JP2005278636A - Brassinosteroid biosynthesis gene d11 participating in semi-dwarfness of plant, and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a d11 gene participating in the semi-dwarfness of a plant, to provide a method for dwarfing a plant with the gene as a target, and to provide a molecule used for the method. <P>SOLUTION: A single gene d11 causing a d11 mutation bringing the abnormality of semi-dwarfness on rice was identified and separated from a huge chromosome domain by a method of map base cloning. The base sequence of the isolated d11 gene was determined, and in order to specify a gene structurally related to the same, a data base was retrieved. Consequently, it was found that the d11 gene encods a d11 protein which is a new cytochrome P450 participating in a brassinosteroid biosynthesis route. It was found that a plant can be bwarfed by utilizing the d11 gene. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a brassinosteroid biosynthetic gene involved in plant semi-dwarfing and to dwarfing plants targeting the gene.

  Downsizing a plant body has various meanings in agriculture and horticulture. For example, it is possible to create ornamental plants with new aesthetic value by reducing the plant height or length, and new product value such as bite size by reducing the size of vegetables and fruits. You can also make crops that have In addition to such industrial use, for example, downsizing of experimental plants is important not only from the viewpoint of ease of handling but also from the viewpoint of effective use of experimental spaces due to a decrease in plant cultivation space.

  In the miniaturization of a plant, the characteristic that the plant height or length of the plant is shorter than the wild type (normal type) is called fertility. Many rice varieties and lines exhibiting this characteristic have been found in the mutagenesis treatment with γ-rays and chemicals, and their genetic analysis has been advanced for a long time. Up to now, it is known that more than 60 genes are involved in this fertility (Non-patent Document 1). However, there is little knowledge about why fertility traits are expressed, and it has become clear that a small number of fertile mutants are caused by mutations in the genes of proteins involved in the biosynthesis of brassinosteroids, which are plant growth hormones. (Non-Patent Documents 2 and 3).

  Among the rice dwarf mutants, dwarf11 (d11), in particular, suppresses elongation between the 2nd node, and as a result, it not only makes the plant height less than half that of the normal type, but also has the feature that the grains become small circles It is known (Non-Patent Documents 4-6). Further, as a result of genetic analysis, it has been revealed that this d11 is controlled by a single recessive gene d11 on the rice chromosome 4 (Non-patent Document 7).

  In d11 gene mutants, phenotypes that are similar to the phenotypes such as fertility, dark green leaves, upright leaves, and head extension exhibited by mutants of the brassinosteroid signaling pathway or biosynthetic pathway, which is a plant hormone Thus, it is presumed that the d11 gene is a locus of a protein involved in the brassinosteroid signal transduction pathway or biosynthetic pathway in the plant body.

  Therefore, isolating the d11 gene and clarifying the function of the gene product is not only important for clarifying the morphogenesis process in plants, but also using the knowledge to determine the size of the plant. This is considered to be extremely important for artificial control.

Rice Genetic Newsletter Vol.12: 30-32 (1995) BRD1: The Plant Journal vol.32: 495-508 (2002) D2: The Plant Cell vol.15: 2900-2910 (2003) Akimine Masao, JSPS Report Volume 1: 108-314 (1925) Nakayama Bao, Bulletin of Faculty of Humanities and Science, Shinshu Volume 4: 1-31 (1954) Takahashi Soemon, Takeda Kazuyoshi, Hokkaido University Nobu Kunibun Bulletin 7: 32-43 (1969) Yoshimura et al., Plant. Mol. Biol., 35: 49-60 (1997)

  An object of the present invention is to identify and isolate the d11 gene involved in plant semi-fertility. Another object of the present invention is to provide a method for dwarfing a plant by controlling the expression of an endogenous d11 gene, and a molecule for controlling the expression of the endogenous d11 gene.

  The rice fertility gene d11 has been known as a gene encoding a protein involved in rice fertility so far as existing in one of the vast regions of rice chromosome 4. In order to isolate the d11 gene from a vast chromosomal region, the present inventors have conducted intensive research using a map-based cloning technique, elucidated the region where the d11 gene exists, and finally found the desired gene. Successfully isolated. Specifically, first, linkage analysis using a molecular marker was performed, and the region on the chromosome where the d11 gene was present was narrowed down to a region sandwiched between specific molecular markers. Next, a physical map was created by aligning the PAC clones for the region around the narrowed down gene, and the base sequence in the candidate region was decoded. By comparing the d11 mutant with the normal rice nucleotide sequence, one cDNA clone (002-107-G06) was successfully identified as the d11 gene.

  The base sequence (2031 base pairs) of the identified clone has been analyzed in a rice genome analysis study. As a result of similarity search on the database, the base sequence of the identified clone has homology to the gene encoding cytochrome P450 (cytochrome P450) involved in the biosynthesis of Arabidopsis and rice brassinosteroids It became clear. From these facts, the present inventors have found that the identified gene is a novel cytochrome P450 gene.

  In plants, many cytochrome P450s are involved in the biosynthesis of plant hormones auxin, gibberellin and brassinosteroids. The d11 gene isolated according to the present invention is a gene encoding a novel cytochrome P450 involved in the brassinosteroid biosynthesis pathway. Accordingly, the present inventors have found that it is possible to dwarf a wide range of plants by suppressing the expression of the d11 gene.

That is, the present invention relates to a method for dwarfing a plant by suppressing the expression of a d11 gene involved in plant dwarfism or a homologous gene thereof, and a molecule used for suppressing the expression of these genes. More specifically, the following invention is provided.
[1] The DNA according to any one of the following (a) to (c), which is used for dwarfing a plant
(A) DNA encoding an RNA complementary to a transcription product of a plant endogenous d11 gene
(B) DNA encoding an RNA having ribozyme activity that specifically cleaves a transcription product of a plant endogenous d11 gene
(C) DNA encoding an RNA that inhibits the endogenous d11 gene expression in plants by a co-suppression effect
[2] The DNA according to [1], wherein the plant d11 gene is a rice d11 gene
[3] A vector comprising the DNA according to [1] or [2] [4] A transformed plant cell that holds the DNA according to [1] or [2] in an expressible manner [5] The plant is rice. Transformed plant cell according to [4] [6] Transformed plant body comprising transformed plant cell according to [4] or [5] [7] Offspring or clone of transformed plant body according to [6] The transformed plant body [8] The plant is rice, the transformed plant body according to [6] or [7] [9] The dwarfed transformation according to [6] or [7] Plant [10] Reproduction material of transformed plant according to any of [6] to [9] [11] A method for producing a transformed plant according to any of [6] to [9]. Then, the DNA according to [1] or [2] is introduced into a plant cell, and the plant body is regenerated from the plant cell. [12] A method for dwarfing a plant [13], characterized by suppressing the expression of an endogenous d11 gene in cells of the plant body [13] The DNA according to [1] or [2] [14] The method according to any one of [11] to [13], wherein the plant is rice.

  According to the present invention, a d11 gene of a plant, a method for dwarfing a plant targeting the gene, and a molecule for dwarfing a plant are provided. Utilization of d11 gene in plants enables high yields of plants such as useful crops and ornamental plants, addition of aesthetic value by dwarfing, and high yielding by marker selection and efficient breeding of dwarfed plants. It is highly expected to become.

  The present invention relates to a method of dwarfing a plant by suppressing the expression of the d11 gene, and a molecule used for suppressing the expression of the gene. The base sequence of the cDNA of rice d11 gene, the base sequence of genomic DNA is shown in SEQ ID NO: 3, and the protein encoded by these genes whose relationship with fertility was clarified by the present inventors. The amino acid sequence is shown in SEQ ID NO: 1.

  The rice d11 gene has been known as a gene that imparts a fertile phenotype to rice and exists in one of the vast regions of rice chromosome 4. The present inventors have narrowed down the region of the d11 gene in rice chromosome 4 by using a map-based cloning technique, and finally succeeded in identifying it as a single gene.

  In the present specification, the “d11 gene” refers to a step from 3-dehydro-6-deoxotearone to 6-dehydrotefasterol in the late C6-oxidation pathway and early C6-oxygen in the brassinosteroid biosynthesis pathway. It refers to the gene encoding the d11 protein, a cytochrome P450, that catalyzes the step of 3-dehydrotesterone to tefasterol in the foundation pathway.

  The form is not particularly limited as long as it can encode d11 protein, and “d11 gene” includes genomic DNA, chemically synthesized DNA, etc. in addition to cDNA. In addition, DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included as long as it can encode d11 protein. In particular, the term “endogenous d11 gene” means genomic DNA that exists naturally in plant cells.

  Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from a plant, a genomic library (a plasmid, phage, cosmid, BAC, PAC, etc. can be used as a vector) is developed and expanded, and a d11 gene (for example, It is possible to prepare by conducting colony hybridization or plaque hybridization using a probe prepared based on SEQ ID NO: 2). It is also possible to prepare by preparing a primer specific to the d11 gene (for example, SEQ ID NO: 2) and performing PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from a plant, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization in the same manner as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

  The “d11 gene” includes not only the rice d11 gene but also homologs thereof, and also includes mutants and variants thereof.

  Furthermore, since the d11 gene is considered to exist widely in the plant kingdom, the “d11 gene” includes not only rice but also homologous genes existing in various plants. Here, the “homologous gene” refers to a gene encoding a protein having physiological functions similar to those of the d11 gene product in rice in various plants.

  Methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki). , RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988). That is, for those skilled in the art, oligonucleotides that specifically hybridize to the d11 gene (eg, SEQ ID NO: 2) using the nucleotide sequence of the rice d11 gene (eg, SEQ ID NO: 2) or a part thereof as a probe. As a primer, it is usually possible to isolate a homologous gene of d11 gene from various plants.

  In order to isolate DNA encoding such a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5 × SSC, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, and 0.1 × SSC. The sequence of the isolated DNA can be determined by a known method. The homology of the isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire amino acid sequence. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  Whether or not the isolated DNA is a DNA encoding the d11 protein depends on whether the sequence homology is high and the expression of an endogenous gene corresponding to the isolated d11 gene is suppressed. It can be evaluated by dwarfing.

  More specifically, the d11 gene of the present invention includes (a) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, and (b) the base sequence code set forth in SEQ ID NO: 2 or 3. DNA containing a region, (c) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2 or 3. d) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 3 is included.

  In the present specification, “dwarfing” means that the plant height or culm length is shorter than that of a wild-type plant. In the present invention, there is no particular limitation on the plant into which the dwarfing trait is introduced by suppressing the expression of the endogenous d11 gene, and a desired plant to be dwarfed can be used. Or ornamental plants are preferred. Examples of useful agricultural products include monocotyledonous plants such as rice, corn, wheat and barley, and dicotyledonous plants such as rapeseed, soybean, cotton, tomato and potato. Examples of ornamental plants include flower plants such as chrysanthemum, roses, carnations, and cyclamen. Since the d11 gene is thought to exist widely in the plant kingdom (see FIGS. 3 and 4), it is possible to dwarf a wide range of plants by targeting the d11 gene.

  When producing a dwarfed plant in the method of the present invention, a DNA for suppressing the expression of the d11 gene is inserted into an appropriate vector, introduced into a plant cell, and the resulting transformation Regenerate plant cells. The DNA and vector become agents for dwarfing plants.

  In the present specification, “suppression of d11 gene expression” includes suppression of gene transcription and suppression of protein translation. Also included is a decrease in expression as well as complete cessation of DNA expression.

  Suppression of the expression of a specific endogenous gene in a plant can be performed using, for example, DNA encoding an RNA complementary to a transcription product of the endogenous d11 gene.

  One embodiment of “DNA encoding RNA complementary to the transcription product of DNA encoding the protein of the present invention” is DNA encoding antisense RNA complementary to the transcription product of DNA encoding the protein of the present invention. It is. The antisense effect in plant cells was demonstrated for the first time by the antisense RNA introduced by electroporation using a transient gene expression method to exert an antisense effect in plants (Ecker and Davis, Proc. Natl Acad. USA, 83: 5372, 1986). Subsequently, tobacco and petunia have been reported to decrease the expression of target genes by expressing antisense RNA (Krol et. Al., Nature 333: 866, 1988), and currently suppress gene expression in plants. Established as a means to

  There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit transcription, splicing, or translation processes and suppress target gene expression (Hirashima and Inoue, "Neurochemistry Experiment Course 2, Nucleic Acid IV Gene Replication and Expression", edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin , pp.319-347, 1993).

  The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.

  Antisense DNA can be prepared, for example, by the phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988) based on the sequence information of DNA set forth in SEQ ID NO: 2. is there. The prepared DNA can be transformed into a desired plant by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the transcription product of the endogenous gene of the plant to be transformed, but may not be completely complementary as long as the gene expression can be effectively inhibited. . The transcribed RNA preferably has a complementarity of 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. In order to effectively inhibit the expression of the target gene using the antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.

  Another embodiment of the “DNA encoding RNA complementary to the transcript of the DNA encoding the protein of the present invention” is a dsRNA (double stranded RNA) complementary to the transcript of the endogenous d11 gene. It is DNA to encode. A phenomenon called RNAi (RNA interference), in which the expression of the introduced foreign gene and target endogenous gene is both suppressed by introducing dsRNA having the same or similar sequence to the target gene into the cell. Can cause. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, a RNaseIII-like nuclease called Dicer with a helicase domain is present in the presence of ATP, and dsRNA is about 21 to 23 base pairs from the 3 ′ end. Cut out and produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the transcript (mRNA) of the target gene at the center of the siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  RNAi was first discovered in nematodes (Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, 1998), but now only nematodes It has been observed in various organisms such as plants, linear animals, Drosophila and protozoa (Fire, A. RNA-triggered gene silencing. Trends Genet. 15, 358-363 (1999), Sharp, PA RNA interference 2001 Genes Dev. 15, 485-490 (2001), Hammond, SM, Caudy, AA & Hannon, GJ Post-transcriptional gene silencing by double-stranded RNA.Nature Rev. Genet. 2, 110-119 (2001), Zamore , PD RNA interference: listening to the sound of silence. Nat Struct Biol. 8, 746-750 (2001)). In these organisms, it has been confirmed that the expression of the target gene is suppressed by actually introducing dsRNA from a foreign body, and is also being used as a method for creating knockout individuals.

  At the beginning of RNAi, dsRNA was thought to be ineffective unless it was longer than a certain length (40 bases), but US Rockefeller University Tuschl et al. Used a single-stranded dsRNA (siRNA) of around 21 base pairs. When introduced into cells, it has been reported that the antiviral response by PKR does not occur in mammalian cells and RNAi is effective (Tuschl, Nature, 411, 494-498 (2001)). As a technology that can be applied to mammalian cells, it has attracted a great deal of attention.

  The DNA of the present invention comprises an antisense code DNA encoding an antisense RNA for any region of a target gene transcription product (mRNA) and a sense code DNA encoding a sense RNA of any region of the mRNA. The antisense RNA and the sense RNA can be expressed from the antisense code DNA and the sense code DNA. Moreover, dsRNA can also be produced from these antisense RNA and sense RNA.

  In the case where the dsRNA expression system of the present invention is retained in a vector or the like, there are a case where antisense RNA and sense RNA are expressed from the same vector, and a case where antisense RNA and sense RNA are expressed from different vectors, respectively. is there. For example, antisense RNA and sense RNA can be expressed from the same vector as antisense RNA in which an antisense coding DNA and a promoter capable of expressing a short RNA such as polIII are linked upstream of the sense coding DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction.

  It is also possible to construct an expression system in which antisense code DNA and sense code DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. In this palindromic style expression system, the two promoter types are preferably different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the sense coding DNA. An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.

  In RNAi, siRNA may be used as dsRNA. “SiRNA” means a double-stranded RNA consisting of short strands in a range that does not show toxicity in cells, and is not limited to the total length of 21 to 23 base pairs reported by Tuschl et al. The length is not particularly limited as long as the length is not shown. For example, the length can be 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Alternatively, the siRNA to be expressed is transcribed and the length of the final double-stranded RNA portion is, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, more preferably 21 to 30 base pairs. be able to.

  As the DNA of the present invention, an appropriate sequence (preferably an intron sequence) is inserted between inverted repeats of a target sequence to produce a double-stranded RNA having a hairpin structure (self-complementary 'hairpin' RNA (hpRNA)). Such constructs (Smith, NA, et al. Nature, 407: 319, 2000, Wesley, SV et al. Plant J. 27: 581, 2001, Piccin, A. et al. Nucleic Acids Res. 29: E55, 2001 ) Can also be used.

  The DNA used for RNAi need not be completely identical to the target gene, but at least 70% or more, preferably 80% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98% , 99% or more). The sequence identity can be determined by the method described above.

  The part of the double-stranded RNA in which the RNAs in dsRNA are paired is not limited to a perfect pair, but mismatch (corresponding base is not complementary), bulge (no base corresponding to one strand) ) Or the like may include an unpaired portion. In the present invention, both bulges and mismatches may be included in the double-stranded RNA region where RNAs in dsRNA pair with each other.

  Suppression of endogenous d11 gene expression can also be carried out using a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type or a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 35: 2191, 1990).

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, it has been shown to be cleaved by A or U (Koizumi et. Al., FEBS Lett. 228: 225, 1988). If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. (Koizumi et.al., FEBS Lett. 239: 285, 1988, Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 35: 2191, 1990, Koizumi et. Al., Nucleic. Acids. Res. 1989).

  Hairpin ribozymes are also useful for the purposes of the present invention. Hairpin ribozymes are found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, Nature 323: 349, 1986). It has been shown that this ribozyme can also be designed to cause target-specific RNA cleavage (Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751, 1992, and Hiroshi Kikuchi, Chemistry and Biology 30: 112, 1992).

  A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. However, at that time, if an extra sequence is added to the 5 ′ end or 3 ′ end of the transcribed RNA, the ribozyme activity may be lost. In such a case, in order to accurately cut out only the ribozyme part from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts in cis for trimming is placed on the 5 'side or 3' side of the ribozyme part. (Taira et.al., Protein Eng. 3: 733, 1990, Dzianott and Bujarski, Proc. Natl. Acad. Sci. USA, 86: 4823, 1989, Grosshans and Cech, Nucleic Acids Res. 19 : 3875, 1991, Taira et. Al., Nucleic Acids Res. 19: 5125, 1991).

  In addition, such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved, thereby further enhancing the effect (Yuyama et al., Biochem. Biophys. Res. Commun. 186: 1271 , 1992). Such a ribozyme can be used to specifically cleave the transcription product of the target gene in the present invention, thereby suppressing the expression of the gene.

  Suppression of endogenous gene expression can also be achieved by “co-suppression” resulting from transformation of DNA having a sequence identical or similar to the target gene sequence. “Co-suppression” refers to a phenomenon in which the expression of both a foreign gene and a target endogenous gene to be introduced is suppressed when a gene having the same or similar sequence as that of the target endogenous gene is introduced into a plant by transformation. Say. Details of the mechanism of co-suppression are not clear, but are often observed in plants (Curr. Biol. 7: R793, 1997, Curr. Biol. 6: 810, 1996).

  For example, in order to obtain a plant body in which the d11 gene is co-suppressed, a vector DNA prepared so that the d11 gene or a DNA having a sequence similar thereto can be expressed is transformed into the target plant, and the resulting plant is obtained. A plant having a d11 mutant trait, that is, a dwarfed plant may be selected from the body. The gene used for co-suppression need not be completely identical to the target gene, but is at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95%, 96%, 97%, 98 %, 99% or more).

  Furthermore, suppression of the expression of the endogenous gene in the present invention can also be achieved by transforming a gene having a dominant negative trait of the target gene into a plant. A gene having a dominant negative trait refers to a gene having a function of eliminating or reducing the activity of an endogenous wild-type gene inherent in a plant by expressing the gene.

  The present invention also provides a vector containing a DNA used to suppress the expression of the endogenous gene, a transformed plant cell that retains the DNA so that it can be expressed, a transformed plant containing the transformed plant cell, and the transformed plant. Provided is a transformed plant body that is a progeny or clone of the body, and a propagation material for the transformed plant body.

  Furthermore, the present invention relates to a method for producing the above-mentioned transformed plant, which comprises a step of introducing a DNA used for suppressing the expression of an endogenous gene into a plant cell and regenerating the plant from the plant cell. .

  Those skilled in the art can introduce the DNA of the present invention into plant cells by a known method such as the Agrobacterium method, electroporation method (electroporation method), or particle gun method.

  When the Agrobacterium method is used, for example, the method of Nagel et al. (Microbiol. Lett. 67: 325, 1990) is used. According to this method, the recombinant vector is transformed into Agrobacterium bacteria, and then the transformed Agrobacterium is introduced into plant cells by a known method such as a leaf disk method. The vector contains an expression promoter so that the DNA of the present invention is expressed in a plant after being introduced into the plant, for example. In general, the DNA of the present invention is located downstream of the promoter, and a terminator is located downstream of the DNA. The recombinant vector used for this purpose is appropriately selected by those skilled in the art depending on the method of introduction into the plant or the type of plant. Examples of the promoter include CaMV35S derived from cauliflower mosaic virus, corn ubiquitin promoter (Japanese Patent Laid-Open No. 2-79983), and the like.

  Examples of the terminator include a terminator derived from cauliflower mosaic virus or a terminator derived from a nopaline synthase gene. However, the terminator is not limited to these as long as it is a promoter or terminator that functions in a plant body.

  Moreover, the plant into which the DNA of the present invention is introduced may be explants, and cultured cells may be prepared from these plants and introduced into the obtained cultured cells. Examples of the “plant cell” of the present invention include plant cells such as scutellum in leaves, roots, stems, flowers and seeds, callus, suspension culture cells and the like.

  Further, in order to efficiently select plant cells transformed by introduction of the DNA of the present invention, the above recombinant vector is introduced into plant cells together with a suitable selection marker gene or a plasmid vector containing a selection marker gene. Is preferred. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene that is resistant to the antibiotic hygromycin, the neomycin phosphotransferase that is resistant to kanamycin or gentamicin, and the acetyltransferase that is resistant to the herbicide phosphinothricin. Genes and the like.

  Plant cells into which the recombinant vector has been introduced are placed on a known selection medium containing a suitable selection agent according to the type of the introduced selection marker gene and cultured. As a result, transformed plant cultured cells can be obtained.

  Next, the plant body is regenerated from the transformed cell introduced with the DNA of the present invention. Plant regeneration can be performed by methods known to those skilled in the art depending on the type of plant cells (Toki et. Al., Plant Physiol. 100: 1503-1507, 1995). For example, in rice, a method for producing a transformed plant is to introduce a gene into protoplasts using polyethylene glycol and regenerate the plant (indian rice varieties are suitable) (Datta et. Al., In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) Pp66-74, 1995). Gene transfer to protoplasts by electric pulses to regenerate plants (Japanese rice varieties are suitable) (Toki et. Al. , Plant Physiol. 100: 1503-1507, 1992), a method of regenerating plants by directly introducing genes into cells by the particle gun method (Christou et. Al., Bio / technology, 9: 957-962, 1991) And several techniques have already been established, such as a method for introducing a gene via Agrobacterium and regenerating a plant (Hiei et. Al., Plant J. 6: 271-282, 1994). Widely used in the technical fieldIn the present invention, these methods can be suitably used.

  The plant body regenerated from the transformed cells is then cultured in an acclimation medium. Thereafter, when the acclimatized regenerated plant body is cultivated under normal cultivation conditions, a dwarfed plant body can be obtained, and matured and fruited to obtain seeds.

  In addition, the presence of introduced foreign DNA in a transformed plant that has been regenerated and cultivated in this way can be analyzed by a known PCR method or Southern hybridization method, or by analyzing the base sequence of the DNA in the plant body. Can be confirmed.

  In this case, extraction of DNA from the transformed plant body can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

  When the foreign gene comprising the DNA of the present invention present in the regenerated plant body is analyzed using the PCR method, the amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction is carried out in a reaction mixture in which the DNA of the present invention or a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA modified according to the present invention is used as a primer. You can also. In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the DNA sequence of the present invention. When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and the DNA fragments correspond to the DNA of the present invention.

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

  In the present invention, as described above, a plant can be dwarfed by suppressing the expression of the plant d11 gene. The plant produced by the method of the present invention is highly expected that, for example, a high yield can be stably obtained for useful crops, and a new aesthetic value is added to ornamental plants.

  Furthermore, by introducing an exogenous d11 gene into a plant, it is conceivable to promote the growth of the plant, particularly to increase the foliage elongation. Production of a transformant of a plant into which an exogenous d11 gene has been introduced can be performed by the method described above. That is, a target transformed plant can be produced by inserting the gene into a vector that functions in a plant cell, introducing the vector into the plant cell, and regenerating the plant from the plant cell.

  Furthermore, DNA encoding antisense RNA, DNA encoding dsRNA, and DNA encoding ribozyme activity are used to suppress the expression of endogenous d11 gene in plants based on the sequence information of d11 gene. Furthermore, it is possible to prepare DNA encoding RNA having a co-suppression effect. The produced DNA can be used to dwarf plants.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

[Example 1]
450 F2 populations were bred with the rice type cultivar Kasalath and the Japanese gene line HO556 carrying the d11 gene, and DNA was extracted for each individual. Since the d11 gene was found to sit on the 4th chromosome, linkage analysis between the d11 gene and the RFLP marker seated on the 4th chromosome was performed to create a linkage map. As a result, it was revealed that the d11 gene was linked to RFLP markers W390 and L353 at a recombination frequency of about 1.8%. Four types of CAPS markers (S10628, R1721, R278, C377) were designed to create a high-density linkage map from these linkage maps.

[Example 2]
A high-precision linkage map of the fertile gene d11 was made. In order to reliably obtain DNA marker polymorphism, SL14 was selected as the parental line of the segregating population. This strain is a strain in which only the fourth chromosome of the genetic background (12 types of chromosomes) of the rice cultivar Nipponbare is replaced with the chromosome of the Indian variety Kasalath. After seeding about 15000 F2 seeds obtained by crossing SL14 and HO556, 3020 fertile individuals (recessive homozygous) were selected from the seedling stage using the CAPS marker and transplanted to the field. Leaf sampling was performed, DNA was extracted from 330 plants, and subjected to RFLP analysis. As a result, the d11 gene was located in a region (0.02 cM) sandwiched between RFLP markers G7008 or C559, W390, or L353 located near the d11 locus of chromosome 4 (FIG. 2).

[Example 3]
PAC libraries were screened using G7008 and L353 to select PAC clones containing the fertility gene d11, create their physical maps, and identify genomic clones containing the d11 locus. As a result, 6 and 4 types of PAC clones containing G7008 and L353 nucleotide sequences could be selected, respectively. In addition, using the terminal DNA fragment of each PAC clone prepared using the cassette-PCR method, we confirmed the duplication of PAC clones, performed chromosome walking, and mapped them onto a linkage map. However, since it was found that these PAC clones could not cover the d11 gene, further PAC clones were screened. PAC clones were similarly aligned by the method described above. As a result, the d11 gene was found to be located between two PAC clones (P424E6 and P471B10).

[Example 4]
From Example 3, the fertile gene d11 gene was located between two PAC clones (P424E6 and P471B10). Furthermore, by using STS markers and amplicon markers, we succeeded in narrowing the d11 gene candidate region to 98 kb. Gene prediction predicted the presence of 18 genes in this 98 kb candidate region. Using the genomic DNA of the d11 mutant, a 98 kb candidate region was amplified by PCR, and the nucleotide sequence was decoded. In this region, the nucleotide sequences of the wild type and d11 mutant were compared and searched for mutation sites (FIG. 2), and it was revealed that mutations were found in the three candidate genes.

  In order to investigate more detailed mutation sites, rice full-length cDNA databases were searched to obtain cDNA base sequences corresponding to these three candidate genes. RT-PCR was performed using primers prepared based on these nucleotide sequence information to obtain PCR products. The nucleotide sequences of the obtained PCR products were compared to search for detailed mutation sites. As a result, mutations were found in all d11 alleles within the cDNA that appears to encode cytochrome P450 (FIG. 3). This revealed that the d11 gene encodes cytochrome P450.

  By the protein coding region estimation program, regions encoding 18 kinds of proteins were shown in the genomic DNA in the 98 kb candidate region. The base sequences of these 18 kinds of candidate proteins were compared between the wild type (Nipponbare) and the mutant (d11 mutant). The present inventors collected eight types of d11 mutants, and verified that these d11 mutants were alleles by an allelism test. First, we randomly selected 5 types of d11 alleles and deleted candidate proteins that had the same base sequence as the wild type. A mutation was detected in one protein candidate region (d11 gene). Furthermore, mutations were also observed in this region in the remaining four d11 alleles. Finally, when the nucleotide sequences of these candidate genes were compared using the cDNAs of these eight types of alleles, differences in the nucleotide sequences from the wild type were confirmed (FIG. 3).

Mutations in the causative genes of the eight types of d11 mutants were classified as groups 1 to 4 as follows.
Group 1 (HO556 and T65d11): Mutation due to the deletion of one base in the second exon.
Group 2 (Tos3220): Mutation due to a 5 base deletion in the third exon.
Group 3 (TCM2410, TCM268, and TCM269): Mutation due to amino acid conversion by substitution of one base in the fourth exon. This region is one of the active centers of P450 (domain A region).
Group 4 (Id11 and N28d11): Mutation caused by the insertion of a single base in the seventh exon.
Groups 1, 2, and 4 are all mutations that cause a frame shift and generate a stop codon. Thus, in these d11 mutants, the cause of the mutation was found such that an accurate translation product was not translated or an essential sequence for forming the domain structure of the active center could not be formed.

  In the expression analysis of d11 mutant, the high expression in internode and pre-flowering tissues is consistent with the dwarf, short grain phenotype of d11 mutant (Fig. 1).

[Example 5]
The amino acid sequence predicted from the cDNA base sequence of the d11 gene was compared with the amino acid sequence of cytochrome P450 known in Arabidopsis and rice to examine which plant hormone biosynthetic pathway is involved. As a result, it became clear that it belongs to the same family as cytochrome P450 which seems to catalyze the brassinosteroid biosynthesis pathway. Furthermore, since the amino acid sequence was not the same as that of known cytochrome P450, it was shown that cytochrome P450 encoded by the d11 gene is a novel gene involved in the brassinosteroid biosynthesis pathway (FIG. 4).

[Example 6]
The PAC clone (P424E6) was treated with restriction enzymes XbaI and EcoRI to obtain a 6.2 Kb fragment of the full-length d11 gene containing a promoter region of about 2 Kb. This 6.2 Kbp fragment was ligated to the cloning site (XbaI and EcoRI) of the binary vector pPZP2H-lac. This vector was introduced into Agrobacterium EHE101, and rice callus was transformed according to a conventional method. The transformant (Transformant-D11) showed a phenotype similar to that of the wild type, while the ears were elongated and drooped (FIG. 5). Therefore, it was revealed that a 6.2 Kb gene fragment containing the d11 gene complements the d11 mutation.

It is a photograph which shows the phenotype of a wild type and d11 mutant. A; Shows rice at heading time. B; Shown between each mutant and wild type second section. C; Each mutant and wild type matured seed is shown. (A) It is a figure which shows the estimation area | region and physical map of d11 locus by a high-density linkage analysis. (B) shows the structure of cytochrome P450 encoded by the d11 gene and the mutation site in the d11 allele. It is a figure which shows the amino acid sequence of the other cytochrome P450 which had homology with the amino acid sequence of d11 gene. Mutation sites found in various d11 alleles are also shown. It is a figure which shows the phylogenetic tree of cytochrome P450 which d11 gene codes, and known Arabidopsis and rice cytochrome P450. It is a photograph which shows that d11 mutation is complemented by introduction | transduction of a wild-type d11 gene. A; Shows rice at heading time. B; shows wild-type and mutant ripening seeds. Left: Wild type rice (WT, T65) is shown. Center: shows a transformant (Transformant-D11) in which the d11 gene is introduced into the d11 mutant. Right: A transformant (Transformant-control vector) in which the binary vector pPZP2H-lac is introduced into the d11 mutant.

Claims (14)

The DNA according to any one of the following (a) to (c), which is used for dwarfing a plant.
(A) DNA encoding an RNA complementary to a transcription product of a plant endogenous d11 gene
(B) DNA encoding an RNA having a ribozyme activity that specifically cleaves a transcription product of a plant endogenous d11 gene
(C) DNA encoding RNA that inhibits the endogenous d11 gene expression in plants by co-suppression effect   The DNA according to claim 1, wherein the plant d11 gene is a rice d11 gene.   A vector comprising the DNA according to claim 1 or 2.   A transformed plant cell that retains the DNA of claim 1 or 2 in an expressible manner.   The transformed plant cell according to claim 4, wherein the plant is rice.   A transformed plant comprising the transformed plant cell according to claim 4 or 5.   A transformed plant which is a descendant or clone of the transformed plant according to claim 6.   The transformed plant according to claim 6 or 7, wherein the plant is rice.   The transformed plant according to claim 6 or 7, which is dwarfed.   The propagation material of the transformed plant body in any one of Claims 6-9.   A method for producing a transformed plant according to any one of claims 6 to 9, comprising a step of introducing the DNA according to claim 1 or 2 into a plant cell and regenerating the plant from the plant cell. Method.   A method for dwarfing a plant, comprising suppressing the expression of an endogenous d11 gene in cells of the plant body.   The method according to claim 12, wherein the DNA according to claim 1 or 2 is introduced into a plant.   The method according to any one of claims 11 to 13, wherein the plant is rice.