Plants with improved deep rooting

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

[Technical Field]

[0001] The present invention relates to a plant with improved deep rooting ability that has a gene encoding a mutant qSOR1 protein. [Background technology]

[0002] Currently, the world's population is increasing, particularly in developing countries, and increasing food production to feed this population is essential. However, due to recent global climate change, extreme rainfall phenomena such as droughts and heavy rains are occurring all over the world. Furthermore, soil degradation and salt accumulation due to years of over-cultivation are also becoming problems. In order to ensure stable and sustainable food production in such an unstable environment, it is necessary to develop crops that are resistant to environmental stress.

[0003] Because roots are the only organs that plants use to acquire and absorb nutrients and water from the soil, root system morphology is extremely important for crop growth. However, the root system morphology suitable for crop production varies depending on the soil environment. For example, deep rooting (deep rooting) is advantageous for drought tolerance, nitrogen absorption, and heavy metal avoidance, while shallow rooting (shallow rooting) is advantageous for phosphorus deficiency tolerance, oxygen deficiency avoidance in waterlogged conditions, and heavy metal phytoremediation. Therefore, improving root system morphology to suit the soil environment is important for developing crops that are resistant to environmental stress.

[0004] Cross breeding is a commonly used method for developing crops. However, cross breeding requires phenotypic selection, and evaluating the root morphology underground usually requires digging up the roots for investigation, which requires a great deal of time and effort. In addition, in the case of grains, roots can only be investigated after the aboveground parts have been harvested. For these reasons, little progress has been made in improving crop roots through cross breeding.

[0005] Meanwhile, in the model plant Arabidopsis, mutant analysis has identified numerous genes involved in root morphogenesis and root morphogenesis under environmental stress, further elucidating the mechanisms underlying root morphology. For example, with regard to tropism, which significantly influences root system morphology, several gravitropism genes (Non-Patent Document 1) and hydrotropism genes (Non-Patent Document 2) have been identified. Furthermore, in monocotyledonous plants, including rice and maize, mutant analysis has also identified many genes involved in root development. For example, in rice, mutants of DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) and LARGE ROOT ANGLE1, which encodes OsPIN2, have been shown to develop shallow roots (Non-Patent Documents 3 and 4), and mutants of Rice Morphology Determinant (RMD), which encodes an actin-binding protein, have been shown to develop deep roots under low-phosphate conditions, which favor shallow rooting (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 as breeding materials as they are.

[0006] In recent years, quantitative trait locus (QTL) analysis using rice has led to the isolation of the DRO1 (DEEPER ROOTING 1) gene and its homologous gene, qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1), as QTLs involved in root elongation angle, and it has been reported that these genes are involved in gravitropism (Patent Document 1, Non-Patent Documents 6 and 7).

[0007] Homologous genes of the rice DRO1 gene and the rice qSOR1 gene are widely present in angiosperms and are known to form a large gene group called the DRO1 family (Non-Patent Document 7). Furthermore, DRO1 family proteins are known to contain five domains that are highly conserved among plants (Non-Patent Document 8), and studies using Arabidopsis and rice have shown that the fifth domain (CCL domain) is involved in gravitropism (Non-Patent Documents 6, 7, and 9). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5791049 [Non-patent literature]

[0009] [Non-Patent Document 1] Nakamura M. et al., Journal of Experimental Botany, 2019, 70(14): 3495-3506 [Non-patent document 2] Dietrich D., Journal of experimental botany, 2018, 69(11): 2759-2771 [Non-patent document 3] Bettembourg M. et al., Rice, 2017, 10(1): 50 [Non-patent document 4] Wang L. et al., Journal of Experimental Botany, 2018, 69(3): 385-397 [Non-patent document 5] Huang G. et al., Nature Communication, 2018, 9(1): 2346 [Non-patent document 6] Uga Y. et al., Nature Genetics, 2013, 45(9): 1097-1102 [Non-Patent Document 7] Kitomi Y. et al., PNAS, 2020, 117(35): 21242-21250 [Non-patent document 8] Guseman JM et al., The Plant Journal, 2017, 89(6): 1093-1105 [Non-Patent Document 9] Taniguchi M. et al., The Plant Cell, 2017, 29: 1984-1999 Summary of the Invention [Problem to be solved by the invention]

[0010] Since the qSOR1 gene is widely present in both monocotyledonous and dicotyledonous plants, it is thought that useful alleles of the qSOR1 gene can be widely used to improve the root systems of not only monocotyledonous crops such as wheat and corn, but also dicotyledonous crops such as soybean and rapeseed. However, the number of qSOR1 gene alleles useful as breeding materials is limited, and currently, technology to improve plant deep rooting has not been fully developed. [Means for solving the problem]

[0011] We searched for lines with nonsynonymous substitutions in the qSOR1 gene from randomly induced mutant rice lines and examined the root phenotypes of the resulting mutant lines. Surprisingly, we found that mutant lines carrying a gene encoding a mutant qSOR1 protein with an amino acid substitution in the third domain (domain III), the function of which was previously unknown, exhibited improved deep-rooting ability compared to the parental cultivar. We backcrossed these mutant lines to the parental cultivar and examined the root morphology of the resulting lines, demonstrating that the improved deep-rooting ability was due to the amino acid substitutions in the qSOR1 protein. Furthermore, we examined the root morphology of Arabidopsis transformants expressing the Arabidopsis LZY3 protein (corresponding to the rice qSOR1 protein; sometimes referred to herein as the "qSOR1 protein") with the same amino acid substitutions, revealing significantly improved deep-rooting ability.

[0012] Based on the above findings, the present inventors have completed the present invention. That is, the present invention includes the following. [1] A plant with improved deep rooting ability that has 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. [2] The plant described in [1], wherein 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 amino acid in the amino acid sequence shown in SEQ ID NO: 2 and leucine at the position corresponding to the 141st amino acid in the amino acid sequence shown in SEQ ID NO: 2. [3] A plant described in [1] or [2], wherein the amino acid substitution is a substitution of proline with serine at the position corresponding to the 140th amino acid in the amino acid sequence shown in SEQ ID NO: 2, or a substitution of leucine with phenylalanine at the position corresponding to the 141st amino acid in the amino acid sequence shown in SEQ ID NO: 2. [4] The mutant qSOR1 protein is (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, 6, 14 or 16; (ii) a protein having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12, containing an amino acid substitution at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having an amino acid sequence having 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 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, and exhibiting activity of improving deep rooting ability of plants. The plant according to any one of [1] to [3], [5] The gene encoding the mutant qSOR1 protein is (iv) a base sequence shown in SEQ ID NO: 3, 5, 13 or 15; (v) a nucleotide sequence encoding a protein that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 or 11, 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 exhibits activity to improve the deep rooting ability of plants; or (vi) a base sequence encoding a protein having an insertion, deletion, substitution, and / or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1 or 11, containing a nucleotide mutation that causes an amino acid substitution in the sequence of positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity to improve the 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 monocotyledonous plant or a dicotyledonous plant. [7] A method for producing a plant with improved deep rooting ability, comprising the step of introducing 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 into the plant's qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene. [8] A method for producing a plant with improved deep rooting ability, comprising the step of introducing into a plant a vector containing a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein having an amino acid substitution in the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2. [9] A method for producing a plant with improved deep rooting ability, comprising the steps of: crossbreeding plants using a plant described in any one of [1] to [6] as a breeding parent to obtain offspring plants; and selecting offspring plants into which a gene encoding the mutant qSOR1 protein has been introduced.

[10] A method for selecting plants with improved deep rooting ability, comprising the steps of: performing nucleic acid amplification of all or part of the qSOR1 gene using DNA derived from a test plant as a template; and identifying plants having a gene encoding a mutant qSOR1 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 based on the results of nucleic acid amplification. This specification includes the disclosure of Japanese Patent Application No. 2022-068983, from which the present application claims priority. [Effects of the Invention]

[0013] According to the present invention, a plant with improved deep rooting ability and a method for producing the same can be provided. [Brief explanation of the drawings]

[0014] [Figure 1A]Figure 1A shows the amino acid sequence comparison of qSOR1 proteins from monocotyledonous plants (rice qSOR1, maize qSOR1 (ZmqSOR1), sorghum qSOR1 (SbqSOR1), wheat qSOR1 (TaqSOR1), and wheatgrass qSOR1 (BdqSOR1)) with those from dicotyledonous plants (Arabidopsis thaliana LZY3 (AtLZY3), soybean NGR2 (GmNGR2), Lotus japonicus NGR (LjNGR), Medicago sativa NGR (MtNGR), poplar NGR (PtNGR), and peach NGR (PpeNGR)). Five highly conserved domains (domains I–V) across multiple plant species are indicated by boxes. [Figure 1B] Continuation of Figure 1A. [Figure 2] FIG. 2 shows the amino acid sequence of the Koshihikari qSOR1 protein. [Figure 3] FIG. 3 shows the mutation sites in the amino acid sequences of the qSOR1 protein of four rice qSOR1 mutant lines compared to the original variety. [Figure 4] FIG. 4 shows the mutation sites of the qSOR1 gene CDS of four rice qSOR1 mutant lines compared to the original variety. [Figure 5] Figure 5 shows the results of a comparison of the amino acid sequences of the rice qSOR1 protein, the Arabidopsis LZY2 protein, the Arabidopsis LZY3 protein, and the Medicago NGR protein. Five domains (domains I to V) that are highly conserved across multiple plant species are indicated by boxes. [Figure 6]Figure 6 shows the root phenotypes of the original variety (Koshihikari) and the qSOR1 mutant rice lines evaluated using the cup method. A is a representative photograph showing the roots of each line cultivated using the cup method. The scale bar represents 1 cm. θrga indicates the root elongation angle. B is a graph showing the root elongation angle of each line cultivated using the cup method. Data are expressed as the mean ± standard deviation. * (asterisk) indicates a significant difference at the 0.1% level based on Dunnett's test using the original variety (Koshihikari) as the control group. [Figure 7] Figure 7 shows the root phenotypes of the original variety (Koshihikari) and the qSOR1 mutant rice lines evaluated using the basket method. (A) Representative photographs showing the roots of each line cultivated using the basket method. The scale bar indicates 1 cm. (B) Graph showing the deep root ratio of each line cultivated using the basket method. Data are expressed as mean ± standard deviation. * indicates a significant difference at the 0.1% level in a Dunnett test using the original variety (Koshihikari) as the control group. [Figure 8] Figure 8 shows the root bending angle of the original variety (Koshihikari) and the rice qSOR1 mutant line. A is a representative photograph showing the gravitropic response of each line 4 hours after the square plate was rotated 90°. The scale bar represents 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 the mean ± standard deviation. * indicates a significant difference from the original variety (Koshihikari) at the 0.1% level by Student's t-test. n indicates the number of individuals measured. [Figure 9] 9 shows a comparison of yield between the original variety (Koshihikari) and the qSOR1 mutant rice line (line name 2792M). Data are shown as the mean ± standard deviation of three replicates of polished rice dry weight (total of 24 plants per plot). [Figure 10] FIG. 10 shows the amino acid sequence of the Arabidopsis thaliana LZY3 protein. [Figure 11] FIG. 11 shows the mutation sites from the wild type in the amino acid sequences of Arabidopsis LZY3 mutant proteins (dLZY3(P130S) and dLZY3(L131F)). [Figure 12] FIG. 12 shows mutation sites from the wild type in the nucleotide sequence encoding the Arabidopsis thaliana LZY3 mutant proteins (dLZY3(P130S) and dLZY3(L131F)). [Figure 13] Figure 13 shows photographs showing the root phenotypes of Arabidopsis thaliana wild-type, lzy2 single mutant, lzy2lzy3 double mutant, dLZY3(P130S) / lzy2lzy3 mutant, and dLZY3(L131F) / lzy2lzy3 mutant. The root phenotypes are shown 14 days 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. The scale bar indicates 5 mm. g indicates the direction of gravity. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will be described in detail below.

[0016] (1) Plants with improved deep rooting The present invention relates to a plant (also referred to as "the plant of the present invention") with improved deep rooting ability, which has a gene (mutant qSOR1 gene) encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein containing an amino acid substitution that improves deep rooting ability.

[0017] As used herein, "deep rooting" refers to the property of roots growing in the direction of gravity (typically underground) at a deep angle relative to a horizontal plane (typically the earth's surface). Deep rooting can be evaluated using, for example, the root growth angle, deep root ratio, or the degree of root gravitropic response as an index. As used herein, "root growth angle" refers to the angle at which roots grow in the direction of gravity relative to the horizontal plane. As used herein, "deep root ratio" refers to the ratio of the number of deep roots to the total number of roots. As used herein, "deep roots" refers to roots that grow in the direction of gravity at an angle greater than a certain angle (e.g., an appropriate angle within the range of 20 to 70 degrees, e.g., 20, 30, 40, 50, 60, or 70 degrees) relative to the horizontal plane. As used herein, "gravitropic response" refers to a plant's ability to sense the direction of gravity and change the growth direction of its roots to the direction of gravity and the growth direction of its aboveground parts to the direction opposite to gravity.

[0018] As used herein, "improved deep rooting ability" refers to improved deep rooting ability compared to the original variety or lineage of the plant (e.g., a wild-type plant). "Improved deep rooting ability" includes an increased root elongation angle (e.g., an increase of 10 degrees or more in the average root elongation angle, or a statistically significant increase in the root elongation angle), an increased deep root ratio (e.g., an increase of 10% or more in the average deep root ratio, or a statistically significant increase in the deep root ratio), and an enhanced root gravitropic response. As used herein, "original variety" and "original lineage" refer to the variety and lineage 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 has a nucleotide mutation in the qSOR1 gene on its genome that improves deep rooting ability, the "original variety" and "original lineage" may refer to the variety or lineage before the nucleotide mutation that improves deep rooting ability was introduced or developed. When the mutant qSOR1 gene carried by the plant of the present invention is an exogenous gene, the "original variety" and "original line" may refer to the variety or line before the mutant qSOR1 gene was introduced.

[0019] Whether or not a test plant (e.g., a plant of the present invention) has improved deep-rootedness can be determined, for example, by evaluating the root elongation angle using the cup method (Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86). Specifically, for example, seeds of the test plant and its original variety or line are sown in a cup-shaped container filled with culture soil and cultivated for a predetermined period (e.g., 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 line, the test plant can be determined to have improved deep-rootedness.

[0020] The improvement of deep rooting ability of a test plant can also be determined by assessing the deep rooting rate using the basket method (Patent No. 5791049; Kitomi Y. et al., Rice, 2015, 8: 16). Specifically, for example, seeds of the test plant and its original variety or lineage are sown in a stainless steel mesh strainer filled with culture soil and cultivated in a hydroponic solution for a predetermined period (e.g., about 1.5 months). The number of deep roots and the total number of roots are then measured, and the deep rooting rate is calculated. If the deep rooting rate of the test plant is increased as described above compared to the original variety or lineage, the test plant can be determined to have improved deep rooting ability.

[0021] The improvement of deep-rootedness of a test plant can also be determined by evaluating the gravitropic response of the roots. Specifically, for example, germinated seeds of the test plant and its original variety or line are sown on agarose gel-filled plates and grown on their sides in the dark at a predetermined temperature for several days (e.g., 2 days). The plate is then rotated 90 degrees vertically, and the root bending angle (the angle between the root extension direction before bending and the root extension direction after bending) is measured several hours later (e.g., 4 hours). If the root bending angle of the test plant is increased compared to that of the original variety or line (e.g., the average root bending angle is increased by 5 degrees or more, preferably 10 degrees or more, or the root bending angle is statistically significantly increased), the test plant can be determined to have an enhanced root gravitropic response and improved deep-rootedness.

[0022] In plants, roots first differentiate as radicles during embryogenesis, which then develop into primary roots. In dicotyledonous plants, the primary roots develop into taproots, from which lateral roots arise, forming a root system called the taproot system. On the other hand, in monocotyledonous plants, the primary roots do not develop much, and instead numerous nodal roots arise from the nodes of the stem, forming a root system called fibrous roots (mainly consisting of seminal roots and nodal roots). The radicles and roots derived therefrom are called adventitious roots (seamless roots), while roots arising from parts other than the radicle (such as the stem) are called adventitious roots. Adventitious roots growing from the stems of plants such as rice and corn are called crown roots. In the present invention, the term "root" may refer to any type of root and is not limited to, but includes, for example, the aforementioned adventitious roots, adventitious roots, roots of dicotyledonous plants (e.g., taproots and lateral roots), roots of monocotyledonous plants (e.g., seminal roots and nodal roots), crown roots, etc.

[0023] Deep rooting is generally known to be advantageous for drought tolerance. For example, a near-isogenic line (Dro1-NIL) in the IR64 background, which introduced the DRO1 gene from the deep-rooted upland rice Kinandang Patong, has been shown to have improved deep rooting compared to the shallow-rooted paddy rice IR64. When IR64 and Dro1-NIL were cultivated under drought conditions, Dro1-NIL produced significantly higher yields than IR64 (Patent No. 5791049; Uga Y. et al., Nature Genetics, 2013, 45(9): 1097-1102).

[0024] Therefore, the plants of the present invention, which have improved deep rooting ability compared to the original variety or line, have higher drought tolerance than the original variety or line, and are thought to be particularly useful for cultivation under drought conditions.

[0025] As used herein, "mutant qSOR1 protein" refers to a protein that has an amino acid mutation (e.g., an amino acid substitution that improves deep rooting ability) that alters protein function relative to the amino acid sequence of wild-type qSOR1 protein. As used herein, "wild-type qSOR1 protein" refers to a qSOR1 protein that does not have the amino acid substitution that improves deep rooting ability. As used herein, "amino acid mutation" includes insertion, deletion, substitution, addition, etc.

[0026] As used herein, "qSOR1 protein" refers to a protein encoded by the qSOR1 gene. The qSOR1 protein is a protein involved in gravitropism, particularly root gravitropism, and has highly conserved sequences (domains I to V, respectively) at positions corresponding to positions 1 to 12, 58 to 64, 140 to 145, and 224 to 241 of the amino acid sequence set forth in SEQ ID NO: 2. The wild-type qSOR1 protein has the amino acid sequence PLDRFL in domain III. Examples of qSOR1 proteins include, but are not limited to, rice qSOR1, maize qSOR1 (ZmqSOR1) protein, sorghum qSOR1 (SbqSOR1) protein, wheat qSOR1 (TaqSOR1) protein, wheatgrass qSOR1 (BdqSOR1) protein, Arabidopsis thaliana LZY2 (AtLZY2) protein, Arabidopsis thaliana LZY3 (AtLZY3) protein, soybean NGR2 (GmNGR2) protein, Lotus japonicus NGR (LjNGR) protein, Medicago sativa NGR (MtNGR) protein, poplar NGR (PtNGR) protein, and peach NGR (PpeNGR) protein (Figures 1 and 5).

[0027] As used herein, the "qSOR1 gene" (a gene encoding the qSOR1 protein) encompasses the rice qSOR1 gene (also referred to as the DRL1 gene or OsNGR2 gene) and its homologous genes. Homologous genes of the rice qSOR1 gene are present in a wide range of plant species, including monocotyledonous plants such as sorghum, maize, barley, wheat, and rice bran, and dicotyledonous plants such as Arabidopsis thaliana, alfalfa, cucumber, lotus, tomato, poplar, soybean, lotus grass, and peach. Examples of homologous genes of the rice qSOR1 gene include, but are not limited to, qSOR1 genes such as the sorghum qSOR1 gene (SbqSOR1; SORBI_3002G373700), maize qSOR1 gene (ZmqSOR1; Zm00001d022133), barley qSOR1 gene (HvqSOR1), wheat qSOR1 genes (TaAqSOR1, TaBqSOR1, TaDqSOR1), and Japanese bracken qSOR1 gene (BdqSOR1); LZY genes such as the Arabidopsis thaliana LZY2 gene (AtLZY2), the Arabidopsis thaliana LZY3 gene (AtLZY3), and the Arabidopsis thaliana LZY4 gene (AtLZY4); and Medicago sativa NGR (NEGATIVE GRAVITROPIC RESPONSE OF These include NGR genes such as the MtNGR (MtROOTS) gene, soybean NGR2 (GmNGR2), lotus NGR (LjNGR), poplar NGR (PtNGR), and peach NGR (PpeNGR), as well as the DRL1 gene.

[0028] The rice qSOR1 (quantitative trait locus for soil surface rooting 1) gene was discovered on rice chromosome 7 as a QTL involved in root elongation angle through QTL analysis using a cross between the rice variety Gemdjah Beton, which forms surface roots (a phenotype in which some crown roots are shallow-rooted and extend to the soil surface), and the rice variety Sasanishiki, which does not form surface roots (Uga Y. et al., Theoretical and Applied Genetics, 2012, 124: 75-86). The rice qSOR1 protein has been reported to be involved in gravitropism and to control root elongation angle. The amino acid sequence of the native rice (Koshihikari) qSOR1 protein is shown, for example, in SEQ ID NO: 2. Furthermore, the CDS encoding the native rice (Koshihikari) qSOR1 protein is shown, for example, in SEQ ID NO: 1.

[0029] It has been reported that the Arabidopsis LZY4 protein is involved only in root gravitropism, while the 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 native Arabidopsis LZY2 and LZY3 proteins are shown, for example, in SEQ ID NOs: 17 and 12, respectively. Furthermore, the CDS encoding the native Arabidopsis LZY3 protein is shown, for example, in SEQ ID NO: 11.

[0030] It has been reported that the Medicago sativa 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 native Medicago sativa NGR protein is shown, for example, in SEQ ID NO: 18.

[0031] In addition, the amino acid sequences of wild-caught corn qSOR1 (ZmqSOR1), sorghum qSOR1 (SbqSOR1), wheat qSOR1 (TaqSOR1), wheatgrass qSOR1 (BdqSOR1), soybean NGR2 (GmNGR2), lotus grass NGR (LjNGR), poplar NGR (PtNGR), and peach NGR (PpeNGR) are shown, for example, in SEQ ID NOs: 24 to 31, respectively.

[0032] 1A and 1B show the results of a comparison of the amino acid sequences of rice qSOR1 protein (SEQ ID NO: 2), maize qSOR1 (ZmqSOR1) protein (SEQ ID NO: 24), sorghum qSOR1 (SbqSOR1) protein (SEQ ID NO: 25), wheat qSOR1 (TaqSOR1) protein (SEQ ID NO: 26), wheatgrass qSOR1 (BdqSOR1) protein (SEQ ID NO: 27), Arabidopsis thaliana LZY3 (AtLZY3) protein (SEQ ID NO: 12), soybean NGR2 (GmNGR2) protein (SEQ ID NO: 28), Lotus japonicus NGR (LjNGR) protein (SEQ ID NO: 29), Medicago sativa NGR (MtNGR) protein (SEQ ID NO: 18), poplar NGR (PtNGR) protein (SEQ ID NO: 30), and peach NGR (PpeNGR) protein (SEQ ID NO: 31). Figure 5 shows the results of a comparison of the amino acid sequences of the rice qSOR1 protein (SEQ ID NO: 2), Arabidopsis thaliana LZY2 protein (SEQ ID NO: 17), Arabidopsis thaliana LZY3 protein (SEQ ID NO: 12), and Medicago sativa NGR protein (SEQ ID NO: 18). As shown in Figures 1 and 5, the amino acid sequences of these qSOR1 proteins, particularly the amino acid sequences of domains I to V, share a very high similarity. In particular, all of these qSOR1 proteins share the amino acid sequence PLDRFL in domain III. Domain III is located at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, positions 130 to 135 of the amino acid sequence shown in SEQ ID NO: 12, positions 128 to 133 of the amino acid sequence shown in SEQ ID NO: 17, positions 109 to 114 of the amino acid sequence shown in SEQ ID NO: 18, positions 127 to 132 of the amino acid sequence shown in SEQ ID NO: 24, positions 131 to 136 of the amino acid sequence shown in SEQ ID NO: 25, positions 121 to 126 of the amino acid sequence shown in SEQ ID NO: 26, positions 122 to 127 of the amino acid sequence shown in SEQ ID NO: 27, positions 111 to 116 of the amino acid sequence shown in SEQ ID NO: 28, positions 105 to 110 of the amino acid sequence shown in SEQ ID NO: 29, positions 112 to 117 of the amino acid sequence shown in SEQ ID NO: 30, and positions 110 to 115 of the amino acid sequence shown in SEQ ID NO: 31.

[0033] As used herein, the term "gene" includes a protein-coding sequence (CDS). A gene may optionally include an untranslated region (UTR), exons and introns, and may include a promoter, enhancer, insulator, terminator, and / or polyA sequence. A gene encompasses not only double-stranded nucleic acid but also single-stranded nucleic acids such as the positive strand (sense strand) or complementary strand (antisense strand) that constitute the double-stranded nucleic acid, and further encompasses genomic DNA, DNA, RNA, mRNA, cDNA, and the like unless otherwise specified. A gene may be, for example, a polynucleotide that encodes a protein. As used herein, the term "polynucleotide" includes both DNA and RNA, and in the case of DNA, it may be single-stranded or double-stranded.

[0034] In the present invention, an amino acid substitution contained in the mutant qSOR1 protein that improves deep rooting ability is, for example, an amino acid substitution in the sequence at positions 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 at positions 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 (e.g., 1, 2, 3, 4, 5, or 6 amino acids) in the sequence at positions corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2.

[0035] The amino acid substitution may be a conservative or non-conservative amino acid substitution, and may be, for example, a substitution of an amino acid (e.g., a non-conservative amino acid substitution) 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, 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 of the amino acid sequence shown in SEQ ID NO: 2, arginine at the position corresponding to the 143rd position of the amino acid sequence shown in SEQ ID NO: 2, phenylalanine at the position corresponding to the 144th position of the amino acid sequence shown in SEQ ID NO: 2, and leucine at the position corresponding to the 145th position of the amino acid sequence shown in SEQ ID NO: 2. Examples of substitutions for the proline at position 140 include substitutions with polar, uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), acidic amino acids (glutamic acid or aspartic acid), or basic amino acids (lysine, arginine, or histidine), but substitutions with serine, threonine, glutamine, asparagine, or cysteine ​​are preferred, substitutions with serine or threonine are more preferred, and substitutions with serine are most preferred. Examples of substitutions for the leucine at position 141 include substitutions with polar, uncharged amino acids (serine, threonine, glutamine, asparagine, or cysteine), aromatic amino acids (phenylalanine, tyrosine, or tryptophan), acidic amino acids (glutamic acid or aspartic acid), or basic amino acids (lysine, arginine, or histidine), but substitution with phenylalanine or tryptophan is preferred, and substitution with phenylalanine is more preferred.

[0036] As used herein, "a position corresponding to the 140th amino acid in the amino acid sequence shown in SEQ ID NO: 2" refers to the position of an amino acid in any amino acid sequence (any amino acid sequence of a qSOR1 protein) aligned with the amino acid sequence shown in SEQ ID NO: 2 that is aligned with the proline at position 140 in the amino acid sequence shown in SEQ ID NO: 2. Similar expressions such as "a position corresponding to position 'x' in the amino acid sequence shown in SEQ ID NO: 2" and "positions corresponding to positions 140 to 145 in 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 containing the above-mentioned amino acid substitution that improves deep rooting ability.

[0037] In the present invention, the gene encoding the mutant qSOR1 protein (mutant qSOR1 gene) may be an endogenous qSOR1 gene on the genome in which a nucleotide mutation that improves deep rooting ability has occurred or has been introduced (e.g., artificially). As used herein, "nucleotide mutation that improves deep rooting ability" refers to a nucleotide mutation that causes an amino acid substitution that improves deep rooting ability. As used herein, "nucleotide mutation" contained in the qSOR1 gene refers to a mutation in the base sequence of the wild-type qSOR1 gene, and includes nucleotide insertion, deletion, substitution, addition, etc.

[0038] In the present invention, the mutant qSOR1 gene may also be an exogenous gene. As used herein, the term "exogenous" gene refers to a gene that has been artificially introduced into a host plant by genetic manipulation such as transformation.

[0039] The amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 4 is the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 (the amino acid sequence of the original variety qSOR1 protein) in which the proline at position 140 is substituted with serine. The amino acid sequence of the mutant qSOR1 protein shown in SEQ ID NO: 6 is the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 in which the leucine at position 141 is substituted with phenylalanine. The amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 14 is the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 12 (the amino acid sequence of the wild-type LZY3 protein) in which the proline at position 130 (corresponding to position 140 in the amino acid sequence shown in SEQ ID NO: 2) is substituted with serine. The amino acid sequence of the mutant LZY3 protein shown in SEQ ID NO: 16 is the amino acid sequence of the amino acid sequence of SEQ ID NO: 12 in which the leucine at position 131 (corresponding to position 141 in the amino acid sequence shown in SEQ ID NO: 2) is substituted with phenylalanine.

[0040] 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 may also be a protein that 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, 98% or more, 99%, or 99.5% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, and 24 to 31, and contains amino acid substitutions in the sequence at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and that exhibits activity of improving plant deep rooting. The mutant qSOR1 protein may also be a protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 50, 1 to 25, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acid in the amino acid sequence shown in any one of SEQ ID NOs: 2, 12, 17, 18, 24 to 31, and including an amino acid substitution in the sequence at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity that improves deep rooting of plants.

[0041] The nucleotide 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, respectively. Specifically, the nucleotide sequence shown in SEQ ID NO: 3 is a nucleotide sequence in which the codons CCG at positions 418 to 420 of the nucleotide sequence shown in SEQ ID NO: 1 (CDS encoding the original variety qSOR1 protein) are substituted with TCG. The nucleotide sequence shown in SEQ ID NO: 5 is a nucleotide sequence in which the codons CTC at positions 421 to 423 of the nucleotide sequence shown in SEQ ID NO: 1 are substituted with TTC. The nucleotide sequence shown in SEQ ID NO: 13 is a nucleotide sequence in which the codons CCT at positions 388 to 390 of the nucleotide sequence shown in SEQ ID NO: 11 (CDS encoding the wild-type LZY3 protein) are substituted with TCT. The nucleotide sequence shown in SEQ ID NO: 15 is a nucleotide sequence in which the codons TTG at positions 391 to 393 of the nucleotide sequence shown in SEQ ID NO: 11 are substituted with TTC.

[0042] In the present invention, the gene encoding the mutant qSOR1 protein may comprise the nucleotide sequence shown in SEQ ID NO: 3, 5, 13, or 15. The gene encoding the mutant qSOR1 protein may also comprise a nucleotide sequence encoding a protein that 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, 98% or more, 99%, or 99.5% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 or 11, 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 exhibits activity of improving plant deep rooting. The gene encoding the mutant qSOR1 protein may also have an insertion, deletion, substitution, and / or addition of 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 base in the base sequence shown in SEQ ID NO: 1 or 11, contain 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 contain a base sequence encoding a protein that exhibits activity that improves deep rooting of plants.

[0043] The plants used in the present invention are typically, but not limited to, angiosperms. The plants may be either annual or perennial plants, and may be either monocotyledonous or dicotyledonous plants. Examples of monocotyledonous plants include, but are not limited to, plants from the Poaceae, Liliaceae, Bromeliaceae, Palmaceae, Araceae, Zingiberaceae, and Orchidaceae families. Examples of dicotyledonous plants include, but are not limited to, Cucurbitaceae, Brassicaceae, Fabaceae, Asteraceae, Lamiaceae, Solanaceae, Rosaceae, Apiaceae, Convolvulaceae, Nelumbaceae, and Salicaceae families.

[0044] The plants may be ornamental flowering plants, agricultural crops such as edible vegetables and fruits, etc. Examples of ornamental flowering plants include morning glories, sunflowers, cosmos, sweet peas, marigolds, pansies, violas, daisies, snapdragons, gerberas, bellflowers, clematis, cannas, cyclamen, chrysanthemums, tulips, roses, carnations, petunias, gypsophila, lilies, and orchids. Examples of agricultural crops include rice, wheat, barley, rye, oats, Job's tears, corn, millet, foxtail millet, barnyard millet, sorghum, finger millet, pearl millet, teff, sugarcane, Arabidopsis, rapeseed, cabbage, komatsuna, radish, Chinese cabbage, broccoli, soybean, kidney bean, broad bean, leek, rapeseed, cabbage, lettuce, tobacco, tomato, strawberry, eggplant, carrot, potato, cotton, onion, garlic, potato, taro, yam, sweet potato, cucumber, lotus, peach, etc. Plants may also be plants used as roadside trees, such as poplar, plane tree, weeping willow, black locust, cherry blossom, and Chinese parasol tree.

[0045] The plants used in the present invention are preferably plants of the Poaceae, Brassicaceae, or Leguminosae families. Examples of Poaceae plants include, but are not limited to, rice, wheat, barley, rye, oats, Job's tears, corn, millet, foxtail millet, barnyard millet, sorghum, finger millet, pearl millet, teff, sugarcane, timothy, Kentucky bluegrass, orchardgrass, Italian ryegrass, perennial ryegrass, tall fescue, bahiagrass, and wheatgrass. Examples of Brassicaceae plants include, but are not limited to, Arabidopsis thaliana, rapeseed, cabbage, komatsuna (Japanese mustard spinach), radish, Chinese cabbage, and broccoli. Examples of Leguminosae plants include, but are not limited to, soybean, kidney bean, broad bean, adzuki bean, pea, alfalfa, and lotus grass.

[0046] The plant used in the present invention is more preferably rice. As used herein, "rice" refers to any plant belonging to the genus Oryza in the family Poaceae. Rice includes cultivated and wild rice. Cultivated rice includes Asian rice (Oryza sativa) and African rice (Oryza glaberrima), and Asian rice includes 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 this specification, the term "plant" includes the whole plant body or parts thereof (leaves, stems, roots, shoot tips, anthers, pollen, embryos, calluses, cells, etc.), seeds, etc.

[0047] Plants may have multiple genomes. For example, wild species of rice include allotetraploids that have two genomes, genome B and genome C. The plant of the present invention may have the mutant qSOR1 gene on at least one genome. The plant of the present invention may also have the mutant qSOR1 gene on at least one genome in a homozygous or heterozygous state.

[0048] Generally, when mutations are introduced into plant genes to improve the plant's stress tolerance (including drought tolerance, etc.), this can have adverse effects on yield, plant type, etc. However, the inventors have found that when nucleotide mutations that improve the above-mentioned deep rooting ability are introduced into the plant's qSOR1 gene, yield does not decrease compared to the original variety, and tends to increase, and plant type does not change.

[0049] Therefore, the plant of the present invention may have an unchanged plant type. As used herein, "an unchanged plant type" means that the plant type has not changed compared to the original variety or line. As used herein, "plant type" refers to the general shape of the above-ground part of the plant, as determined by characteristics such as stem and branch spread. For example, when the plant of the present invention is rice, the plant type can be evaluated by the height of the rice plant, the number of panicles, the panicle length, the rachis length, the number of primary rachis branches per panicle, the number of secondary rachis branches per panicle, the number of seeds per panicle, etc.

[0050] The plant of the present invention may have a maintained or increased yield, particularly 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 (e.g., 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 line (e.g., the yield is increased by 5% or more). For example, when the plant of the present invention is rice, the yield can be measured as the dry matter weight of polished rice grains.

[0051] (2) Method for producing mutant plants The plants of the present invention can be produced, for example, by introducing a nucleotide mutation that improves deep rooting into the qSOR1 gene on the plant genome. Therefore, the present invention provides a method for producing plants with improved deep rooting (plants of the present invention) (hereinafter also referred to as the "method for producing a mutant plant of the present invention"), which includes a step of introducing a nucleotide mutation that improves deep rooting into the qSOR1 gene of a plant.

[0052] In the above-described method for producing a mutagenized plant of the present invention, terms such as "plant," "qSOR1 gene," "nucleotide mutation that improves deep rooting," and "improved deep rooting" are as defined in the description of the plant of the present invention. In the above-described method for producing a mutagenized plant of the present invention, the plant into which the mutation is introduced may be a plant (e.g., a wild-type plant) that does not have a nucleotide mutation in the qSOR1 gene that improves deep rooting, or a plant that has a nucleotide mutation in the qSOR1 gene that improves deep rooting. The plant may be, for example, a monocotyledonous or dicotyledonous plant, such as a grass or Brassicaceae plant, such as rice. In the method for producing a mutagenized plant of the present invention, the nucleotide mutation that improves deep rooting may be, 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, such as a substitution of proline with serine at position 140 of the amino acid sequence shown in SEQ ID NO: 2, or a nucleotide mutation that causes a substitution of leucine with phenylalanine at position 141 of the amino acid sequence shown in SEQ ID NO: 2. In the method for producing a mutant plant of the present invention, the mutant qSOR1 gene generated by introducing a nucleotide mutation that improves deep rooting ability is, for example, (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, 6, 14 or 16; (ii) a protein having 90% or more sequence identity with any one of the amino acid sequences shown in SEQ ID NOs: 2, 12, 17, 18, and 24 to 31, and containing an amino acid substitution at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity of improving plant deep rooting; or (iii) A protein having an amino acid sequence having 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, and 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, and exhibiting activity of improving deep rooting ability of plants. may be coded.

[0053] In the method for producing a mutagenized plant of the present invention, nucleotide mutations that improve deep rooting can be introduced by, for example, introducing random mutations into the plant genome and then selecting from the resulting mutants those that have introduced or accumulated nucleotide mutations that improve deep rooting. Random mutations can be introduced, for example, by irradiation with radiation such as X-rays or gamma rays, or by treatment with mutagenic chemicals such as nitroso compounds (e.g., nitrosoguanidine), base analogs (e.g., bromodeoxyuridine), or alkylating agents (e.g., ethylnitrosourea (ENU) or ethyl methanesulfonate (EMS)).

[0054] Nucleotide mutations that improve deep rooting can also be introduced by introducing site-specific mutations into the genome of a plant. Site-specific mutations can be introduced using, for example, Gateway (R) This can be performed by various site-specific mutagenesis techniques, such as site-specific mutagenesis based on homologous recombination, PCR-based site-specific mutagenesis, or genome editing techniques using transcription activator-like effector nuclease (TALEN) (JP Patent Publication Nos. 2012-514976 and 2013-513389), zinc finger nuclease (JP Patent Nos. 4350907 and 4555292), CRISPR / Cas9 (Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821. (2012)).

[0055] The method for producing a mutant plant of the present invention may include a step of selecting mutants that have introduced or accumulated nucleotide mutations that improve deep rooting. The selection of mutants can be carried out, for example, by determining the nucleotide sequence of the qSOR1 gene and selecting mutants that have the nucleotide mutations that improve deep rooting in the determined nucleotide sequence.

[0056] (3) Method for producing transformed plants The plants of the present invention can also be produced by transformation. Accordingly, the present invention provides a method for producing a plant with improved deep rooting ability (the plant of the present invention) (hereinafter also referred to as the "transgenic plant production method of the present invention"), which comprises the step of introducing into a plant a vector containing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting ability. The present invention also provides the above-mentioned gene and vector used in such a transgenic plant production method. In the above-described method for producing a transformed plant of the present invention, terms such as "amino acid substitution that improves deep rooting ability," "mutant qSOR1 protein," "gene," "improved deep rooting ability," and "plant" are as defined in the description of the plant of the present invention. In the above-described method for producing a transformed plant of the present invention, the plant into which the vector is introduced may also be a plant (e.g., a wild-type plant) into which the vector has not been introduced. The plant may be, for example, a monocotyledonous or dicotyledonous plant, such as a grass or a Brassicaceae plant, such as rice. In the method for producing a transformed plant of the present invention, the amino acid substitution that improves deep rooting ability may be, for example, an amino acid substitution at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, such as a substitution of proline with serine at position 140 of the amino acid sequence shown in SEQ ID NO: 2, or a substitution of leucine with phenylalanine at position 141 of the amino acid sequence shown in SEQ ID NO: 2. In the above-mentioned method for producing a transformed plant of the present invention, the mutant qSOR1 protein is (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, 6, 14 or 16; (ii) a protein having 90% or more sequence identity with any one of the amino acid sequences shown in SEQ ID NOs: 2, 12, 17, 18, and 24 to 31, and containing an amino acid substitution at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity of improving plant deep rooting; or (iii) A protein having an amino acid sequence having 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, and 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, and exhibiting activity of improving deep rooting ability of plants. It could be. 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.

[0057] The vector can be introduced into a plant by any of the techniques commonly used in the art, and suitable methods include the Agrobacterium method, the particle gun method, the electroporation method, etc. The plant used for the vector introduction may be a whole plant, a plant organ, or a plant tissue fragment, or a callus or protoplast may be prepared and used.

[0058] The above-mentioned method for producing a transformed plant of the present invention may include a step of selecting a transformed plant into which the above-mentioned vector has been introduced.

[0059] Plants into which a vector has been introduced can be selected, for example, by utilizing the presence or absence of expression of a selection 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. Suitable antibiotic resistance genes include, but are not limited to, kanamycin resistance genes, neomycin resistance genes, ampicillin resistance genes, hygromycin resistance genes, etc.

[0060] Introduction of a vector into a plant can also be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like.

[0061] (4) Method for producing plants with improved deep rooting through hybridization The present invention also provides a method for producing a plant with improved deep rooting ability (a plant of the present invention) (also referred to as the "breeding method of the present invention"), which includes the steps of crossbreeding plants using a plant of the present invention as a breeding parent to obtain progeny plants, and selecting progeny plants into which a gene encoding the mutant qSOR1 protein has been introduced.

[0062] "Plant crossing using" a plant of the present invention as a breeding parent refers to crossing plants of the present invention with each other, or with a plant of the same or closely related species. Crossing may be performed 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 resulting progeny plant may be crossed with the recurrent parent (backcrossing), and the resulting progeny plant may be crossed with another plant of the same or closely related species, and this process may be repeated (sequential backcrossing). Alternatively, a plant of the present invention may be crossed with a plant of the same or closely related species, and the resulting progeny plant may be crossed with another plant of the same or closely related species.

[0063] The selection of progeny plants into which the gene encoding the mutant qSOR1 protein has been introduced can be carried out by the methods described in relation to the method for producing a mutant plant and the method for producing a transformed plant of the present invention.

[0064] (5) Method for selecting plants with improved deep rooting ability The present invention also provides a method for selecting plants with improved deep-rooting ability (also referred to as the "selection method of the present invention"), which includes the steps of performing nucleic acid amplification of all or part of the qSOR1 gene using DNA derived from a test plant as a template, and identifying plants having a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep-rooting ability based on the results of the nucleic acid amplification.

[0065] In the selection method of the present invention, the term "subject plant" refers to a plant subjected to the selection method of the present invention. In the selection method of the present invention, the term "plant" is as defined above in the description of the plant of the present invention. The plant may be, for example, a monocotyledonous or dicotyledonous plant, such as a grass or a Brassicaceae plant, such as rice. In the selection method of the present invention, the subject plant may be, for example, a mutant obtained by introducing random mutations into a plant, or a progeny plant obtained by crossbreeding a plant using a plant of the present invention as a breeding parent. 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," and "improved deep rooting ability" are also as defined above in the description of the plant of the present invention. In the selection method of the present invention, an amino acid substitution that improves deep rooting ability can be, for example, an amino acid substitution in the sequence at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, for example, a substitution of proline with serine at position corresponding to 140 of the amino acid sequence shown in SEQ ID NO: 2, or a substitution of leucine with phenylalanine at position corresponding to 141 of the amino acid sequence shown in SEQ ID NO: 2. In the above-mentioned method for producing a transformed plant of the present invention, the mutant qSOR1 protein is (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, 6, 14 or 16; (ii) a protein having 90% or more sequence identity with any one of the amino acid sequences shown in SEQ ID NOs: 2, 12, 17, 18, and 24 to 31, and containing an amino acid substitution at positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, and exhibiting activity of improving plant deep rooting; or (iii) A protein having an amino acid sequence having 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, and 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, and exhibiting activity of improving deep rooting ability of plants. It could be.

[0066] In the present invention, any nucleic acid amplification technique can be used, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), transcription mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), or ligase chain reaction (LCR).

[0067] 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 be 15 or more bases long, or 20 or more bases long, or 50 or less bases long, or 30 or less bases long. The primer may also be, for example, 15 to 50 bases long, 20 to 50 bases long, or 20 to 30 bases long.

[0068] In the selection method of the present invention, the "part of the qSOR1 gene" may be any region that includes the base sequence encoding domain III of the qSOR1 protein (the sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2).

[0069] The selection method of the present invention can be carried out by, for example, direct sequencing by the Sanger method, the HRM (High Resolution Melting) method, or the KASP method. TM This can be performed using Kompetitive Allele Specific PCR (Kompetitive Allele Specific PCR) genotyping assay (LGC Biosearch Technologies), dCAPS (derived amplified polymorphic sequence) method, etc.

[0070] The selection method of the present invention may further include a step of evaluating the root elongation angle, deep root rate, or gravitropism of the test plant. The method for evaluating the root elongation angle, deep root rate, or gravitropism is as described for the plant of the present invention.

[0071] In one embodiment of the screening method of the present invention, nucleic acid amplification is carried out using genomic DNA derived from a test plant as a template, (i) a forward primer comprising a nucleotide sequence of at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 19; and (ii) a reverse primer containing a nucleotide sequence of at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 20 or 21; This may be carried out using a primer set comprising:

[0072] The above primer set may be, for example, (i) a forward primer comprising the nucleotide sequence shown in SEQ ID NO: 19, and (ii) a reverse primer containing the nucleotide sequence shown in SEQ ID NO: 20 or 21 It may include:

[0073] The nucleotide sequence shown in SEQ ID NO: 19 corresponds to the nucleotide sequence in the second intron of the rice qSOR1 gene (nucleotides 629 to 652 of the nucleotide sequence of SEQ ID NO: 23), and the nucleotide sequences shown in SEQ ID NOs: 20 and 21 correspond to the nucleotide sequences in the third intron of the rice qSOR1 gene (nucleotides 1453 to 1478 and 1301 to 1327 of the nucleotide sequence of SEQ ID NO: 23, respectively). Therefore, by performing nucleic acid amplification using the above primer set, a region containing the third exon (including the region encoding domain III of the qSOR1 protein) is amplified.

[0074] After nucleic acid amplification using the primer set, the presence or absence of a nucleotide mutation that improves deep rooting in the qSOR1 gene can be determined by determining the base sequence of the amplified product using the Sanger method, etc. Plants determined to have a nucleotide mutation that improves deep rooting in the qSOR1 gene can be identified as plants having a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting.

[0075] The primer used for nucleic acid amplification in the selection method of the present invention preferably contains the above-defined nucleotide sequence at its 3' end.

[0076] The selection method of the present invention may include a step of preparing a test plant prior to the nucleic acid amplification step. The step of preparing a test plant may include, for example, introducing a nucleotide mutation that improves deep rooting into the plant's qSOR1 gene to produce the test plant; introducing a vector containing a gene encoding a mutant qSOR1 protein containing an amino acid substitution that improves deep rooting into the plant to produce the test plant; or crossbreeding a plant of the present invention as a breeding parent to obtain progeny plants as test plants. These steps can be performed as described in "(2) Method for Producing Mutant Plants," "(3) Method for Producing Transgenic Plants," and "(4) Method for Producing Plants with Improved Deep Rooting by Crossbreeding."

[0077] The selection method of the present invention can be used, for example, in the method of producing a mutagenized plant, the method of producing a transformed plant, or the breeding method of the present invention. [Example]

[0078] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0079] Example 1: Identification of qSOR1 mutant lines from rice mutant lines Rice mutant lines were screened for lines with nonsynonymous substitutions in the qSOR1 gene. The region flanking exon 3 of the qSOR1 gene (Os07g0614400) was amplified by polymerase chain reaction (PCR) using genomic DNA from rice (Oryza sativa) cultivar Koshihikari (M1 generation, n = 3,072), which was obtained by treating the zygotes with ethylnitrosourea (ENU). The forward primer qSOR1-p1-F1 (5'-tggatatattttgcatggtttttg-3') (SEQ ID NO: 19) and the reverse primer qSOR1-p1-R1 (5'-caacatctacgacgtcaaattagtct-3') (SEQ ID NO: 20) were used as a template. Prime STAR GXL DNA polymerase (Takara Bio) was used for PCR amplification.

[0080] The resulting PCR product was heated to 98°C and then cooled to dissociate and reassociate the double-stranded DNA. It was then treated with Cel-I nuclease extracted from celery and subjected to agarose gel electrophoresis. Since Cel-I nuclease cleaves double-stranded DNA at mismatch sites, strains that exhibit cleavage fragments during agarose gel electrophoresis can be determined to have a substitution mutation in the qSOR1 gene.

[0081] For strains in which cleavage fragments were detected by agarose electrophoresis, the qSOR1 gene was sequenced using the forward primer qSOR1-p1-F1 (5'-tggatatattttgcatggtttttg-3') (SEQ ID NO: 19) and the reverse primer qSOR1-p1-R2 (5'-gaaatggagtgagtagatgataacttg-3') (SEQ ID NO: 21). Analysis of the sequence revealed four strains with nonsynonymous substitutions in the qSOR1 gene. The CDSs of the qSOR1 gene determined for these four qSOR1 mutant strains (strain names 0951M, 2792M, 0909M, and 2574M) are shown in SEQ ID NOs: 3, 5, 7, and 9, respectively. The encoded amino acid sequences are shown in SEQ ID NOs: 4, 6, 8, and 10, respectively. Comparison of the CDS of the qSOR1 gene of these four qSOR1 mutant lines with the CDS (SEQ ID NO: 2) of the qSOR1 gene of the original variety (Koshihikari) revealed the following mutations (see Figures 3 and 4). Line name 0951M: A mutation (missense mutation) resulting in a substitution of proline with serine at position 140 (P140S). Line name 2792M: A mutation resulting in a substitution of leucine with phenylalanine at position 141 (L141F). Line name 0909M: A mutation resulting in a substitution of arginine with cysteine ​​at position 204 (R204C). Line name 2574M: A mutation resulting in a substitution of arginine with histidine at position 204 (R204H). 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.

[0082] The three-dimensional structure of the qSOR1 protein was predicted using I-TASSER. It was suggested that arginine 204 forms salt bridges with aspartic acid 17 and glutamic acid 226, which may contribute to the stabilization of the three-dimensional structure of the qSOR1 protein. In particular, the line 0909M, in which the basic amino acid arginine at position 204 is replaced by a neutral amino acid cysteine, was predicted to exhibit altered root phenotypes compared to the parent cultivar. On the other hand, the amino acid substitutions P140S and L141F did not alter the structure of the qSOR1 protein and were therefore not expected to affect the root phenotype.

[0083] Comparison of the amino acid sequences encoded by the rice (Koshihikari) qSOR1 gene and its isolated homologous genes, the Arabidopsis LZY2 gene, the Arabidopsis LZY3 gene, and the Medicago sativa NGR gene (SEQ ID NOs: 2, 17, 12, and 18, respectively), revealed that proline 140 and leucine 141 are located in domain III, which is highly conserved among different species. On the other hand, arginine 204 is conserved in rice and Medicago sativa, but is a different amino acid in Arabidopsis (Figure 5).

[0084] Example 2. Root phenotype of qSOR1 mutant lines To examine whether the root elongation angle of the qSOR1 mutant line obtained in Example 1 was changed compared to the original variety (Koshihikari), the root elongation angle was measured by the cup method.

[0085] Plastic cups measuring 3.7 cm in diameter and 4 cm in height were filled to the brim with synthetic granular fertilizer, and then sterilized seeds of the original cultivar or the qSOR1 mutant line (M3 generation, homozygous for the qSOR1 gene mutation) were sown in the cups. The cups containing the seeds were placed on a stainless steel tray, and unfertilized fertilizer was added to cover the entire cup. The cups were then cultivated for approximately three weeks. 20 individuals were grown for each of the original cultivar and the four qSOR1 mutant lines. After the cultivation period, the plants were removed from the cups, the roots were washed, and the root elongation angle was measured with a protractor.

[0086] The results are shown in Figure 6. As expected in Example 1, the root elongation angle of line 0909M was significantly reduced compared to the original variety. Furthermore, the root elongation angle of line 2574M was comparable 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.

[0087] These results suggest 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) unexpectedly have the effect of deepening rice roots.

[0088] Example 3. Effect of mutations in the qSOR1 gene on root phenotype The qSOR1 mutant lines used in the cup method were generated by mutagen treatment, so they may have other mutations in their genomes. Therefore, to further demonstrate that the changes in root phenotype observed in the cup method were caused by mutations in the qSOR1 gene, we performed the following experiment.

[0089] The qSOR1 mutant lines (lines 0909M, 0951M, and 2792M) that showed altered root elongation angles in Example 2 were backcrossed three times to the original cultivar and selfed. The resulting three BC3F3 lines (near-isogenic lines in the Koshihikari background) were confirmed by direct sequencing to retain the mutations in the qSOR1 gene.

[0090] The root phenotypes of these three BC3F3 lines were investigated using a modified basket method using a stainless steel mesh strainer. Sterilized seeds of the original cultivar or BC3F3 line were sown in a custom-made stainless steel strainer with a diameter of 7.5 cm and filled with fertilizer-free soil. The plants were then grown in a hydroponic solution for approximately one and a half months. After the cultivation period, roots extending downward at an angle of more than 30 degrees relative to the soil surface were defined as deep roots. The number of deep roots and the total number of roots were counted. The deep root ratio (RDR30; Ratio of Deeper Root than 30 degrees) was calculated by dividing the number of deep roots by the total number of roots. A higher value of the deep root ratio indicates deeper roots. The deep root ratio was investigated for 20 individuals each of the original cultivar and the three BC3F3 lines.

[0091] The results are shown in Figure 7. The deep root ratio of the BC3F3 lines derived from 0909M was significantly lower than that of the parent varieties, whereas the deep root ratio of the BC3F3 lines derived from 0951M and 2792M was significantly higher than that of the parent varieties. These results were consistent with those obtained using the cup method.

[0092] These results indicate that the changes in root phenotype of the qSOR1 mutant lines are due to mutations occurring within the qSOR1 gene. In other words, the R204C substitution in the amino acid sequence of the rice qSOR1 protein has the effect of shallowing rice roots, while the P140S and L141F substitutions (substitutions in domain III) have the effect of deepening rice roots, contrary to predictions from the original amino acid sequence.

[0093] 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.

[0094] For each of the three BC3F3 lines obtained in Example 3 and the original variety (Koshihikari), 30 seeds were dehusked to obtain brown rice. The brown rice was washed three times with 10 ml of sterilized water and then treated with 1% PPM (Plant Preservative Mixture) as a fungicide. TM The seeds were placed in a petri dish containing 10 ml of medium containing 1% PEG-400 (Plant Cell Technology) and left at 30°C for one day. The germinated seeds were sown on a rectangular plate containing 0.4% agarose gel and left at 28°C in the dark for two days. The rectangular plate was then rotated 90°, and after four hours, the roots were photographed and the root bending angle was measured.

[0095] The results are shown in Figure 8. Compared to the original variety, the BC3F3 line derived from 0909M had a significantly smaller root curvature angle, while the BC3F3 lines derived from 0951M and 2792M had significantly larger root curvature angles.

[0096] These results indicate that rice plants carrying a gene encoding a qSOR1 protein containing the R204C substitution have shallower roots due to a weaker gravitropic response than the original cultivar, whereas rice plants carrying a gene encoding a qSOR1 protein containing the P140S and L141F substitutions (substitutions in domain III) have deeper roots due to a stronger gravitropic response than the original cultivar.

[0097] Example 5. Effect of substitutions in domain III of qSOR1 protein on yield To clarify whether substitutions in domain III of the rice qSOR1 protein have a negative effect on agricultural traits such as yield, yield surveys were conducted for the 2792M-derived BC3F3 line obtained in Example 3 and its original variety (Koshihikari) over two years, in 2020 and 2021.

[0098] Both strains were cultivated in paddy fields within the National Agriculture and Food Research Organization (NARO) in Tsukuba City, Ibaraki Prefecture in 2020 and 2021. Each strain was cultivated in a paddy field of 1 m 2 The plants were planted individually at a density of 22.2 plants per plot (15cm x 30cm). Germinated seeds were sown in April, raised in a paddy nursery for one month, and then transplanted into the paddy fields. The field design consisted of three replicates of 42 plants (6 x 7) randomly arranged in each plot for each line. The paddy fields were fertilized with 12g m of P2O5 as chemical fertilizer. -2 , 9g m of K2O -2 , N to 12g m -2 Each was applied according to conventional methods.

[0099] The heading date (the date when 50% of the plants in each plot emerged) for the original cultivar and the 2792M-derived BC3F3 line in 2020 was August 6-7 in all three plots, meaning that heading occurred almost simultaneously for both lines. The heading date for the original cultivar in 2021 was July 30-31 in all three plots, and the heading date for the 2792M-derived BC3F3 line in 2021 was July 28 in all three plots. Because summer temperatures in 2021 were higher than in 2020, it is thought that the heading dates for both lines in 2021 were about one week earlier than in 2020. The difference in heading dates between lines was small compared to the inter-year differences.

[0100] After the cultivation period, 24 plants were harvested from each plot, naturally dried, and then threshed. The matured grains were then selected by wind sorting (2020) or water sorting (2021). The selected grains were then air-dried in a dryer at 80°C for three days, and the dry weight of the polished grains was measured.

[0101] The results are shown in Figure 9. The dry weight of polished grains of the 2792M-derived BC3F3 line increased by 6.7% in 2020 and 12.7% in 2021 compared to the original variety. These results indicate that substitutions in domain III of the rice qSOR1 amino acid sequence do not adversely affect yield, but rather result in increased yield. The plant type of the 2792M-derived BC3F3 line was similar to that of the original variety, and no particular abnormalities were observed.

[0102] Example 6: Effects of Arabidopsis LZY3 mutant proteins with substitutions in domain III on root phenotypes In this example, to investigate whether substitutions in domain III of the Arabidopsis thaliana LZY3 protein also enhance root depth in Arabidopsis, we evaluated the effects of LZY3 mutant proteins (dLZY3(P130S) and dLZY3(L131F)) with a proline-to-serine substitution at position 130 (P130S) and a leucine-to-phenylalanine substitution at position 131 (L131F) of SEQ ID NO: 12 on root phenotype (Figures 10-12). 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.

[0103] LZY3p:LZY3-mCherry is a vector that expresses a fusion protein of LZY3 protein and mCherry (red fluorescent protein) under the control of the LZY3 promoter (Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999, provided by the National Institute for Basic Biology, Inter-University Research Institute Corporation, National Institutes of Natural Sciences; the nucleotide sequence of the construct contained in this vector is shown in SEQ ID NO: 22). The 388th C in the LZY3 gene was substituted with a T to create a vector (LZY3p:dLZY3(P130S)-mCherry) that expresses a fusion protein of dLZY3(P130S) mutant protein and mCherry under the control of the LZY3 promoter. Furthermore, by substituting G at position 393 of the LZY3 gene with C in the LZY3p:LZY3-mCherry vector, we created a vector (LZY3p:dLZY3(L131F)-mCherry) that expresses a fusion protein of the dLZY3(L131F) mutant protein and mCherry under the control of the LZY3 promoter.

[0104] Agrobacterium GV3101 was used to introduce LZY3p:dLZY3(P130S)-mCherry and LZY3p:dLZY3(L131F)-mCherry into the Arabidopsis lzy2lzy3 double mutant (a mutant of Arabidopsis Columbia lacking both the LZY2 and LZY3 genes). Seeds from the resulting T1 generation of transformed plants were sown on 1 / 2 MS agar medium containing 20 μg / ml hygromycin, and plants exhibiting hygromycin resistance were selected. Selected plants were then transferred to new 1 / 2 MS agar medium and grown vertically, allowing for observation of root morphology.

[0105] As controls, seeds of Arabidopsis thaliana Columbia (wild type), a mutant of Arabidopsis thaliana Columbia lacking the LZY2 gene (lzy2 single mutant), and a mutant of Arabidopsis thaliana Columbia lacking both the LZY2 and LZY3 genes (lzy2lzy3 double mutant) were sown on agar medium, and then grown on agar plates upright to observe the root morphology.

[0106] The results are shown in Figure 13. The roots of the wild-type and lzy2 single mutants were similar in morphology, whereas the lateral roots of the lzy2lzy3 double mutant were more upwardly elongated than those of the wild-type. This is consistent with previous results (Taniguchi M. et al., The Plant Cell, 2017, 29:1984-1999). In contrast, the lzy2lzy3 double mutant (in which LZY3p:dLZY3(P130S)-mCherry was introduced) (dLZY3(P130S) / lzy2lzy3 mutant) and the lzy2lzy3 double mutant (in which LZY3p:dLZY3(L131F)-mCherry was introduced) (dLZY3(L131F) / lzy2lzy3 mutant) showed more downwardly elongated roots than the parental line (lzy2lzy3 double mutant), complementing the lzy2lzy3 double mutant phenotype. Furthermore, the dLZY3(P130S) / lzy2lzy3 and dLZY3(L131F) / lzy2lzy3 mutants showed significantly downward root elongation compared with the wild-type expressing the wild-type LZY3 protein and the lzy2 single mutant.

[0107] These results indicate that LZY3 mutant proteins with substitutions in domain III have the effect of increasing root depth compared to wild-type LZY3 proteins, i.e., that substitutions in domain III of the LZY3 protein also have the effect of increasing root depth in Arabidopsis thaliana.

[0108] These results suggest that mutations in domain III of the qSOR1 protein have the effect of increasing root depth not only in monocotyledonous plants such as rice, but also in dicotyledonous plants such as Arabidopsis.

[0109] In Figure 13, the roots of the dLZY3(P130S) / lzy2lzy3 and dLZY3(L131F) / lzy2lzy3 mutants appear slightly shorter than those of the wild type, lzy2 single mutant, and lzy2lzy3 double mutant. This is likely due to the influence of hygromycin in the medium. Furthermore, the plant morphology of the dLZY3(P130S) / lzy2lzy3 and dLZY3(L131F) / lzy2lzy3 mutants did not change significantly compared to the lzy2lzy3 double mutant.

[0110] array SEQ ID NO: 1 CDS encoding Koshihikari qSOR1 protein SEQ ID NO: 2 Amino acid sequence of Koshihikari qSOR1 protein SEQ ID NO: 3 CDS encoding the qSOR1 protein of the 0951M strain SEQ ID NO: 4: Amino acid sequence of qSOR1 protein of strain 0951M SEQ ID NO: 5 CDS encoding the qSOR1 protein of the 2792M strain SEQ ID NO: 6: Amino acid sequence of qSOR1 protein of strain 2792M SEQ ID NO: 7 CDS encoding the qSOR1 protein of the 0909M strain SEQ ID NO: 8 Amino acid sequence of qSOR1 protein of the 0909M strain SEQ ID NO: 9 CDS encoding the qSOR1 protein of the 2574M strain SEQ ID NO: 10 Amino acid sequence of qSOR1 protein of strain 2574M SEQ ID NO: 11 CDS encoding Arabidopsis LZY3 protein SEQ ID NO: 12: Amino acid sequence of Arabidopsis LZY3 protein SEQ ID NO: 13: Nucleotide sequence encoding dLZY3 (P130S) mutant protein SEQ ID NO: 14: Amino acid sequence of dLZY3(P130S) mutant protein SEQ ID NO: 15: Nucleotide sequence encoding dLZY3 (L131F) mutant protein 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 Medicago sativa 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: Nucleotide sequence of the construct contained in the LZY3p:LZY3-mCherry vector (including the promoter and the polynucleotide encoding the fusion protein) SEQ ID NO: 23 Genomic DNA sequence of Koshihikari qSOR1 gene SEQ ID NO: 24: Amino acid sequence of maize qSOR1 (ZmqSOR1) protein 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 Brachypodium 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 patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A plant with improved deep rooting ability, which has a gene encoding a mutant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) protein containing an amino acid substitution in a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid substitution is a substitution of leucine with phenylalanine at 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: 6 or 16; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having 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 comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; plant.

2. The gene encoding the mutant qSOR1 protein is (iv) a base sequence shown in SEQ ID NO: 5 or 15; (v) A nucleotide sequence encoding a protein that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 or 11, contains a nucleotide mutation that causes the amino acid substitution, and exhibits activity that improves plant deep rooting; or (vi) a base sequence encoding a protein having an insertion, deletion, substitution, and / or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1 or 11, containing a nucleotide mutation that causes the amino acid substitution, and exhibiting activity that improves plant deep rooting; The plant of claim 1 , comprising:

3. 2. The plant of claim 1, which is a monocotyledonous or dicotyledonous plant.

4. A method for producing a plant with improved deep rooting ability, comprising the step of introducing a nucleotide mutation that causes an amino acid substitution in a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2 into a qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene of the plant, wherein the amino acid substitution is a substitution of leucine with phenylalanine at position 141 of the amino acid sequence shown in SEQ ID NO: 2; The mutant qSOR1 gene generated by the introduction of the nucleotide mutation is a mutant qSOR1 protein as follows: (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 16; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having 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 comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. Code the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; method.

5. A method for producing a plant with improved deep rooting ability, comprising the step of introducing into a 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 a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid substitution is a substitution of leucine with phenylalanine at 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: 6 or 16; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having 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 comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; method.

6. A method for producing a plant with improved deep rooting ability, comprising the steps of: crossbreeding plants using a plant described in any one of claims 1 to 3 as a breeding parent to obtain offspring plants; and selecting offspring plants into which a gene encoding the mutant qSOR1 protein has been introduced.

7. A method for selecting a plant with improved deep rooting ability, comprising the steps of: performing nucleic acid amplification of the entire or a part of the qSOR1 gene using DNA derived from a test plant as a template; and identifying a plant having a gene encoding a mutant qSOR1 protein containing an amino acid substitution in a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2 based on the results of nucleic acid amplification, wherein the amino acid substitution is a substitution of leucine with phenylalanine at 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: 6 or 16; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or 12, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having 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 comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; method.

8. A plant with improved deep rooting ability, having 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 the 140th to 145th amino acids of the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid substitution is a substitution of proline with serine at the position corresponding to the 140th amino acid 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 set forth in SEQ ID NO: 4; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2, and comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; The plant is a monocotyledonous plant. plant.

9. The gene encoding the mutant qSOR1 protein is (iv) the base sequence shown in SEQ ID NO: 3; (v) A nucleotide sequence encoding a protein that has 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1, contains a nucleotide mutation that causes the amino acid substitution, and exhibits activity that improves plant deep rooting ability; or (vi) a base sequence encoding a protein having an insertion, deletion, substitution, and / or addition of 1 to 10 bases in the base sequence shown in SEQ ID NO: 1, containing a nucleotide mutation that causes the amino acid substitution, and exhibiting activity that improves plant deep rooting; The plant of claim 8, comprising:

10. A method for producing a plant with improved deep-rooting ability, comprising the step of introducing a nucleotide mutation into a plant qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene, the nucleotide mutation causing an amino acid substitution in a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid substitution is a substitution of proline with serine at position 140 of the amino acid sequence shown in SEQ ID NO: 2, The mutant qSOR1 gene generated by the introduction of the nucleotide mutation is a mutant qSOR1 protein as follows: (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2, and comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. Code the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; The plant is a monocotyledonous plant. method.

11. A method for producing a plant with improved deep rooting ability, comprising the step of introducing into a 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 a sequence corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid substitution is a substitution of proline with serine at position 140 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 set forth in SEQ ID NO: 4; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2, and comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; The plant is a monocotyledonous plant. method.

12. A method for producing a plant with improved deep rooting ability, comprising the steps of: crossbreeding plants using the plant described in claim 8 or 9 as a breeding parent to obtain offspring plants; and selecting offspring plants into which the gene encoding the mutant qSOR1 protein has been introduced.

13. A method for selecting plants with improved deep rooting ability, comprising the steps of: performing nucleic acid amplification of all or part of the qSOR1 gene using DNA derived from a test plant as a template; and identifying, based on the results of nucleic acid amplification, plants having a gene encoding a mutant qSOR1 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, wherein the amino acid substitution is a substitution of proline with serine at position 140 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 set forth in SEQ ID NO: 4; (ii) a protein having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, including the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants; or (iii) A protein having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2, and comprising the amino acid sequence containing the amino acid substitution, and exhibiting activity of improving deep rooting ability of plants. and the mutant qSOR1 protein does not contain any amino acid mutations other than the amino acid substitutions at positions corresponding to positions 140 to 145 of the amino acid sequence shown in SEQ ID NO: 2; The plant is a monocotyledonous plant. method.

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