JP3605633B2 - Novel plant gene, plant modification method using the gene, and plant obtained by the method - Google Patents

Description

【図面の簡単な説明】
【図１】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の玄米の写真である。（Ａ）はｓｇ１の玄米、そして（Ｂ）は野生型の玄米である。
【図２】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の幼植物の写真である。（Ａ）はｓｇ１の幼植物、そして（Ｂ）は野生型の幼植物である。
【図３】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の幼植物の側根を示す写真である。（Ａ）はｓｇ１の幼植物、そして（Ｂ）は野生型の幼植物である。
【図４】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の草丈の経日変化を示す図である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型の草丈を示す。
【図５】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の茎数の経日変化を示す図である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型の茎数を示す。
【図６】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の圃場移植後３０日目の植物体の写真である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型である。
【図７】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の圃場移植直後の植物体の写真である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型である。
【図８】あきたこまちの野生型およびその胚乳形成突然変異体ｓｇ１の成熟期の農業形質の比較を示す図である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型である。
【図９】ＳＧ１遺伝子のプラスミドｐＢｌｕｅｓｃｒｉｐｔ ＳＫ（−）中にクローニングされたｃＤＮＡ断片の概略を示す図である。
【図１０】ＳＧ１遺伝子の塩基配列および推定されるアミノ酸配列を示す図である。
【図１１】ｓｇ１系統と野生型植物体の各器官におけるｍＲＮＡの発現を示す図である。（Ａ）はｓｇ１系統、そして（Ｂ）は野生型である。
【図１２】ＳＧ１遺伝子（センス）およびアンチセンスＳＧ１遺伝子を含む形質転換ベクターの一部を示す図である。（Ａ）は、センスＳＧ１遺伝子を、（Ｂ）は、アンチセンスＳＧ１遺伝子をそれぞれ含む形質転換ベクターの一部を示す図である。
【図１３】アンチセンスＳＧ１遺伝子を導入した形質転換イネの玄米（Ａ）と非形質転換イネの玄米（Ｂ）の写真である。
【図１４】アンチセンスＳＧ１遺伝子を導入した形質転換イネ（Ｓ−１、Ｓ−２、Ｓ−３）と非形質転換イネ（Ｂ）におけるＳＧ１遺伝子のコピー数の比較を示す図である。
【図１５】アンチセンスＳＧ１遺伝子を導入した形質転換イネと非形質転換イネ（Ｂ）にの葉身におけるｍＲＮＡの発現の比較を示す図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel plant gene. More specifically, the present invention relates to genes that control plant morphology. The present invention also relates to a method for modifying a plant using the gene and a plant obtained by the method.
[0002]
[Prior art]
Many genes have been isolated from rice and their nucleotide sequences have been determined. However, the functions of many genes contained in rice DNA have not yet been elucidated, and they have not been used effectively. There is a need to isolate and identify useful genes in plants and to develop efficient methods for isolating them.
[0003]
Transposons are mutagenic genes known to be ubiquitous in the genomes of animals, yeast, bacteria and plants. Transposons have been classified into two classes according to their transposition mechanism. Transposons belonging to class II transpose in the form of DNA without replication. As transposons belonging to class II, Ac / Ds, Spm / dSpm and Mu element of maize (Zea mays) (Fedoroff, 1989, Cell 56, 181-191; Fedoroff et al., 1983, Cell 35, 235-242; Schiefelbin et al. Natl. Acad. Sci. USA 82, 4783-4787, and the Tam element of Antirrhinum majus (Bonas et al., 1984, EMBOJ, 3, 1015-1019). Transposons belonging to class II are widely used for gene isolation using transposon tagging. This technique takes advantage of the fact that transposons are translocated on the genome and inserted into a gene, causing physiological and morphological mutations in the gene and changing the phenotype controlled by the gene. The genes affected by detecting this change are isolated (Bancofft et al., 1993, The Plant Cell, 5, 631-638; Colasanti et al., 1998, Cell, 93, 593-603; Gray et al., 1997; Cell, 89, 25-31; Keddie et al., 1998, The Plant Cell, 10, 877-887; Whitham et al., 1994, Cell, 78, 1101-1115).
[0004]
Transposons belonging to class I, also called retrotransposons, replicate and translocate via RNA intermediates. Class I transposons were initially identified and characterized in Drosophila and yeast, but recent studies have shown that they are ubiquitous in plant genomes and account for a significant portion thereof (Bennetzen). , 1996, Trends Microbiolo., 4, 347-353; Voytas, 1996, Science, 274, 737-738). Most retrotransposons appear to be immobile, integrated units. Recent studies have shown that some of these are activated under stress conditions such as wounding, pathogen attack and cell culture (Grandbastien, 1998, Trends in Plant Science, 3, 181-187; Wessler, 1996, Curr. Biol. 6, 959-961; Wessler et al., 1995, Curr. Opin. Genet. Devel. 5, 814-821. For example, in tobacco, Tnt1A and Tto1 (Pouteau et al., 1994, Plant J., Takeda et al., 1988, Plant Mol. Biol., 36, 365-376), and Tos17 in rice (Hirochika et al., 1996, Proc. Natl. Acad. Sci. US). For 93,7783-7788) activation has been reported in the stress conditions.
[0005]
The rice retrotransposon Tos17 is the most studied class I element in plants. Tos17 was cloned by RT-PCR using degenerate primers created based on the conserved amino acid sequence of the reverse transcriptase domain between the Ty1-copia group retroelements (Hirochika et al., 1992, Mol. Gen.). Genet., 233, 209-216). Tos17 has a 4.3 kb long, two identical 138 bp LTR (long terminal repeat) and a PBS (primer binding site) complementary to the 3 ′ end of the initiating methionine tRNA (Hirochika et al., 1996, supra). ). Tos17 transcript is strongly activated by tissue culture and increases its copy number with culture time. In Nipponbare, a model japonica cultivar for genome research, the initial copy number of Tos17 is 2, but in the regenerated plants after tissue culture, the number is increased to 5 to 30 copies (Hirochika et al., 1996, supra). Unlike class II transposons characterized in yeast and Drosophila, Tos17 transposes in a random manner in the chromosome and causes stable mutations, and therefore reverse genetics of functional analysis of genes in rice ( Reverse Genetics) (Hirochika, 1997, Plant Mol. Biol. 35, 231-240; 1999, Molecular Biology of Rice (K. Shimamoto, Springer-Verlag, 43-58).
[0006]
[Problems to be solved by the invention]
The present invention provides a novel plant gene provided using Tos17. The present inventors have repeated systematic analysis of the phenotype of a plant having a newly transferred Tos17 copy in rice and the sequence adjacent to the Tos17 target site. As a result, Tos17 insertion resulted in short roots, narrow leaves, small grains, etc. A rice mutant having the following characteristics was discovered, and the gene responsible for this mutation was isolated, thereby completing the present invention.
[0007]
[Means for Solving the Problems]
The present invention relates to a polynucleotide encoding a gene that regulates plant morphology, which polynucleotide encodes an amino acid sequence from Met at position 1 to Val at position 689 of SEQ ID NO: 2, or It includes a polynucleotide encoding an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence.
[0008]
The present invention, in one aspect, relates to a protein comprising the amino acid sequence encoded by the above polynucleotide.
[0009]
The invention, in one aspect, relates to a vector comprising a control sequence and a polynucleotide as described above operably linked to the control sequence.
[0010]
In one aspect, the present invention relates to a vector comprising the antisense sequence of the gene encoded by the above polynucleotide.
[0011]
The present invention, in one aspect, relates to a method for modifying a plant, the method comprising the steps of: introducing the above vector into a plant tissue to obtain a transformant; regenerating the transformant to obtain a plant And selecting the plant for a desired trait.
[0012]
The desired trait may be selected from the group consisting of small grains, short culms, and dark green leaves.
[0013]
The present invention relates in one aspect to a plant transformed with the above vector.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
According to the present invention, there is provided a polynucleotide encoding a plant gene that controls plant morphology. As used herein, the term "controls the morphology of a plant" refers to the usefulness that can be used to improve plant varieties, such as changing plant height, root length, stem number, leaf and seed morphology, and seed yield in plants. Creating agricultural traits. The term "plant" includes monocots and dicots.
[0015]
The polynucleotide encoding the plant gene controlling the morphology of the plant of the present invention is typically a polynucleotide encoding an amino acid from Met at position 1 to Val at position 689 of SEQ ID NO: 2, or the amino acid A polynucleotide comprising a polynucleotide encoding an amino acid sequence in which one or several amino acid sequences have been deleted, substituted or added in the sequence.
[0016]
The polynucleotide encoding the plant gene that controls the shape of the plant of the present invention has at least 80% of the amino acid sequence from Met at position 1 to Val at position 689 in SEQ ID NO: 2 as long as the shape of the plant is controlled. Having a sequence identity of preferably at least 85% sequence identity, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity and most preferably at least 99% sequence identity. Nucleotides. The term "sequence identity" means that the two compared polynucleotides are the same, and the percent sequence identity between the two compared polynucleotide sequences is the percentage of the two compared polynucleotide sequences. After optimally aligning the polynucleotide sequences, the number of matched positions where identical nucleobases (eg, A, T, C, G, or I) occur in both sequences is taken to be the number of matched positions, The number of positions determined is divided by the total number of comparison polynucleotides and the result is calculated by multiplying by 100. Sequence identity can be calculated, for example, using the following tools for sequence analysis: Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Magazine, 5th Edition, 5th Edition). Rice, P. (1996) Program Manual for EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1ERQ, England, England, England, England, England, Wisconsin, USA 53711; (Invertership Hospitality and University of Geneva, Geneva, Switzerland).
[0017]
As used herein, the term "control sequence" refers to a DNA sequence having a functional promoter and any associated transcriptional elements (eg, enhancers, CCAAT boxes, TATA boxes, SPI sites, etc.).
[0018]
As used herein, the term "operably linked" refers to a polynucleotide that regulates its expression, such as a promoter, an enhancer, and various regulatory elements such as enhancers, so that the gene can be expressed in a host cell so that the gene can be expressed. It means being connected.
[0019]
It is well known to those skilled in the art that the types and types of control sequences may vary depending on the host cell. For example, the CaMV 35S promoter, the nopaline synthase promoter, and the like are well known to those skilled in the art. For introducing a gene into a plant, a method known to those skilled in the art is used. For example, methods via Agrobacterium and methods for direct introduction into plant cells are well known. As a method via Agrobacterium, for example, the method of Nagel et al. (Microbiol. Lett., 67, 325 (1990)) can be used. In this method, first, for example, an expression vector is introduced into Agrobacterium, and then the transformed Agrobacterium is transformed into a plant by the method described in Plant Molecular Biology Manual (SB Gelvin et al., Academic Press Publishers). This is a method for introduction into plant cells or plant tissues. Here, “plant tissue” includes callus obtained by culturing plant cells. As a method for directly introducing a gene into a plant cell or plant tissue, an electroporation method and a gene gun method are known.
[0020]
The cell or plant tissue into which the gene has been introduced is first selected by drug resistance such as hygromycin resistance, and then regenerated into a plant by a conventional method.
[0021]
The names used herein below, and the laboratory procedures described below, use procedures that are well known and commonly used in the art. Standard techniques are used for recombinant methods, polynucleotide synthesis, and microbial culture and transformation (eg, electroporation). This technique and procedure are generally described in the art and various general references provided throughout this document (in general, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold See Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference.
[0022]
【Example】
Hereinafter, the present invention will be described with reference to examples. The following examples illustrate, but do not limit, the invention.
[0023]
(Example 1: Tos17 activation by culture)
Ripe seeds of rice cultivar “Akitakomachi” were tested, and callus culture and cell suspension culture were performed. Activation of Tos17 was performed according to the method of Tsugawa and Suzuki (2000, Plant Cell Reports 19, 371-375). In short, the mature rice seeds were added to 1 mg / ml of MS medium supplemented with 2,4-D (2,4-dichlorophenoxyacetic acid) (Murashige and Skoog, 1962, Physiol. Plant., 15, 473-479). After culturing at 25 ° C. for 1 week on the above, callus induction was performed by culturing for 3 weeks in a KSP medium (Tsugawa and Suzuki, supra). The obtained callus was subjected to suspension culture for about three months in a KSP liquid medium (Tsugakawa and Suzuki, supra) supplemented with 1 mg / l of 2,4-D. Furthermore, the cells were transferred to a PR medium (Tsugawa and Suzuki, supra), cultured for about one week, and then transferred to a redifferentiation R medium (Tsugawa and Suzuki, supra), and transferred to a regenerated rice (R). ₀ Generation plant).
[0024]
(Example 2: Selection of small mutants and their characteristics)
The first generation (R ₁ ) A second generation (R) of about 380 strains obtained by obtaining a plant and further selfing it ₂ ) Was used as a test material to select a mutant line for endosperm formation. One of the mutant lines, the brown rice of a line named sg1 (small grain 1), was smaller than the wild-type brown rice (FIG. 1 (B)) as shown in FIG. 1 (A). .
[0025]
The sg1 line was cultivated on a 1% agar medium (Ryo and Ichii, 1996, Nisshokki, 65, 473-478), and the growth characteristics and morphological characteristics of the plants were investigated. As shown in FIG. 2 (A), at the seedling stage, this sg1 line had almost the same plant height as the wild type (FIG. 2 (B)), but the seed root length and crown Root length was shorter than wild type. Also, as shown in FIG. 3, the lateral root length was shorter than that of the wild type (FIG. 3 (A): sg1 strain, and FIG. 3 (B): wild type).
[0026]
Next, the rice was transplanted and cultivated in a field, and the growth characteristics, morphological characteristics, and characteristics of brown rice after maturation were examined over time. FIG. 4 shows the plant height of the sg1 line, FIG. 5 shows the number of tillers (the number of stems), FIG. 6 shows the root length, FIG. 7 shows the leaf blade, and FIG. In each figure, A and B show the results of the sg1 strain and the wild type, respectively.
[0027]
As shown in FIG. 4, the difference in plant height between the sg1 mutant line (A) and the wild type (B) (average of 10 strains) slightly increased as the number of days after transplantation increased, and reached when the plant height reached the maximum ( (About 80 days after transplantation), the plant height of the sg1 mutant strain was about 10% shorter than that of the wild type.
[0028]
As shown in FIG. 5, the number of tillers in the sg1 mutant strain (A) was about 10 to 20% smaller than that in the wild type (B) throughout the entire survey period (average of 10 strains each). FIGS. 6 and 7 show sg1 mutant (A) and wild-type (B) plants 30 days after transplantation in the field. As shown in FIG. 6, the root length of the sg1 mutant line (A) was shorter than that of the wild type (B). In addition, as shown in FIG. 7, in the sg1 mutant line (A), the expanded leaf blade was wound slightly inward and the blade width was slightly narrower than the wild type (B).
[0029]
As shown in FIG. 8, in the investigation of the agricultural traits during the mature stage, the spike length of the sg1 mutant strain was almost the same as that of the wild type, but the number of grains per spike was smaller than that of the wild type, and The yield was rather low, the weight of a thousand brown rice grains was about 15% less, and the yield per strain was about 50% less. In addition, the characteristic value of the agricultural trait shown in FIG. 8 is an average value of the values measured for 10 strains according to a conventional method.
[0030]
(Example 3, Isolation of full-length cDNA of SG1 gene and expression of mRNA)
In order to identify and isolate the causative gene of the sg1 mutation, individuals showing the sg1 mutation and normal individuals were identified from the heterogeneous population from which the sg1 mutation was separated, and the CTAB method (Murray and Thompson, 1980, Nucleic acid was extracted by Nucleic Acids Res. 8, 4321-4325). The extracted DNA was cleaved with restriction enzyme XbaI, and genomic Southern analysis (Southern, 1975, J. Mol. Biol., 98, 503) was performed using Tos17 as a probe. The presence of the gene of interest was identified by this genomic Southern analysis, and the DNA base sequence adjacent to the Tos17 insertion site was determined by TAIL-PCR using a DNA fragment containing this gene as template DNA.
[0031]
Briefly, DNAs obtained from an individual exhibiting the sg1 mutation and a normal individual were cut with restriction enzyme XbaI, and after agarose electrophoresis, were adsorbed to a nylon membrane. Southern hybridization was carried out using the Tos17 partial sequence (XbaI-BamHI fragment about 1000 bp) as a probe. As a result, in the individual showing the sg1 mutation, the band of the newly inserted Tos17 (about 6800 bp) was observed as homozygous, but not in the normal individual, and the band of the inserted Tos17 was completely linked to the sg1 mutant phenotype. I knew I was doing it. From these results, it can be seen that the DNA represented by the band hybridizing with the standard Tos17 probe contains the causative gene causing the sg1 mutation, and that Tos17 is inserted into the genomic region represented by this band and the genotype becomes homozygous. It was concluded that sg1 mutants occurred. Then, this DNA was used as a template to TAs-PCR (Liu Y-G. Et al., 1995, Genomics, 25, 674-681, Liu Y-G. Et al., 1995, Plant J., 8, 457-463) to Tos17. The adjacent sequence, that is, a part of the gene responsible for the sg1 mutation was isolated.
[0032]
First, total DNA from a regenerated plant having a new Tos17 target site was extracted, cut with a restriction enzyme XbaI, and then subjected to electrophoresis to cut out a nuclear DNA fragment of the Tos17 target site. An amplification reaction was performed by TAIL-PCR three times using the three sets of primers shown. First: Tos17-specific primer T1, AGTCGCTGATTTCTTTCACCAAGG and optional primer A1, NGTCGA (G / C) (A / T) GANA (A / T) GAA. Second round: Primer T2 specific for Tos17, GAGAGCATCATCGGTTACATCTTCTC and optional primer A1. 3rd: Primer T3, ATCCACCTTGAGTTTGAGGGG and A1 specific for Tos17. Next, the third TAIL-PCR product was electrophoresed on agarose to purify the target DNA fragment. Using a commercially available labeling kit (PE Biosystems) using the DyeCycle method, a sequencing reaction solution using this DNA as a template was prepared, and PCR (96 ° C., 3 minutes, 1 cycle; 96 ° C., 30 seconds, 50 C., 15 seconds, 60 ° C., 4 minutes, 25 cycles), and the DNA sequence of the target gene adjacent to Tos17 was determined using a 373S DNA Sequencer (PE Biosystems).
[0033]
Based on the obtained base sequence, primers SP1, TTGGACATTGCCGGATTGCCTAT and SP2, and ACAAGGGGAAACTGGGAGTTGCTGA specific to the target gene were designed, and a fragment of the target gene was amplified by a PCR method according to a conventional method. Using the obtained fragment (284 bp) as a probe, a cDNA library (λZAPII) prepared from mRNA expressed in rice seed callus of The library cloned in XhoI-EcoRI) was screened three times, and the cDNA of the target gene was cloned into the multiple cloning site (XhoI-EcoRI) of the plasmid pBluescript SK (-) (FIG. 9). Then, the entire nucleotide sequence of the cloned cDNA was determined according to a conventional method, and the gene encoding the amino acid sequence deduced from this cDNA was named SG1. This cDNA clone insert had a total length of 2576 bases and encoded an amino acid translation region having 2070 bases (corresponding to 689 amino acid residues) (FIG. 10).
[0034]
Using this rice cDNA as a probe, Southern analysis was performed on a wild-type nuclear DNA extract. As a result, only one copy of the SG1 gene was present in the rice nuclear genome, and all genes registered in the Japan Data Bank (DDBJ) were registered. As a result of conducting a homology search for, no gene having homology with this cDNA base sequence was found. Therefore, it was concluded that the SG1 gene was a novel gene.
[0035]
When the expression of mRNA of the SG1 gene in the sg1 line and the wild-type plant was examined by Northern analysis, the wild-type showed a particularly high expression level in the root, and its expression in the seed callus, roots, leaves and young panicles. (FIG. 11: A is a sg1 line and B is a lane showing the results of wild type Northern analysis). On the other hand, its expression was slightly detected in calli and leaves of the sg1 line. For mature brown rice, no SG1 gene mRNA was detected (data not shown).
(Example 4: Expression of transformed plant into which SG1 gene was introduced)
In order to confirm the function of the cloned SG1 gene, the SG1 gene and the antisense sequence of the SG1 gene were introduced into a binary vector pPZP202 to construct a transformation vector (FIG. 12). As the SG1 gene, a 2645 bp nucleotide sequence containing a part of the multicloning site of the plasmid pBluescript SK (-) in the nucleotide sequence of 1 to 2576 of SEQ ID NO: 1 was used. As the SG1 gene antisense sequence, Multicloning of plasmid pBluescript SK (-) into nucleotide sequence 1-2576 Part of one The 2605 bp nucleotide sequence including the part was inserted into the KpnI-SacI site and the BamHI-KpnI site in the binary vector pPZP202, respectively. Each gene was inserted in frame with the promoter CaMV35S. In FIG. 12A, HPTset represents the hygromycin resistance gene, 35S represents the CaMV 35S promoter, and NOS represents the NOS terminator.
[0036]
Using the resulting transformation vector, Agrobacterium tumefaciens strain EHA101 was transformed by electroporation under selection of 50 mg / l kanamycin and hygromycin. The obtained Agrobacterium strain was cryopreserved until use.
[0037]
From the wild-type seeds, the grain is removed to make brown rice, which is sterilized with 70% ethanol for 3 minutes, and sterilized distilled water. so After washing three times, it was further sterilized with a 50% sodium hypochlorite solution for 30 minutes and washed five times with sterile distilled water. This brown rice is added with 30 g / l sucrose, 0.3 g / l casamino acid, 2.8 g / l proline, and 2.0 mg / l 2,4-D, and solidified with 4.0 g / l gellite. Placed on a callus induction medium containing allowed N6 medium (Chu et al., 1975, Sci. Sinica, 18, 659-668). The pH of the medium was adjusted to 5.8 before autoclaving. Brown rice was grown at 28 ° C. for 4 weeks in the light, and calli having a size of about 5 mm were obtained. This callus was used for Agrobacterium infection.
[0038]
The above Agrobacterium cryopreserved in glycerol was treated with 20 mg / l kanamachiin, 50 mg / l hygromycin, 100 mg / l of 28 medium in darkness, adjusted to pH 7.2 containing speccomycin and solidified on 15 g / l agar-solidified AB medium (Chilton et al., 1974, Proc. Natl. Acad. Sci. USA, 71, 3672-3676). Culturing was carried out at a temperature of 3 days. Agrobacterium cells were collected and suspended in a liquid AAM medium (Hiei et al., 1994) containing 10 mg / l acetone syringone (Hiei et al., 1994, Plant J., 6, 271-282). After the callus was immersed in the obtained suspension for 2 minutes, excess water was removed with a sterilized paper towel, and the callus was placed in the above callus induction medium containing 10 mg / l acetone syringone. Co-cultivation was performed at 28 ° C. for 3 days to infect Agrobacterium. The obtained callus was washed 10 times with sterilized distilled water, and finally once with sterilized distilled water containing 500 mg / l carbenicillin, and excess water was removed with a sterilized paper towel. This callus was cultured at 28 ° C. for 2 weeks in the above-described callus induction medium containing 10 mg / l acetone syringone, 50 mg / l hygromycin, and 300 mg / l carbenicillin, and further 50 mg / l hygromycin and 100 mg / l carbenicillin were added. The cells were cultured for 4 weeks in a callus induction medium containing the cells. Hygromycin resistant calli were selected and 30 g / l sucrose, 30 g / l sorbitol, 2 g / l casamino acid, 2.2 mg / l kinetin, 1.0 mg / l NAA, 100 mg / l carbenicillin, Transferred to regeneration medium containing 50 mg / l hygromycin and 4 g / l gellite at pH 5.8 MS basal medium (Murashige and Skoog, 1962, Physiol. Plant., 15, 473-497).
[0039]
Transformants were easily regenerated in a regeneration medium containing hygromycin, transferred to soil and cultivated.
[0040]
As a result of introducing the SG1 gene antisense sequence into wild-type rice, the regenerated cells (T ₀ ), The growth of the transformed rice was equivalent to that of the untransformed rice, ₀ (Brown rice) obtained by self-breeding rice (T ₁ ) Showed smaller graininess than the wild type (FIG. 13 (A): FIG. 13 (B) shows the control wild type seeds).
[0041]
This brown rice (T ₁ ) Were germinated and grown in a medium supplemented with hygromycin, and separated into resistant individuals and non-resistant individuals. Individuals (transformants) having hygromycin resistance were cultivated and the morphological characteristics of the transformed plants were examined. As a result, individuals having various plant heights were observed, some of which exhibited the same characteristic trait as the sg1 mutant.
[0042]
Transformed rice (T) showing a phenotype different from the wild type ₁ In the same manner as described above, genomic DNA was extracted from the leaf blade, and Southern analysis was performed using the SG1 gene fragment as a probe. The copy number of the introduced SG1 gene antisense sequence varied among individuals and was investigated. In three individuals (S-1, S-2, S-3), the number was found to be 1 to 3 (FIG. 14: treatment with four types of restriction enzymes having no restriction enzyme site inside the cDNA of SG1 gene). This is the result of Southern analysis.The wild type (B) has one band in each of lanes 1 to 4, and the copy number of the SG1 gene is 1. In the transformed rice S-1 strain, the number of bands is 4. , The S-2 strain was 2, and the S-3 strain was 3, so that the number of newly introduced SG1 gene antisense sequences was 3, 1, and 2, respectively.) In addition, the transformed rice (R ₁ ), The total RNA was extracted from the leaf blade, and Northern analysis was performed using the SG1 gene fragment as a probe. As a result, a large amount of the introduced SG1 gene antisense sequence mRNA was expressed (A in FIG. 15). Is Transformed rice, and FIG. 15B is a Northern analysis of a control, untransformed rice).
[0043]
As described above, it has been found that the introduction of the SG1 gene antisense sequence changes various traits including plant height in plant transformants. Therefore, it was confirmed that the SG1 gene greatly affects various traits including plant morphogenesis. The transformed plant provided by the present invention can be used as a material for developing new varieties.
[0044]
【The invention's effect】
A novel gene that is highly expressed in plant roots and panicles is provided. By utilizing this gene, plants having various forms are provided.
[0045]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a photograph of brown rice of Akitakomachi wild type and its endosperm-forming mutant sg1. (A) is sg1 brown rice and (B) is wild-type brown rice.
FIG. 2 is a photograph of a young plant of Akitakomachi wild type and its endosperm-forming mutant sg1. (A) is an sg1 seedling, and (B) is a wild-type seedling.
FIG. 3 is a photograph showing lateral roots of a young plant of Akitakomachi wild type and its endosperm-forming mutant sg1. (A) is an sg1 seedling, and (B) is a wild-type seedling.
FIG. 4 is a graph showing daily changes in plant height of wild type of Akitakomachi and its endosperm-forming mutant sg1. (A) shows the sg1 line, and (B) shows the wild type plant height.
FIG. 5 is a graph showing the daily change in the number of stems of Akitakomachi wild type and its endosperm-forming mutant sg1. (A) shows the number of sg1 lines, and (B) shows the number of stems of the wild type.
FIG. 6 is a photograph of a plant 30 days after field transplantation of a wild type of Akitakomachi and its endosperm-forming mutant sg1. (A) is the sg1 strain and (B) is the wild type.
FIG. 7 is a photograph of a plant immediately after transplantation of a wild type of Akitakomachi and its endosperm-forming mutant sg1 into a field. (A) is the sg1 strain and (B) is the wild type.
FIG. 8 is a diagram showing a comparison of agronomic traits of Akitakomachi wild type and its endosperm-forming mutant sg1 during maturation. (A) is the sg1 strain and (B) is the wild type.
FIG. 9 is a diagram showing an outline of a cDNA fragment cloned into a plasmid pBluescript SK (−) of the SG1 gene.
FIG. 10 is a diagram showing the base sequence and deduced amino acid sequence of the SG1 gene.
FIG. 11 is a diagram showing the expression of mRNA in each organ of the sg1 line and wild-type plants. (A) is the sg1 strain and (B) is the wild type.
FIG. 12 is a view showing a part of a transformation vector containing an SG1 gene (sense) and an antisense SG1 gene. (A) shows a part of a transformation vector containing a sense SG1 gene, and (B) shows a part of a transformation vector containing an antisense SG1 gene.
FIG. 13 is a photograph of brown rice (A) of transformed rice into which an antisense SG1 gene has been introduced and brown rice (B) of non-transformed rice.
FIG. 14 is a diagram showing a comparison of the copy number of the SG1 gene between the transformed rice (S-1, S-2, S-3) into which the antisense SG1 gene has been introduced and the non-transformed rice (B).
FIG. 15 is a diagram showing a comparison of mRNA expression in the leaf blade between a transformed rice into which an antisense SG1 gene has been introduced and a non-transformed rice (B).

Claims (8)

A polynucleotide, polynucleotide or one or several amino acids are deleted in the amino acid sequence, encoding the amino acid sequence from Met at position 1 of SEQ ID NO: 2 to 689 of the Val, substituted or look containing a polynucleotide encoding the additional amino acid sequence, by deleting the gene comprising the polynucleotide in a plant, phenotype plant height is reduced, phenotype root length becomes shorter, expressions leaf narrows A polynucleotide that produces at least one phenotype selected from the group consisting of a phenotype, a phenotype with reduced stem number, and a phenotype with reduced seed number . A protein comprising an amino acid sequence encoded by the polynucleotide of claim 1. A vector comprising a control sequence and the polynucleotide of claim 1 operably linked to the control sequence. A vector comprising the antisense sequence of the gene encoded by the polynucleotide of claim 1. A method of modifying a plant,
A step of introducing the vector according to claim 4 into a plant tissue to obtain a transformant;
Regenerating the transformant to obtain a plant; and selecting the plant for a desired trait,
A method comprising: The method according to claim 5, wherein the desired trait is selected from the group consisting of small grain, short culm, and dark green leaves. A plant transformed with the vector according to claim 4. A polynucleotide, complement of a polynucleotide encoding the amino acid sequence from Met at position 1 of SEQ ID NO: 2 to 689 of the Val or one or several amino acids in the amino acid sequence, deletion , look containing the complement of a polynucleotide encoding a substitution or addition of amino acid sequence, by the polynucleotide into a plant, phenotype plant height is reduced, root length is short becomes phenotype, leaves narrow A polynucleotide that results in at least one phenotype selected from the group consisting of a phenotype that reduces stalk number, and a phenotype that reduces seed number .

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