JP2007330163A - Method for modifying light interception of rice plant by gene engineering technique - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a gene Spk(t) associated with tiller spreading properties of rice plant and to provide a method for modifying light interception, or plant type of rice plant by using the gene Spk(t) associated with spreading stub in rice plant. <P>SOLUTION: The nucleic acid has a genome sequence containing a Spk(t) locus. The Spk(t) cDNA and the Spk(t) protein are obtained. The method for modifying light interception, or plant type of rice plant by a gene engineering technique comprises using a nucleic acid having a genome sequence containing the Spk(t) locus or a nucleic acid containing the Spk(t) cDNA. The transgenic plant has a plant type of spreading stub. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a rice strain openability-related gene Spk (t). The present invention also relates to a method for improving the light receiving state or grass type of rice using the Spk (t) gene.

  Improving the plant type in rice is an important breeding target because it directly affects cultivation suitability and yield. The openness of the strain is one of the important components of improved grass-type breeding because it greatly affects the light receiving system.

  Conventional grass-type breeding has been carried out by selection of breeders in the field. However, since the plant type is determined by the action of a large number of loci, selection by phenotype requires a lot of labor and time. In addition, it has been impossible in conventional breeding to artificially modify the grass type by genetic engineering techniques, such as changing the expression pattern of the gene involved in the grass type.

It has been reported that the rice strain openness-related gene Spk (t) (derived from the indica variety Kasalath) sits between the C62163 locus and the E4055 locus on chromosome 9 (Yamamoto, T., et al. al., Breeding Science, 47: 141-144 (1997); Miyata, M., et al., Ikushugakukenkyu, 4 (suppl.1): 69 (2002); and Miyata, M., et al., Breeding Science, 55: 237-239 (2005);). However, the gene sequence has not been specified yet. In addition, by QTL analysis using the progeny of cross between Japonica cultivar Lemont and Indica cultivar Teqing (see Li, Z., et al., Euphytica, 109: 79-84 (1999)) It has been shown that there are loci involved in the angle, and it is possible that they are the same loci as the Spk (t) locus, but the gene sequence is not specified.
Yamamoto, T., et al., Breeding Science, 47: 141-144 (1997) Miyata, M., et al., Ikushugakukenkyu, 4 (suppl.1): 69 (2002) Miyata, M., et al., Breeding Science, 55: 237-239 (2005)

  The subject of the present invention is that the Spk (t) gene is related to the rice strain openness-related gene Spk (t) known to be located between the C62163 locus and the E4055 locus on chromosome 9 of the indica variety Kasalath. It is an object of the present invention to specify a locus to be seated in a narrower range to which gene recombination technology can be applied, and to specify and provide Spk (t) cDNA.

  The present invention also includes introducing a nucleic acid containing a genomic sequence of a locus on which the Spk (t) gene provided by the present invention or a nucleic acid containing Spk (t) cDNA into rice by genetic recombination technology, It is an object to provide a method for improving the light receiving state or grass type of rice.

 As a result of diligent research, the present inventors have identified the locus where the Spk (t) gene is located in a very narrow range on the 9th chromosome of the indica variety Kasalath and succeeded in identifying Spk (t) cDNA. The present invention has been completed.

Thus, the present invention is a nucleic acid comprising the genomic sequence of the Spk (t) locus, comprising:
a) a nucleic acid comprising the base sequence represented by base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1;
b) a part of the nucleic acid of a) above, which is involved in the rice strain openability;
c) a nucleic acid having at least 70% identity with the nucleic acid of a) or b) above and participating in the rice strain openability;
d) a nucleic acid in which one or more bases are deleted, substituted, inserted or added in the base sequence of the nucleic acid of a) or b) above and which is involved in the rice strain openability;
e) a nucleic acid that hybridizes with the complementary strand of the nucleic acid of a) or b) under stringent conditions and that is involved in the openability of rice strains; and f) any of the nucleic acids of a) to e) above. Complementary nucleic acids;
The nucleic acid is selected from the group consisting of:

The present invention also relates to a nucleic acid comprising Spk (t) cDNA,
a) a nucleic acid comprising the base sequence represented by bases 39-818 of SEQ ID NO: 2;
b) a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3;
c) a nucleic acid having at least 70% identity with the nucleic acid of a) above and being involved in the rice book openability;
d) a nucleic acid in which one or more bases are deleted, substituted, inserted or added in the base sequence of the nucleic acid of a) above and which is involved in rice strain opening;
e) a nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid of a) above and that is involved in rice strain openability; and
f) a nucleic acid complementary to the nucleic acid of any one of a) to e) above;
The nucleic acid is selected from the group consisting of:

  In another aspect, the present invention provides a method for improving the light reception status or plant type of rice, comprising introducing into the rice a nucleic acid comprising the Spk (t) locus of the present invention or a nucleic acid comprising Spk (t) cDNA. To do.

  The present invention also provides rice having a plant-opening grass type obtained by the method of the present invention.

The present invention further relates to a Spk (t) protein comprising:
a) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
b) a protein having at least 70% identity with the amino acid sequence represented by SEQ ID NO: 3 and involved in the rice strain openability; and c) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 3 A protein which is deleted, substituted, inserted or added and which is involved in the rice strain openability;
The protein is selected from the group consisting of:

  The present invention further provides antibodies against the amino acids of the present invention.

  Hereinafter, the present invention will be described in detail.

Nucleic Acids Containing the Spk (t) Locus The present invention provides nucleic acids comprising the genomic sequence of the rice strain openering gene (Spk (t)) locus.

  The Spk (t) gene has previously been found to be located between the C62163 locus and the E4055 locus on chromosome 9 of the indica variety Kasalath, but this region is very wide, about 120 kb, It was not suitable for use in engineering grass-type modifications.

  As described in Example 2, the present inventors first identified the base sequence of SEQ ID NO: 1, which is narrower than the conventionally specified range, as the genomic sequence on which the Spk (t) gene sits. Further, as described in Examples 3 and 5, in the base sequence of SEQ ID NO: 1, the region containing the Spk (t) gene is represented by bases 13230-35205 (22.0 kb fragment) of SEQ ID NO: 1, base 1888- It was identified as 34894 (15.0 kb fragment), bases 24567-35389 (10.8 kb fragment), bases 25591-31644 (6.1 kb fragment), and bases 27356-31107 (3.8 kb fragment).

  The inventors further identified Spk (t) cDNA as described in Example 4, and that the Spk (t) gene sits in a region represented by bases 2741-28687 of SEQ ID NO: 1. Revealed.

  Accordingly, the nucleic acid containing the genome sequence of the Spk (t) locus of the present invention is a nucleotide sequence represented by the base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1, or a part thereof, and is a rice strain openability. A nucleic acid comprising a base sequence involved in

  Here, the nucleic acid containing the base sequence represented by the base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1 is a sequence other than the base sequence represented by the base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1. A nucleotide sequence represented by nucleotides 25591-31644 (6.1 kb fragment) of No. 1; a nucleotide sequence represented by nucleotides 24567-35389 (10.8 kb fragment) of SEQ ID NO: 1; bases 88888-34894 (15. A base sequence selected from the group consisting of: a base sequence shown by SEQ ID NO: 1; a base sequence shown by bases 13230-35205 (22.0 kb fragment) of SEQ ID NO: 1; and a base sequence shown by SEQ ID NO: 1. It may be a nucleic acid containing or a nucleic acid comprising the base sequence.

  In addition, a nucleic acid that is a part of the base sequence represented by base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1 and includes a base sequence involved in rice strain openability is at least base 27412 of SEQ ID NO: 1. It is a nucleic acid containing the base sequence shown by -28687. Since the Spk (t) gene has been shown to sit in the region indicated by bases 2741-28687 of SEQ ID NO: 1 as described in Example 4 herein, at least a nucleic acid having this sequence is , It is thought to be involved in the opening of rice strains.

  However, the nucleic acid containing the genomic sequence of the Spk (t) locus of the present invention is shorter than the genomic sequence between the C62163 locus and the E4055 locus on chromosome 9 of the Indica variety Kasalath.

  Hereinafter, in the present specification, the nucleic acid containing the genomic sequence of the Spk (t) locus of the present invention described above is represented by the term “nucleic acid containing the Spk (t) locus”. The present invention also includes nucleic acid variants containing the Spk (t) locus. Therefore, in the present specification, the term “nucleic acid containing an Spk (t) locus” is used with the intention of widely including variants of nucleic acids containing an Spk (t) locus unless otherwise specified.

  Nucleic acid variants containing the Spk (t) locus of the present invention are involved in rice strain openability. The term "nucleic acid is involved in rice line opening" means that the plant into which the nucleic acid has been introduced has an activity of expressing the line opening characteristic. Alternatively, the term “nucleic acid is involved in rice openability” means that the nucleic acid encodes a protein having an activity to give a plant openability phenotype similar to the Spk (t) protein shown in SEQ ID NO: 3. Say.

  The variant of the nucleic acid containing the Spk (t) locus of the present invention is a base comprising one or more nucleotide deletions, substitutions, insertions and / or additions in the base sequence described above for the nucleic acid containing the Spk (t) locus. A nucleic acid comprising a sequence, which may be a nucleic acid involved in rice strain openability. Regarding the nucleic acid variant containing the Spk (t) locus of the present invention, the “one or more nucleotides” is not particularly limited as long as it is a nucleic acid involved in the rice strain openability as long as it is one or more nucleotides. “One or more nucleotides” may be described as one or several nucleotides. The specific range of “one or more nucleotides” is 1 to thousands, 1 to 1,000, 1 to 500, 1 to 100, 1 to 50, 1 to 20, 1 to 10, Alternatively, it may be 1 to 5 nucleotides.

  Nucleic acid variants containing the Spk (t) locus of the present invention may also have at least 70%, at least 80%, at least 90%, at least 95%, at least 98% of the base sequence described above for nucleic acids containing the Spk (t) locus. , Nucleic acids having at least 99%, or at least 99.5% identity and may be involved in rice strain openability.

  Here, the percent identity between two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing the sequence information using a computer program. The A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux, et al., 1984, Nucl. Acids Res ., 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters for the “GAP” program are: (1) GCG run of unary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), Schwartz and Gribskov and Burgess, Nucl. Acids Res., 14 as described by Dayhoff, “Atlas of Polypeptide Sequence and Structure” National Biomedical Research Foundation, pages 353-358, 1979. : Weighted amino acid comparison matrix of 6745, 1986; or other comparable comparison matrix; (2) 30 penalties for each gap in amino acids and one additional penalty for each symbol in each gap; or each gap in nucleotide sequence Added 50 penalties for each symbol in each gap 3 penalty; (3) no penalty for end gaps: and (4) no maximum penalty to long gaps include. Other sequence comparison programs used by those skilled in the art are available, for example, at the National Library of Medicine website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html The BLASTN program, version 2.2.7, or UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet site: http://blast.wustl.edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) a segment of query sequence with low composition complexity (Wootton and Federhen's SEG program (Computers and Woistton and Federhen, 1996 “Analysis of compositionally biased regions in sequence databases” Methods Enzymol., 266: 544-71) Or include a filter to mask segments consisting of short-periodic internal repeats (determined by the XNU program of Claverie and States (Computers and Chemistry, 1993)), and (B) match against database sequences Statistical significance threshold for reporting, and According to the statistical model of the E-score (Karlin and Altschul, 1990), the expected probability of a match found by chance; if the statistically significant difference due to a match is greater than the E-score threshold, the fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

  The variant of the nucleic acid containing the Spk (t) locus of the present invention further comprises a nucleic acid that hybridizes under stringent conditions to the complementary strand of the base sequence described above for the nucleic acid containing the Spk (t) locus, It may be a nucleic acid involved in strain openability.

Here, “under stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA (pH 8.0), and / or about 40-50 ° C., 2 × SSC-6 × SSC, and may or may not contain about 50% formamide Hybridization conditions for the hybridization solution (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42 ° C.), as well as for example about 40 ° C.-60 ° C., 0 Use of wash conditions with a wash solution containing 5-6 × SSC and may or may not contain 0.1% SDS. Preferably, moderately stringent conditions are about 50 ° C., 6 × SSC to 2 × SSC, preferably about 50 ° C., 6 × SSC, more preferably about 50 ° C., 2 × SSC hybridization conditions (and Cleaning conditions). Highly stringent conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, highly stringent conditions may be hybridized at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, more preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg, hybridization conditions as described above, and approximately 65 ° C.-68 ° C., 0. Defined with washing with a washing solution that contains 2 × SSC and may or may not contain 0.1% SDS. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete.

In order to achieve the desired degree of stringency by applying the basic principles known to those skilled in the art and governing the hybridization reaction and duplex stability, as further described below, the wash temperature It should be understood that the wash salt concentration can be adjusted as needed (see, eg, Sambrook et al., 2001). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the length of the hybrid can be determined by juxtaposing the nucleic acid sequences and identifying the region or regions with optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be the length of less than 50 base pairs, not should be 5-25 less ℃ than hybrid melting temperature (T _m), T m is determined by the following equation Is done. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (A + T base number) +4 (G + C base number). For hybrids longer than 18 base pairs, T _m = 81.5 ° C. + 16.6 (log ₁₀ [Na ⁺ ]) + 41 (molar fraction [G + C]) − 0.63 (% formamide) −500 / n, where N is the number of bases in the hybrid, and [Na ⁺ ] is the sodium ion concentration in the hybridization buffer (1 × SSC [Na ⁺ ] = 0.165 M). Preferably, each such hybridizing nucleic acid is at least 8 nucleotides (or more preferably at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 1% of the length of the nucleic acid to which it hybridizes (more preferably at least 25%, or at least 50%, or at least 70%, and most preferably at least 80) At least 70% (more preferably at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, with the nucleic acid to which it hybridizes, Most preferably Te has a sequence identity of at least 99.5%). Here, sequence identity, as described in more detail above, by comparing the sequences of hybridizing nucleic acids aligned in parallel to maximize identity with overlapping portions while minimizing sequence gaps. It is determined.

  The nucleic acid containing the Spk (t) locus of the present invention may include a complementary strand of the nucleic acid described above. The nucleic acid of the present invention includes single-stranded and double-stranded nucleic acids. In the present specification, the nucleic acid may be used with the intention of including DNA and / or RNA.

  The nucleic acid containing the Spk (t) locus of the present invention is synthesized by genomic DNA obtained from plants, DNA chemically synthesized by methods known to those skilled in the art, DNA polymerase, reverse transcriptase, etc. (by PCR method etc.) DNA or a combination thereof is included.

  Whether or not a nucleic acid variant containing the Spk (t) locus is involved in rice strain openability is not limited, but can be examined as follows, for example. According to the method of Hiei et al. (Plant J., 6: 271-82, (1994)), the nucleic acid fragment to be tested is introduced into Nipponbare. After the transformed plant is grown to a suitable time for transplantation, a PCR test is performed to select individuals considered to possess the introduced fragment in a complete form, and cultivated according to the conventional method. Then, the phenotype of the cultivated transformed plant is compared with the phenotype of the Spk strain (a strain having a Spak (t) region derived from Kasalath with a genetic background of Nipponbare). When the phenotype of the transformed plant shows the same open grass type as that of the Spk strain, the nucleic acid fragment to be tested is a nucleic acid involved in the openability of rice strains, whereas the closed plant type similar to that of Nipponbare is used. When indicated, it is determined that the nucleic acid fragment to be tested is not involved in the rice strain openability.

Nucleic acids containing Spk (t) cDNA The present invention provides nucleic acids containing Spk (t) cDNA.

  The nucleic acid containing Spk (t) cDNA of the present invention is a nucleic acid containing the base sequence represented by bases 39-818 of SEQ ID NO: 2.

  Moreover, the nucleic acid containing Spk (t) cDNA of the present invention includes a variant of nucleic acid containing Spk (t) cDNA. In the present specification, the term “nucleic acid containing Spk (t) cDNA” is used with the intention of including variants of nucleic acid containing Spk (t) cDNA unless otherwise specified.

  The nucleic acid variant containing the Spk (t) cDNA of the present invention is involved in the rice strain openability. The term "nucleic acid is involved in rice line opening" means that the plant into which the nucleic acid has been introduced has an activity of expressing the line opening characteristic. Alternatively, the term “nucleic acid is involved in rice openability” means that the nucleic acid encodes a protein having an activity to give a plant openability phenotype similar to the Spk (t) protein shown in SEQ ID NO: 3. Say.

  A variant of a nucleic acid containing the Spk (t) cDNA of the present invention is a nucleic acid that does not completely match the nucleotide sequence represented by nucleotides 39 to 818 of SEQ ID NO: 2, but encodes the amino acid sequence represented by SEQ ID NO: 3. It may be. The amino acid sequence represented by SEQ ID NO: 3 is an amino acid sequence encoded by the nucleic acid represented by bases 39-818 of SEQ ID NO: 2. One or more codons may encode the same amino acid, which is called the degeneracy of the genetic code. For this reason, a protein in which the sequence that does not completely match the base sequence represented by bases 39-818 of SEQ ID NO: 2 has the same amino acid sequence as the base sequence represented by bases 39-818 of SEQ ID NO: 2 Can be coded. The base sequence of such variants may arise from silent mutations (eg, occurring during PCR amplification) or may be the product of intentional mutagenesis of the native sequence.

  In addition, the nucleic acid variant containing the Spk (t) cDNA of the present invention is a base sequence containing one or more nucleotide deletions, substitutions, insertions and / or additions in the base sequence described above for the Spk (t) cDNA. It may be a nucleic acid comprising a nucleic acid involved in rice strain opening. Regarding the nucleic acid variant containing the Spk (t) cDNA of the present invention, the “one or more nucleotides” is not particularly limited as long as it is a nucleic acid involved in rice strain openability as long as it is one or more nucleotides. “One or more nucleotides” may be described as one or several nucleotides. A specific range of “one or more nucleotides” is 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 20, 1 to 10, or It may be 1 to 5 nucleotides.

  Nucleic acid variants comprising the Spk (t) cDNA of the present invention may also have at least 70%, preferably at least 80%, at least 90%, at least 95%, at least 98% of the base sequence described above for Spk (t) cDNA, It may be a nucleic acid having at least 99%, or at least 99.5% identity, and involved in rice strain openability. The percent identity between two base sequences can be determined by the method described above.

  The variant of the nucleic acid containing the Spk (t) cDNA of the present invention is further a nucleic acid that hybridizes under stringent conditions to the complementary strand of the base sequence described above for the Spk (t) cDNA, It may be a nucleic acid involved in Here, “under stringent conditions” means under the previously defined conditions.

  The nucleic acid containing the Spk (t) cDNA of the present invention may also include its complementary strand.

  The nucleic acid containing the Spk (t) cDNA of the present invention is synthesized by cDNA obtained from plants, DNA chemically synthesized by methods known to those skilled in the art, DNA polymerase, reverse transcriptase, etc. (by PCR method etc.). DNA, or a combination thereof.

  Whether or not a variant of a nucleic acid containing the Spk (t) cDNA of the present invention is involved in rice strain openability is not limited, but is transformed as described above for the nucleic acid containing the Spk (t) locus. Plants can be created and examined by comparing them with the Spk (t) line and the Nipponbare phenotype.

Method for Improving Rice Light-Reception Status or Grass Type The present invention is directed to introducing rice nucleic acid containing the Spk (t) locus of the present invention or nucleic acid containing Spk (t) cDNA of the present invention into rice. A method of improving the light receiving posture or grass shape is provided. Here, the nucleic acid containing the Spk (t) locus of the present invention or the nucleic acid containing the Spk (t) cDNA of the present invention is as defined above.

In another embodiment, the method of the present invention comprises the following steps:
(1) constructing a recombinant vector comprising a nucleic acid comprising the Spk (t) locus of the present invention or a nucleic acid comprising the Spk (t) cDNA of the present invention;
(2) introducing the recombinant vector into rice;
(3) selecting transformed plant material; and
(4) Redifferentiating the selected transformant to obtain transformed rice;
A method for improving the light receiving state or grass type of rice.

  The method for introducing a nucleic acid into rice in the method of the present invention is not particularly limited, and a known method can be used. The nucleic acid of the present invention may be introduced by a known genetic engineering method.

Any suitable recombinant vector may be used for transduction by genetic engineering techniques. Such recombinant vectors containing the nucleic acids of the invention are also within the scope of the invention.
The recombinant vector of the present invention is not particularly limited as long as it is a vector containing the nucleic acid of the present invention, but may be a recombinant expression vector with a sequence such as a promoter or terminator that controls the expression of the protein encoded by the nucleic acid. .

  Examples of the method for incorporating the DNA fragment of the nucleic acid of the present invention into a vector such as a plasmid include the method described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1.53 (1989). Etc. For convenience, a commercially available ligation kit (for example, manufactured by Takara Shuzo Co., Ltd.) can also be used. A recombinant vector (for example, a recombinant plasmid) thus obtained is introduced into rice which is a host cell.

  A vector can be conveniently prepared by ligating a desired gene to a recombination vector (for example, plasmid DNA) available in the technical field by a conventional method. Plant transformation vectors are particularly useful when using the nucleic acid fragment of the present invention to confer rice-opening traits to rice. The plant vector is not particularly limited as long as it has the ability to express a gene contained in the nucleic acid in a plant cell and produce a protein encoded by the gene. For example, pBI221, pBI121 (hereinafter referred to as Clontech). And vectors derived therefrom. In particular, for transformation of rice, which is a monocotyledonous plant, pIG121Hm, pTOK233 (above Hiei et al., Plant J., 6, 271-282 (1994)), pSB424 (Komari et al., Plant J., 10, 165- 174 (1996)).

  A transformed plant can be prepared by constructing a plant transformation vector by replacing the nucleic acid fragment of the present invention at the site of the β-glucuronidase (GUS) gene of the above-described vector, and introducing the vector into a plant. The plant transformation vector preferably contains at least a promoter, a translation initiation codon, a desired gene (the nucleic acid of the present invention or a part thereof), a translation termination codon, and a terminator. Further, it may appropriately contain a DNA encoding a signal peptide, an enhancer sequence, untranslated regions on the 5 'side and 3' side of the desired gene, a selection marker region, and the like. The promoter and terminator are not particularly limited as long as they function in plant cells. The promoter and terminator included in the Spak (t) genomic sequence derived from Kasalath may be used as they are, or those derived from other sources may be incorporated. When incorporating other sources, a promoter that constitutively expresses may be used, or an inducible promoter may be used. Examples of the promoter for constitutive expression include the actin and ubiquitin gene promoters in addition to the 35S promoter previously incorporated in the vector. Control sequences such as promoters, enhancers, terminators and the like are operably linked so that these control sequences are functionally related to the introduced nucleic acid fragment.

  Of the nucleic acids containing the Spk (t) locus of the present invention, when a nucleic acid having at least the nucleotides 25591-31644 (6.1 kb fragment) of SEQ ID NO: 1 is incorporated into a recombinant expression vector as a transgene, the nucleic acid is a promoter, translation Since it contains an initiation codon, a desired gene, a translation termination codon, and a terminator, it is not necessary to incorporate a control sequence such as another promoter or terminator into the expression vector, unless otherwise required.

  Among the nucleic acids containing the Spk (t) locus of the present invention, it has at least the base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1 and is longer than the bases 25591-31644 (6.1 kb fragment) of SEQ ID NO: 1. When a non-nucleic acid is incorporated as a transgene into a recombinant expression vector, it is preferable to incorporate an appropriate promoter into the recombinant expression vector separately. This is because the transgene contains a translation initiation codon, a desired gene, a translation termination codon, and a terminator, but may not contain a promoter.

  Among the nucleic acids containing the Spk (t) locus of the present invention, a part of the base 27356-31107 (3.8 kb fragment) of SEQ ID NO: 1, for example, the bases 2741-28687 of SEQ ID NO: 1, and the Spk (t ) When a nucleic acid containing cDNA is incorporated into a recombinant expression vector as a transgene, it is preferable to incorporate an appropriate promoter and terminator separately into the recombinant expression vector. This is because the transgene includes a translation initiation codon, a desired gene, and a translation termination codon, but may not include a promoter and terminator.

  In general, a recombinant vector contains a selection marker gene for identifying a transformant. As the selection marker, a commonly used marker can be used by a conventional method. Examples include tetracycline, ampicillin, or an antibiotic resistance gene such as kanamycin or neomycin, hygromycin or spectinomycin.

  As a method for introducing a plasmid into a host cell, the calcium phosphate method or calcium chloride / rubidium chloride described in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1.74 (1989) is generally used. Method, electroporation method, electroinjection method, a method by chemical treatment such as PEG, a method using a gene gun and the like. In the case of plant cells, it can be transformed by, for example, the leaf disc method (Science, 227, 129 (1985)) or the electroporation method (Nature, 319, 791 (1986)).

  In particular, as a method for introducing a gene into a plant, a method using Agrobacterium (Horsch, et al., Science, 227, 129 (1985), Hiei, et al., Plant J., 6, 271-282 (1994) ), Electroporation (Fromm, et al., Nature, 319, 791 (1986)), PEG (Paszkowski et al., EMBO J., 3, 2717 (1984)), microinjection (Crossway, et al., Mol. Gen. Genet., 202, 179 (1986)), and micro object collision method (McCabe et al., Bio / Technology, 6, 923 (1988)). The method is not particularly limited as long as it is a method for introducing a nucleic acid into a desired plant.

  Although not limited, methods for transducing rice using Agrobacterium include, for example, Hiei, et al., Plant J., 6, p.271-282 (1994), Komari, et al., Plant J., 10, p.165-174 (1996), Ditta, et al., Proc. Natl. Acad. Sci. USA, 77: p.7347-7351 (1980).

  Although not limited, the introduction of the nucleic acid of the present invention into rice can be specifically performed as follows. First, a plasmid vector containing the desired nucleic acid fragment to be introduced is prepared. As the plasmid vector, for example, pSB11, pSB22 and the like described in the plasmid map in Komari, et al., Plant J., 10, p.165-174 (1996) can be used. Alternatively, those skilled in the art can construct appropriate vectors themselves based on plasmid vectors such as pSB11 and pSB22. In the examples described later in this specification, an intermediate vector pSB200 having a hygromycin resistance gene cassette was prepared and used based on pSB11. Specifically, first, a terminator (Tnos) of nopaline synthase was connected to a ubiquitin promoter and a ubiquitin intron (Pubi-ubiI). By inserting a hygromycin resistance gene (HYG (R)) between the ubiI-Tnos of the Pubi-ubiI-Tnos obtained from this, a connector comprising Pubi-ubiI-HYG (R) -Tnos is obtained. Obtained. PKY205 was obtained by connecting this ligation to the HindIII / EcoRI fragment of pSB11 (Komari et al., Supra). By inserting a linker sequence for adding restriction sites of NotI, NspV, EcoRV, KpnI, SacI, and EcoRI into the HindIII site existing upstream of Pubi of pKY205, pSB200 having a hygromycin resistance gene cassette was obtained. .

  Next, E. coli (for example, DH5α, JM109, MV1184, etc., all of which can be purchased from TAKARA, for example) is transformed with the recombinant vector containing the inserted nucleic acid.

  Furthermore, using transformed E. coli, the Agrobacterium strain is preferably combined with a helper E. coli strain, for example, as described by Ditta et al. (Proc. Natl. Acad. Sci. USA, 77: p. 7347-7351 (1980)). Perform triparential mating according to the method. Although not necessarily limited, Agrobacterium tumefaciens strain LBA4404 / pSB1, LBA4404 / pNB1, LBA4404 / pSB3 etc. can be used, for example. In any case, plasmid maps are described in the above-mentioned Komari, et al., Plant J., 10, p.165-174 (1996), and those skilled in the art can use them by constructing a vector themselves, for example. Although not limited, for example, HB101 / pRK2013 (available from Clontech) can be used as the helper E. coli. In addition, although less commonly, there is a report that E. coli carrying pRK2073 can also be used as a helper E. coli (Lemas, et al., Plasmid, 1992, 27, p.161-163).

  Next, using Agrobacterium in which the desired hybridization occurred, for example, rice was transformed in accordance with the method of Hiei et al. (Plant J., 6, p.271-282 (1994)). The introduced nucleic acid fragment is introduced into rice.

  In the method of the present invention, the rice into which the nucleic acid of the present invention is introduced is not particularly limited as long as it is rice, but is preferably a rice having a strain-closing grass type. Examples of rice having a closed plant type include japonica rice, preferably Nihonbare, Asominori, Koshihikari, Kinuhikari, Dontokoi, and Hinohikari.

  In the method of the present invention, the form of rice into which the nucleic acid of the present invention is introduced is not particularly limited. For example, cells, leaves, roots, stems, buds, flowers (including stamens, pistils, etc.), berries, seeds, Germinated seed or any other part of plant tissue, growth point, explant, immature embryo, callus or somatic embryo-like tissue (hereinafter referred to as callus or the like in this specification, or simply callus), or complete plant body, All forms of rice are included. Preferred rice forms into which the nucleic acid of the present invention is introduced are immature embryos or callus, and most preferred are immature embryos.

  Further, in the method of the present invention, after introducing the desired introduced nucleic acid fragment into rice, it is cultivated according to the conventional method, whereby transformed rice having a plant-opening grass type can be obtained. If necessary, an individual carrying the introduced nucleic acid fragment in a complete form can be selected by performing a PCR assay or the like.

  Transformed rice obtained by the method of the present invention is also within the scope of the present invention. The transformed rice of the present invention has a plant-opening grass type.

Spk (t) protein The present invention provides Spk (t) protein.

  The Spk (t) protein of the present invention is a protein comprising the amino acid sequence represented by SEQ ID NO: 3. In addition, the Spk (t) protein of the present invention includes mutants of the Spk (t) protein. In the present specification, the term “Spk (t) protein” is used with the intention of including variants of the Spk (t) protein unless otherwise specified.

  A variant of the Spk (t) protein is a protein comprising an amino acid sequence containing one or more amino acid deletions, substitutions, insertions and / or additions in the amino acid sequence of SEQ ID NO: 3, It may be a protein involved. The protein involved in the rice line openability means that the protein has the same activity as that of the Spk (t) protein shown in SEQ ID NO: 3 to give the plant openability phenotype to plants.

  The substitution may be a conservative substitution, which is the replacement of a particular amino acid residue with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitution between aliphatic group-containing amino acid residues, such as substitution between Ile, Val, Leu or Ala, Lys and Arg, Glu and Asp, Gln and Asn Substitution between polar residues such as substitution is included.

  Mutations resulting from amino acid deletions, substitutions, insertions and / or additions may be performed on the DNA encoding the wild-type protein, for example by site-directed mutagenesis (eg, Nucleic Acid Research, Vol. 10, No. 20), which is a well-known technique. , p. 6487-6500, 1982, the entire contents of which are incorporated herein by reference). As used herein, “one or more amino acids” means amino acids that can be deleted, substituted, inserted and / or added by site-directed mutagenesis. “One or more amino acids” may be described as one or several amino acids. A specific range of “one or more amino acids” may be 1 to 80, 1 to 50, 1 to 25, 1 to 15, 1 to 10, or 1 to 5.

  Site-directed mutagenesis can be performed, for example, using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated, in addition to the specific mismatch that is the desired mutation, as follows: . That is, the synthetic oligonucleotide is used as a primer to synthesize a strand complementary to the phage, and a host cell is transformed with the obtained double-stranded DNA. Transformed bacterial cultures are plated on agar and plaques are formed from single cells containing phage. Then, theoretically 50% of the new colonies contain phages with mutations as single strands and the remaining 50% have the original sequence. The obtained plaque is hybridized with a synthetic probe labeled by kinase treatment at a temperature that hybridizes with the DNA having the desired mutation and that does not hybridize with DNA having the original strand. . Next, plaques that hybridize with the probe are picked up and cultured to recover DNA.

  In addition to the above-mentioned site-directed mutagenesis, a method for performing deletion, substitution, insertion and / or addition of one or more amino acids while maintaining the activity of the amino acid sequence of a biologically active peptide There are also methods of treating the gene with a mutagen and methods of selectively cleaving the gene, then removing, substituting, inserting or adding selected nucleotides and then ligating.

  A variant of the Spk (t) protein also has at least 70%, preferably at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99. It may be a protein containing an amino acid sequence having 5% identity, and involved in the rice strain openability.

  The percent identity between two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences is based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol., 48: 443-453, 1970), and the University of Wisconsin Genetics Computer Group (UWGCG) It may be determined by comparing the sequence information using a more available GAP computer program. Preferred default parameters for the GAP program include: (1) Scoring matrix as described in Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). , Blosum62; (2) 12 gap weights; (3) 4 gap length weights; and (4) no penalty for end gaps.

  Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). The program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website. Various conditions (parameters) for identity search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values. Alternatively, the percent identity between two amino acid sequences can be determined by genetic information processing software GENETYX Ver. The program may be determined using a program such as 7 (manufactured by Genetics) or the FASTA algorithm. At that time, the default value may be used for the search.

  The Spk (t) protein of the present invention may be a protein encoded by a nucleic acid containing the Spk (t) cDNA of the present invention.

  The Spk (t) protein of the present invention is a recombinant protein production method known to those skilled in the art, that is, an expression vector containing a nucleic acid encoding the protein is introduced into a host cell, and the host cell is used for expression of the recombinant protein. It can be obtained by culturing under suitable conditions and recovering the expressed recombinant protein.

  Whether or not the Spk (t) protein of the present invention is involved in the rice strain openability is not limited, but using the gene encoding the Spk (t) protein of the present invention, It is possible to examine by making a converted plant and comparing it with the Spk (t) line and the Nipponbare phenotype.

Antibody The present invention provides an antibody against the Spk (t) protein of the present invention.

  The antibodies of the present invention may be raised against the Spk (t) protein of the present invention or a fragment thereof. Here, the fragment of the Spk (t) protein of the present invention is a fragment having a sequence containing at least 6, at least 10, at least 20, or at least 30 amino acids in the amino acid sequence of the protein.

  The antibody may be produced by subjecting the Spk (t) protein of the present invention or a fragment thereof to animals, such as, but not limited to, mice, rats, rabbits, guinea pigs, goats, etc. used in the art for antibody production. It may be prepared by immunization. The antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies can be prepared based on antibody production methods well known to those skilled in the art.

  The antibody of the present invention can also be used to detect the Spk (t) protein of the present invention in assays such as Western blotting and ELISA. The antibody of the present invention can also be used to recover the Spk (t) protein of the present invention by affinity purification.

  The present invention provides a nucleic acid containing the Spk (t) locus of the present invention and Spk (t) cDNA, thereby imparting a stock-opening property, which is a plant type greatly related to the light receiving state, by a genetic engineering technique. Contribute in terms of providing a method.

Example 1 Identification of Spk (t) region by linkage analysis ( materials and methods )
From the BC ₃ F ₃ populations of Nipponbare and Kasalath, 6 inbred progenies (3840 individuals) in which the Spk (t) region (C62163 to E4055) was heterogeneous were tested, and the genes of the C62163 locus and E4055 locus The type was investigated and recombinant individuals between both loci were searched. About the obtained recombinant type | mold individual, the Spk (t) locus genotype was estimated by cultivating a self-bred next generation line (60 individuals of each line) in a field, and observing the phenotype regarding a stock openability.

The marker used for linkage analysis was prepared as follows. A PCR primer for amplifying a part of the genomic fragment between C62163 and E4055 was designed using the nucleotide sequence information of Nipponbare PAC clone P0012B6 (obtained from National Institute of Agrobiological Sciences). PCR is performed using Kasalath total DNA or BAC clone B104D11 (Kasalath-derived BAC located at a similar position to P0012B6, obtained from National Institute of Agrobiological Sciences) as a template, and the base sequence of the PCR product is decoded by the direct sequencing method. did. The decoded Kasalath base sequence was compared with the Nipponbare base sequence (P0012B6) to search for polymorphisms.
( Results and discussion )
As a result of examining the genotypes of the C62163 locus and the E4055 locus, it was found that 16 of the 3840 individuals were recombinant individuals between the two loci. Previous analysis of 1316 individuals (Miyata, M., et al., Ikushugakukenkyu, 4 (suppl.1): 69 (2002); and Miyata, M., et al., Breeding Science, 55: 237-239 (2005);), 6 recombinant individuals between both loci were obtained, so a total of 22 individuals were subjected to linkage analysis.

Ten PCR markers were generated in this region by partially decoding the Kasalath genomic sequence between C62163-E4055 and comparing it with the Nipponbare sequence. As a result of investigating the genotype of each marker locus for the 22 individuals, it was revealed that the Spk (t) locus sits between the 11860MseI locus and the 143976ALP locus (FIG. 1).

Example 2 Complete decoding of Kasalath base sequence of candidate region ( materials and methods )
In order to clarify the Kasalath base sequence of the Spk (t) candidate region clarified in Example 1, using primers (33 pairs) designed based on the corresponding Nipponbare sequence (P0012B6), the total DNA of Kasalath Alternatively, PCR was performed using BAC (B104D11) DNA as a template, and the base sequence of the product was decoded.
( Results and discussion )
By joining the 33 decoded base sequences, the base sequence (SEQ ID NO: 1) of the 40.8 kb region considered to include Spk (t) was clarified.

Example 3 Complementarity Test ( Materials and Methods )
(1) 15.9 kb fragment (bases 53-15926 of SEQ ID NO: 1)
After complete digestion of Kasalath BAC clone B104D11 with PacI and BglII, DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and smoothed with a DNA Blunting Kit (TAKARA). The reaction solution was subjected to electrophoresis on an agarose gel, and the separated 15.9 kb fragment was purified using QIAEXII (QIAGEN).

  Plasmid vector pSB200 (an intermediate vector having a hygromycin resistance gene cassette) was completely digested with EcoRV, and then DNA was collected by ethanol precipitation. The recovered DNA was dissolved in a TE solution and then dephosphorylated with CIAP (TAKARA). After subjecting the reaction solution to electrophoresis on an agarose gel, the vector fragment was purified from the gel using QIAEXII (QIAGEN).

  The two fragments prepared as described above were tested, and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). After the reaction, DNA was collected by ethanol precipitation. The recovered DNA was dissolved in pure water (prepared by an apparatus manufactured by Millipore), mixed with E. coli DH5α, and subjected to electroporation. The solution after electroporation was cultured with shaking in LB medium (37 ° C., 1 hour), spread on an LB plate containing spectinomycin, and incubated (37 ° C., 16 hours). About 12 of the resulting colonies, a plasmid was isolated, and the desired E. coli was selected by examining the restriction enzyme fragment length pattern and the boundary base sequence.

(2) 19.3 kb fragment (bases 5637-24930 of SEQ ID NO: 1)
After complete digestion of Kasalath's BAC clone B104D11 with PacI, SwaI and PflMI, DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and smoothed with a DNA Blunting Kit (TAKARA). The reaction solution was subjected to electrophoresis on an agarose gel, and the separated 19.3 kb fragment was purified using QIAEXII (QIAGEN).

  This fragment was tested with the EcoSB-CIAP-treated pSB200 fragment used in (1), and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired E. coli was selected according to the method described in (1).

(3) 22.0 kb fragment (bases 13230-35205 of SEQ ID NO: 1)
Kasalath BAC clone B104D11 was completely digested with KpnI and subjected to electrophoresis on an agarose gel. The isolated 22.0 kb fragment was purified using QIAEXII (QIAGEN).

  Plasmid vector pSB200 was completely digested with KpnI, and then DNA was collected by ethanol precipitation. The recovered DNA was dissolved in a TE solution and then dephosphorylated with CIAP (TAKARA). After subjecting the reaction solution to electrophoresis on an agarose gel, the vector fragment was purified from the gel using QIAEXII (QIAGEN).

  The two fragments prepared as described above were tested, and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired E. coli was selected according to the method described in (1).

(4) 15.0 kb fragment (bases 1888-34894 of SEQ ID NO: 1)
Kasalath BAC clone B104D11 was completely digested with SmaI, HpaI and PacI, and subjected to electrophoresis on an agarose gel. The isolated 15.0 kb fragment was purified using QIAEXII (QIAGEN).

  This fragment was tested with the EcoSB-CIAP-treated pSB200 fragment used in (1), and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired E. coli was selected according to the method described in (1).

(5) 10.8 kb fragment (bases 24567-35389 of SEQ ID NO: 1)
After complete digestion of Kasalath BAC clone B104D11 with BglII and ScaI, DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and smoothed with a DNA Blunting Kit (TAKARA). The reaction solution was subjected to electrophoresis on an agarose gel, and the separated 10.8 kb fragment was purified using QIAEXII (QIAGEN).

  This fragment was tested with the EcoSB-CIAP-treated pSB200 fragment used in (1), and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired E. coli was selected according to the method described in (1).

(6) 11.4 kb fragment (bases 29156-40600 of SEQ ID NO: 1)
After complete digestion of Kasalath BAC clone B104D11 with SphI and BstXI, DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and smoothed with a DNA Blunting Kit (TAKARA). The reaction solution was subjected to electrophoresis through an agarose gel, and the separated 11.4 kb fragment was purified using QIAEXII (QIAGEN).

  This fragment was tested together with the EcoRV-CIAP-treated pSB200 fragment used in (1), and DNA Ligation Kit Ver. 1 (TAKARA) was used for the ligation reaction. Thereafter, desired E. coli was selected according to the method described in (1).

(7) from Production (1) of the transformed plants _{(T 0} generation) Six E.coli were selected by (6), Agrobacterium tumefaciens strain LB4404 / pSB1 (Komari, T., et al., Plant J., 10 : 165-174 (1996)) and helper E. coli HB101 / pRK2013 (see Ditta, G., et al., Proc. Natl. Acad. Sci. USA, 77: 7347-7351 (1980)) In accordance with the method of Ditta et al. For 6 of the colonies generated on AB plates containing spectinomycin, plasmids were isolated and the desired Agrobacterium of each construct was selected by examining the restriction enzyme fragment length pattern.

  Using Agrobacterium selected as described above, Nipponbare was transformed according to the method of Hiei et al. (Plant J., 6: 271-282 (1994)). Transformed plants were transferred to the greenhouse after acclimation. After growing until the appropriate transplantation period, 20 plants for each construct were transplanted into 1/5000 are Wagner pots (1 to 4 individuals / pot) and cultivated according to the conventional method.

(8) Perform a PCR test on the transformed plant (T ₀ ) produced in the T ₁ generation test (7), select 4 individuals per construct that are considered to have the introduced fragment in their complete form, (20 individuals / strain) was transplanted into 1/5000 aren Wagner pots (1 to 4 individuals / pot) and cultivated according to the conventional method.
( Results and discussion )
Result of observation of the phenotype in _{T 0} generation of each construct, 22.0Kb fragment, when introduced the 15.0kb fragment or 10.8kb fragment from Kasalath in genetic background Spk system (Nipponbare Spk (t ) An individual showing a strain open grass type similar to that of the strain having the region was found. On the other hand, all of the individuals into which the 15.9 kb fragment, 19.3 kb fragment, or 11.4 kb fragment had been introduced showed a closed grass type similar to Nipponbare.

Also in T ₁ generation, 22.0Kb fragment, only when introduced the 15.0kb fragment or 10.8kb fragment was found individuals exhibiting grass type Spk system type.

Interpreting the above results in consideration of the positional relationship of each fragment (FIG. 2), it was considered that the Spk (t) gene was present in the 10.8 kb fragment. Therefore, try subjected _{T 1} strains 10.8kb fragment (derived from a single _{T 0} individuals), it was examined the association between the transgene and the phenotype. As a result, the population determined to possess the introduced fragment by PCR assay showed a strain open grass type, and the population determined not to carry the introduced fragment showed a closed grass type (FIG. 3). This confirmed that the Spk (t) gene was present in the 10.8 kb fragment.

Example 4 cDNA Screening ( Materials and Methods )
Spk strain obtained by continuous backcrossing of Nipponbare to Kasalath (strain with Kasalath-derived Spk (t) region in the genetic background of Nipponbare) is cultivated in a greenhouse according to the customary method, and the base of leaf sheath before heading is sampled did. After extracting total RNA by SDS-phenol method (Watanabe, A., and Price, CA, Proc. Natl. Acad. Sci. USA, 79: 6304-6308 (1982), QuickPrep mRNA Purification Kit (Amersham Pharmacia Biotech) The poly (A) ⁺ RNA was purified by the following procedure: The purified poly (A) ⁺ RNA was tested, and a cDNA library was prepared using the ZAP-cDNA Synthesis Kit (Stratagene).

In order to screen cDNA corresponding to ID1367, which is a Nipponbare full-length cDNA, from this cDNA library (FIG. 2), the following two kinds of primers:
5′-GAACCAAAGTCGATCTCTTGATGC-3 ′ (SEQ ID NO: 4)
5′-ACATACTCTTGTCAGTTATCACTG-3 ′ (SEQ ID NO: 5)
Was used and PCR was performed using the Kasalath BAC clone B104D11 as a template. After electrophoresis, an amplification product of about 1100 bp was recovered from the agarose gel using the QIAEX II Gel Extraction Kit (QIAGEN). The recovered fragment was ³² P-labeled (Probe A) using Rediprime II DNA labeling system (Amersham Pharmacia Biotech).

In order to screen a cDNA library on a 10.8 kb fragment and upstream from ID 1367 (FIG. 2), the following two kinds of primers:
5′-TCCTTCAATATACCATGGGAAGGAC-3 ′ (SEQ ID NO: 6)
5′-AATGTTCTCAACCACACTAACTGC-3 ′ (SEQ ID NO: 7)
Was used and PCR was performed using the Kasalath BAC clone B104D11 as a template. After electrophoresis, an amplification product of about 700 bp was recovered from the agarose gel by the method described above. The recovered fragment was ³² P-labeled by the method described above (probe B).

Using the cDNA library prepared as described above, 10 agar media in which about 15000 plaques appeared were prepared. Plaque lift was performed twice for each agar medium and transferred to Hybond-N ⁺ (Amersham Pharmacia Biotech). One membrane was used for hybridization with probe A, and the other membrane was used for hybridization with probe B. The series of operations was performed according to the manufacturer's manual. The positive plaque was subcloned into pBluescript according to the manufacturer's manual (Stratagene), and then the nucleotide sequence was decoded.
( Results and discussion )
As a result of screening using the A fragment as a probe, 7 cDNA clones were isolated. When the base sequence of each clone was decoded, it was found that all were clones of the same gene. By comparing the base sequence of the longest clone (SEQ ID NO: 2) with the Kasalath genomic base sequence (SEQ ID NO: 1) of the Spk (t) candidate region, the gene structure was revealed (FIG. 4). Specifically, the Spk (t) cDNA shown in SEQ ID NO: 2 corresponds to base 27374-27423, base 27587-27653, base 27757-28373, base 28604-28693, and base 30245-30550 in SEQ ID NO: 1. The sequence was a sequence of exons. Further, it was estimated that bases 39 to 41 of SEQ ID NO: 2 were the start codon and bases 816 to 818 of SEQ ID NO: 2 were the stop codon. These correspond to the bases 2741-27414 (start codon) and bases 2885-28687 (stop codon) of SEQ ID NO: 1, respectively, on the genome sequence. Therefore, it was estimated that the Spk (t) gene sits at the locus corresponding to bases 2741-28687 of SEQ ID NO: 1 in the Kasalath genomic sequence.

  Moreover, although the Spk (t) gene of the present invention corresponds to the gene ID 1367 registered in the Nipponbare full-length cDNA database, a difference was observed in the splicing pattern. As a result, compared with the deduced amino acid of Kasalath (SEQ ID NO: 3), Nipponbare was considered to encode an incomplete amino acid. A protein database was searched, but no protein with known function showing high homology with this gene product was found.

When the B fragment was used as a probe, no positive clone appeared.

Example 5 Additional Complementarity Test ( Materials and Methods )
Based on the results of Examples 3 and 4, the fragment region containing the Spk (t) gene was further limited, and a complementation test was performed.

(1) 6.1 kb fragment (bases 25591-31644 of SEQ ID NO: 1)
The plasmid clone isolated from the desired Escherichia coli obtained by ligation in Example 3 (5) was completely digested with BsaWI and RsrII, and then DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and smoothed with a DNA Blunting Kit (TAKARA). The reaction solution was subjected to electrophoresis on an agarose gel, and the separated 6.1 kb (FIG. 2) fragment was purified using QIAEXII (QIAGEN).

  This fragment was tested together with the EcoSB-CIAP-treated pSB200 fragment used in (3) of Example 3, and ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired Escherichia coli was selected according to the method described in Example 3 (1).

(2) Constant expression of 3.8 kb fragment (base 27356-31107 of SEQ ID NO: 1) Using Kasalath BAC clone B104D11 as a template, PCR was performed using the following two types of primers.
5′-TGCCATGCATTTTGAAGATCTGAGCACTGT-3 ′ (SEQ ID NO: 8)
5′-TCAAGCGGAGCTCCAACTGAAATCTCCATC-3 ′ (SEQ ID NO: 9)
For PCR, Pyrobest DNA polymerase (TAKARA) was used, and a cycle consisting of heat denaturation (94 ° C., 1 minute), annealing (58 ° C., 1 minute) and extension reaction (72 ° C., 3 minutes) was performed for 30 cycles. Repeated times. The PCR reaction solution was completely digested with BglII and SacI and subjected to electrophoresis on an agarose gel. The isolated 3.8 kb fragment was purified using QIAEXII (QIAGEN).

  Next, in order to obtain a fragment containing the promoter and intron of the ubiquitin gene for constitutive expression of this fragment, pSB200 was completely digested with HindIII and BamHI and electrophoresed on an agarose gel. The isolated 2.1 kb fragment (including the promoter and intron of the ubiquitin gene) was purified using QIAEXII (QIAGEN).

  Plasmid vector pSB200 was completely digested with HindIII and SacI, and then DNA was recovered by ethanol precipitation. The recovered DNA was dissolved in a TE solution and then dephosphorylated with CIAP (TAKARA). After the reaction solution was subjected to electrophoresis on an agarose gel, a 9.6 kb vector fragment was purified from the gel using QIAEXII (QIAGEN).

  The three fragments prepared as described above were tested, and a ligation reaction was performed using DNA Ligation Kit Ver. 1 (TAKARA). Thereafter, desired Escherichia coli was selected according to the method described in Example 3 (1).

(3) using the Agrobacterium were selected by producing the above transformant plant (T ₀ generation), Hiei et al. (Hiei, Y., et al, Plant J., 6:. 271-282 (1994)) According to this method, Nipponbare was transformed. Transformed plants were transferred to the greenhouse after acclimation. After growing to the appropriate time for transplantation, 12 individuals selected for each construct are selected by PCR assay and transplanted into 1/5000 are Wagner pots (1 to 4 individuals / pot). Cultivated according to the law.

(4) From each of the next-generation individual lines of the transformed plant (T ₀ ) produced in the T ₁ generation test (3), two lines were selected for each construct in which the presence or absence of the transgene was separated by PCR test. did. About the selected lines, 6 individuals were selected for each of the individuals with and without the introduced gene, transplanted to 1/5000 are Wagner pots (1 individual / pot), and cultivated according to the conventional method.
( Results and discussion )
Result of observation of the phenotype in _{T 0} generation of both constructs, when introduced the 6.1kb fragment, the same strain as the (system with Spk (t) regions from Kasalath in genetic background of Nipponbare) Spk strains When the 3.8 kb fragment was constitutively expressed, the strain opener grass type was stronger than that of the Spk line (FIG. 3).

For the T ₁ generation phenotypic 6.1kb fragment construct, whereas the population carrying the transgene showed similar strains exposition of plant type and Spk lineage population possesses no transgene and Nipponbare Similar closed grass type was shown. With respect to the T ₁ generation phenotypic 3.8kb fragment constitutive expression constructs, populations carrying the transgene whereas showed intense strain exposition of plant type than Spk system, it does not carry the transgene The population showed a closed grass pattern similar to Nipponbare.

  From the above results, it was revealed that the Spk (t) gene is an allele of the ID1367 locus. It was also shown that the stock opening property can be manipulated by changing the expression pattern of the Spk (t) gene using genetic engineering techniques.

FIG. 1 is a schematic diagram showing mapping of the Spk (t) locus. The numbers below the schematic diagram indicate the number of recombinant individuals between each marker locus and the Spk (t) locus. FIG. 2 is a schematic diagram showing the positioning of fragments examined in the complementation test. ID2802, ID4557, ID1367, and ID4898 are Nipponbare full-length cDNAs registered in the public database. FIG. 3 is a photograph showing a comparison of grass types. FIG. 4 is a schematic diagram showing the structure of the Spk (t) gene. A gray box indicates an exon, the number of each of which is listed below.

Claims (8)

A nucleic acid, the following:
a) a nucleic acid comprising the base sequence represented by base 27356-31107 of SEQ ID NO: 1;
b) a part of the nucleic acid of a) above, which is involved in the rice strain openability;
c) a nucleic acid having at least 70% identity with the nucleic acid of a) or b) above and participating in the rice strain openability;
d) a nucleic acid in which one or more bases are deleted, substituted, inserted or added in the base sequence of the nucleic acid of a) or b) above and which is involved in the rice strain openability;
e) a nucleic acid that hybridizes with the complementary strand of the nucleic acid of a) or b) under stringent conditions and that is involved in the openability of rice strains; and f) any of the nucleic acids of a) to e) above. Complementary nucleic acids;
The nucleic acid selected from the group consisting of: A nucleic acid comprising the base sequence represented by bases 27356-31107 of SEQ ID NO: 1 is the following:
The base sequence represented by bases 25591-31644 of SEQ ID NO: 1;
The base sequence represented by bases 24567-35389 of SEQ ID NO: 1;
The base sequence represented by bases 1888-34894 of SEQ ID NO: 1;
A base sequence represented by bases 13230-35205 of SEQ ID NO: 1; and a base sequence represented by SEQ ID NO: 1;
The nucleic acid according to claim 1, wherein the nucleic acid comprises a base sequence selected from the group consisting of:   A part of a nucleic acid comprising the base sequence represented by bases 27356-31107 of SEQ ID NO: 1, wherein the nucleic acid involved in rice strain openability comprises at least the base sequence represented by bases 2741-28687 of SEQ ID NO: 1 The nucleic acid according to claim 1, wherein   A method for improving the light receiving state or grass type of rice, comprising introducing the nucleic acid according to any one of claims 1 to 3 into rice.   Rice having a plant-opening grass type obtained by introducing the nucleic acid according to any one of claims 1 to 3 into rice. a) a nucleic acid comprising the base sequence represented by bases 39-818 of SEQ ID NO: 2;
b) a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 3;
c) a nucleic acid having at least 70% identity with the nucleic acid of a) above and being involved in the rice book openability;
d) a nucleic acid in which one or more bases are deleted, substituted, inserted or added in the base sequence of the nucleic acid of a) above and which is involved in rice strain opening;
e) a nucleic acid that hybridizes under stringent conditions with the complementary strand of the nucleic acid of a) above and that is involved in rice strain openability; and
f) a nucleic acid complementary to the nucleic acid of any one of a) to e) above;
A nucleic acid selected from the group consisting of:   A method for improving the light receiving state or grass type of rice, comprising introducing the nucleic acid according to claim 6 into rice.   Rice having a plant-opening grass type obtained by introducing the nucleic acid according to claim 6 into rice.