JP2010081839A - Gene making seed large by overexpression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To isolate a gene corresponding to signal transduction of BR in rice. <P>SOLUTION: The isolation of a gene increasing the expression levels after 3 h and 24 h is tried by adding brassinolide (BL) of a typical brassinosteroid to a brd1 variant having a low BR endogenous amount. As a result of microarray analysis, a gene AK071601 in which the expression levels are increased to two or more times after both of 3 h and 24 h compared to the controls of not adding the brassinolide is discovered. The growth of OsBU3-overexpression callus of T0 generation is confirmed to be extremely promoted compared to the control as a result that whether the change in the form or the like is observed or not is confirmed by forming a rice overexpressing the AK071601. The T1 seed has a longer length and a wider width according to the transcription amount of the OsBU3. Further, in the growth later stage, abnormal elongation of internode is observed in the OsBU3 overexpression rice. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a callus or plant having at least one trait selected from the group consisting of enlargement of seeds, promotion of callus growth, and abnormal elongation between nodes, and callus and plants obtained using the same The body and its use.

  The plant hormone brassinosteroid (BR) is known to be involved in plant morphogenesis and response to environmental stress. In terms of chemical structure, BR is characterized by having a steroid skeleton, and more than 40 types of plants including angiosperms, gymnosperms, algae and ferns are represented by brassinolide (BL) and castasterone. Over 40 types of free form have been isolated and identified (Fujioka and Sakurai, 1997). Among them, BL is considered to play the most important role because it is widely distributed in the plant kingdom and exhibits the strongest activity among natural BR.

The present inventors have already isolated a BR biosynthetic mutant brd1 ( brassinosteroid-dpendent 1 ) in rice, showing that the brd1 mutant is extremely dwarf and has a very short leaf sheath and rolled leaf blades. (Mori et al. 2002, FIG. 1). The present inventors have also shown that BR is involved in many morphological and physiological processes in rice, such as leaf blade elongation and development, tiller formation, root development, and reproductive growth. (Mori et al. 2002).

Prior art documents of the present invention are shown below.
Fujioka, S. and Sakurai, A. (1997) Natural Products Report, 14, 1-10 Livak, KJ, Schmittgen, TD (2001) Methods 25: 402-408 Mori, M et al., (2002) Plant Physiol. 130: 1152-1161. Nakamura, H et al., (2007) Plant Mol Biol. 65: 357-371 Sato, H et al., (1996) Plant Sci. 119: 39-47. Toki, S et al., (2006) Plant J. 47: 969-76. Yazaki, J et al., (2004) Physiol Genomics. 17 (2): 87-100.

  As described above, BR is known to be involved in plant morphogenesis and response to environmental stress. Therefore, the present invention has been made to isolate genes involved in BR signaling in rice for the purpose of utilizing the useful action of BR in agriculture and the like.

The present inventors Upon solving the above-genes involved in signal transduction of BR is expressed in low BR endogenous amount brd1 variants are suppressed, in addition after the initial stage and the addition of BL to brd1 We thought that expression might increase. Therefore, the inventors added brassinolide (BL), which is a representative brassinosteroid, to the brd1 mutant, and attempted to isolate a gene whose expression level increased after 3 hours and 24 hours. The present inventors have found the microarray analysis, 3 hours after, genes increase in expression more than twice as compared to 24 hours after both brassinolide non loading control (DDBJ Accession No. AK071601, OsBU3 ( Oryza sative Brassinosteroid Upregulated 3 ))) was successfully isolated.

Next, the present inventors produced rice that overexpresses AK071601, and confirmed whether or not changes in morphology were observed. As a result, OsBU3 growth overexpression calli T0 generation, it was confirmed to significantly accelerated compared to the control.
In addition, when the regenerated plant body was compared with the wild type (WT), remarkable bending of the lamina joint was observed. The seeds increased in length and width according to the amount of OsBU3 transferred. Furthermore, in the late growth stage, abnormal elongation between nodes was observed in OsBU3 overexpressing rice.
This is the first time that the relationship between the promotion of rice callus growth, seed enlargement, abnormal internode elongation in the light, and enhancement of the BR signal has been found.

The present invention relates to a gene having an increase in seed size, promotion of callus growth, abnormal elongation between nodes in addition to bending of a lamina joint, and a callus and a plant body into which the gene is introduced. Provides the following [1] to [6].
[1] A method for imparting to a plant at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes, including the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). Forming a callus from a cell or regenerating a plant body,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(Ii) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (iv) SEQ ID NO: 1 DNA that hybridizes under stringent conditions with DNA comprising the base sequence described in 1.
[2] A plant cell into which the DNA according to any one of (a) to (d) below or a vector containing the DNA has been introduced, which increases seed size, promotes callus growth, and abnormal elongation between nodes. A plant cell capable of forming a callus having at least one trait selected from the group consisting of:
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (d) SEQ ID NO: 1 DNA that hybridizes under stringent conditions with DNA comprising the base sequence described in 1.
[3] Callus formed from the plant cell according to [2] or a regenerated plant body, at least selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes Callus or plant having one trait,
[4] The callus according to [3], wherein the callus has at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal growth of internodes. Or plant body,
[5] Plant propagation material according to [3] or [4],
[6] A callus or plant having at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes, including the following steps (a) and (b): Production method;
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). Forming a callus from a cell or regenerating a plant body,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(Ii) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (iv) SEQ ID NO: 1 DNA which hybridizes under stringent conditions with DNA comprising the base sequence described in 1.

According to the present invention, a gene (OsBU3) that promotes callus growth, enlarges seeds, and has an abnormal elongation action between nodes in a bright place has been provided. In rice in which the gene identified in the present invention was overexpressed, in addition to bending of the lamina joint, an increase in seed size, promotion of callus growth, and abnormal elongation between nodes were observed.
By changing the expression level of OsBU3 or its homolog gene and changing the seed size, it is possible to give a new commercial value mainly to plants (cereal grains etc.) whose seeds are edible. Moreover, it became possible to give new commercial value to crops and foliage plants by changing the plant type and internode length by overexpression and suppression of expression. In addition, since overexpression of OsBU3 promotes callus growth, the callus introduced with OsBU3 may be used for production of substances such as pigments, alkaloids, and drugs.

[Embodiment of the Invention]
The present invention provides at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes using OsBU3 ( Oryza sative Brassinosteroid Upregulated 3 , DDBJ Accession No. AK071601). A method of applying to plants is provided.
In the present invention, “seed enlargement” means that the size (length or width) of the seed is slightly increased as compared with the wild-type plant body. Whether or not the seed size of the plant body obtained by the method of the present invention has increased compared to the wild type can be determined by measuring, for example, the length and width of the seed, It is not limited.
Further, in the present invention, “promotion of callus growth” means that the growth rate is slightly increased as compared with wild-type callus.
In the present invention, “abnormal extension between nodes” means that the nodes other than the upper four nodes under the head extend longer than WT. In addition, although it is rarely possible to divide from a node between long elongated nodes, it is also included in the “abnormal elongation between nodes” of the present invention that it is easy to divide from a node between elongated nodes. .

  The form of the DNA used in the present invention (hereinafter sometimes referred to as “the DNA of the present invention”) is not particularly limited, and may be cDNA or genomic DNA. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For example, genomic DNA is designed by designing an appropriate primer pair from the known base sequence information (SEQ ID NO: 1) of OsBU3 (DDBJ Accession No. AK071601), performing PCR using the genomic DNA prepared from the target plant as a template, It can be prepared by screening a genomic library using the obtained amplified DNA fragment as a probe. Similarly, by designing a primer pair, performing PCR using cDNA or mRNA prepared from the target plant as a template, and screening the cDNA library using the resulting amplified DNA fragment as a probe, the DNA of the present invention Can be prepared. Furthermore, if a commercially available DNA synthesizer is used, the target DNA can be prepared by synthesis.

  As long as the DNA of the present invention has a function of imparting to plants at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes, OsBU3 derived from rice Not only DNA encoding a protein (SEQ ID NO: 2) but also DNA encoding a protein structurally similar to the protein (for example, mutant, derivative, allele, variant and homolog) can be used. Such DNA includes, for example, DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2. .

  Methods well known to those skilled in the art for preparing DNA encoding proteins with modified amino acid sequences include, for example, the site-directed mutagenesis method (Kramer, W. and Fritz, HJ Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology. 154, 1987, 350-367.). In addition, the amino acid sequence of the encoded protein may be mutated in nature due to the mutation of the base sequence. As described above, even if the DNA encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence (SEQ ID NO: 2) of the natural type OsBU3 protein, As long as it has a function of imparting to plants at least one trait selected from the group consisting of promotion of callus growth and abnormal elongation between nodes, it is included in the DNA of the present invention.

  The number of amino acids to be modified is not particularly limited, but is generally within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids). The amino acid modification is preferably a conservative substitution. The hydropathic index (Kyte, J. and Doolittle, RF J Mol Biol. 157 (1), 1982, 105-132.) And Hydrophilicity value (US Pat. No. 4,554,101) for each amino acid before and after modification are as follows: , Preferably within ± 2, more preferably within ± 1, and most preferably within ± 0.5.

  Moreover, even if the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the DNA of the present invention.

  Examples of DNA encoding a protein structurally similar to rice-derived OsBU3 protein (SEQ ID NO: 2) include hybridization techniques (Southern, EM Journal of Molecular Biology. 98, 1975, 503.) and polymerase chain reaction (PCR). ) Technology (Saiki, RK et al. Science. 230, 1985, 1350-1354 .; Saiki, RK et al. Science, 239, 1988, 487-491.) Can also be used. is there. That is, the DNA of the present invention includes DNA that hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1. In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, “stringent conditions” refers to conditions of 6M urea, 0.4% SDS, 0.5 × SSC, or hybridization conditions of stringency equivalent thereto, but are not particularly limited to these conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, 6M urea, 0.4% SDS, and 0.1xSSC.

  A plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize optimum stringency by appropriately selecting these factors. The isolated DNA is considered to have high homology with the amino acid sequence (SEQ ID NO: 2) of the rice-derived OsBU3 protein at the amino acid level. High homology means a sequence of at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence. Refers to the identity of The identity of amino acid sequences and nucleotide sequences is determined by the algorithm BLAST (Karlin, S. and Altschul, SF Proc Natl Acad Sci US A. 87 (6), 1990, 2264-2268 .; Karlin, S. and Altschul, SF Proc Natl Acad Sci US A. 90 (12), 1993, 5873-5877.). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul, S.F. et al. J Mol Biol. 215 (3), 1990, 403-410.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  The DNA of the present invention may be in a form inserted into a vector. The vector is not particularly limited as long as it can express the inserted gene in plant cells. For example, a vector having a promoter for constitutive gene expression in plant cells (for example, potato chitinase gene SK2 promoter, cauliflower mosaic virus 35S promoter, etc.) or inducibly activated by external stimulation It is also possible to use a vector having a promoter.

  The coding region of the protein in the base sequence described in SEQ ID NO: 1 is from the 107th base to the 367th base. An amino acid sequence generated from the 107th base to the 367th base in the base sequence described in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

  The above-mentioned DNA having a function of imparting to a plant at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes is introduced into a plant cell. Callus or plant having at least one trait selected from the group consisting of enlargement of seeds, promotion of callus growth, abnormal elongation between nodes by forming callus from plant cell or regenerating plant Can be manufactured. Therefore, the present invention provides a method for producing a callus or plant having at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes.

  Examples of plant cells into which the DNA or vector is introduced include monocotyledonous plants such as rice, wheat, barley, corn and sorghum, dicotyledonous plants such as Arabidopsis thaliana, rapeseed, tomato, soybean, potato, tobacco, and purple. However, it is not limited to these.

  The form of the plant cell into which the DNA or vector is introduced is not particularly limited as long as it can regenerate the plant body, and includes, for example, suspension culture cells, protoplasts, leaf sections, and callus.

  The introduction of the DNA or vector into plant cells can be carried out by methods known to those skilled in the art such as polyethylene glycol method, electroporation method, Agrobacterium-mediated method, particle gun method and the like. it can. In the method involving Agrobacterium, for example, according to the method of Nagel et al. (Nagel, R. et al. FEMS Microbiol Lett. 67, 1990, 325-328.) The DNA can be introduced into a plant cell by infecting the plant cell with the Agrobacterium by direct infection method or leaf disk method.

The regeneration of plant bodies from plant cells can be performed by methods known to those skilled in the art depending on the type of plant. For example, for rice, the method of Fujimura et al. (Fujimura. Et al. Tissue Culture Lett. 2, 1995, 74.) can be mentioned, and for wheat, Harris et al. (Harris, R. et al. Plant Cell Reports. 7 1988, 337-340) and Ozgen et al. (Ozgen, M. et al. Plant Cell Reports. 18, 1998, 331-335). For barley, Kihara and Funatsuki (Kihara, M. and Funatsuki, H. Breeding Sci. 44, 1994, 157-160.) and Lurs and Lorz (Lurs, R. and Lorz, H. Theor. Appl. Genet. 75, 1987, 16-25.) In the case of maize, the method of Shillito et al. (Shillito, RD, et al. Bio / Technology, 7, 1989, 581-587.) And Gordon-Kamm et al. (Gordon-Kamm, WJ et al. Plant Cell. 2 (7), 1990, 603-618.). For sorghum, the method of Wen et al. (Wen, FS, et al. Euphytica. 52, 1991, 177-181.) And Hagio (Hagio, T. Breeding Sci. 44, 1994, 121-126.), But is not limited thereto.
For Arabidopsis, the method of Akama et al. (Akama. Et al. Plant Cell Reports. 12, 1992, 7-11.) Can be mentioned. For rape, Wang et al. (Wang, YP et al. Plant Breeding. 124, 2005, 1-4.), And for tomato, Koblitz and Koblitz (Koblitz, H and Koblitz, D. Plant Cell Reports. 1, 1982, 143-146.) And Morgan and Cocking (Morgan , A. and Cocking, ECZPflanzenpysiol. 106, 1982, 97-104.), And for soybeans, Lazzeri et al. (Lazzeri, PA et al., Plant Mol. Biol. Rep. 3, 1985, 160- 167.) and the method of Ranch et al. (Ranch, JP et al., In Vitro Cell Dev. Biol. 21, 1985, 653-658.). For potato, Visser et al. (Visser, RGF et al. Theor. Appl. Genet. 78, 1989, 594-600.). For tobacco, Horsch et al. (Horsch, RB et al., Science, 227, 1985, 1229-1231.) For example, but not limited to.

  Once a transformed plant into which the DNA or vector is introduced into the genome is obtained, offspring or clones can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible.

  Whether or not the callus or plant has at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes can be determined by comparing with a control. I can do it. In the present invention, the control means a callus or plant of the same kind as the callus or plant of the present invention, in which the DNA of the present invention is not overexpressed. The control in the present invention is not limited as long as it is a plant of the same species as the callus or plant of the present invention and the DNA of the present invention is not overexpressed. Accordingly, the control of the present invention includes, for example, a callus or a plant body into which DNA other than OsBU3 derived from the rice of the present invention has been introduced. Examples of such a plant body include, for example, a callus or a plant body of the same species as the transformed plant body of the present invention, which is transformed with a DNA other than the DNA of the present invention, the present invention, Callus or plant transformed with DNA introduced with a function-deficient mutation of the DNA of the present invention, callus or plant transformed with the DNA of the present invention transformed into the function-suppressed type, functional expression of the DNA of the present invention Examples include callus or plant transformed with a DNA fragment of an insufficient region, but are not limited thereto.

Thus, the present invention regenerates a callus or plant body having at least one trait selected from the group consisting of seed enlargement, callus growth promotion, abnormal elongation between nodes, and callus formation or plant body. There is also provided a callus or plant that is a progeny plant cell, a descendant or clone of the callus or plant, and a propagation material for the callus or plant.
Note that the callus or plant body, plant cell, offspring or clone plant body and propagation material of the present invention may have all the traits of seed enlargement, callus growth promotion, and abnormal elongation between nodes. Good.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not restrict | limited to a following example at all.

1. Materials and methods
1-1. Hydroponics of rice
The seedlings 2 weeks after sowing in 1/2 Murashige-Skoog medium (containing 3% sucrose, 0.4% gellite) were transferred to hydroponics, and grown under light conditions for 16 hours in the gross chamber. The hydroponic solution used was Kimura B solution (Sato et al. 1996).

1-2. BL processing and sampling sowing 50 days after were BL (Wako) process. About 3 hours after the start of light irradiation, BL (40 nM) was added to one hydroponic solution, and the other was left as it was in a gloss chamber. Sampling of the plant body (5 young leaves and 5 leaves each) with and without BL (control) was performed 3 hours and 24 hours after BL treatment and quickly frozen in liquid nitrogen.

1-3. Total RNA extraction and purification
After extraction using ISOGEN (Nippon Gene), RNA purified using RNeasy Mini kit (QIAGEN) was used as RNA.

1-4. Microarray analysis
CDNA was synthesized and amplified from total RNA extracted from brd1 with and without BL added (control), and labeled with Cy3 and Cy5 simultaneously with reverse transcription synthesis to cRNA. Each labeled cRNA (500 ng) was hybridized (60 ° C., 17 hours) to 22K rice array (Yazaki et al. 2004), and the intensity of the signal was read with a scanner. In the experiment, for the BL addition (Cy3) vs. control (Cy5) and the BL addition (Cy5) vs. control (Cy3), the same total RNA samples were compared with fluorescent dyes and compared by a Dye-swap experiment. In addition, from the dye-swap experiment, it was considered that the genes having a fluorescence intensity ratio of 2 or more increased and genes of 0.5 or less were decreased in both 2 slides.

1-5. Real time-PCR
The 1st-strand synthesis was performed using 4 μg of total RNA and using the 1st-strand cDNA Synthesis Kit (Amersham Biosciences). The iCycle iQ real-time PCR analysis system MC (Bio-Rad) was used for Real time-PCR. Cyber Green used iQ SYBR Green Supermix (Bio-Rad). Polyubiquitin ( RUBQ2 , Genbank Accession No. AF184280) was used as a standard. Incidentally, the primer pair of RUBQ2 5'-GGTCGTCCCGAGCCTCTGTT-3 '(SEQ ID NO: 3) and 5'-GCAAATGAGCAAATTGAGCA-3' (SEQ ID NO: 4) The primer pair of the OsBU3 (DDBJ Accession No. AK071601) 5 '-ggaggagatcaacgagctcat-3' (SEQ ID NO: 5) and 5'-cggtgcaggctcttgatgta-3 '(SEQ ID NO: 6) were used. PCR temperature conditions and reaction time / number of cycles were 95 ° C for 15 minutes for 1 cycle, 94 ° C for 15 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds for 45 cycles, and 72 ° C for 7 minutes for 1 cycle. Expression levels of OsBU3 was calculated by Comparative C _T method (Livak and Schmittegen, 2001).

1-6. Varieties produce rice transgenic rice was used Nipponbare. The rice expression vector was pRiceFOX (Nakamura et al. 2007). A full-length cDNA (AK071601) obtained from the Rice Genome Resource Center of the National Institute of Agrobiological Resources was cleaved with SfiI and ligated to the pRiceFOX vector. Rice transformation was performed by high-speed transformation method (Toki et al. 2006) using Agrobacterium strain EH105. Transformed rice was cultivated in an isolated greenhouse at 28 ° C for 13.5 hours.

2. result
2-1. Gene expression analysis by microarray and Real-time PCR When the morphology was observed over time after adding brassinolide (BL) to hydroponically brd1 mutant, there was almost no change after 3 and 24 hours. After 96 hours, the youngest leaves were elongated (FIG. 2). In order to examine the initial gene expression after addition of BL, leaves were sampled for 3 hours and 24 hours after addition, RNA was extracted, and expression was comprehensively compared with the sample without addition of BL using a 22K microarray. Among them, genes whose expression was increased by 2 times or more were selected after 3 hours and after 24 hours compared with the non-added control. The gene of DDBJ Accession No. AK071601, which has one of the selected genes and has the bHLH motif, was named OsBU3 ( Oryza sative Brassinosteroid Upregulated 3 ). When OsBU3 expression was confirmed by real-time PCR, it was shown that the expression was significantly increased at least 24 hours and 96 hours after BL treatment.

(Explanation of Table 1)
Real-time PCR was used. It shows how many times the transcription amount of OsBU3 increased when BL treatment was performed on the brd1 mutant compared to the untreated sample. (1) and (2) show biological repeats in which all steps of cultivation, BL treatment, and RNA preparation were performed separately.

2-2. Phenotypes in overexpressed rice callus
Since OsBU3 expression increased in the early stage after BL addition, it was considered that OsBU3 may be involved in BR signaling. Therefore, we decided to make rice that overexpresses OsBU3, and to examine whether there was any change in morphology. Then, at the stage of OsBU3 overexpression calli T0 generation, a significant growth promotion was observed compared to controls (Figure 3).

2-3. Compared to WT phenotypically regenerated plant bodies in overexpressing rice plants, significant bending of the lamina joint was observed, it exhibited a plant type which leaf is opened (Fig. 4). The seeds increased in both length and width in proportion to the amount of OsBU3 transferred. The seeds of OsBU3 : OX-2 expressed at a high level were about 15% larger (Fig. 5). However, fertility was reduced compared to WT. Furthermore, in the late growth period, abnormal internode elongation was observed in OsBU3 overexpressing rice (FIG. 6).

The present inventors have identified the BL-inducible gene OsBU3 using the brd1 mutant. Overexpression of OsBU3 in Nipponbare (WT) caused lamina joint bending, a typical reaction observed when BL was added, resulting in an open leaf-shaped plant. In addition, seed enlargement, callus growth promotion, and abnormal growth of internodes were observed. From the above results, this gene can be a material for imparting similar properties to various plants by over-expression using genetic recombination technology.

It is a photograph which shows the brd1 mutant (Mori et al., 2002) of rice. A. Plants 3 weeks after germination, B. Plants 80 days after germination. The left is WT (Nihonbare), and the right is an enlarged view of brd1 of brd1 CB. It is a photograph showing recovery of phenotype by adding brassinolide (BL). When the brd1 mutant is hydroponically cultivated and treated with BL, the phenotype is restored in young leaves (arrows). After 96 hours, there is considerable recovery and leaf elongation is observed. After about 3 weeks, it returns to almost normal form. It is a photograph which shows the phenotype of OsBU3 overexpression callus. T0 generation callus. On regeneration medium. OsBU3 : OX is an overexpressed callus of OsBU3 . It is a photograph which shows the phenotype of the OsBU3 overexpression rice plant body. OsBU3 : OX is an overexpression of OsBU3 . Lamina joint is bent compared to WT (Nippon Hare) and shows a grass shape with open leaf blades. The amount of transcription was examined by Real-time PCR. It is the photograph and graph which show the phenotype of the seed in OsBU3 overexpression body. The top photo is WT (Nihonbare) from the left, OsBU3 : OX-1, OsBU3 : OX-2. OsBU3 : OX-1, OsBU3 : OX-2 are independent overexpressors. The amount of transcription was examined by Real-time PCR. The lower graph shows the length and width of each seed. It is a photograph which shows the phenotype between nodes in an OsBU3 overexpressing body. The internodes of OsBU3 : OX-2 (see FIG. 5) are shown. Arrows indicate nodes. You can see how the tiller extends from the nodes between the expanded nodes.

Claims (6)

A method for imparting to a plant at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes, comprising the steps of (a) and (b) below:
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). Forming a callus from a cell or regenerating a plant body,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(Ii) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (iv) SEQ ID NO: 1 DNA which hybridizes under stringent conditions with DNA comprising the base sequence described in 1. A plant cell into which the DNA according to any one of (a) to (d) below or a vector containing the DNA is introduced, the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes A plant cell capable of forming a callus having at least one trait selected from or regenerating a plant body;
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1,
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (d) SEQ ID NO: 1 DNA which hybridizes under stringent conditions with DNA comprising the base sequence described in 1. A callus formed from a plant cell according to claim 2 or a regenerated plant, wherein the callus is at least one trait selected from the group consisting of seed enlargement, callus growth promotion, and abnormal elongation between nodes. Callus or plant body having Callus or plant body having at least one trait selected from the group consisting of seed enlargement, callus growth promotion, abnormal internode elongation, which is a descendant or clone of the callus or plant body according to claim 3 . Callus or plant propagation material according to claim 3 or 4. A method for producing a callus or a plant having at least one trait selected from the group consisting of enlargement of seeds, promotion of callus growth, and abnormal elongation between nodes, including the following steps (a) and (b):
(A) a step of introducing a DNA selected from the group consisting of (i) to (iv) below or a vector containing the DNA into a plant cell; and (b) a plant into which the DNA or vector has been introduced in step (a). Forming a callus from a cell or regenerating a plant body,
(I) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
(Ii) DNA containing the coding region of the base sequence set forth in SEQ ID NO: 1,
(Iii) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 2, and (iv) SEQ ID NO: 1 DNA which hybridizes under stringent conditions with DNA comprising the base sequence described in 1.