JP5515060B2 - How to develop rice roots - Google Patents

Description

  The present invention relates to a method for producing rice with a developed side root and a method for developing a side root of rice by culturing under low nitrogen conditions as compared to wild-type rice.

  Nitrogen is one of the important nutrients that support plant growth. Nitrogen works to promote plant growth. For example, seven times as much nitrogen fertilizer has been used to double global crop production over the past 40 years. However, there are various problems in consuming a large amount of nitrogen to promote plant growth.

  A large amount of nitrogen introduced into the soil flows into lakes and oceans via groundwater and causes environmental pollution such as the occurrence of plankton abnormalities. In addition, large amounts of nitrogen input to the soil can cause livestock damage. For example, livestock such as cows that have ingested grass grown on soil that has absorbed a large amount of nitrogen produces N-nitroso compounds in the stomach, increasing the possibility of developing cancer, and the nitrate state in milk. As the amount of nitrogen increases, infants drinking milk from dairy cows grown under high nitrogen are more likely to cause hemoglobinemia. Furthermore, in recent years, nitrogen fertilizers have soared due to rising prices of fossil fuels as raw materials, and farming methods that require the use of large amounts of nitrogen fertilizers reduce the profits of farmers.

  As an attempt to solve the above-mentioned problems related to mass consumption of nitrogen, research is currently being conducted to improve the yield of plants including biomass under low nitrogen conditions by increasing the nitrogen utilization efficiency. In order to increase the nitrogen utilization efficiency of plants, it is required to improve the nitrogen absorption ability and assimilation ability of plants. Among them, as an approach for increasing the nitrogen absorption capacity, there is an attempt to improve the root architecture, which is a field of nitrogen absorption.

  Plant roots are broadly divided into main and side roots. In general, in plants containing rice, when there are too few nitrogen sources, the side roots are not formed and only the main roots are elongated, but when nitrogen is present, the side roots differentiate and extend from the main roots in order to absorb nitrogen efficiently. So far, the present inventors have used 20.6 mM ammonium sulfate, and rice that expresses mutant EL5 in which a part of the amino acid sequence of EL5, which is a RING-H2 type ubiquitin ligase localized in cell membrane, is mutated. When cultured in MS medium containing 8 mM potassium nitrate and 3.0 mM calcium chloride, the roots turn brown and do not grow (Non-Patent Document 1), but then root the roots by cultivating them in tap water or pure water for 4 days. The method was reported (nonpatent literature 2). Furthermore, it has been reported that the side roots of Arabidopsis thaliana can be elongated by culturing Arabidopsis thaliana in the presence of auxin (Non-patent Document 3).

H. Koiwai, A. Tagiri, S. Katoh, E. Katoh, H. Ichikawa, E. Minami, and Y. Nishizawa, (2007), The Plant J. 51, 92-104. Hanae Koiwai, Emi Nakajima, Kutaro Kishimoto, Kyoko Kato, Eiichi Minami, Yoko Nishizawa (2008) Abstracts of the 49th Annual Meeting of the Japanese Society of Plant Physiology p.172 Yoko Okushima, Hidehiro Fukaki, Makoto Onoda, Athanasios Theologis, and Masao Tasaka, (2007), Plant Cell 19: 118-130.

  A plant with long side roots is expected to have a large surface area of the whole root and a high nitrogen-absorbing capacity, and growth is not inhibited even under low nitrogen conditions. In addition, by reducing the amount of nitrogen fertilizer used for plants, it is possible to carry out agriculture with less burden on the environment and livestock and at a lower cost. Therefore, there is a demand for a method for producing a long plant having a large number of side roots that can be cultivated under low nitrogen conditions.

  However, among the conventionally known techniques, the method described in Non-Patent Document 2 is a method of forming a main root of rice, and is not a method of forming a side root of rice. Moreover, as described in Non-Patent Document 3, the side roots of Arabidopsis thaliana can be extended by auxin. However, it has not been known so far that the lateral root of rice is elongated by auxin, and even if the lateral root of rice is elongated by auxin, auxin suppresses the elongation of the main root. It is predicted that the overall surface area cannot be increased.

  Therefore, in order to efficiently absorb nitrogen in the soil, it is possible to produce rice with a longer side root or a larger number of side roots compared to wild-type rice, that is, rice with developed side roots, without suppressing the elongation of the main root. How to do is not known so far.

  Accordingly, an object of the present invention is to provide a method for producing rice having a developed side root and a method for developing a side root of rice, compared with wild-type rice, with little suppression of elongation of the main root.

  As a result of diligent research, the inventors of the present invention have used, as a nitrogen source, less than 1 mM of a transformed rice expressing a V162A mutant EL5 in which the valine at position 162 is substituted with alanine in the amino acid sequence of domain IV of EL5. By culturing using a medium containing potassium nitrate or ammonium sulfate of less than 10 mM, rice having a larger number of lateral roots than wild type rice was successfully obtained. The phenomenon that cultivated rice under this low nitrogen condition yields rice with developed lateral roots is that when there are few nitrogen sources in the soil, the lateral root primordium, which is the part where lateral roots begin to grow, is almost differentiated and elongated. Although it is not a phenomenon that contradicts the previous knowledge that lateral root formation is promoted when the nitrogen source is increased, it was a surprising phenomenon for the present inventors. It is also known that main root elongation and lateral root development are controlled by separate mechanisms. As described in Non-Patent Document 3, side roots are developed by auxin treatment, but elongation of main roots is suppressed. Moreover, when rice is cultivated in an environment rich in nitrogen source, as described above, lateral roots develop, but elongation of main roots is suppressed. However, the above phenomenon promoted the development of lateral roots without affecting the elongation mechanism of the main roots. That is, the above phenomenon is a phenomenon that can promote the development of the lateral root without suppressing the elongation of the main root.

  Furthermore, the present inventors control the expression of mutant EL5 with an EL5 gene promoter that controls the expression of wild-type EL5, thereby expressing a mutant EL5 having a different aspect from the above (for example, W165A mutant EL5). It has been found that converted rice can develop lateral roots under low nitrogen conditions. The present invention has been completed based on these findings.

  Therefore, according to the present invention, transformed rice expressing EL5 having a mutation is used under conditions suitable for growth of wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. A rice having a longer side root and / or a larger number of side roots than wild-type rice cultivated under such conditions is provided.

  According to another aspect of the present invention, transformed rice expressing EL5 with a mutation is suitable for growing wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. A method is provided for developing rice lateral roots, comprising culturing under conditions.

  Preferably, the EL5 having the mutation is EL5 having a mutation in domain IV of EL5.

  Preferably, EL5 having a mutation in domain IV has at least one mutation selected from the group consisting of deletion, substitution, inversion, addition and insertion of one to a plurality of amino acids in the amino acid sequence of domain IV EL5.

  Preferably, EL5 having a mutation in domain IV is at least selected from the group consisting of valine at position 162, leucine at position 138, tryptophan at position 165, and cysteine at position 153 in the amino acid sequence of EL5. EL5 in which one amino acid residue is substituted with an amino acid residue different from the wild type.

  Preferably, EL5 having a mutation in domain IV is EL5 in which the valine at position 162 in the amino acid sequence of EL5 is substituted with alanine.

  Preferably, the transformed rice contains a gene encoding EL5 having a mutation under the control of at least one promoter selected from the group consisting of an EL5 gene promoter, a cauliflower mosaic virus-derived 35S promoter, and a corn-derived ubiquitin promoter. .

  Preferably, the nitrate is at least one nitrate selected from the group consisting of potassium nitrate, sodium nitrate, potassium nitrite and sodium nitrite.

  Preferably, the ammonium salt is at least one ammonium salt selected from the group consisting of ammonium sulfate, ammonium chloride, ammonium phosphate and ammonium acetate.

  Preferably, the culture is performed for 5 to 10 days.

  According to the present invention, rice having a longer side root and / or a larger number of side roots than wild-type rice can be obtained under low nitrogen conditions. Usually, if the nitrogen concentration is increased, lateral roots can be developed, but elongation of the main roots is suppressed. However, the present invention utilizes the reverse phenomenon, and by reducing the nitrogen concentration, the lateral root can be developed with almost no suppression of the main root elongation. By cultivating rice under such a low nitrogen condition, it is possible to reduce the impact on the environment and livestock compared to wild-type rice cultivation, and to realize agriculture that is economically advantageous.

It is the figure which showed the schematic diagram of the protein encoded by various EL5 genes, and the ubiquitin ligase activity. Symbols indicate I: transmembrane region, II: basic amino acid region, III: conserved region, IV: RING-H2 domain, IV ': amino acid mutation-introduced RING-H2 domain, and V: C-terminal region, respectively. The average length of crown roots formed on the 10th day of non-nitrogen-containing hydroponics (A), the number of lateral root formation per crown root unit length (B), and a photograph (C) of the 6th day of cultivation were shown. FIG. Symbols indicate NT: non-transformed (wild type) rice (variety: Nipponbare), EL5V162A; EL5V162A expression rice, respectively. It is the figure which showed the side root formation degree of hydroponic cultivation 10 days after hydroponics of the wild-type rice and the transformed rice which expresses EL5V162A, and the photograph of those typical roots. It is a schematic diagram of pENTR-EL5V162A and pENTR-EL5W165A, which are vectors containing a mutant EL5 gene. It is a schematic diagram of pBI333-EN4-EL5V162A and pBI333-EN4-EL5W165A which are binary vectors containing a mutant EL5 gene. It is a schematic diagram of pBI333-EN4-RCG3 which is a binary vector used for preparing a binary vector containing a mutant EL5 gene. It is the schematic diagram of the cloning vector which cloned EL5 promoter. It is a schematic diagram of pBI333-PEL5-EL5W165A which is a binary vector containing a mutant EL5 gene having an EL5 promoter. It is a photograph 10 days after rooting test of rice transformed with pBI333-PEL5-EL5W165A and wild-type rice.

Details of the present invention will be described below.
The present invention relates to a method for producing rice having developed lateral roots and a method for developing rice lateral roots. The method for producing a rice having developed lateral roots according to the present invention comprises transforming rice expressing EL5 having a mutation into a wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. It includes culturing under conditions suitable for growth and obtaining rice having a longer side root or a larger number of side roots than wild-type rice cultivated under the condition. Hereinafter, a method for producing rice with developed side roots according to the present invention will be described step by step.

1. Transformed rice EL5 that expresses mutated EL5 is a kind of cell membrane-localized ubiquitin ligase (hereinafter also referred to as E3) registered under the accession number: AB045120 in GenBank and the like. A gene encoding EL5 (hereinafter also referred to as EL5 gene) is an endogenous gene of rice, and is registered as the accession number in GenBank and the like.

  As shown in FIG. 1, wild-type EL5 has 5 domains (I: 36th to 64th: transmembrane region, II: 65th to 97th: basic amino acid region, III: 98th position) -128th: conserved region, IV: 129th-181st: RING-H2 domain, V: 182nd-325th: C-terminal region). The EL5 having a mutation (hereinafter also referred to as a mutant EL5) means one having one or two or more amino acid residues in any one or two or more of domains 1 to 5 having a mutation. There are no particular restrictions on the number and mode of mutations.

  As a specific example of the mutation EL5, FIG. 1 shows a schematic diagram of EL5 having a mutation in domain IV. In FIG. 1, EL5C153A, EL5V162A, and EL5W165A are mutant EL5s in which cysteine at position 153, valine at position 162, and tryptophan at position 165 of EL5 in domain IV are respectively substituted with alanine.

  The in vitro E3 activity of EL5 is determined by the degree of interaction between domain IV RING-H2 domain and ubiquitin-conjugating enzyme (E2) and is reported by Katoh et al. (JBC, 280, 41015, 2005) Has been. That is, as shown in FIG. 1, EL5C153A and EL5W165A have substantially lost E3 activity, and EL5V162A has E3 activity reduced with respect to the wild type. The E3 activity of EL5L138A is further reduced than EL5V162A. As a speculation that does not affect the scope of the present invention, in order to obtain rice having developed lateral roots, EL5V162A and EL5L 138A are obtained rather than obtaining rice expressing mutant EL5 in which E3 activity is substantially lost. For example, it may be preferable to obtain rice that expresses mutant EL5 with reduced E3 activity. E3 activity can be qualitatively measured by the following method.

  The in vitro E3 activity of EL5 is based on the method of Takai et al. (Plant Journal 30, 447, 2002) based on the method of Matsuda et al. (J. Cell Sci., 114, 1949, 2001), Katoh et al. (JBC, 280 , 41015, 2005). That is, a recombinant protein (MBP-EL5) in which the region containing the RING-H2 domain from glycine at position 96 to valine at position 181 of EL5 is fused to the C-terminus of maltose binding protein (MBP) is used in E. coli. Prepare. This MBP-EL5 is mixed with ubiquitin, ubiquitin-conjugating enzyme, ubiquitin-activating enzyme, ATP, etc., and incubated at 30 ° C. or 35 ° C. for 1-2 hours. When the reaction product was subjected to normal Western analysis using an anti-MBP antibody or anti-ubiquitin antibody, depending on the RING-H2 domain of EL5 used, ubiquitin-ligating enzyme activity (also called ubiquitin ligase activity or E3 activity), MBP A band shifted to the polymer side by electrophoresis due to the increase in molecular weight due to ubiquitin ligation to the lysine residue was detected. If there is no E3 activity, no band shift is observed, and if the E3 activity is reduced, the intensity of the shifted band becomes lighter than when the original MBP-EL5 was reacted.

  Transformed rice that expresses mutant EL5 is not particularly limited. Rice that has been introduced in a state in which the mutant EL5 gene can be expressed, rice that has undergone mutation in the endogenous EL5 gene, or rice that has both of these characteristics Either is acceptable. In this specification, rice that does not express mutant EL5 is referred to as wild-type rice compared to transformed rice that expresses mutant EL5.

  As a method for introducing a mutant EL5 gene into rice in a state where it can be expressed, any known method for introducing a foreign gene into rice can be used without limitation. Examples of such a method include a method in which a vector in which a mutant EL5 gene is linked between a promoter that functions in rice and a terminator is prepared, and then this vector is introduced into rice. The molecular biological technique in this specification is, for example, Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997).

  In the above method, for example, an EL5 gene promoter that is a promoter that functions in rice, a cauliflower mosaic virus-derived 35S (CaMV 35S) promoter, a corn-derived ubiquitin promoter, and the like can be used. Examples of the terminator include a terminator derived from a cauliflower mosaic virus and a terminator derived from a nopaline synthase gene. Moreover, as will be described later, an enhancer may be included between the promoter and the mutant EL5 gene.

  There is no particular limitation on the method for introducing mutation into the endogenous EL5 gene of rice, and a known method for introducing mutation into a gene in rice, for example, a drug that induces mutation of a gene in rice There is no limitation on the method of adding or the method of irradiating UV or radioactivity.

2. Method for Producing Transformed Rice Expressing EL5 with Mutation As a specific example of a method for producing transformed rice expressing mutated EL5, the method described in Examples described later will be described.

(1) Method for obtaining mutant EL5 gene First, a mutant EL5 gene or a vector containing the mutant EL5 is obtained. A vector containing the EL5 gene cDNA prepared according to a conventional method based on the nucleotide sequence information described in Accession No. AB045120 is prepared. Using this vector as a template, PCR using a primer set that can introduce mutations at a desired site and a commercially available kit (eg, Quickchange site-directed mutagenesis kit, Stratagene) resulted in site-specific mutation. A vector containing the EL5 gene can be obtained. A specific example of the vector containing the mutation EL5 is shown in FIG. About the obtained vector, it is desirable to sequence the EL5 gene and confirm that the mutation has been introduced at the desired site and that the other nucleotide sequences have no mutation.

(2) Preparation of binary vector into which mutant EL5 gene is inserted Once mutant EL5 gene is obtained, various methods known so far, such as Agrobacterium method, particle gun method, polyethylene glycol method, etc. are used. Thus, an EL5 gene having a mutation can be introduced into rice, and then EL5 can be expressed in rice. For example, if an EL5 gene having a mutation is inserted into a T-DNA region in a binary vector, and then this T-DNA region is introduced into rice via a bacterium belonging to the genus Agrobacterium , the mutant EL5 is transformed into rice. Can be expressed.

  Specifically, a fragment containing an EL5 gene having a mutation excised from the vector obtained in (1) above is located in a T-DNA region of a binary vector, between a promoter functioning in rice and a terminator. Insert into the foreign gene transfer site. The binary vector is not particularly limited as long as it has a T-DNA region having the above-described configuration. For example, in addition to a commercially available binary vector such as pB1121 (Clontech), a commercially available binary vector having various modifications is used. be able to.

  The T-DNA region preferably contains a selectable marker gene cassette capable of expressing a selectable marker in order to efficiently select the rice into which this region has been introduced, or in the rice together with the DNA containing the selectable marker gene cassette. It is preferably introduced. Examples of the selectable marker gene include, but are not limited to, a hygromycin phosphotransferase gene that provides resistance to the antibiotic hygromycin, a neomycin phosphotransferase gene that provides resistance to kanamycin, and the like.

  As a binary vector obtained by modifying a commercially available product, for example, in the T-DNA region of a commercially available binary vector, in addition to the above selectable marker gene cassette, one or more regions that promote the expression of mutant EL5 such as an enhancer You can use things that contain pieces.

  For example, pBI333-EN4-RCG3 shown in FIG. 6 has a CaMV 35S promoter :: hygromycin phosphotransferase (HPT) :: CaMV terminator as a selectable marker cassette in the T-DNA region of commercially available pB1121. Furthermore, the promoter in the foreign gene expression region is changed to EN4 which is an artificial promoter obtained by repeating the enhancer region of the CaMV 35S promoter four times. The details of pBI333-EN4-RCG3 can be referred to the literature of the present inventors (Nishizawa et al., Theor. Appl. Genet., 99, 383-390, 1999).

(3) Gene introduction method into Agrobacterium A method for introducing a binary vector containing a T-DNA region into which a mutated EL5 gene has been inserted into an Agrobacterium genus, Any method for introducing and transforming bacteria can be used without limitation. For example, an electroporation method, a calcium ion method, a triple parent method, or the like can be used, but the electroporation method is preferably used. Specifically, when the electroporation method according to the method described in Nagel et al. (Nagel et al., Microbiol. Lett., 67, 325, 1990) is used, the binary vector prepared in (2) above is used. Can be introduced into Agrobacterium. After introducing the binary vector into the genus Agrobacterium, transformed Agrobacterium can be selected using the selection marker in the binary vector.

(4) Gene transfer method to rice By bringing the above transformed Agrobacterium into contact with rice, transformed rice capable of expressing the mutant EL5 can be obtained. Rice usually refers to plants belonging to the genus Gramineae, and is called Oryza sativa in scientific name. Rice has several types of roots, for example, seed roots, crown roots, lateral roots, root hairs and the like. In general, seed root refers to the development of larvae already formed in embryos in seeds, which are also called main roots in some plants and are known to have only one in rice; Crown roots are roots that originate from the shoot base and are also known as node roots and adventitious roots; lateral roots are roots that originate from the inner sheath cell layer of seed roots and crown roots, also called branch roots Root; root hair refers to a hairy root in which a single cell growing from a seed root, a crown root, or a lateral root is elongated.

  As a method for obtaining transformed rice, a generally known method of contacting a genus Agrobacterium with a plant can be used without limitation. For example, Toki S (Plant Mol. Biol. Rep. 1997) and Toki S ( Although it can implement according to the method as described in literature, such as Plant J. 2006), it is not limited to these. Specific examples of the method of infecting rice with transformed Agrobacterium are as follows, but are not limited thereto.

  The ripened ripe seed is washed with a predetermined concentration of ethanol, sodium hypochlorite, sterilized water, and the like. The sterilized seed is placed in a medium containing auxin (for example, N6D medium), and seed preculture is performed. The transformed Agrobacterium obtained in (3) above is infected with seeds after pre-culture under conditions suitable for infection. After infection, Agrobacterium is sterilized from the seed using a sterilizing agent such as meropen, and then transformed cells are selected using the selection marker.

  The selected transformed cells can be transferred to a regeneration medium containing an appropriate plant regulator (eg, MS medium containing cytokinin) and incubated for an appropriate period of time. In order to regenerate rice, the redifferentiated transformant is transferred to a rooting medium (for example, MS medium not containing a plant regulator). As a method of regenerating a transformant into rice, for example, a method of regenerating rice from callus (Toki, et al., Plant Journal, 47, 969-976, 2006), or using an electroporation method, A method of regenerating rice from protoplasts (Toki, et al., Plant Physiol., 100, 1503-1507, 1992) can be used. If a transformant in which the mutant EL5 gene is introduced into the genome is obtained, it is possible to mass-produce rice cultured tissues, rice shoots, and the like. After root development is confirmed, the transformed rice can be potted. Regarding these methods, reference can be made to paragraphs 0028 to 0030 of Japanese Patent No. 3141084. However, since transformed rice expressing EL5 with mutations hardly forms roots in MS medium, in order to make a pot, it is cultivated in tap water or the like and rooted.

3. Method for culturing transformed rice expressing EL5 with mutation The method for culturing transformed rice expressing EL5 with mutation for the purpose of obtaining rice with developed lateral roots is a nitrate source of less than 1 mM nitrate or 10 mM. There is no particular limitation as long as transformed rice expressing EL5 having a mutation is cultured under conditions suitable for growth of wild-type rice using a medium containing less ammonium salt.

  The transformed rice used for the culture may be rice having such a size that a crown root can develop. For example, shoots (shoots) of 2 cm or more are preferable, shoots of 3 cm to 10 cm are more preferable, and 3 cm to 5 cm. The shoot is more preferable.

  The medium used for culturing transformed rice contains less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. The nitrate is not particularly limited as long as it is contained in the medium at a concentration of less than 1 mM, preferably 0.1 mM or less, more preferably 0.05 mM or less, and even more preferably 0.01 mM or less. For example, potassium nitrate, sodium nitrate, Although potassium nitrite and sodium nitrite can be used alone or in combination, potassium nitrate is preferred. Similarly, the ammonium salt is not particularly limited as long as the concentration in the medium is less than 10 mM, preferably 5 mM or less, more preferably 2 mM or less, and even more preferably 0.2 mM or less. For example, ammonium sulfate, ammonium chloride, phosphorus Ammonium acid and ammonium acetate can be used alone or in combination, but ammonium sulfate is preferred. The concentration ranges of nitrate and ammonia salt contained in the medium are not particularly limited as long as they are less than 1 mM and less than 10 mM, respectively. For example, a concentration of 0 mM, that is, a medium containing no nitrogen source may be used.

  As the composition of the medium other than the nitrogen source, a medium containing a known medium composition suitable for rice can be used without limitation. For example, MS medium (Murashige-Skoog medium) or B5 medium (OLGamborg et al, (1968), Experimental Cell Research, 50, 151-158). For example, in order to develop the lateral root in addition to the growth of the crown root, it is preferable to reduce the amount of calcium salt, and it is more preferable that the concentration of the calcium salt in the medium is 100 μM or less.

  The form of the medium is not particularly limited, and can be, for example, a liquid, semi-solid or solid medium. More specifically, a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source can be used as a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source.

  The temperature, period, irradiation amount, and other conditions for cultivating the transformed rice are conditions suitable for growth of wild-type rice having no mutation in the EL5 gene, for example, 5-35 ° C., preferably 25-30 ° C .; It can be cultured for several days to several tens of days, preferably 5 to 10 days; irradiated with white light without irradiation or with several kilolux to several tens of kilolux, preferably 2 to 5 kilolux.

4). Rice with developed side roots Rice with developed side roots obtained by the production method of the present invention is rice with a longer side root and / or a larger number of side roots than wild-type rice. The wild-type rice to be compared is cultured under the same conditions as the culture conditions of the transformed rice expressing EL5 having a mutation. The timing of culturing transformed rice and wild-type rice that express EL5 with mutation may be the same or different. Whether the side roots are longer than the wild type rice and / or the number of side roots is greater than 1 mM nitrate or less than 10 mM ammonium as a nitrogen source by visual inspection or using an instrument such as a microscope or a digital microscope. Rice obtained by culturing transformed rice expressing EL5 having a mutation under conditions suitable for growth of wild-type rice using a medium (hereinafter also referred to as rice obtained by the method of the present invention) And by measuring the length of the side roots and / or the number of side roots of wild-type rice (hereinafter referred to as control rice) cultured under the same conditions.

  For example, by comparing the control rice with a predetermined length, for example, root root from a crown root, and confirming that the number of side roots with a length of 0.5 mm or more per 1 cm crown root is larger, It can be confirmed that it is a developed rice. In the rice with the developed side roots obtained by the method of the present invention, when the body length is 10 to 15 cm, the number of side roots having a length of 0.5 mm or more per 1 cm crown root is usually compared with the control rice. 1.2 times, 1.5 times, 2 times, 2.5 times, 3 times, or 4 times or more.

  For example, in the case of using rice soil with less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source, the rice with developed side roots obtained by the method of the present invention can be used as is or without replanting. Nitrogen source can be added to continue the culture. In addition, for example, when cultured using an MS medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source, it is preferable to replant rice in which lateral roots have developed into rice cultivation soil. In this case, it can be expected that the rice having developed side roots is cultivated under a condition in which the nitrogen source is reduced as compared with the cultivation condition of wild-type rice.

5). Method for Developing Side Roots of Rice As described above, transformed rice expressing EL5 having a mutation is used to grow wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. When cultured under suitable conditions, the side roots of the transformed rice can be developed more than the side roots of wild-type rice. The present invention also includes a method of developing a lateral root of rice, comprising culturing under a condition suitable for growth of wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. included.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

1. Production of modified EL5 gene
In order to obtain a mutant EL5 gene clone encoding EL5V162A in which the 162nd valine residue of EL5 protein was replaced with alanine, the cDNA of the EL5 gene (accession number: cDNA of the amino acid coding region of AB045120) was cloned. The pENTR vector (Invitrogen) was used as a template, and PCR was performed using Quickchange site-directed mutagenesis kit (Stratagene) according to the protocol attached to the kit. As an oligo DNA primer, 5′-CCACGCCGAGTGC GCG GACATGTGGCTCG-3 ′ (SEQ ID NO: 1 in the sequence listing) and 5′-CGAGCCACATGTC CGC GCACTCGGCGTGG-3 ′ (SEQ ID NO: 2 in the sequence listing) are used for the preparation of EL5V162A. For the preparation of EL5W165A, 5′-CGTCGACATG GCG CTCGGCTCCCACTCC-3 ′ (SEQ ID NO: 3 in the sequence listing) and 5′-GGAGTGGGAGCCGAG CGC CATGTCGACG-3 ′ (SEQ ID NO: 4 in the sequence listing) were used. The underlined portion of each primer indicates the base into which the mutation has been introduced. Sequencing the EL5 gene part of the resulting plasmid, confirming that the mutation has been introduced at the target site and that there are no mutations in other nucleotide sequences, pENTR-EL5V162A and pENTR -EL5W165A (see FIG. 4) was obtained.

2. Construction of vector for plant introduction In order to express the mutant EL5 gene obtained as described above in rice, artificial promoter EN4 (independent administrative agency agricultural organism) in which the 35S promoter enhancer region of cauliflower mosaic virus (CaMV) is repeated four times. Binary vectors pBI333-EN4-EL5V162A and pBI333-EN4-EL5W165A (see Fig. 5) linked downstream of the Resource Research Institute (assigned from Dr. Hirohiko Tsuji) were constructed. Specifically, it was carried out as follows.

  pBI333-EN4-RCG3 (prepared according to the method described in Nishizawa et al., Theor. Appl. Genet., 99, 383-390, 1999, see FIG. 6) is a binary vector pBI121 (Clontech, Accession). In addition to the CaMV 35S promoter :: hygromycin phosphotransferase (HPT) :: CaMV terminator as a selection marker cassette in the T-DNA region of the number: AF485783), the above-mentioned EN4 and RCG3 ( Rice chitinase gene Cht-3; accession number: D16223) and nopaline synthase terminator (NOS3 ′). This pBI333-EN4-RCG3 was opened with SacI, blunt-ended with T4 DNA polymerase, and then cleaved with XbaI to remove RCG3, from which XbaI derived from pENTR-EL5V162A or pENTR-EL5W165A previously cloned was removed. -EcoRV fragments were ligated to complete pBI333-EN4-EL5V162A and pBI333-EN4-EL5W165A.

  In order to express EL5W165A using EL5's own promoter, the EL5 promoter (PEL5) was cloned by the following method. Specifically, PCR was performed using 5'-GCGGGCCCGCAAATAGGGTCTCTCGTTAGC-3 '(SEQ ID NO: 5 in the sequence listing) and 5'-CCTCTAGATGCATATGTGACCGCGCGCACT-3' (SEQ ID NO: 6 in the sequence listing) using rice (variety: Nipponbare) DNA as a template. Then, after cutting with ApaI and XbaI, ligation was performed between ApaI and XbaI of pBlueScript SK (Stratagene) to obtain SK-PEL5 (see FIG. 7). Next, the fragment from ApaI to XbaI containing PEL5 of SK-PEL5 was cleaved with pBI333-EN4-EL5W165A prepared earlier with ApaI and XbaI, and ligated with the fragment excluding the EN4 promoter, and pBI333-PEL5- EL5W165A was completed (see FIG. 8).

3. Production of transgenic rice (1) Gene transfer to Agrobacterium
According to the method of Nagel et al. (See Microbiol. Lett., 67, 325, 1990), pBI333-EN4-EL5V162A or pBI333-PEL5-EL5W165A was introduced into Agrobacterium tumefacience (EHA105 strain) by electroporation. Then, transformed Agrobacterium was obtained by culturing at 28 ° C. for 2 days on LB medium containing 50 μg / mL kanamycin and 50 μg / mL hygromycin.

(2) Gene introduction into rice Transformation of rice was carried out according to an ultra-rapid transformation method (see Japanese Patent No. 3141084 or Toki et al., Plant Journal, 47, 969-976, 2006). However, meropen (Dainippon Sumitomo Pharma Co., Ltd.) was used to disinfect Agrobacterium. Specifically, it was carried out as follows.

The transformed Agrobacterium suspension prepared in (1) above and rice ( Oryza sativa variety: Nipponbare) pre-cultured according to the ultra-rapid transformation method were added to 2N6-AS medium (30 g / L). Sucrose, 10 g / L glucose, 0.3 g / L casamino acid, 2 mg / L 2,4-D, 10 mg / L acetosyringone, 4 g / L gellite, pH 5.2) in the dark for 3 days, 28 Co-cultured at 0 ° C. After washing Agrobacterium from the seeds using sterilized water containing 25 mg / L meropen, the seeds were 12.5 mg / L meropen, 50 mg / L hygromycin as a selection marker, and 4 g / L gellite. It was placed on the added N6 medium (selection medium) and cultured in the dark at 28 ° C. for about 10 days to proliferate hygromycin-resistant cells to obtain callus.

  The selected hygromycin-resistant callus was redifferentiated (MS medium supplemented with 6.25 mg / L meropene and 50 mg / L hygromycin: 30 g / L sucrose, 30 g / L sorbitol, 2 g / L casamino acid, 2 mg / The cells were transplanted to L kinetin, 0.002 mg / L NAA, 4 g / L gellite, pH 5.8) and continued to be cultured at 28 ° C. in the light until redifferentiation.

  Redifferentiated individuals were placed on hormone-free medium (hormone-free MS medium supplemented with 6.25 mg / L meropene and 25 mg / L hygromycin). About 10 days later, transplanted to a new hormone-free medium every shoot, and after about one week, when the transformed plant became large, the sterile culture was finished. The rice introduced with pBI333-PEL5-EL5W165A was conditioned for 3 days, In addition, pBI333-EN4-EL5V162A-introduced rice is rooted in tap water for 10 days, transplanted to a pot filled with “Kureha granular soil-D” (trade name; Kureha Chemical), and grown in a greenhouse. Next-generation self-propagating seeds (R1 generation) were obtained.

4). Rooting test under nitrogen-free conditions (see Fig. 2 for results)
Rice that expresses the EL5V162A gene is markedly inhibited from rooting in the above-mentioned hormone-free medium. However, by continuing cultivation in the hormone-free medium, it is possible to distribute and increase the shoot.

The shoots of EL5V162A gene-introduced rice (EL5V162A rice) that were subcultured on a hormone-free medium and became 3-5 cm long 3-4 weeks after the subculture were used. As a control, non-transformed rice (variety: Nipponbare) was removed, sterilized with 50% hypochlorous acid for 30 minutes, washed 3-5 times with sterile water, and then diluted with 4-fold MS agar for 7 days. Rice that had germinated and resected endosperm and roots was used. Hydroponic solution 100ml were placed respectively in mayonnaise bottle _{(70μM CaCl 2, 36μM FeSO 4} , 9μM MnSO 4, 3μM ZnSO 4, 0.2μM CuSO 4, 4.5μM H 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na ₂ HPO ₄ , 16 μM KCl, 150 μM MgCl ₂ , 3% Sucrose) planted with 12 shoots per bottle using a plastic cage and cultured aseptically for 10 days under white light irradiation of about 3 kilolux (28 ° C.), the length of the rooted root, and the number of side roots per unit length of the crown root were measured.

  As a result, the number of all lateral roots rooted from the crown root surface was 3.95 ± 0.33 / cm in the control rice, whereas it was 16.35 ± 1.11 / cm in EL5V162A rice, which was about 4 times the number of lateral roots. An increase was seen. The crown root length was 1.94 ± 0.73 cm for control rice and 2.23 ± 1.11 cm for EL5V162A rice.

5. Rooting test under various nitrogen concentrations (See Figure 3 for results)
The shoots of EL5V162A gene-introduced rice (EL5V162A rice) that were subcultured on a hormone-free medium and became 3-5 cm long 3-4 weeks after the subculture were used. As a control, non-transformed rice (variety: Nipponbare) was removed, sterilized with 50% hypochlorous acid for 30 minutes, washed 3-5 times with sterile water, and then diluted with 4-fold MS agar for 7 days. Rice that had germinated and resected endosperm and roots was used. Hydroponic solution 100ml were placed respectively in mayonnaise bottle _{(70μM CaCl 2, 36μM FeSO 4} , 9μM MnSO 4, 3μM ZnSO 4, 0.2μM CuSO 4, 4.5μM H 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na ₂ HPO ₄ , 16 μM KCl, 150 μM MgCl ₂ , 3% Sucrose, 10 mM MES (pH 5.8) and various nitrogen sources), plant 12 shoots per bottle using a plastic basket Incubation at 28 ° C aseptically for 10 days under white light irradiation of about 3 kilolux, and the degree of lateral root formation by comparing the number of lateral roots of about 0.5 mm or more at the root 1 cm and root tip 1 cm with the control rice Was described. Nitrogen sources in each test section were added at the following concentrations.
Ammonium sulfate (final concentration: 0.2 mM, 2 mM, 5 mM, 10 mM)
Potassium nitrate (final concentration: 0.01 mM, 0.05 mM, 0.1 mM, 1 mM, 10 mM)

The hydroponic liquid preparation method is as follows. Final concentration _{70μM CaCl 2, 36μM FeSO 4,} 9μM MnSO 4, 3μM ZnSO 4, 0.2μM CuSO 4, 4.5μM H 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na 2 HPO 4, 16μM KCl, 150μM MgCl 2, The solution prepared to 3% Sucrose was autoclaved, and 1/100 volume of 1M MES (pH 5.8) sterilized by filter was added. This was used as a nitrogen-free hydroponic solution, and an autoclaved ammonium sulfate or potassium nitrate solution was added to the above final concentration.

  As a result, EL5V162A rice formed many lateral roots in the presence of 1 mM or less of potassium nitrate or 5 mM or less of ammonium sulfate relative to the control rice in each nitrogen concentration test group.

6). EL5W165A introduction rice rooting test (See Fig. 9 for results)
Brown rice of self-propagating next-generation seeds of rice transformed with pBI333-PEL5-EL5W165A grown in an isolated greenhouse was sterilized with 50% hypochlorous acid for 30 minutes, washed with sterilized water 3-5 times, and then 15 mg / L containing hygromycin, final concentration _{70μM CaCl 2, 36μM FeSO 4,} 9μM MnSO 4, 3μM ZnSO 4, 0.2μM CuSO 4, 4.5μM H 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na 2 HPO 4, 16μM KCl, 150 μM MgCl ₂ , 10 mM MES (pH 5.8), placed on 1.5% agar medium and cultured at 28 ° C. for 10 days. As a control, brown rice of non-transformed rice (variety: Nihonbare) was sterilized with 50% hypochlorous acid for 30 minutes, washed with sterilized water 3 to 5 times, and final concentrations of 70 μM CaCl ₂ , 36 μM FeSO ₄ , 9 μM _{MnSO 4, 3μM ZnSO 4, 0.2μM} CuSO 4, 4.5μM H 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na 2 HPO 4, 16μM KCl, 150μM MgCl 2, 10 mM MES (pH5.8), 1. It was placed on a 5% agar medium and cultured for 10 days at 28 ° C. in the light.

After removing endosperm and roots of hygromycin-resistant pBI333-PEL5-EL5W165A and control rice, 100 ml of hydroponic solution in each mayonnaise bottle (70 μM CaCl ₂ , 36 μM FeSO ₄ , 9 μM MnSO ₄ , 3 μM ZnSO _{4, 0.2μM CuSO 4, 4.5μM H} 3 BO 4, 0.05μM Na 2 MoO 4, 7μM Na 2 HPO 4, 16μM KCl, to _{150μM MgCl 2, 3% Sucrose,} 10 mM MES (pH5.8)), plastic Twelve shoots were planted per bottle using a cocoon made of the product, and aseptically cultured under white light irradiation of about 3 kilolux for 10 days at 28 ° C., the lateral root formation was observed. As a result, PEL5-EL5W165A rice formed more lateral roots than control rice.

  The method for producing rice having developed side roots and the method for developing rice side roots according to the present invention can be used, for example, in the field of agriculture and biomass production using rice roots. According to the present invention, the amount of nitrogen in the soil is reduced to produce rice with lateral roots developed, and the use of nitrogen fertilizer is suppressed because this rice is expected to continue to grow under low nitrogen conditions. It can be expected that agriculture with low cost and low environmental impact will be possible. In addition, unlike the prior art, the rice produced according to the present invention does not require a rooting agent treatment, so that it can be expected to reduce costs when cultivating rice roots to produce substances.

Claims (7)

Transformed rice expressing EL5 with a mutation is cultured under conditions suitable for growth of wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source, in lateral roots than a wild-type rice cultured involves obtaining a long and / or the number of lateral roots often rice, a method for producing a rice lateral roots have developed,
The EL5 having the mutation is EL5 in which at least one amino acid residue selected from the group consisting of positions 138, 153, 162, and 165 in the amino acid sequence of EL5 is substituted with alanine The way . Culturing transformed rice expressing mutated EL5 under conditions suitable for growth of wild-type rice using a medium containing less than 1 mM nitrate or less than 10 mM ammonium salt as a nitrogen source. a method of developing the lateral roots,
The EL5 having the mutation is EL5 in which at least one amino acid residue selected from the group consisting of positions 138, 153, 162, and 165 in the amino acid sequence of EL5 is substituted with alanine The way . The method according to claim 1 or 2 , wherein EL5 having the mutation is EL5 in which valine at position 162 in the amino acid sequence of EL5 is substituted with alanine. The transformed rice contains a gene encoding EL5 having a mutation under the control of at least one promoter selected from the group consisting of an EL5 gene promoter, a cauliflower mosaic virus-derived 35S promoter, and a corn-derived ubiquitin promoter. The method according to any one of 1 to 3 . The method according to any one of claims 1 to 4 , wherein the nitrate is at least one nitrate selected from the group consisting of potassium nitrate, sodium nitrate, potassium nitrite, and sodium nitrite. The method according to any one of claims 1 to 4 , wherein the ammonium salt is at least one ammonium salt selected from the group consisting of ammonium sulfate, ammonium chloride, ammonium phosphate, and ammonium acetate. The method according to any one of claims 1 to 6 , wherein the culturing is performed for 5 to 10 days.