plants include, for example, mutants obtained by introducing random mutations into plants, progeny plants obtained by crossbreeding plants using the plants of the present invention as breeding parents, etc. It can be. In the selection method of the present invention, terms such as "DNA", "qSOR1 gene", "amino acid substitution that improves deep rooting ability", "mutant qSOR1 protein", "gene", "improved deep rooting ability", etc. as defined in the description of the plants of the present invention. In the selection method of the present invention, amino acid substitutions that improve deep rooting ability include, for example, amino acid substitutions in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; This may be a substitution of proline with serine at the position corresponding to position 140 of the amino acid sequence shown in SEQ ID NO: 2, or a substitution of phenylalanine with leucine at the position corresponding to position 141 of the amino acid sequence shown in SEQ ID NO:2.
In the above method for producing a transformed plant of the present invention, the mutant qSOR1 protein is
(i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16,
(ii) having 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 12, 17, 18, and 24 to 31, and positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; or
(iii) has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31; It can be a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in No. 2, and exhibiting an activity for improving deep rooting ability of plants.
 本発明において、核酸増幅技術としては、ポリメラーゼ連鎖反応(PCR)法、ランプ(Loop-Mediated Isothermal Amplification; LAMP)法、TMA(Transcription Mediated Amplification)法、NASBA(Nucleic Acid Sequence-Based Amplification)法、LCR(Ligase Chain Reaction)法等の任意の方法を用いることができる。 In the present invention, nucleic acid amplification techniques include polymerase chain reaction (PCR) method, LAMP (Loop-Mediated Isothermal Amplification) method, TMA (Transcription Mediated Amplification) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, LCR Any method such as the (Ligase Chain Reaction) method can be used.
 本発明の選抜方法において、核酸増幅の鋳型は、被験植物由来のゲノムDNA又はcDNAであり得る。本発明の選抜方法において、核酸増幅に用いるプライマーは、15塩基長以上若しくは20塩基長以上であってよく、50塩基長以下若しくは30塩基長以下であってよい。上記プライマーはまた、例えば、15~50塩基長、20~50塩基長、若しくは20~30塩基長であってもよい。 In the selection method of the present invention, the template for nucleic acid amplification may be genomic DNA or cDNA derived from a test plant. In the selection method of the present invention, the primer used for nucleic acid amplification may have a length of 15 bases or more, or 20 bases or more, and may have a length of 50 bases or less or 30 bases or less. The primers may also be, for example, 15-50 bases long, 20-50 bases long, or 20-30 bases long.
 本発明の選抜方法において、「qSOR1遺伝子の一部」は、qSOR1タンパク質のドメインIIIをコードする塩基配列(配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列)を含む領域であればよい。 In the selection method of the present invention, "part of the qSOR1 gene" is a region containing the nucleotide sequence encoding domain III of the qSOR1 protein (sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2). Good to have.
 本発明の選抜方法は、例えば、サンガー法等によるダイレクトシーケンス、HRM(High Resolution Melting)法、KASPTM(Kompetitive Allele Specific PCR)ジェノタイピングアッセイ(LGC Biosearch Technologies)、dCAPS(derived amplified polymorphic sequence)法等を用いて行うことができる。 The selection method of the present invention includes, for example, direct sequencing using the Sanger method, HRM (High Resolution Melting) method, KASP TM (Kompetitive Allele Specific PCR) genotyping assay (LGC Biosearch Technologies), dCAPS (derived amplified polymorphic sequence) method, etc. This can be done using
 本発明の選抜方法は、被験植物の根伸長角度、深根率又は重力屈性を評価する工程をさらに含んでもよい。根伸長角度、深根率又は重力屈性を評価する方法については、本発明の植物に関して記載した通りである。 The selection method of the present invention may further include the step of evaluating the root elongation angle, deep root ratio, or gravitropism of the test plant. The methods for evaluating root elongation angle, deep root ratio, or gravitropism are as described for the plants of the present invention.
 本発明の選抜方法の一実施形態では、核酸増幅は、被験植物由来のゲノムDNAを鋳型として、
(i) 配列番号19に示す塩基配列上の連続した少なくとも15塩基の塩基配列を含むフォワードプライマー、及び
(ii) 配列番号20又は21に示す塩基配列上の連続した少なくとも15塩基の塩基配列を含むリバースプライマー
を含むプライマーセットを用いて行ってもよい。
In one embodiment of the selection method of the present invention, nucleic acid amplification is performed using genomic DNA derived from a test plant as a template.
(i) A forward primer containing a base sequence of at least 15 consecutive bases on the base sequence shown in SEQ ID NO: 19, and
(ii) A primer set including a reverse primer containing at least 15 consecutive bases of the base sequence shown in SEQ ID NO: 20 or 21 may be used.
 上記のプライマーセットは、例えば、
(i) 配列番号19に示す塩基配列を含むフォワードプライマー、及び
(ii) 配列番号20又は21に示す塩基配列を含むリバースプライマー
を含むものであり得る。
The above primer set is, for example,
(i) A forward primer containing the base sequence shown in SEQ ID NO: 19, and
(ii) It may contain a reverse primer containing the base sequence shown in SEQ ID NO: 20 or 21.
 配列番号19に示す塩基配列は、イネqSOR1遺伝子の第2イントロン上の塩基配列(配列番号23の塩基配列の629番目~652番目)に対応し、配列番号20及び21に示す塩基配列は、イネqSOR1遺伝子の第3イントロン上の塩基配列(それぞれ、配列番号23の塩基配列の1453番目~1478番目、1301~1327番目)に対応する。したがって、上記プライマーセットを用いて核酸増幅を行うことにより、第3エクソンを含む領域(qSOR1タンパク質のドメインIIIをコードする領域を含む)が増幅される。 The nucleotide sequence shown in SEQ ID NO: 19 corresponds to the nucleotide sequence on the second intron of the rice qSOR1 gene (positions 629 to 652 of the nucleotide sequence in SEQ ID NO: 23), and the nucleotide sequences shown in SEQ ID NO: 20 and 21 correspond to the nucleotide sequence on the second intron of the rice qSOR1 gene. Corresponds to the nucleotide sequence on the third intron of the qSOR1 gene (respectively, positions 1453 to 1478 and 1301 to 1327 of the base sequence of SEQ ID NO: 23). Therefore, by performing nucleic acid amplification using the above primer set, the region including the third exon (including the region encoding domain III of the qSOR1 protein) is amplified.
 上記プライマーセットを用いて核酸増幅後、サンガー法等により増幅産物の塩基配列を決定することにより、qSOR1遺伝子に深根性を向上させるヌクレオチド変異が存在するか否か判定することができる。qSOR1遺伝子に深根性を向上させるヌクレオチド変異が存在すると判定された植物を、深根性を向上させるアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を有する植物として同定することができる。 After nucleic acid amplification using the above primer set, the nucleotide sequence of the amplified product is determined by the Sanger method or the like to determine whether or not the qSOR1 gene has a nucleotide mutation that improves deep rootability. A plant determined to have a nucleotide mutation that improves deep rooting ability in the qSOR1 gene can be identified as a plant that has a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability.
 本発明の選抜方法において核酸増幅に用いるプライマーは、上記で規定した塩基配列を3'末端に含むことが好ましい。 The primer used for nucleic acid amplification in the selection method of the present invention preferably contains the base sequence defined above at its 3' end.
 本発明の選抜方法は、核酸増幅を行う工程の前に、被験植物を用意する工程を含んでいてもよい。被験植物を用意する工程は、例えば、植物のqSOR1遺伝子に深根性を向上させるヌクレオチド変異を導入して被験植物とする工程、深根性を向上させるアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を含むベクターを植物に導入して被験植物とする工程、又は本発明の植物を育種親として用いて植物の交配を行い、子孫植物を被験植物として取得する工程であり得る。これらの工程は、「(2)変異導入植物の作出方法」、「(3)形質転換植物の作出方法」、「(4)交配による深根性が向上した植物の作出方法」に記載の通りに行うことができる。 The selection method of the present invention may include a step of preparing a test plant before the step of performing nucleic acid amplification. The step of preparing a test plant includes, for example, a step of introducing a nucleotide mutation that improves deep rooting ability into the qSOR1 gene of the plant to make it a test plant, and a step of introducing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability. This may be a step of introducing a vector containing the vector into a plant to obtain a test plant, or a step of cross-breeding the plants using the plant of the present invention as a breeding parent to obtain a progeny plant as a test plant. These steps were performed as described in "(2) Method for producing mutated plants," "(3) Method for producing transformed plants," and "(4) Method for producing plants with improved deep rooting ability by hybridization." It can be carried out.
 本発明の選抜方法は、例えば、本発明の変異導入植物作出方法、形質転換植物作出方法又は育種方法において使用することができる。 The selection method of the present invention can be used, for example, in the method of producing a mutated plant, the method of producing a transformed plant, or the breeding method of the present invention.
 以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.
<実施例1.イネ突然変異系統からのqSOR1突然変異系統の同定>
 イネ突然変異系統から、qSOR1遺伝子中に非同義置換を有する系統を探索した。
 イネ(Oryza sativa)品種コシヒカリを受精卵法によりエチルニトロソウレア(ENU)で処理することによって得られたイネ突然変異系統(M1世代、n=3,072)のゲノムDNAを鋳型とし、フォワードプライマーqSOR1-p1-F1(5’- tggatatattttgcatggtttttg -3’)(配列番号19)及びリバースプライマーqSOR1-p1-R1(5’- caacatctacgacgtcaaattagtct -3’)(配列番号20)を用いて、qSOR1遺伝子(Os07g0614400)のエクソン3を挟む領域をポリメラーゼ連鎖反応(polymerase chain reaction;PCR)法により増幅した。PCR法による増幅には、Prime STAR GXL DNA polymerase(タカラバイオ)を使用した。
<Example 1. Identification of qSOR1 mutant lines from rice mutant lines>
Among the rice mutant lines, we searched for lines with nonsynonymous substitutions in the qSOR1 gene.
Genomic DNA of a rice mutant line (M1 generation, n=3,072) obtained by treating rice (Oryza sativa) cultivar Koshihikari with ethylnitrosourea (ENU) using the fertilized egg method was used as a template, and the forward primer qSOR1-p1 was used as a template. -F1 (5'- tggatatattttgcatggtttttg -3') (SEQ ID NO: 19) and reverse primer qSOR1-p1-R1 (5'- caacatctacgacgtcaaattagtct -3') (SEQ ID NO: 20) were used to target exon 3 of the qSOR1 gene (Os07g0614400). The region between the two was amplified by polymerase chain reaction (PCR). Prime STAR GXL DNA polymerase (Takara Bio) was used for amplification by PCR method.
 得られたPCR産物を98℃に加熱後冷却して二本鎖DNAを解離・再会合させた後、セロリより抽出したCel-Iヌクレアーゼで処理し、アガロース電気泳動した。Cel-Iヌクレアーゼは、二本鎖DNAをミスマッチ部位で切断するため、上記アガロース電気泳動において切断断片が検出された系統は、qSOR1遺伝子に置換変異を有する系統であると判断することができる。 The obtained PCR product was heated to 98°C and cooled to dissociate and reassociate the double-stranded DNA, and then treated with Cel-I nuclease extracted from celery and subjected to agarose electrophoresis. Since Cel-I nuclease cleaves double-stranded DNA at mismatch sites, strains in which cleavage fragments were detected in the agarose electrophoresis described above can be determined to be strains with substitution mutations in the qSOR1 gene.
 上記アガロース電気泳動において切断断片が検出された系統について、さらに、フォワードプライマーqSOR1-p1-F1(5’- tggatatattttgcatggtttttg -3’)(配列番号19)及びリバースプライマーqSOR1-p1-R2(5’- gaaatggagtgagtagatgataacttg -3’)(配列番号21)を用いてqSOR1遺伝子の塩基配列を決定した。決定した塩基配列を解析した結果、qSOR1遺伝子中に非同義置換を有する系統が4つ見つかった。これら4つのqSOR1突然変異系統(系統名0951M、2792M、0909M及び2574M)について決定したqSOR1遺伝子のCDSを、それぞれ配列番号3、5、7、9に示す。また、これらにコードされるアミノ酸配列を、それぞれ配列番号4、6、8、10に示す。これら4つのqSOR1突然変異系統のqSOR1遺伝子のCDSは、原品種(コシヒカリ)のqSOR1遺伝子のCDS(配列番号2)との比較の結果、以下の変異を有することが明らかとなった(図3及び4参照)。系統名0951M; 140番目のプロリンのセリンへの置換(P140S)をもたらす突然変異(ミスセンス変異)が生じている。系統名2792M; 141番目のロイシンのフェニルアラニンへの置換(L141F)をもたらす突然変異が生じている。系統名0909M; 204番目のアルギニンのシステインへの置換(R204C)をもたらす突然変異が生じている。系統名2574M; 204番目のアルギニンのヒスチジンへの置換(R204H)をもたらす突然変異が生じている。ここで、上記アミノ酸置換の位置は、配列番号2のアミノ酸配列に従って付番される。以下、実施例1~5において、同様である。 For the strains in which cleavage fragments were detected in the above agarose electrophoresis, forward primer qSOR1-p1-F1 (5'- tggatatattttgcatggtttttg -3') (SEQ ID NO: 19) and reverse primer qSOR1-p1-R2 (5'- gaaatggagtgagtagatgataacttg -3') (SEQ ID NO: 21) to determine the nucleotide sequence of the qSOR1 gene. Analysis of the determined nucleotide sequences revealed four strains with non-synonymous substitutions in the qSOR1 gene. The CDS of the qSOR1 gene determined for these four qSOR1 mutant lines ( strain names 0951M, 2792M, 0909M, and 2574M) are shown in SEQ ID NOs: 3, 5, 7, and 9, respectively. Furthermore, the amino acid sequences encoded by these are shown in SEQ ID NOs: 4, 6, 8, and 10, respectively. As a result of comparing the CDS of the qSOR1 gene of these four qSOR1 mutant lines with the CDS of the qSOR1 gene of the original variety (Koshihikari) (SEQ ID NO: 2), it was revealed that they had the following mutations (Figure 3 and (see 4). Strain name 0951M; A mutation (missense mutation) resulting in the substitution of proline at position 140 with serine (P140S) has occurred. Strain name 2792M; A mutation resulting in the substitution of leucine at position 141 with phenylalanine (L141F) has occurred. Strain name 0909M; A mutation has occurred that results in the substitution of cysteine for arginine at position 204 (R204C). Strain name 2574M; A mutation resulting in the substitution of arginine at position 204 with histidine (R204H) has occurred. Here, the positions of the above amino acid substitutions are numbered according to the amino acid sequence of SEQ ID NO: 2. The same applies to Examples 1 to 5 below.
 I-TASSERによるqSOR1タンパク質の立体構造予測を行ったところ、204番目のアルギニンは17番目のアスパラギン酸及び226番目のグルタミン酸と塩架橋を形成しており、qSOR1タンパク質の立体構造の安定化に寄与している可能性があることが予想された。特に、204番目のアミノ酸が塩基性アミノ酸であるアルギニンから中性アミノ酸であるシステインに置換されている系統名0909Mは、原品種と比較して根の表現型に変化を示すことが予想された。一方、P140S及びL141Fのアミノ酸置換はqSOR1タンパク質の構造に変化を及ぼすものではなく、根の表現型には影響はしないことが予想された。 When predicting the three-dimensional structure of the qSOR1 protein using I-TASSER, the arginine at position 204 forms a salt bridge with aspartic acid at position 17 and glutamic acid at position 226, which contributes to stabilizing the three-dimensional structure of the qSOR1 protein. It was expected that there might be a possibility that In particular, strain name 0909M, in which the basic amino acid arginine was replaced with the neutral amino acid cysteine at position 204, was expected to show a change in root phenotype compared to the original variety. On the other hand, the amino acid substitutions of P140S and L141F did not change the structure of the qSOR1 protein and were not expected to affect the root phenotype.
 イネ(コシヒカリ)qSOR1遺伝子、並びに遺伝子単離された相同遺伝子であるシロイヌナズナLZY2遺伝子、シロイヌナズナLZY3遺伝子、及びタルウマゴヤシNGR遺伝子によりコードされるアミノ酸配列(それぞれ、配列番号2、17、12、18)を比較したところ、140番目のプロリン及び141番目のロイシンは、異種間でアミノ酸配列が高く保存されているドメインIIIに存在した。一方、204番目のアルギニンはイネ及びタルウマゴヤシでは保存されていたが、シロイヌナズナでは異なるアミノ酸であった(図5)。 The amino acid sequences (SEQ ID NOs: 2, 17, 12, 18, respectively) encoded by the rice (Koshihikari) qSOR1 gene and the isolated homologous genes, the Arabidopsis LZY2 gene, the Arabidopsis LZY3 gene, and the Taruuma alfalfa NGR gene. As a result of comparison, proline at position 140 and leucine at position 141 were found to be present in domain III, whose amino acid sequence is highly conserved among different species. On the other hand, arginine at position 204 was conserved in rice and alfalfa, but it was a different amino acid in Arabidopsis (Figure 5).
<実施例2.qSOR1突然変異系統の根の表現型>
 実施例1で得られたqSOR1突然変異系統の根伸長角度が原品種(コシヒカリ)と比較して変化しているかどうかを調べるため、カップ法により根伸長角度を測定した。
<Example 2. Root phenotype of qSOR1 mutant line>
In order to investigate whether the root elongation angle of the qSOR1 mutant line obtained in Example 1 had changed compared to the original variety (Koshihikari), the root elongation angle was measured by the cup method.
 底にドリルで給水用の穴をあけた直径3.7 cm、高さ4 cmのプラスチック製のカップに合成粒状培土を擦切りいっぱいまで入れ、そこに消毒した原品種又はqSOR1突然変異系統(M3世代、qSOR1遺伝子突然変異についてホモ接合性)の種子を播種した。種子を播種したカップをステンレス製のトレーに置き、カップ全体が隠れるように無肥料培土を敷き詰め、約3週間程度栽培した。原品種と4つのqSOR1突然変異系統の各々について20個体栽培した。栽培期間終了後、カップから植物を取り出し、根を洗い流して根伸長角度を分度器で測定した。 Fill a plastic cup with a diameter of 3.7 cm and a height of 4 cm with a hole drilled in the bottom for water supply, and fill it with synthetic granular soil until it reaches its full capacity. Seeds homozygous for the gene mutation were sown. The cup containing the seeds was placed on a stainless steel tray, and fertilizer-free soil was spread over the cup so that the entire cup was covered, and the seeds were cultivated for about 3 weeks. Twenty individuals of each of the original variety and four qSOR1 mutant lines were grown. At the end of the cultivation period, the plants were removed from the cups, the roots were washed away, and the root elongation angle was measured using a protractor.
 結果を図6に示す。実施例1での予想の通り、系統名0909Mの根伸長角度は原品種と比較して有意に低下していた。また、系統名2574Mの根伸長角度は原品種と同程度であった。一方、表現型への影響はないと考えていた系統名0951Mと系統名2792Mの根伸長角度は、原品種と比較して有意に増大していた。 The results are shown in Figure 6. As expected in Example 1, the root elongation angle of strain 0909M was significantly lower than that of the original variety. In addition, the root elongation angle of strain 2574M was similar to that of the original variety. On the other hand, the root elongation angles of line 0951M and line 2792M, which were thought to have no effect on the phenotype, were significantly increased compared to the original variety.
 これらの結果から、イネqSOR1アミノ酸配列におけるR204C置換はイネの根を浅くする作用を有すること、P140S置換及びL141F置換(ドメインIIIにおける置換)は、意外なことに、イネの根を深くする作用を有することが示唆された。 These results show that the R204C substitution in the rice qSOR1 amino acid sequence has the effect of shallowing rice roots, and that the P140S and L141F substitutions (substitutions in domain III) surprisingly have the effect of deepening rice roots. It was suggested that the
<実施例3.qSOR1遺伝子内の突然変異の根の表現型に対する効果>
 上記のカップ法に用いたqSOR1突然変異系統は、変異原処理により作製されたものであるため、上記の突然変異以外の突然変異をゲノム中に有している可能性もある。そこで、カップ法で見出された根の表現型の変化がqSOR1遺伝子内の突然変異によって引き起こされたものであることをさらに証明すべく、以下の実験を行った。
<Example 3. Effect of mutations in the qSOR1 gene on root phenotype>
Since the qSOR1 mutant strain used in the cup method described above was created by mutagen treatment, it is possible that it has mutations other than the above mutations in its genome. Therefore, in order to further prove that the changes in root phenotype found by the cup method were caused by mutations in the qSOR1 gene, we conducted the following experiment.
 実施例2で根伸長角度に変化のあったqSOR1突然変異系統(系統名0909M、0951M及び2792M)をそれぞれ原品種に3回戻し交配し、自殖した。得られた3つのBC3F3系統(コシヒカリ背景の準同質遺伝子系統)が上記のqSOR1遺伝子内の突然変異を維持していることをダイレクトシークエンスにより確認した。 The qSOR1 mutant lines ( strain names 0909M, 0951M, and 2792M) with changes in root elongation angle in Example 2 were each backcrossed to the original variety three times and selfed. It was confirmed by direct sequencing that the three BC3F3 lines obtained (quasi-isogenic lines with Koshihikari background) maintained the above-mentioned mutation in the qSOR1 gene.
 これら3つのBC3F3系統の根の表現型を、ステンレス製のメッシュ状のザルを用いた改良型バスケット法により調査した。直径7.5 cmのステンレス製の特注ザルに無肥料培土を充填し、消毒した原品種又はBC3F3系統の種子を播種し、水耕液中で約1か月半程度栽培した。栽培期間終了後、地表面に対して30度を超える角度で下に向かって伸長した根を深根とし、深根の数と総根数を測定した。深根の数を総根数で割った値を深根率(RDR30; Ratio of Deeper Root than 30 degree)として算出した。深根率はその値が大きいほど深根であることを示す。深根率は、原品種及び3つのBC3F3系統それぞれについて20個体ずつ調査した。 The root phenotypes of these three BC3F3 lines were investigated using a modified basket method using a stainless steel mesh colander. A custom-made stainless steel colander with a diameter of 7.5 cm was filled with fertilizer-free culture soil, and sterilized seeds of the original variety or BC3F3 strain were sown and cultivated in hydroponic solution for about a month and a half. After the cultivation period ended, roots that extended downward at an angle exceeding 30 degrees to the ground surface were considered deep roots, and the number of deep roots and the total number of roots were measured. The number of deep roots divided by the total number of roots was calculated as the deep root ratio (RDR30; Ratio of Deeper Root than 30 degrees). The larger the value of the deep root ratio, the deeper the root. The deep root rate was investigated for 20 individuals each of the original variety and three BC3F3 lines.
 結果を図7に示す。0909M由来BC3F3系統の深根率は、原品種と比較して有意に低下していたのに対し、0951M及び2792M由来BC3F3系統の深根率は、原品種と比較して有意に増加していた。これらの結果はカップ法による結果と一致していた。 The results are shown in Figure 7. The deep root percentage of the 0909M-derived BC3F3 line was significantly decreased compared to the original variety, whereas the deep root percentage of the 0951M and 2792M-derived BC3F3 lines was significantly increased compared to the original variety. . These results were consistent with those obtained by the cup method.
 以上の結果から、qSOR1突然変異系統の根の表現型の変化はqSOR1遺伝子内に生じた突然変異に起因すること、すなわち、イネqSOR1タンパク質のアミノ酸配列におけるR204C置換はイネの根を浅くする作用を有し、P140S置換及びL141F置換(ドメインIIIにおける置換)は、当初のアミノ酸配列からの予測に反し、イネの根を深くする作用を有することが示された。 From the above results, the change in the root phenotype of the qSOR1 mutant line is due to the mutation that occurred in the qSOR1 gene, that is, the R204C substitution in the amino acid sequence of the rice qSOR1 protein has the effect of shallowing the roots of rice. Contrary to predictions from the original amino acid sequence, the P140S and L141F substitutions (substitutions in domain III) were shown to have the effect of deepening the roots of rice.
<実施例4.qSOR1遺伝子内の突然変異の重力屈性に対する効果>
 qSOR1は重力屈性に関与する遺伝子であることから、上記のqSOR1遺伝子内の突然変異の重力屈性応答への影響を調べた。
<Example 4. Effect of mutations in the qSOR1 gene on gravitropism>
Since qSOR1 is a gene involved in gravitropism, we investigated the effects of the above mutations in the qSOR1 gene on gravitropic responses.
 実施例3で得られた3つのBC3F3系統及び原品種(コシヒカリ)それぞれについて、30粒の種子から籾殻を取り除き玄米とした。玄米を滅菌水10mlで3回洗った後、殺菌剤として1% PPM(PLANT PRESERVATIVE MIXTURETM、Plant Cell Technology)を含有する培地10mlが入ったシャーレに入れて、30℃で1日静置した。発芽した種子を0.4%アガロースゲルが入った角形プレートに播種し、28℃で2日間、暗黒条件に静置した。その後、角形プレートを90度回転させ、4時間後に根を撮影し根の屈曲角度を計測した。 For each of the three BC3F3 lines and the original variety (Koshihikari) obtained in Example 3, the rice husks were removed from 30 seeds to obtain brown rice. After washing the brown rice three times with 10 ml of sterilized water, it was placed in a petri dish containing 10 ml of a medium containing 1% PPM (PLANT PRESERVATIVE MIXTURE TM , Plant Cell Technology) as a disinfectant, and allowed to stand at 30°C for 1 day. Germinated seeds were sown on square plates containing 0.4% agarose gel and left standing in the dark at 28°C for 2 days. Thereafter, the square plate was rotated 90 degrees, and after 4 hours, the roots were photographed and the bending angle of the roots was measured.
 結果を図8に示す。原品種と比較して0909M由来BC3F3系統は根屈曲角度が有意に小さく、0951M及び2792M由来BC3F3系統は根屈曲角度が有意に大きかった。 The results are shown in Figure 8. Compared to the original variety, the root bending angle of the 0909M-derived BC3F3 line was significantly smaller, and the root bending angle of the 0951M- and 2792M-derived BC3F3 lines was significantly larger.
 これらの結果から、R204C置換を含むqSOR1タンパク質をコードする遺伝子を有するイネは、重力屈性応答が原品種に比べて弱いために根が浅くなり、P140S置換及びL141F置換(ドメインIIIにおける置換)を含むqSOR1タンパク質をコードする遺伝子を有するイネは、重力屈性応答が原品種に比べて強いために根が深くなることが示された。 From these results, rice that has the gene encoding the qSOR1 protein containing the R204C substitution has shallower roots due to a weaker gravitropic response than the original variety, and the P140S and L141F substitutions (substitutions in domain III). Rice that has the gene encoding the qSOR1 protein has been shown to have deeper roots due to a stronger gravitropic response than the original variety.
<実施例5.qSOR1タンパク質のドメインIIIにおける置換の収量への影響>
 イネqSOR1タンパク質のドメインIIIにおける置換が収量などの農業形質に悪影響を与えるかどうかを明らかにするため、実施例3で得られた2792M由来BC3F3系統及びその原品種(コシヒカリ)について、2020年と2021年の2年間、収量調査を行った。
<Example 5. Effect of substitutions in domain III of qSOR1 protein on yield>
In order to clarify whether the substitution in domain III of the rice qSOR1 protein has a negative effect on agronomic traits such as yield, we investigated the 2792M-derived BC3F3 line obtained in Example 3 and its original variety (Koshihikari) in 2020 and 2021. A yield survey was conducted for two years.
 両系統を、2020年と2021年に茨城県つくば市の農研機構内の水田で栽培した。各系統は1m2あたり22.2株(15cm×30cm)の密度で1株1個体植えとした。発芽した種子は4月に播種し、水田苗代で1か月間育苗した後に、水田に移植した。圃場設計は、各系統1区画当たり42株(6×7)の無作為配置の3反復とした。水田には、化学肥料として、P2O5を12g m-2、K2Oを9g m-2、Nを12g m-2をそれぞれ慣行法に従い施用した。 Both lines were cultivated in rice fields within the National Agriculture and Food Research Organization in Tsukuba City, Ibaraki Prefecture in 2020 and 2021. Each line was individually planted at a density of 22.2 plants per 1 m2 (15 cm x 30 cm). Germinated seeds were sown in April, grown in paddy seedlings for one month, and then transplanted to paddy fields. The field design was three replicates with 42 plants (6 x 7) randomly arranged per plot for each line. As chemical fertilizers, 12 g m -2 of P 2 O 5 , 9 g m -2 of K 2 O, and 12 g m -2 of N were applied to the rice fields according to the conventional method.
 2020年の原品種及び2792M由来BC3F3系統の出穂日(各区画の50%の個体が出穂した日)は、3区画すべてにおいて8月6~7日であり、両系統の出穂はほぼ同時であった。2021年の原品種の出穂日は3区画すべてにおいて7月30~31日であり、2021年の2792M由来BC3F3系統の出穂日は3区画すべてにおいて7月28日であった。2021年は2020年より夏の気温が高かったため、両系統とも2021年の出穂日が2020年の出穂日より1週間程度早くなったと考えられる。出穂日の系統間差はこのような年次間差に比べて小さかった。 The heading dates of the original variety and the 2792M-derived BC3F3 line in 2020 (the date when 50% of individuals in each plot started heading) were August 6th to 7th in all three plots, and the heading dates of both lines were almost simultaneous. Ta. The heading date of the original variety in 2021 was July 30th to 31st in all three plots, and the heading date of the 2792M-derived BC3F3 line in 2021 was July 28th in all three plots. Because the summer temperature in 2021 was higher than in 2020, it is thought that the heading date in 2021 was about one week earlier than the heading date in 2020 for both lines. The difference between lines in heading date was smaller than this difference between years.
 栽培期間終了後、各区画から24株を収穫し、自然乾燥後、脱穀した。次いで、風選(2020年)又は水選(2021年)により登熟籾を選別した後、選別した籾を80℃で3日間乾燥器にて風乾し、精籾乾物重を計測した。 After the cultivation period ended, 24 plants were harvested from each plot, dried naturally, and then threshed. Next, after sorting the ripened paddy by wind selection (2020) or water selection (2021), the selected paddy was air-dried in a dryer at 80°C for 3 days, and the dry weight of the refined paddy was measured.
 結果を図9に示す。2792M由来BC3F3系統の精籾乾物重は、原品種に比べて2020年は6.7%、2021年は12.7%増加した。この結果から、イネqSOR1アミノ酸配列のドメインIIIにおける置換は収量に悪影響を及ぼさず、むしろ、収量の増加をもたらすことが示された。なお、2792M由来BC3F3系統の草型は原品種と同様であり、特に異常は見られなかった。 The results are shown in Figure 9. The dry weight of wheat of the BC3F3 line derived from 2792M increased by 6.7% in 2020 and 12.7% in 2021 compared to the original variety. These results showed that substitutions in domain III of the rice qSOR1 amino acid sequence did not adversely affect yield, but rather resulted in an increase in yield. The plant type of the 2792M-derived BC3F3 line was similar to the original variety, and no particular abnormality was observed.
<実施例6.ドメインIIIに置換を有するシロイヌナズナLZY3変異体タンパク質の根の表現型に対する効果>
 本実施例では、シロイヌナズナ(Arabidopsis thaliana)LZY3タンパク質のドメインIIIにおける置換がシロイヌナズナでも根を深くする作用を有するかどうかを調べるために、配列番号12の130番目のプロリンのセリンへの置換(P130S)及び131番目のロイシンのフェニルアラニンへの置換(L131F)を有するLZY3変異体タンパク質(それぞれ、dLZY3(P130S)、dLZY3(L131F))の根の表現型に対する効果を評価した(図10~12)。なお、dLZY3(P130S)及びdLZY3(L131F)変異体タンパク質のアミノ酸配列は、それぞれ配列番号14、16に示し、それをコードする塩基配列は、それぞれ配列番号13、15に示す。
<Example 6. Effect of Arabidopsis LZY3 mutant protein with substitution in domain III on root phenotype>
In this example, in order to investigate whether substitution in domain III of Arabidopsis thaliana LZY3 protein has the effect of deepening roots in Arabidopsis, we replaced proline at position 130 of SEQ ID NO: 12 with serine (P130S). and the effect of LZY3 mutant proteins (dLZY3(P130S) and dLZY3(L131F), respectively) having the substitution of leucine at position 131 with phenylalanine (L131F) on the root phenotype was evaluated (FIGS. 10 to 12). The amino acid sequences of the dLZY3(P130S) and dLZY3(L131F) mutant proteins are shown in SEQ ID NOs: 14 and 16, respectively, and the nucleotide sequences encoding them are shown in SEQ ID NOs: 13 and 15, respectively.
 LZY3タンパク質及びmCherry(赤色蛍光タンパク質)の融合タンパク質をLZY3プロモーター下で発現するベクターであるLZY3p:LZY3-mCherry(Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999、大学共同利用機関法人自然科学研究機構・基礎生物学研究所から供与、当該ベクターに含まれる構築物の塩基配列を配列番号22に示す)において、LZY3遺伝子の388番目のCをTに塩基置換することによって、dLZY3(P130S)変異体タンパク質及びmCherryの融合タンパク質をLZY3プロモーター下で発現するベクター(LZY3p:dLZY3(P130S)-mCherry)を作製した。また、上記LZY3p:LZY3-mCherryベクターにおいて、LZY3遺伝子の393番目のGをCに塩基置換することによって、dLZY3(L131F)変異体タンパク質及びmCherryの融合タンパク質をLZY3プロモーター下で発現するベクター(LZY3p:dLZY3(L131F)-mCherry)を作製した。 LZY3p:LZY3-mCherry, a vector that expresses a fusion protein of LZY3 protein and mCherry (red fluorescent protein) under the LZY3 promoter (Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999, University Joint Use The nucleotide sequence of the construct contained in the vector, which was provided by the National Institute for Basic Biology of the National Institutes of Natural Sciences, is shown in SEQ ID NO: 22), by substituting C at position 388 of the LZY3 gene with T A vector (LZY3p:dLZY3(P130S)-mCherry) expressing a fusion protein of the (P130S) mutant protein and mCherry under the LZY3 promoter was created. In addition, in the above LZY3p:LZY3-mCherry vector, by substituting the 393rd G of the LZY3 gene with C, a vector (LZY3p: dLZY3(L131F)-mCherry) was produced.
 アグロバクテリウムGV3101株を用いて、LZY3p:dLZY3(P130S)-mCherry及びLZY3p:dLZY3(L131F)-mCherryの各々をシロイヌナズナのlzy2lzy3二重変異体(シロイヌナズナColumbia株のLZY2遺伝子及びLZY3遺伝子が欠失した変異体)に導入した。得られた形質転換植物のT1世代の種子を20μg/mlのハイグロマイシンを含む1/2MS寒天培地に播種し、ハイグロマイシンに耐性を示す植物体を選抜した。選抜した植物体を新たに1/2MS寒天培地に移植し、寒天培地を垂直に立てて栽培し、根系形態を観察した。 Using Agrobacterium GV3101 strain, LZY3p:dLZY3(P130S)-mCherry and LZY3p:dLZY3(L131F)-mCherry were isolated from Arabidopsis lzy2lzy3 double mutant (Arabidopsis Columbia strain in which LZY2 and LZY3 genes are deleted). mutant). Seeds of the T1 generation of the obtained transformed plants were sown on a 1/2MS agar medium containing 20 μg/ml of hygromycin, and plants showing resistance to hygromycin were selected. The selected plants were newly transplanted onto a 1/2 MS agar medium, grown on the agar medium vertically, and the morphology of the root system was observed.
 コントロールとして、シロイヌナズナColumbia株(野生型)、シロイヌナズナColumbia株のLZY2遺伝子が欠失した変異体(lzy2単一変異体)、シロイヌナズナColumbia株のLZY2遺伝子及びLZY3遺伝子が欠失した変異体(lzy2lzy3二重変異体)の種子を寒天培地に播種後、寒天培地を垂直に立てて栽培し、根系形態を観察した。 As controls, Arabidopsis Columbia strain (wild type), Arabidopsis Columbia strain mutant in which the LZY2 gene has been deleted (lzy2 single mutant), and Arabidopsis Columbia strain in which the LZY2 and LZY3 genes have been deleted (lzy2lzy3 double mutant) After sowing the seeds of the mutant) on an agar medium, the agar medium was grown vertically and the morphology of the root system was observed.
 結果を図13に示す。野生型及びlzy2単一変異体の根は同様の形態であり、lzy2lzy3二重変異体の側根は野生型に比べて上方に向かって伸長していた。これは、先行研究の結果(Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999)と一致していた。これに対し、LZY3p:dLZY3(P130S)-mCherryが導入されたlzy2lzy3二重変異体(dLZY3(P130S)/lzy2lzy3変異体)及びLZY3p:dLZY3(L131F)-mCherryが導入されたlzy2lzy3二重変異体(dLZY3(L131F)/lzy2lzy3変異体)はいずれも、原系統であるlzy2lzy3二重変異体と比較して根が下方に向かって伸長しており、lzy2lzy3二重変異体の表現型を相補していた。さらに、dLZY3(P130S)/lzy2lzy3変異体及びdLZY3(L131F)/lzy2lzy3変異体は、野生型LZY3タンパク質を発現する野生型及びlzy2単一変異体と比べても、顕著に根が下方に向かって伸長していた。 The results are shown in Figure 13. The roots of the wild type and the lzy2 single mutant had similar morphology, and the lateral roots of the lzy2lzy3 double mutant extended upward compared to the wild type. This was consistent with the results of a previous study (Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999). On the other hand, the lzy2lzy3 double mutant (dLZY3(P130S)/lzy2lzy3 mutant) into which LZY3p:dLZY3(P130S)-mCherry was introduced and the lzy2lzy3 double mutant into which LZY3p:dLZY3(L131F)-mCherry was introduced ( dLZY3(L131F)/lzy2lzy3 mutant) all had roots elongated downward compared to the original line, the lzy2lzy3 double mutant, and complemented the phenotype of the lzy2lzy3 double mutant. . Furthermore, the roots of the dLZY3(P130S)/lzy2lzy3 mutant and the dLZY3(L131F)/lzy2lzy3 mutant significantly elongate downward, even compared to the wild type and lzy2 single mutant that express the wild-type LZY3 protein. Was.
 これらの結果は、ドメインIIIにおける置換を有するLZY3変異体タンパク質は、野生型LZY3タンパク質に比べて根を深くする作用を有すること、即ちLZY3タンパク質のドメインIIIにおける置換はシロイヌナズナでも根を深くする作用があることを示す。 These results indicate that the LZY3 mutant protein with a substitution in domain III has the effect of deepening roots compared to the wild-type LZY3 protein, that is, the substitution in domain III of the LZY3 protein also has the effect of deepening roots in Arabidopsis. Show that something is true.
 以上より、qSOR1タンパク質のドメインIIIにおける変異は、イネ等の単子葉植物だけでなく、シロイヌナズナ等の双子葉植物でも根を深くする作用を有することが示された。 From the above, it was shown that mutations in domain III of the qSOR1 protein have the effect of deepening roots not only in monocotyledonous plants such as rice but also in dicotyledonous plants such as Arabidopsis.
 なお、図13では、dLZY3(P130S)/lzy2lzy3変異体及びdLZY3(L131F)/lzy2lzy3変異体の根が、野生型、lzy2単一変異体及びlzy2lzy3二重変異体と比べて若干短く見えるが、これは培地中のハイグロマイシンによる影響であると考えられる。また、上記のdLZY3(P130S)/lzy2lzy3変異体及びdLZY3(L131F)/lzy2lzy3変異体の草型は、lzy2lzy3二重変異体と比べて顕著な変化はなかった。 In addition, in Figure 13, the roots of the dLZY3(P130S)/lzy2lzy3 mutant and dLZY3(L131F)/lzy2lzy3 mutant appear to be slightly shorter than those of the wild type, lzy2 single mutant, and lzy2lzy3 double mutant; This is considered to be an effect of hygromycin in the medium. Furthermore, the grass types of the above-mentioned dLZY3(P130S)/lzy2lzy3 mutant and dLZY3(L131F)/lzy2lzy3 mutant were not significantly changed compared to the lzy2lzy3 double mutant.
配列
配列番号1 コシヒカリqSOR1タンパク質をコードするCDS
配列番号2 コシヒカリqSOR1タンパク質のアミノ酸配列
配列番号3 0951M系統のqSOR1タンパク質をコードするCDS
配列番号4 0951M系統のqSOR1タンパク質のアミノ酸配列
配列番号5 2792M系統のqSOR1タンパク質をコードするCDS
配列番号6 2792M系統のqSOR1タンパク質のアミノ酸配列
配列番号7 0909M系統のqSOR1タンパク質をコードするCDS
配列番号8 0909M系統のqSOR1タンパク質のアミノ酸配列
配列番号9 2574M系統のqSOR1タンパク質をコードするCDS
配列番号10 2574M系統のqSOR1タンパク質のアミノ酸配列
配列番号11 シロイヌナズナLZY3タンパク質をコードするCDS
配列番号12 シロイヌナズナLZY3タンパク質のアミノ酸配列
配列番号13 dLZY3(P130S)変異体タンパク質をコードする塩基配列
配列番号14 dLZY3(P130S)変異体タンパク質のアミノ酸配列
配列番号15 dLZY3(L131F)変異体タンパク質をコードする塩基配列
配列番号16 dLZY3(L131F)変異体タンパク質のアミノ酸配列
配列番号17 シロイヌナズナLZY2タンパク質のアミノ酸配列
配列番号18 タルウマゴヤシNGRタンパク質のアミノ酸配列
配列番号19 フォワードプライマーqSOR1-p1-F1
配列番号20 リバースプライマーqSOR1-p1-R1
配列番号21 リバースプライマーqSOR1-p1-R2
配列番号22 LZY3p:LZY3-mCherryベクターに含まれる構築物(プロモーター及び融合タンパク質をコードするポリヌクレオチドを含む)の塩基配列
配列番号23 コシヒカリqSOR1遺伝子のゲノムDNA配列
配列番号24 トウモロコシqSOR1(ZmqSOR1)タンパク質のアミノ酸配列
配列番号25 ソルガムqSOR1(SbqSOR1)タンパク質のアミノ酸配列
配列番号26 コムギqSOR1(TaqSOR1)タンパク質のアミノ酸配列
配列番号27 ミナトカモジグサqSOR1(BdqSOR1)タンパク質のアミノ酸配列
配列番号28 ダイズNGR2(GmNGR2)タンパク質のアミノ酸配列
配列番号29 ミヤコグサNGR(LjNGR)タンパク質のアミノ酸配列
配列番号30 ポプラNGR(PtNGR)タンパク質のアミノ酸配列
配列番号31 モモNGR(PpeNGR)タンパク質のアミノ酸配列
 本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
Sequence SEQ ID NO: 1 CDS encoding Koshihikari qSOR1 protein
SEQ ID NO: 2 Koshihikari qSOR1 protein amino acid sequence SEQ ID NO: 3 CDS encoding 0951M strain qSOR1 protein
SEQ ID NO: 4 Amino acid sequence of qSOR1 protein of 0951M strain SEQ ID NO: 5 CDS encoding qSOR1 protein of 2792M strain
SEQ ID NO: 6 Amino acid sequence of 2792M strain qSOR1 protein SEQ ID NO: 7 CDS encoding 0909M strain qSOR1 protein
SEQ ID NO: 8 Amino acid sequence of qSOR1 protein of 0909M strain SEQ ID NO: 9 CDS encoding qSOR1 protein of 2574M strain
SEQ ID NO: 10 Amino acid sequence of 2574M strain qSOR1 protein SEQ ID NO: 11 CDS encoding Arabidopsis LZY3 protein
SEQ ID NO: 12 Amino acid sequence of Arabidopsis LZY3 protein SEQ ID NO: 13 Base sequence encoding dLZY3(P130S) mutant protein SEQ ID NO: 14 Amino acid sequence of dLZY3(P130S) mutant protein SEQ ID NO: 15 Encoding dLZY3(L131F) mutant protein Base sequence SEQ ID NO: 16 Amino acid sequence of dLZY3(L131F) mutant protein SEQ ID NO: 17 Amino acid sequence of Arabidopsis LZY2 protein SEQ ID NO: 18 Amino acid sequence of Alfalfa NGR protein SEQ ID NO: 19 Forward primer qSOR1-p1-F1
SEQ ID NO: 20 Reverse primer qSOR1-p1-R1
SEQ ID NO: 21 Reverse primer qSOR1-p1-R2
SEQ ID NO: 22 Base sequence of construct (including promoter and polynucleotide encoding the fusion protein) contained in LZY3p:LZY3-mCherry vector SEQ ID NO: 23 Genomic DNA sequence of Koshihikari qSOR1 gene SEQ ID NO: 24 Amino acids of maize qSOR1 (ZmqSOR1) protein Sequence SEQ ID NO: 25 Amino acid sequence of sorghum qSOR1 (SbqSOR1) protein SEQ ID NO: 26 Amino acid sequence of wheat qSOR1 (TaqSOR1) protein SEQ ID NO: 27 Amino acid sequence of Minato chinensis qSOR1 (BdqSOR1) protein SEQ ID NO: 28 Amino acid sequence of soybean NGR2 (GmNGR2) protein SEQ ID NO: 29 Amino acid sequence of Lotus japonicus NGR (LjNGR) protein SEQ ID NO: 30 Amino acid sequence of Poplar NGR (PtNGR) protein SEQ ID NO: 31 Amino acid sequence of Peach NGR (PpeNGR) protein All publications, patents and patents cited herein The application is hereby incorporated by reference in its entirety.

Claims (10)

  1.  配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)タンパク質をコードする遺伝子を有する、深根性が向上した植物。 It has improved deep rooting ability and has a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein that contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. plant.
  2.  前記アミノ酸置換が、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリン及び配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンからなる群から選択されるアミノ酸の置換である、請求項1に記載の植物。 The amino acid substitution is a substitution of an amino acid selected from the group consisting of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 and leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2. , the plant according to claim 1.
  3.  前記アミノ酸置換が、配列番号2に示すアミノ酸配列の140番目に対応する位置のプロリンのセリンへの置換、又は配列番号2に示すアミノ酸配列の141番目に対応する位置のロイシンのフェニルアラニンへの置換である、請求項1に記載の植物。 The amino acid substitution is a substitution of proline at the position corresponding to the 140th position of the amino acid sequence shown in SEQ ID NO: 2 with serine, or a substitution of leucine at the position corresponding to the 141st position of the amino acid sequence shown in SEQ ID NO: 2 with phenylalanine. The plant according to claim 1.
  4.  前記変異型qSOR1タンパク質が、
    (i) 配列番号4、6、14若しくは16に示すアミノ酸配列からなるタンパク質、
    (ii) 配列番号2若しくは12に示すアミノ酸配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質、又は
    (iii) 配列番号2若しくは12に示すアミノ酸配列において1~10個のアミノ酸の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む、アミノ酸配列からなり、かつ植物の深根性を向上させる活性を示すタンパク質
    である、請求項1に記載の植物。
    The mutant qSOR1 protein is
    (i) A protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 14 or 16,
    (ii) An amino acid that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12 and contains an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. a protein consisting of a sequence and exhibiting an activity of improving deep rooting of plants, or
    (iii) Has an insertion, deletion, substitution, and/or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or 12, and corresponds to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. 2. The plant according to claim 1, which is a protein consisting of an amino acid sequence containing an amino acid substitution in the sequence at a position, and which exhibits an activity of improving deep rooting ability of plants.
  5.  前記変異型qSOR1タンパク質をコードする遺伝子が、
    (iv) 配列番号3、5、13若しくは15に示す塩基配列、
    (v) 配列番号1若しくは11に示す塩基配列と90%以上の配列同一性を有し、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列、又は
    (vi) 配列番号1若しくは11に示す塩基配列において1~10個の塩基の挿入、欠失、置換、及び/又は付加を有し、配列番号2に示すアミノ酸配列の140~145番目の配列中にアミノ酸置換を引き起こすヌクレオチド変異を含み、かつ植物の深根性を向上させる活性を示すタンパク質をコードする塩基配列、
    を含む、請求項1に記載の植物。
    The gene encoding the mutant qSOR1 protein is
    (iv) the base sequence shown in SEQ ID NO: 3, 5, 13 or 15,
    (v) A nucleotide mutation that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 or 11 and causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. a base sequence that encodes a protein that contains and exhibits an activity of improving deep rooting of plants, or
    (vi) Has an insertion, deletion, substitution, and/or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1 or 11, and is in the 140th to 145th sequence of the amino acid sequence shown in SEQ ID NO: 2. a nucleotide sequence that contains a nucleotide mutation that causes an amino acid substitution and that encodes a protein that exhibits an activity to improve deep rooting ability of plants;
    The plant according to claim 1, comprising:
  6.  単子葉植物又は双子葉植物である、請求項1に記載の植物。 The plant according to claim 1, which is a monocot or a dicot.
  7.  植物のqSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)遺伝子に、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を引き起こすヌクレオチド変異を導入する工程を含む、深根性が向上した植物を作出する方法。 A deep method that involves introducing into the plant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene a nucleotide mutation that causes an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. A method for producing plants with improved roots.
  8.  配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1(quantitative trait locus for SOIL SURFACE ROOTING 1)タンパク質をコードする遺伝子を含むベクターを植物に導入する工程を含む、深根性が向上した植物を作出する方法。 A vector containing a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein containing an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2 is introduced into plants. A method for producing plants with improved deep rooting, including a process.
  9.  請求項1~6のいずれか1項に記載の植物を育種親として用いて植物の交配を行い、子孫植物を取得する工程、及び前記変異型qSOR1タンパク質をコードする遺伝子が導入された子孫植物を選抜する工程を含む、深根性が向上した植物を作出する方法。 A step of crossbreeding plants using the plant according to any one of claims 1 to 6 as a breeding parent to obtain a progeny plant, and a progeny plant into which the gene encoding the mutant qSOR1 protein has been introduced. A method for producing plants with improved deep rooting, including a selection process.
  10.  被験植物由来のDNAを鋳型としてqSOR1遺伝子の全体又はその一部について核酸増幅を行う工程、核酸増幅の結果に基づいて、配列番号2に示すアミノ酸配列の140~145番目に対応する位置の配列中にアミノ酸置換を含む変異型qSOR1タンパク質をコードする遺伝子を有する植物を同定する工程を含む、深根性が向上した植物を選抜する方法。 A step of performing nucleic acid amplification on the whole or part of the qSOR1 gene using DNA derived from the test plant as a template, and based on the results of the nucleic acid amplification, the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. A method for selecting plants with improved deep rooting ability, the method comprising the step of identifying a plant having a gene encoding a mutant qSOR1 protein containing an amino acid substitution.
PCT/JP2023/013277 2022-04-19 2023-03-30 Plant with improved deep-rootedness WO2023203988A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021064402A1 (en) * 2019-10-01 2021-04-08 University Of Leeds Plants having a modified lazy protein

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021064402A1 (en) * 2019-10-01 2021-04-08 University Of Leeds Plants having a modified lazy protein

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