AU2010202388B2 - Variant Myb-related gene that accelerates plant growth and method for accelerating plant growth using the same - Google Patents

Abstract

This invention provides a method of introducing a variant Myb transcriptional regulator into a target plant to selectively impart target traits thereto without causing adverse effects. This invention also provides a variant Myb-related gene comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region and comprising silent mutations introduced in such nucleotide sequence, so as to prevent miRNA that targets the Myb-related gene involved in plant growth from suppressing expression of the Myb-related gene.

Description

AUSTRALIA FB RICE & CO Patent and Trade Mark Attorneys Patents Act 1990 OJI PAPER CO., LTD COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Variant Myb-related gene that accelerates plant growth and method for accelerating plant growth using the same The following statement is a full description of this invention including the best method of performing it known to us:- VARIANT Myb-RELATED GENE THAT ACCELERATES PLANT GROWTH AND METHOD FOR ACCELERATING PLANT GROWTH USING THE SAME Technical Field The present invention relates to a variant Myb-related gene that can accelerate plant growth, a method for accelerating plant growth using the same, miRNA that targets the Myb-related gene that can suppress plant growth, and a method for suppressing plant growth using such miRNA. Related Application Data This application is an Australian complete application filed on June 8, 2010, which claims priority from Japanese Application No. 163138/2009 filed on July 9, 2009, the contents of which are incorporated herein in their entirety by way of reference. Background The Myb gene is a transcriptional regulator gene that exists in a wide variety of living species, from yeast to animals and plants. At first, the Myb gene was isolated as a cancer gene (c-Myb), independently from the avian myeloblastosis virus (AMV) and the E26 virus (Roussel M., 1979, Nature, 218: 452-455). As a result of later research, the Myb gene was found to encode a transcription factor (Biedenkapp H., et al., 1988, Nature, 335: 835-837), represent a family comprising many related genes, and exist in a wide variety of 1 species, including yeast, nematodes, insects and plants, as well as vertebrates (Masaki Iwabuchi and Kazuo Shinozaki, Shokubutsu genomu kinou no dainamizumu: tensha inshi ni yoru hatsugen seigyo (Dynamism of Plant Genome functions: Expression control by transcriptional factor), Springer Japan, 2001). Genes that belong to the Myb gene family each encode a DNA binding domain consisting of a helix-turn-helix structure of about 50 nucleotides, which 1A is referred to as the "Myb domain." As Myb gene products of vertebrates, a Myb, b-Myb and c-Myb are known. All products each comprise three Myb domains (RI, R2, and R3) and mainly function as transcriptional regulators associated with cell growth and differentiation (Weston K., 1999, Curr. Opin. Genet. Dev. 8, 76-81). In contrast, many Myb genes are present in plants, and such Myb genes are roughly classified into three types based on structural characteristics ; that is, the RlR2R3 type that has three Myb domains, as does the Myb gene of a mammalian and regulates cell growth by regulating the cell cycle (Ito M., 2005, J. Plant Res., 118 (1): 61-69); the R2R3 type that regulates pigment synthesis, morphogenesis, and various vital phenomena such as environmental response (Jin H. and Martin C., 1999, Plant Mol. Biol., 41 (5): 577-588); and a type that has an Ri-like or R3-like domain involved in light mediated regulation, morphogenesis, and disease and damage (Feldbrugge et al., 1997, Plant J. 11, 1079-1093; Kirik V. and Baumlein H., 1996, Gene 183, 109 113; Wang Z. Y., et al., 1997, Plant Cell 9, 491-507). Numerous pieces of genetic information about plants have been accumulated in the present century, and knowledge regarding factors involved in transcriptional regulation of genes associated with fundamental physiological functions, including photosynthesis, primary metabolism, and secondary metabolism, as well as knowledge about the functions of such factors, has been rapidly increasing. As a subsequent step, various molecular genetic techniques for artificial regulation of gene expression have been developed, so as to impart new functions to plants. An example of such a technique is a method in which a promoter region of a transcriptional regulator gene is substituted or modified to introduce a variant transcriptional regulator gene into a target plant, thereby imparting functions of interest to the plant. Even if such objective is attained via such technique, however, the technique would disadvantageously impose unanticipated adverse effects, such as simultaneous induction of other unfavorable or harmful traits. Summary of the Invention The present invention is intended to develop a method of introducing a variant transcriptional regulator gene into a target plant to selectively impart target traits thereto without causing adverse effects. In general, a transcriptional regulator often collectively regulates expression of various genes, and introduction of variation into such transcriptional regulator would significantly influence the expression of many other genes that are regulated by such factor. Accordingly, it is not usually easy to selectively impart target traits via introduction of a variant transcriptional regulator gene. In recent years, however, the presence of small RNA that regulates gene expression at the translational phase, such as miRNA (micro RNA) or siRNA (small (or short) interference RNA), as well as the mechanism of RNA silencing, have been discovered (Lee R. C., et al., 1993, Cell, 75: 843-854; Baulcombe D, 2002, Science, 297: 2002 2003). The discovery of such mechanism demonstrates that gene expression is not regulated at the translational phase alone, but rather as a collective result of regulations separately carried out at transcriptional and translational phases (Jackson R. J. 3 and Standart N., Sci. STKE, 2007, (367) rel). The present inventors have conducted concentrated studies in order to develop a novel method for attaining the above object by using such mechanism of post-transcriptional regulation. As a result, they discovered miRNA comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region and having the ability to regulate expression of a gene encoding the Myb related protein associated with plant growth. The present invention has been completed based on such finding. Specifically, the present invention provides the following. (1) A variant Myb-related gene capable of accelerating plant growth by introducing the gene into a plant cell, the variant Myb-related gene being derived from the Myb-related gene comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region via introduction of a silent mutation(s) into the nucleotide sequence so as to disable expression regulation mediated by miRNA that binds to the nucleotide sequence. (2) The variant Myb-related gene according to (1), wherein the nucleotide sequence as shown in SEQ ID NO: 1 is represented by SEQ ID NO: 2. (3) The variant Myb-related gene according to (1) or (2), wherein at least one of the silent mutations is substitution of T at position 13 in SEQ ID NO: 1 with C. (4) The variant Myb-related gene according to any of (1) to (3), which comprises at least 5 silent mutations. (5) The variant Myb-related gene according to any of (1) to (4), wherein the Myb-related gene comprises the nucleotide sequence as shown in SEQ ID NO: 3 or a nucleotide sequence having 95% or higher identity therewith.

(6) An expression vector comprising the variant Myb-related gene according to any of (1) to (5). (7) A host transformed with the expression vector according to (6), or a progeny thereof. (8) The host or a progeny thereof according to (7), wherein the host is a microorganism or plant. (9) The host or a progeny thereof according to (8), wherein the plant is of the genus Eucalyptus (hereafter it is referred to as "Eucalyptus," which is a common abbreviation for the same, except when a species name is used) or the genus Nicotiana. (10) A method of accelerating the growth of a plant, comprising introducing the expression vector according to (6) into the plant. (11) A miRNA capable of suppressing the growth of a plant by introducing the miRNA into a plant cell, wherein the miRNA comprises the nucleotide sequence as shown in SEQ ID NO: 7 and targets a transcription product of the Myb-related gene according to any of (1) to (5). (12) The miRNA according to (11), wherein the nucleotide sequence as shown in SEQ ID NO: 7 is represented by SEQ ID NO: 8. (13) An expression vector comprising DNA encoding the miRNA according to (11) or (12). (14) A host transformed with miRNA according to (11) or (12) or the expression vector according to (13), or a progeny thereof. (15) The host or a progeny thereof according to (14), wherein the host is a microorganism or plant. (16) The host or a progeny thereof according to (15), wherein the plant is of the genus Eucalyptus or the genus Nicotiana. (17) A method of suppressing the growth of a plant, comprising introducing the expression vector according to (13) into the plant. According to the variant Myb-related gene of the present invention and the method for accelerating plant growth of the present invention, the traits of interest; i.e., growth acceleration as intended, can be imparted to a plant into which such gene has been introduced, without causing other adverse effects. With the use of miRNA of the present invention and the method for suppressing plant growth of the present invention, the inherent mechanism of plant growth suppression may be specifically enhanced, so that the growth of the plant into which miRNA has been introduced can be suppressed. Brief Description of the Drawings Fig. 1 shows the amino acid sequence and the R2- and R3-Myb domains of the EcMyb protein. Fig. 2 shows the nucleotide sequence of the EcMyb-related gene (the EcMyb gene), the amino acid sequence of the EcMyb protein (EcMyb prt), positions of primers used for isolating the gene, and a region corresponding to SEQ ID NO: 2 (the Myb-miRNA-binding site). Fig. 3 (a) schematically shows the results of inspection of the Myb-miRNA cleavage site; Fig. 3 (b) shows the Myb-miRNA-binding site and the Myb-miRNA cleavage site; and Fig. (c) shows an agarose gel electrophoresis showing the transcription product of the EcMyb related gene cleaved with Myb 6 miRNA. Fig. 4 shows 9 silent mutations introduced into the Myb miRNA-binding site as shown in SEQ ID NO: 2; wherein one such variation (6C) is a silent mutation inherent to EcMyb. Fig. 5 shows expression of EcMyb or mEcMyb in a transgenic tobacco. Fig. 6 shows the growth of a transgenic tobacco (1). Fig. 7 shows the growth of a transgenic tobacco (2). Detailed Description 1. Variant Myb-related gene An aspect of the present invention concerns a variant Myb related gene that can accelerate the growth of a plant by being introduced into a plant cell. 1-1. Myb-related gene The term "Myb-related gene" used herein refers to a R2R3 type wild-type Myb-related gene in the plant Myb gene family, comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region and having functions of accelerating plant growth, a natural variant thereof, or a fragment thereof. The term "wild-type Myb-related gene" refers to a Myb-related gene encoding the Myb-related protein existing in nature and having the nucleotide sequence that is most abundantly present in alleles in populations of the same species. The Myb-related protein encoded by such gene has functions inherent to the protein (e.g., a function as a transcriptional regulator). 7 The "R2R3 type" Myb-related gene is an Myb-related gene encoding two Myb domains (i.e., R2 and R3 domains) in its structure, as described above. For example, WEREWOLF (WER), which negatively regulates hair root 7A formation (Lee et al., 1999, Cell, 99: 473-483), GLABRAl (GLI), which is involved in trichome formation (Oppenheimer et al., 1991, Cell 67 (3): 483-493), and TRANSPARENT TESTA 2 (tt2), which accumulates tannin in the seed coat (Nesi et al., 2001, Plant Cell, 13 (9), 2099-2114), are of the R2R3 type. The term "natural variant thereof" refers to a variant of a wild-type Myb related gene existing in nature. Examples thereof include a gene comprising a nucleotide sequence derived from the nucleotide sequence of the wild-type Myb related gene by deletion, substitution or addition of one or several nucleotides, a gene comprising a nucleotide sequence having 95% or higher, preferably 98% or higher and more preferably 99% or higher identity with the aforementioned nucleotide sequence, and a gene hybridizing under stringent conditions to a fragment of a nucleic acid comprising a nucleotide sequence complementary to a partial nucleotide sequence of the wild-type Myb-related gene. All such genes have activity of accelerating plant growth, as do wild-type Myb proteins. The term "identity" used herein refers to the percentage of the identical nucleotides of a nucleotide sequence relative to the entire number of nucleotides in the other nucleotide sequence, when two nucleotide sequences are aligned with or without the introduction of a gap. The term "several nucleotides" refers to 2 to 10 nucleotides, for example, 2 to 7, 2 to 5, 2 to 4, or 2 or 3 nucleotides. The term "under stringent conditions" refers to a condition in which, non-specific hybrids are not formed. In general, hybridization may be carried out under low stringent to high stringent conditions, with high stringent conditions being preferable. Under low stringent conditions, for example, washing following hybridization is carried out at 42*C in the presence of 5x SSC and 0.1% SDS, and preferably at 50*C in the presence of 5x SSC and 0.1% SDS. Under high stringent conditions, for example, washing following hybridization is carried out at 65*C in the presence of 0.lx SSC and 0.1% SDS. Specific examples of natural variants include a variant based on a polymorphism such as a single nucleotide polymorphism (SNP), a splice variant and a variant based on the degeneracy of the genetic code. The term "fragment thereof" refers to a DNA fragment of the wild-type Myb-related gene or a natural variant thereof, which comprises the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region, and part of the Myb related protein encoded by such fragment has biological functions equivalent to those of the Myb-related protein encoded by the wild-type Myb-related gene, i.e., activity as a transcriptional regulator. The nucleotide length of a DNA fragment is not particularly limited, provided that the DNA fragment has such functions. The "nucleotide sequence as shown in SEQ ID NO: 1" corresponds to an miRNA-binding site (i.e., the Myb-miRNA-binding site) targeting the Myb related gene (hereafter referred to as "Myb-miRNA" for descriptive purposes). Specifically, expression of the Myb-related gene according to the present invention is suppressed via a bond of miRNA to the Myb-miRNA-binding site. Nucleotide represented by R, Y and N in SEQ ID NO: 1 indicates A (adenine) or G (guanine), C (cytosine) or T (thymine), or A, T, G or C, respectively. The description regarding Myb-miRNA is omitted here* since it is provided in the section "5. Myb-miRNA" in detail. The term "in its coding region" refers to the inside of the nucleotide sequence region encoding the Myb-related protein. Accordingly, the term "comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region" refers to a condition in which a sequence comprises the Myb-miRNA binding site as shown in SEQ ID NO: 1 in its coding region. The term "plant growth" refers to stem (including tree stem) elongation, root elongation, and an increase in stem diameter (including arbor diameter). Specifically, the term refers to cell growth, differentiation and histogenesis at the growing point and/or in the vascular cambium of a plant. The term "functions of accelerating plant growth" refers to functions that are directly or indirectly associated with acceleration of cell growth (including regulation of cell wall formation) at the growing point and/or in the vascular cambium of a plant. A function that is directly associated with acceleration of cell growth refers to a function which directly acts on cell growth such as a function of a cell cycle regulation factor. A function that is indirectly associated with acceleration of cell growth is, for example, regulation of expression of a gene that directly acts on cell growth, thereby indirectly contributing to cell growth. Specific examples of the R2R3-type Myb-related gene comprising the nucleotide sequence as shown in SEQ ID NO: 1 in its coding region; i.e., the Myb-related gene from which the variant Myb-related gene of the present invention originates, include a wild-type Myb-related gene of Eucalyptus camaldulensis comprising the nucleotide sequence as shown in SEQ ID NO: 3, which was newly isolated by the present inventors (hereafter, such gene is referred to as the "EcMyb gene" for descriptive purposes) and natural variants thereof. The EcMyb gene comprises, as a sequence corresponding to SEQ ID NO: 1, the nucleotide sequence as shown in SEQ ID NO: 2 in a region between residues 310 and 331 (A of the initiation codon is designated as 1, and the same applies hereinafter) and encodes the Myb-related protein comprising 221 amino acids as shown in SEQ ID NO: 9. Also, the Myb-related gene may be the Myb related gene of other plant species, and preferably an EcMyb orthologue, that has 95% or higher, preferably 98% or higher, and more preferably 99% or higher identity with the nucleotide sequence of the EcMyb gene as shown in SEQ ID NO: 3 or hybridizes under stringent conditions to a probe comprising a nucleotide sequence complementary to a partial nucleotide sequence of the EcMyb gene. For example, the Myb-related gene of Eucalyptus gunii as shown in SEQ ID NO: 4 which has 97% sequence identity with the nucleotide sequence of the EcMyb gene (hereafter referred to as the "EgMyb gene" for descriptive purposes) and the Myb-related gene derived from Populus trichocarpa as shown in SEQ ID NO:. 5, which is registered under the accession number: GenBank XM_002302608.1 (hereafter referred to as the "PtMyb gene" for descriptive purposes) are within the scope of the Myb-related gene of the present invention. The present inventors discovered that expression of the Myb-related gene comprising the Myb-miRNA-binding site in its coding region is translationally suppressed by the Myb-miRNA-mediated RNA silencing mechanism. Regarding miRNA, the section "5. Myb-miRNA" below may be referred to. 1-2. Variant Myb-related gene The term "variant Myb-related gene" refers to a gene derived from the aforementioned Myb-related gene via introduction of a silent mutation into the Myb-miRNA-binding site as shown in SEQ ID NO: 1. In the present invention, Myb-miRNA-mediated suppression of Myb related gene expression is considered to be achieved as follows based on the results of Example 2 below. That is, Myb-miRNA binds to the Myb-miRNA binding site on the Myb-related gene transcription product, and a site between T13 and T14 (which is a site between U13 and U14 (U: uridine), since RNA is the actual target molecule) of the nucleotide sequence as shown in SEQ ID NO: 1 is cleaved by the action of RISC (RNA-induced silencing complex)/miRNP, which forms a complex with Myb-miRNA. The mechanism of miRNA mediated suppression of gene expression is described in detail in the section "5. Myb-miRNA" below. Based on such mechanism, the present invention provides a variant Myb-related gene that disables Myb-miRNA-mediated expressional suppression but does not change the amino acid sequence of a protein encoded by the Myb-related gene. The term "silent mutation" refers to a mutation that causes mutation in the nucleotide sequence of the gene but does not cause mutation in the amino acid sequence of the protein encoded by such gene; i.e., variation of nucleotides in a degenerate codon. In the present invention, the Myb-miRNA-binding site encodes an amino acid comprising WNTHLSK (SEQ ID NO: 6). Accordingly, the present invention includes all silent mutations that maintain such amino acid sequence. When SEQ ID NO: I is represented by the nucleotide sequence as shown in SEQ ID NO: 2, for example, variations C6T (substitution of C6 with T), T9A/G/C (substitution of T9 with A, G, or C), T13-G15A/T/C (i.e., G15A/T/C when residue 13 is T) or T13C-G15A (i.e., G15A when T13 is substituted with C), A16-C18T or A16T-G17C-C18A/T/G, and/or G21A can be introduced. The condition described by the expression "...disables Myb-miRNA mediated suppression of expression" refers to a situation in which a variant Myb-related gene acquires Myb-miRNA-tolerance upon introduction of silent mutations and expression thereof is not suppressed via Myb-miRNA-mediated RNA silencing. For example, it refers a situation in which a Myb-miRNA cannot recognize the variant Myb-related gene transcription product as a target molecule or cannot cleave the variant Myb-related gene transcription product even if it can recognize the same. The condition described by the expression "...cannot recognize the variant Myb-related gene transcription product as a target molecule" refers to a situation in which Myb-miRNA cannot bind to the Myb-miRNA-binding site on the target transcription product of the variant Myb-related gene. In such a case, silent mutation may be introduced into the plant into which the variant Myb related gene of the present invention is to be introduced, so that a stable base pair would not be formed between endogenous Myb-miRNA of the plant and the Myb-miRNA-binding site of the variant Myb-related gene transcription product. For example, it is preferable that at least 5, and a maximum of 9, silent mutations be introduced into the nucleotide sequence as shown in SEQ ID NO: 1. When the Myb-miRNA-binding site is the EcMyb gene identified by the nucleotide sequence as shown in SEQ ID NO: 2, such gene may have at least 5 silent mutations (Fig. 4). The condition described by the expression "...cannot cleave the variant Myb-related gene transcription product even if it can recognize the same" refers to a situation in which Myb-miRNA can bind to the Myb-miRNA-binding site of the variant Myb-related gene transcription product but such site is not cleaved by the above-described mechanism. An example thereof is a situation in which silent mutation is introduced into a nucleotide that is highly conserved among organism species at the Myb-miRNA-binding site, such as a situation in which silent mutation (T13C) involving substitution of T at position 13 flanking a site cleaved by Myb-miRNA at the Myb-miRNA-binding site as shown in SEQ ID NO: 1, with C is introduced. By introducing the variant Myb-related gene of the present invention into a target plant body, expression suppression mediated by endogenous Myb miRNA can be specifically avoided, and the Myb-related protein comprising the same amino acid sequence as that of the wild-type Myb-related gene can be stably imparted to such plant. As a result, growth of the plant can be accelerated. 1-3. Preparation of variant Myb-related gene The variant Myb-related gene of the present invention can be prepared by a method known in the art. For example, mRNA is first extracted from the target plant in order to isolate cDNA of the Myb-related gene. After extraction, a library of purified cDNA is prepared. Thereafter, the target Myb-related gene can be isolated from the cDNA library via PCR (e.g., Inverse-PCR, anchor PCR or TAIL-PCR) or hybridization techniques. Primers used for PCR techniques and probes used for hybridization techniques may be prepared based on, for example, SEQ ID NO: 3 (the EcMyb gene), SEQ ID NO: 4 (the EgMyb gene) or SEQ ID NO: 6 (the PtMyb gene) described herein, or nucleotide sequences obtained from the known genetic information on cell wall transcriptional regulator genes that is available on common databases accessed via the internet, such as NCBI (http://www.ncbi.nlm.nih.gov/), Riken Japan (http://www.psc.riken.jp/database/index.html) or the DNA sequence analysis information database of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/j/resources/database.html). Since a relatively high level of conservation of the Myb-miRNA-binding site is observed among plant species, in particular, use of such site as a probe or one of the primers enables isolation of the target Myb-related gene from cDNA libraries of different species. Specific methods for cDNA library preparation and target gene isolation are known in the art. For example, a reference may be made to Sambrook, J. et al., 1989, Molecular Cloning: a Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Subsequently, silent mutation necessary for the nucleotide sequence region as shown in SEQ ID NO: 1 in the resulting Myb-related gene is introduced. Such introduction of variation can be carried out in accordance with a method known in the art. For example, a method involving inverse PCR and a method for introduction of variation involving the use of a commercially available mutagenesis kit (e.g., QuickChange Site-Directed Mutagenesis Kit (Stratagene) and Altered Sites II in vitro Mutagenesis System (Promega)) may be employed. Several manufacturers of life-science-related products (e.g., Takara Bio) provide services that introduce desired variations into genes or the like, and such services may be utilized. 2. Expression vector comprising variant Myb-related gene Another aspect of the present invention relates to an expression vector comprising the aforementioned variant Myb-related gene (hereafter referred to as the "variant Myb-related gene expression vector"). 2-1. Variant Myb-related gene expression vector In the present invention, the term "variant Myb-related gene expression vector" refers to a vector prepared by introducing the variant Myb-related gene into an adequate expression vector. Such vector may be introduced into an adequate host cell, so that the introduced variant Myb-related gene can be expressed under adequate conditions. The backbone portion of the expression vector according to the present invention; i.e., the major component other than the variant Myb-related gene of the present invention, is preferably a plasmid or virus, although the backbone is not particularly limited thereto. Examples of plasmids that can be used include pBI, pPZP, pSMA, pUC, pBR, pBluescript (Stratagene), pTriEXTM (TaKaRa) and binary vectors, such as pBI or pRI, and examples of viruses that can be used include cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV) and tobacco mosaic virus (TMV). Such plasmids or viruses may be adequately selected in accordance with the hosts into which they are to be introduced. Examples of hosts include E. coli, yeast, plant cells (including those of the plant body, plant organs and plant tissue), insect cells, and animal cells. Host cells that are known in the art may be adequately selected in accordance with the intended purposes. In addition to the variant Myb-related gene, the backbone of the variant Myb-related gene expression vector of the present invention can incorporate elements, for example, control regions, such as promoter, enhancer or terminator regions, and label regions, such as a selection marker gene. Types thereof are not particularly limited. In accordance with hosts (e.g., bacteria, yeast, plant cells or insect cells) or purposes to be applied within the hosts (e.g., cloning or gene expression), elements known in the art may be adequately selected. Such elements are described below. Examples of promoters that can operate in E. coli include lac, trp, and tac promoters, and phage rhamda-derived PR or PL promoters. Examples of promoters that can operate in yeast include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, TPIl promoters and ADH2-4c promoters. Examples of promoters that can operate in plant cells include 35S promoters of cauliflower mosaic virus (CaMV), promoters of nopaline synthase genes (Pnos), maize ubiquitin promoters, rice actin promoters and tobacco PR protein promoters. Examples of promoters that can operate in insect cells include polyhedrin promoters, P1O promoters, basic protein promoters of Autographa californica polyhedrosis, baculovirus immediate early gene 1 promoters and baculovirus 39K delayed early gene promoters. When topical expression of the variant Myb-related gene is intended at a given site of a transformant, a site-directed promoter may be used. When acceleration of root growth is intended, for example, a promoter that induces root-specific expression disclosed in JP Patent Publication (kokai) No. 2007-77677 A may be used. Examples of enhancers include an enhancer region including an upstream sequence in the CaMV 35S promoter and a CMV enhancer. Examples of terminators include terminators of noparine synthase (NOS) genes, terminators of octopine synthase (OCS) genes, CaMV 35S terminators, 3' terminators of E. coli lipopolyprotein lpp, trp operon terminators, amyB terminators and ADHI gene terminators. Examples of selection marker genes include drug resistance genes (e.g., tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, hygromycin resistance genes, spectinomycin resistance genes, chloramphenicol resistance genes or neomycin resistance genes), fluorescent or luminescence reporter genes (e.g., luciferase, p-galactosidase, p-glucuronidase (GUS) or green fluorescence protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase genes. 2-2. Preparation of variant Myb-related gene expression vector The variant Myb-related gene may be inserted into a given site of an expression vector by a method known in the art, such as the method described in Sambrook, J. et al., 1989, Molecular Cloning: a Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. In general, the prepared variant Myb-related gene is cleaved with an appropriate restriction enzyme and inserted into a corresponding restriction enzyme site of a preferable expression vector, a multicloning site, or the protruding 5'-T end of a PCR product having the protruding 3'-A end produced with DNA polymerase such as Taq to ligate the gene to the expression vector. When a commercially available system or kit is used, an expression vector of interest can be prepared by a method specific to such system or kit. For example, the Gateway System (Invitrogen) can be employed. With the use of the variant Myb-related gene expression vector according to this embodiment, so that a transformant that expresses a variant Myb-related gene described below can be obtained by introducing such vector into an adequate host. 3. Transformant or progeny thereof Another aspect of the present invention relates to a transformant prepared by introducing an expression vector comprising the variant Myb-related gene into a host or a progeny thereof. 3-1. Transformant In this description, the term "transformant" refers to a host transformed with an expression vector comprising the above variant Myb-related gene.

Hosts to be used are not particularly limited, provided that the variant Myb related gene of the present invention encoded by the introduced expression vector is expressed. In general, hosts that can express genes of interest are limited to some extent, depending on expression vectors. Thus, adequate hosts may be selected in accordance therewith. Specific examples of hosts include: microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis), yeast (e.g., budding yeast such as Saccharomyces cerevisiae), fission yeast (e.g., Schizosaccharomyces pombe), and methanol-assimilating yeast (e.g., Pichia pastoris); fungi (e.g., Aspergillus and Neurospora); plants (including plant bodies, plant organs, plant tissue, and differentiated or undifferentiated plant cells (calluses)); and insect cells (e.g., sf9 and sf21). When plant hosts are used, examples thereof include bryophytes, pteridophytes and spermatophytes, and varieties thereof are not limited. The transformants of the present invention also include clones having the same genetic information. When hosts are unicellular microorganisms that perform asexual reproduction, such as E. coli or yeast, for example, cells that are newly developed via division or budding from the first generation transformants are within the scope of the transformants of the present invention. When hosts to be transgenic are plants, transgenic plants derived from parts of the plant bodies obtained from the first generation transformants via cuttage, grafting or layering, and trophozoites that are newly developed from vegetative propagation organs (e.g., rhizomes, tuberous roots, bulbs and runners) via asexual reproduction from the first generation transformants are within the scope of the transformants of the present invention. 3-2. Progeny of transformant The term "progeny of transformant" used herein refers to an offspring resulting from sexual reproduction of the first generation transformants into which the variant Myb-related gene of the present invention has been introduced, which retains the variant Myb-related gene of the present invention in an expressible state. When transformants are plants, for example, seedlings of the transformants are the progenies of the transformants. The generation of the progeny is not limited, provided that the variant Myb-related genes of the present invention are transferred in an expressible state. 3-3. Method of transformation The variant Myb-related gene expression vector of the present invention can be introduced into a host by a method known in the art. When hosts are bacteria, the heat shock method, a method involving the use of calcium ions (e.g., the calcium phosphate method), electroporation or other techniques may be employed. Such techniques are known in the art and described in various documents, such as Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. When hosts are plant cells and the expression vector is a plasmid vector, the protoplast method, the particle gun method or the Agrobacterium method can be employed as a method of transformation. According to the protoplast method, plant cells (protoplasts) from which cell walls have been removed via an enzymatic treatment with cellulase or the like are used to introduce the target gene into plant cells. The protoplast method can further be divided into electroporation, microinjection or the polyethylene glycol method, depending on the method of gene introduction. According to electroporation, electric pulses are applied to a mixed solution of protoplasts and the target genes in order to introduce the genes into protoplasts. According to microinjection, the target genes are directly introduced into protoplasts under a microscope with the use of a microneedle. According to the polyethylene glycol method, polyethylene glycol is allowed to act on protoplasts in order to introduce the target genes thereinto. According to the particle gun method, the target genes are adhered to microparticles of gold or tungsten, and the resultants are injected into plant tissue cells with the aid of a high-pressure gas to introduce the target genes into such cells. According to this technique, transformants comprising target genes incorporated in the genomic DNA of host cells can be obtained. The Agrobacterium method is a method of plant cell transformation involving the use of, as transforming factors, bacteria of the genus Agrobacterium (e.g., Agrobacterium tumefaciens and Agrobacterium rhizogenes) and Ti plasmids derived therefrom. With this technique, the target gene can be introduced into genomic DNA of a host plant cell. Such techniques are known in the art, and details of such techniques are described in, for example, Shokubutsu Taisha Kogaku Handbook (the Handbook of plant metabolic engineering), 2002, NTS, or Shinban Moderu Shokubutsu no Jikken Protokoru (the experimental protocol for model plants (new edition): Idengakuteki shuhou kara genomu kaiseki made (From genetic engineering to genome analysis), Shujunsha, 2001). When hosts are plant cells and the expression vector is a virus vector (e.g., a CaMV, BGMV or TMV vector described above), the plant cell may be infected with the virus vector, so that the variant Myb-related gene can be introduced thereinto. For example, the plant virus genome is first inserted into a cloning vector, such as an E. coli-derived vector, to prepare a recombinant.

Subsequently, the variant Myb-related gene is inserted and cloned into the virus genome of the recombinant. The plant virus genomic region is then cleaved from the recombinant with the aid of a restriction enzyme, and the target plant cell is infected with the resulting virus genome. Thus, the target gene can be introduced into a plant cell. Such method of gene introduction involving the use of a virus vector is described in detail in, for example, the method of Hohn et al. (Molecular Biology of Plant Tumors, Academic Press, New York, 1982, p. 549) or US Patent No. 4,407,956. When hosts are plant cells, the plant type is not particularly limited. A preferable plant endogenously comprises Myb-miRNA as shown in SEQ ID NO: 7 and the Myb gene havingthe target gene thereof; i.e., the Myb-miRNA-binding site as shown in SEQ ID NO: 1 in its coding region. Examples thereof include plant cells derived from plants of the genus Eucalyptus and plants of the genus Nicotiana. Examples of plants of the genus Eucalyptus include Eucalyptus grandis, Eucalyptus urophylla, Eucalyptus camaludulensis, Eucalyptus globulus, Eucalyptus nitens, Eucalyptus gunni, Eucalyptus radiata, Eucalyptus amplifolia, Eucalyptus archeri, Eucalyptus bridgesiana, Eucalyptus baxteri, Eucalyptus bicostata, Eucalyptus blakelyi and Eucalyptus botryoides. Examples of plants of the genus Nicotiana include Nicotiana tabacum and Nicotiana rustica. Other examples include Arabidopsis thaliana and plants of the genus Populus, such as Populus trichocarpa, Populus alba, Populus nigra, Populus candicans, Populus tremula, and Populus sieboldii. 3-4. Method for obtaining progeny Progenies can be obtained from the transformants of the present invention by a method that is commonly employed for obtaining progenies of organism species that are the hosts of such transformants. If the hosts of the transformants are E. coli or yeast cells, for example, progenies can be obtained via culture in an appropriate known medium. Reference may be made to, for example, Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. If the hosts of the transformants are plants, in general, seedlings can be easily obtained via soil culture (i.e., seeding). For example, seeds are obtained from the transgenic plants of the present invention by allowing the transgenic plants to take root in an adequate medium and transferring such plants to a pot containing hydrous soil. The plants are allowed to grow under adequate cultivation conditions and seeds are prepared by a common method in the art. The resulting seeds are sowed in, for example, adequate hydrous soil and allowed to grow under adequate cultivation conditions. Thus, the progenies of the transformants of the present invention can be obtained. 4. Method for accelerating plant growth Another aspect of the present invention relates to a method of introducing a variant Myb-related gene expression vector into a plant body to accelerate the growth thereof. 4-1. Target plant The principle of the present invention is based on introducing the variant Myb-related gene of the present invention into a target plant to express the gene in the plant cell, thereby providing a variant Myb-related gene protein having a wild-type amino acid sequence to the transformant while avoiding expressional suppression mediated by endogenous Myb-miRNA. Thus, the target plant of this method is not particularly limited, provided that such plant comprises the nucleotide sequence as shown in SEQ ID NO: 1 of the present invention and comprises at least one Myb-related gene that accelerates plant growth and Myb miRNA of the present invention in its genome. Examples thereof include various plants of the genus Eucalyptus, the genus Nicotiana, and the genus Populus described in the aforementioned section "3-2. Method of transformation". 4-2. Method The present invention is achieved by introducing the variant Myb-related gene expression vector described in the section "2. Expression vector comprising variant Myb-related gene" above into a target plant and obtaining the transgenic plant described in the aforementioned section "3. Transformant". The variant Myb-related gene expression vector may be introduced into a target plant in accordance with the method described in the aforementioned section "3-2. Method of transformation". In this case, a variant Myb-related gene expression vector that can express the gene of interest in a plant cell is used, since the transformant is a plant. For example, a plasmid vector, such as a pBI or pRI binary vector, or a virus vector, such as a cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV) or tobacco mosaic virus (TMV) vector, may be used. Naturally, hosts into which such vectors are introduced are target plants. Methods for transforming target plants with the use of the variant Myb related gene expression vector vary depending on types of expression vectors to be used. When an expression vector is a plasmid vector, the protoplast method, the particle gun method or the Agrobacterium method can be employed. Such techniques are known in the art, and detailed descriptions are available in the above-cited documents. When an expression vector is a virus vector, a reference may be made to, for example, the method of Hohn et al. (Molecular Biology of Plant Tumors, Academic Press, New York, 1982, pp. 549) or US Patent No. 4,407,956. Transformation is generally carried out via the in vitro method involving plant tissue culture with the use of a callus or tissue section. In such a case, it is necessary to reproduce a plant body from a transgenic plant cell. Such technique is also known in the art, and reference may be made to the above mentioned Shokubutsu Taisha Kogaku Handbook (the Handbook of plant metabolic engineering), 2002, NTS, or Shinban Moderu Shokubutsu no Jikken Protokoru (the experimental protocol for model plants (new edition): Idengakuteki shuhou kara genomu kaiseki made (From genetic engineering to genome analysis), Shujunsha, 2001). When a target plant is of the genus Eucalyptus, a plant body can be reproduced from the cell of the transgenic Eucalyptus with reference to the method of Dohi et al. (JP Patent Publication (kokai) No. HIl-127025 A (1999)). Alternatively, transformation may be carried out via a method of transformation in which the Myb-related gene of the present invention is directly introduced into a cell of a target plant; a so-called in planta method, without a step of callus or cell culture. 5. Myb-miRNA Another aspect of the present invention relates to Myb-miRNA that can suppress the growth of a plant by being introduced into a plant cell. The term "Myb-miRNA" used herein refers to miRNA comprising a nucleotide sequence consisiting of 21 nucleotides as shown in SEQ ID NO: 7 and targeting the Myb-related gene transcription product of the present invention.

miRNA (micro RNA) is endogenous non-coding RNA (i.e., RNA that does not encode a protein) comprising 19 to 25 nucleotides evolutionarily conserved across organism species. It is small RNA regulating expression of a target gene comprising a nucleotide sequence complementary to such miRNA at the post-transcriptional and pre-translational stage. In general, miRNA is transcribed from the genome as a pri-miRNA (primary miRNA) precursor comprising several hundreds to several thousands of nucleotides, and it is converted into hairpin type pre-miRNA (precursor-miRNA) comprising about 60 to 100 nucleotides with the aid of Drosha, Pasha, and the like in nuclei. Subsequently, it is transported to the cytoplasm, a hairpin loop portion is cleaved by Dicer, and double-stranded miRNA comprising 19 to 25 nucleotides (miRNA duplex) is then formed. Thereafter, a side chain having a sequence complementary to mRNA of the target gene is incorporated into RISC/miRNP as mature single-stranded miRNA, target mRNA is recognized via a bond to the nucleotide sequence of mature miRNA, and it is cleaved and its translation is suppressed by RISC and miRNP. Thus, expression of the target gene can be suppressed (David P. Bartel, Cell, Vol. 116, 281-297, January 23, 2004). The portion described by the expression "nucleotide sequence as shown in SEQ ID NO: 7" corresponds to a mature single-stranded Myb-miRNA portion. Single-stranded Myb-miRNA recognizes the Myb-miRNA-binding site (these sequences may not be completely complementary to each other and may have one or two mismatched nucleotides) as shown in SEQ ID NO: 1, which is a complementary nucleotide sequence thereof, to thereby suppressively regulate expression of the Myb-related gene comprising the same (see Example 2). Nucleotides indicated by R, Y, and N in SEQ ID NO: 7 may be A or G, C or U, or A, U, G or C, respectively. A specific example is Myb-miRNA derived from Eucalyptus grandis comprising the nucleotide sequence as shown in SEQ ID NO: 8 (hereafter referred to as "EgMyb-miRNA" for descriptive purposes). A mechanism of suppression of gene expression by miRNA is known in the art. For example, reference may be made to Eulalio et al., 2008, Cell, 132:9-14. 6. Expression vector comprising DNA encoding Myb-miRNA Another aspect of the present invention relates to an expression vector comprising DNA encoding the aforementioned Myb-miRNA (hereafter such vector is referred to as the "Myb-miRNA expression vector") has been introduced. 6-1. DNA encoding Myb-miRNA The term "DNA encoding Myb-miRNA" used herein refers to DNA encoding Myb-miRNA of the present invention in an operable state after transcription. The term "operable state" refers to a situation in which Myb miRNA of the present invention remains capable of functioning as Myb-miRNA after it has been transcribed. DNA that merely encodes the nucleotide sequence as shown in SEQ ID NO: 7 corresponding to the mature single-stranded miRNA portion would not function as the Myb-miRNA of the present invention. This is because double-stranded RNA would not be formed after transcription. In order to maintain Myb-miRNA of the present invention in an operable state after transcription, it is necessary for DNA to encode Myb-miRNA, so that double stranded RNA having the nucleotide sequence as shown in SEQ ID NO: 7 and complementary sequence thereto is produced from one of its strands. An example of DNA encoding Myb-miRNA of the present invention in an operable state after transcription is DNA encoding Myb-shRNA (short hairpin RNA). shRNA is single-stranded RNA comprising a nucleotide sequence corresponding to part of the target gene (e.g., the Myb-miRNA-binding site of the present invention) ligated to an antisense strand thereof (e.g., Myb-miRNA of the present invention) via a short spacer sequence. In shRNA, a base pair of a sense region and an antisense region is formed within a molecule, the spacer sequence simultaneously forms a loop structure, and thus, the entire shRNA molecule has a hairpin-type stem-loop structure. DNA encoding such Myb shRNA is composed of, for example, a DNA fragment encoding the nucleotide sequence as shown in SEQ ID NO: 7, a DNA fragment having a nucleotide sequence complementary thereto, and a spacer sequence comprising an appropriate nucleotide sequence that ligates the 3' end of a DNA fragment to the 5' end of the other DNA fragment. The spacer sequence may generally comprise 3 to 24 nucleotides, and preferably 4 to 15 nucleotides. Many manufacturers of life-science-related products (e.g., Takara Bio and Invitrogen) provide services to prepare DNA encoding shRNA, and such services may be utilized. Also, DNA encoding Myb-miRNA may encode a Myb-miRNA precursor; i.e., pri-Myb-miRNA or pri-Myb-miRNA. As another example, DNA encoding a nucleotide having the nucleotide sequence as shown in SEQ ID NO: 7 and DNA encoding a nucleotide having a nucleotide sequence complementary thereto may be independently expressed. Each nucleotide may be expressed in cis (e.g., in the same expression vector) or in trans (e.g., in different expression vectors). In order to maintain the expression levels of both nucleotides at equivalent levels, for example, the same promoter is preferably used. In such a case, each DNA may be introduced into and expressed in the target cell, so that double-stranded RNA is formed in the cells and Myb-miRNA of the present invention can be consequently maintained in an operable state. 6-2. Myb-miRNA expression vector In the present invention, the Myb-miRNA expression vector is prepared by introducing DNA encoding Myb-miRNA of the present invention into an adequate expression vector. Via introduction into an adequate host cell, inserted Myb-miRNA can be expressed under adequate conditions. The type of expression vector is not particularly limited. An expression vector may be appropriately selected in accordance with the host into which the expression vector is to be introduced, e.g., E. coli, yeast, plant cells (including those of plant bodies, plant organs, and plant tissue), insect cells or animals. Regarding the backbone portion of the expression vector according to the present invention; i.e., the major component other than Myb-miRNA of the present invention (including control regions, such as promoter, enhancer, or terminator regions, and selection marker genes), expression vectors described in the aforementioned section "2-1. A variant Myb-related gene expression vector" can also be employed. Thus, overlapping descriptions are omitted. When the expression vector of the present invention is used in the section "Method of suppressing plant growth" below, a promoter that can overexpress Myb-miRNA in a plant cell is preferably used, so as to effectively suppress plant growth. For example, a promoter that can specifically express the gene of interest in tissue in which cell division is active or in the thickened xylem tissue involved in cell elongation is preferable. 6-2. Preparation of Myb-miRNA expression vector A method in which DNA encoding Myb-miRNA is inserted into a given site of an expression vector to prepare the Myb-miRNA expression vector can be carried out in accordance with a method known in the art, such as the method described in Sambrook, J. et al., 1989, Molecular Cloning: a Laboratory Manual Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 7. Transformant Another aspect of the present invention relates to a transformant into which the Myb-miRNA expression vector has been introduced. 7-1. Transformant The term "transformant" used herein refers to a host that was transgenic by the Myb-miRNA expression vector and a clone thereof. Any host can be used without particular limitation, provided that it is suitable for the vector used and it can express Myb-miRNA of the present invention. Examples thereof include hosts described in the aforementioned section "3-1. Transformant". Preferable hosts are microorganism or plant cell hosts (including those of plants of the genus Eucalyptus or the genus Nicotiana). 7-2. Progeny of transformant The term "progeny of transformant" used herein refers to an offspring resulting from sexual reproduction of the first generation transformants into which the Myb-miRNA expression vector of the present invention has been introduced, which retains DNA encoding Myb-miRNA of the present invention in an expressible state. When transformants are seed plants, for example, seedlings of the transgenic plants are the progenies of the transformants. The generation of the progeny is not limited, provided that DNA encoding Myb miRNA of the present invention is transferred in an expressible state.

7-3. Method of transformation The Myb-miRNA expression vector of the present invention can be introduced into a host in accordance with the method described in the aforementioned section "3-3. Method of transformation". 7-4. Method for obtaining progeny Progenies can be obtained from the transformants of the present invention in accordance with the method described in the aforementioned section "3-4. Method for obtaining progeny". 8. Method for suppressing plant growth Another aspect of the present invention relates to a method of introducing a variant Myb-related gene or expression vector into a plant body to suppressing the growth thereof. This aspect of the present invention is based on a principle that is opposite to the principle of the aforementioned section "4. Method for accelerating plant growth". Specifically, this aspect of the present invention is based on the principle of DNA encoding Myb-miRNA of the present invention being introduced into a target plant, with Myb-miRNA being overexpressed in the plant body to suppress expression of the target endogenous Myb-related gene. Thus, the target plant of this method is not particularly limited, provided that such plant comprises the nucleotide sequence as shown in SEQ ID NO: 1 of the present invention and comprises at least one Myb-related gene that accelerates plant growth in the genome. Examples thereof include various plants of the genus Eucalyptus, Nicotiana, and Populus described in the aforementioned section "3-2. Method of transformation" A basic procedure of the method according to this aspect is identical to the method described in 4. the section "Method for accelerating plant growth" above. Thus, such method may be carried out in accordance therewith. Examples <Example 1> Acquisition of Eucalyptus Myb-related gene (EcMyb gene) (1) Preparation of total RNA Tree trunk and cambium tissue were sampled from 5-year-old Eucalyptus camaldulensis grown at the Forestry Research Institute, Oji paper Co., Ltd. The tissue samples were ground in liquid nitrogen, and total RNA was then extracted using the Concert Plant RNA Reagent (Invitrogen). The procedure of extraction in accordance with the attached instructions was employed. As a result, 1 mg of total RNA was obtained. (2) Preparation of mRNA mRNA was purified from total RNA using the PolyA Tract mRNA Isolation System (Promega). The procedure of extraction in accordance with the attached instructions was employed. As a result, 6 jg of mRNA was obtained from 1 mg of total RNA. (3) Preparation of cDNA library A cDNA library of E. camaldulensis was prepared in accordance with the instructions of the SMART cDNA Library Construction Kit (Takara Bio). Specifically, an oligo dT primer was annealed to I ig of the purified mRNA, and cDNA was then synthesized using reverse transcriptase. Resulting cDNA was digested with the SfiI restriction enzyme, size fractionation was carried out using a column, and the resultant was ligated to the XTriplEx2 vector.

Subsequently, a X-phage packaging reaction was carried out using Gigapack Gold III (Stratagene), and a cDNA library comprising 5x105 independent clones was prepared. (4) Isolation of EcMyb gene About 20,000 cDNA clones were arbitrarily extracted from the independent clones, and nucleotide sequences of the clones were determined using the CEQ 8000 Genetic Analysis System (Beckman Coulter, Inc.). The determined nucleotide sequences were crosschecked with the gene database of the DNA Data Bank of Japan (DDBJ) via Blast search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). As a result, cDNA clones exhibiting high homology to the WEREWOLF gene (the R2R3-type Myb gene), which is considered to regulate differentiation and morphogenesis in Arabidopsis thaliana, Oryza sativa, Populus, and the like, were discovered. The sequence of the cDNA clone was found to comprise the entire amino acid coding region including the 5'- and 3'-non-translational regions. This newly isolated Myb related gene of E. camaldulensis (the EcMyb gene: SEQ ID NO: 3) encodes the Myb-related protein (EcMyb protein) comprising 121 amino acids (SEQ ID NO: 9). The EcMyb protein was found to comprise two Myb domains (R2 and R3) on the N-terminal side, and it was found to be a transcriptional regulator of the R2R3-type Myb family, as in the case of the Myb66 protein (Fig. 1). <Example 2> Isolation of miRNA involved in suppression of Myb gene expression (Myb miRNA) and examination of activity thereof. (1) Purification and.analysis of Eucalyptus miRNA Flower buds were sampled from 5-year-old Eucalyptus grandis grown at the Forestry Research Institute, Oji paper Co., Ltd. Samples were ground in liquid nitrogen, and total RNA was then extracted using the Concert Plant RNA Reagent (Invitrogen). The total RNA was separated by a conventional technique on 15% polyacrylamide gel, bands of an RNA region equivalent to about 18 to 26 nucleotides in length, which was calculated based on the size of an electrophoresis marker, was cleaved from the gel, and RNA was recovered from the gel using the FrashPAGE Fractinator (Ambion). Subsequently, nucleotide sequences of about 1,000,000 low-molecular-weight RNA molecules were analyzed by the MPSS method (Takara Bio, Bio View, 2005, No. 50: 17 19). As a result of analysis, these sequences were found to be classified into about 130,000 types. Subsequently, these sequences were subjected to homology search with the genome sequences of E. grandis, the sequences that were not consistent with the genome sequences were eliminated, and sequences having a homology to known RNA sequences were further eliminated. As a result, about 30,000 types of low-molecular-weight RNA molecules were separated as miRNA candidates. Subsequently, information on nucleotide sequences of about 500 bp, including regions in front and behind such sequences, was obtained from the Eucalyptus genome sequence. The secondary structures of the candidates within the obtained regions were deduced, and 208 RNA molecules having structures characteristic of miRNA (i.e., the hairpin structure) were identified. These molecules were collectively designated as the group of Eucalyptus miRNA molecules. (2) Isolation of miRNA involved in suppression of EcMyb gene expression miRNA molecules that can recognize the EcMybl genes isolated in Example 1 as target molecules were searched for from the group of Eucalyptus miRNA molecules. As a result, the Eucalyptus miRNA molecule (EgMyb miRNA) comprising the nucleotide sequence as shown in SEQ ID NO: 8 was obtained.. (3) Examination of suppression of Myb-related gene expression by Myb-miRNA Expression of the EcMyb gene isolated in Example 1 was examined to be regulated by EcMyb-miRNA, which is an EgMyb-miRNA orthologue deduced to be endogenous in E. camaldulensis. Total RNA was extracted from an E. camaldulensis leaf and cDNA was synthesized using an oligo dT primer. Subsequently, an adaptor was ligated to the 5' end of the synthesized cDNA using the First Choice RLM-RACE Kit (Ambion) (Fig. 3a). A basic procedure in accordance with the attached instructions was employed. Subsequently, the adaptor-ligated cDNA was used as a template to carry out PCR using primers specific for EcMyb (outer primer A and inner primer B: SEQ ID NOs: 10 and 11, respectively) and primers specific for such adaptor (outer primer C and inner primer D: SEQ ID NOs: 12 and 13,respectively), as shown in Fig. 1 and Fig. 2. A PCR cycle comprising denaturation at 94*C for 30 seconds, annealing at 55*C for 30 seconds and elongation at 68*C for 90 seconds was repeated 35 times, and ten independent PCR reactions were carried out (n = 10). Subsequently, the PCR product was confirmed via electrophoresis on 1.0% agarose gel (Fig. 3b) and obtained using a cloning kit (TA Cloning Kit, Invitrogen). Thereafter, the nucleotide sequence of such product was determined. As a result, EcMyb mRNA was found to be cleaved between 13U and 14U at the Myb-miRNA-binding site corresponding to SEQ ID NO: 2 in all reaction samples (Fig. 3c). The results demonstrate that expression of EcMyb mRNA is suppressed by EcMyb-miRNA.

<Example 3> Preparation of variant EcMyb-related gene expression vector (1) Variant EcMyb-related gene Silent mutation was introduced in order to prevent Myb-miRNA from binding to a site consisting of the nucleotide sequence as shown in SEQ ID NO: 2 contained in EcMyb mRNA (i.e., the Myb-miRNA-binding site). Since EcMyb-miRNA had not yet been identified, 9 silent mutations were introduced, so as to avoid pairing of nucleotides between EgMyb-miRNA, which is deduced to be homologous to EcMyb-miRNA, and the nucleotide sequence as shown in EQ ID NO: 2 in this example (Fig. 4). Actually, synthesis of DNA as shown in SEQ ID NO: 14 was consigned to Takara Bio. (2) Preparation of variant EcMyb-related gene expression vector The obtained variant EcMyb-related gene, mEcMyb, (SEQ ID NO: 14) and the wild-type EcMyb gene prepared in Example 1 were each cloned into the Gatewaylm entry clone pENTR/D/TOPO vector (Invitrogen), and the resultants were introduced into binary vectors (pGWB2; Nakagawa et al., 2007, J Biosci Bioeng, 34-41) by the LR reaction. A procedure utilizing the Gateway system (Invitrogen) was carried out in accordance with the attached instructions. Thus, the target expression vectors, pGWB2-mEcMyb and pGWB2-EcMyb, were prepared. These expression vectors were each introduced into the Agrobacterium LBA4404 strain using the Gene Pulser (BioRad) in accordance with the attached instructions. These expression vectors were stored in glycerol before transformation into plants. <Example 4> Acceleration of plant growth by avoiding expressional suppression mediated by endogenous Myb-miRNA (1) Preparation of transgenic tobacco In order to verify the capacity of mEcMyb to avoid RNAi mediated by endogenous Myb-miRNA and the effects of growth acceleration on plants into which mEcMyb had been introduced, tobacco leaves (N. tabacum BY4) were infected with Agrobacterium carrying pGWB2-EcMyb and pGWB2-mEcMyb prepared in (1) of this example in accordance with a conventional technique (see Shougo Matsumoto and Yasunori Machida, 1990, Shokubutsu Keishitsu Tenkan hou (A technique of plant transformation), Gendai Kagaku (Modern Chemistry), June, 25 to 29), and selection was carried out using hygromycin resistance as an indicator to prepare transgenic tobacco. Tobacco was used herein instead of Eucalyptus mainly for the following reasons. That is, the growth speed of tobacco is faster than that of Eucalyptus, among useful herbaceous plants other than Gramineae, genetic engineering techniques, including transformation, of tobacco have been already established, and transformation of woody plants is generally much more difficult than that of herbaceous plants. Although there has not been any specific reports that any of them can serve as EcMyb orthologues in tobacco, candidate genes for EcMyb orthologues were deduced to be universally present in all plants, as a result of sequence comparison among other plant species, the genome decoding of which has been completed or is in progress. Since the EcMyb orthologue gene was deduced to be present in tobacco, it was used in this example. (2) Confirmation of mEcMyb gene expression In order to inspect EcMyb or mEcMyb expression in a transgenic tobacco carrying pGWB2-EcMyb or pGWB2-mEcMyb obtained in aforementioned (1) of this embodiment, total RNA was extracted from transgenic tobacco leaves, and RT-PCR was carried out in accordance with a conventional technique. The Fw and Rv primers used in PCR have a nucleotide sequence ( SEQ ID NO: 15) corresponding to the sense strand sequence comprising residues 158 to 180 of EcMyb and a nucleotide sequence ( SEQ ID NO: 16) corresponding to the antisense strand sequence comprising residues 385 to 404 of EcMyb, respectively. The Myb-miRNA-binding site is present between primers (from amino acids 310 to 331). A PCR cycle comprising denaturation at 94*C for 30 seconds, annealing at 55*C for 30 seconds, and elongation at 68*C for 90 seconds was repeated 35 times. Three independent RT-PCR reactions were carried out (n = 3). After RT-PCR, samples of 2 ptl each were electrophoresed on 1.0% agarose gel. The results are shown in Fig. 5. Wt represents non-transformed wild type tobacco (BY4), WtEc represents a wild-type EcMyb transformant, and mEc represents a variant EcMyb transformant. The variant EcMyb transformant was found to contain a larger quantity of mRNA carrying the Myb-miRNA binding site than the wild-type EcMyb transformant. This indicates that exogenous mEcMyb mRNA is not affected by the cleavage action of tobacco endogenous Myb-miRNA, i.e., RNAi, and it can stably function in tobacco cells. In contrast, EcMyb mRNA was detected in an amount larger than that of endogenous NtMyb mRNA due to overexpression from the expression vector, although such amount was smaller than that of mEcMyb mRNA. This is considered to result from cleavage of part thereof by tobacco endogenous Myb miRNA. The experiment demonstrates that the variant Myb-related gene of the present invention can avoid RNAi by Myb-miRNA in a plant in which MybmiRNA that targets the transcription product of the variant Myb-related gene of the present invention is endogenously present. (3) Inspection of transgenic tobacco growth The transformant into which pGWB2-mEcMyb obtained in aforementioned (1) had been introduced and a non-transformed wild-type plant (BY4) were allowed to grow for 6 months in accordance with a conventional technique, and the growth conditions thereof were compared. The results are shown in Fig. 6 and in Fig. 7. In Fig. 6, a solid line indicates a transgenic tobacco (BY4+pGWB2-mEcMyb), a broken line indicates a wild-type tobacco plant (BY4), a square plot indicates height (cm) (the left vertical axis), and a round plot represents stem diameter (mm) (the right vertical axis). In Fig. 7, "a" indicates a wild-type tobacco plant (BY4), and b and c indicate transgenic tobacco (BY4+pGWB2-mEcMyb). As a result of introduction of the pGWB2-mEcMyb gene, significant differences in growth with respect to height and stem diameter were observed after 6 months, compared with non-transformed wild-type plants (Fig. 6 and Fig. 7). The results indicate that the use of the variant Myb-related gene of the present invention enables artificial inhibition of RNAi with regard to the Myb related gene inherent to the plant. In particular, the growth of a plant into which the variant gene of the present invention had been introduced was found to be accelerated compared with the general state, and the variant Myb-related gene of the present invention was found to function across plant species. Thus, transformation of trees (e.g., plants of the genus Eucalyptus) for commercial tree plantation with the use of the variant Myb-related gene of the present invention enables effective utilization of such trees for woody biomasses.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. 39A

Claims (16)

1. A variant Myb-related gene capable of accelerating plant growth, wherein the variant Myb-related gene is derived from a Myb-related gene comprising in its coding region the nucleotide sequence set forth in SEQ ID NO: 1 and wherein the variant Myb-related gene comprises a nucleotide sequence set forth SEQ ID NO: 1 having one or more silent mutation(s) so as to disable regulation of expression of a Myb related gene mediated by binding of an miRNA to the nucleotide sequence set forth in SEQ ID NO: 1.

2. The variant Myb-related gene according to claim 1, wherein the nucleotide sequence set forth in SEQ ID NO: 1 is represented by SEQ ID NO: 2.

3. The variant Myb-related gene according to claim 1 or 2, wherein at least one of the silent mutations is a substitution of T at position 13 in SEQ ID NO: 1 with C.

4. The variant Myb-related gene according to any one of claims 1 to 3, comprising a nucleotide sequence set forth SEQ ID NO: 1 having at least 5 silent mutations.

5. The variant Myb-related gene according to any one of claims 1 to 4, wherein the Myb-related gene comprises the nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence having 95% or higher identity to SEQ ID NO: 3. 40

6. The variant Myb-related gene according to any one of claims 1 to 5, wherein the variant Myb-related gene disables regulation of expression of a Myb-related gene mediated by an miRNA that binds to the nucleotide sequence set forth in SEQ ID NO: 1 in a plant cell.

7. An expression vector comprising the variant Myb-related gene according to any one of claims 1 to 6.

8. A host transformed with the expression vector according to claim 7, or a progeny thereof comprising the expression vector.

9. The host or a progeny thereof according to claim 8, wherein the host is a microorganism or plant.

10. The host or a progeny thereof according to claim 9, wherein the plant is of the genus Eucalyptus or the genus Nicotiana.

11. A method of accelerating the growth of a plant, comprising introducing the expression vector according to claim 7 into the plant.

12. A method of suppressing the growth of a plant, comprising introducing into a plant cell an expression vector comprising a nucleic acid encoding an miRNA wherein the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 7 and targets a transcription product of a Myb-related gene comprising in its coding region the nucleotide sequence set forth in SEQ ID NO: 1. 41

13. The method according to claim 12, wherein the Myb-related gene comprises in its coding region the nucleotide sequence set forth in SEQ ID NO: 2.

14. The method according to claim 12 or claim 13, wherein the Myb-related gene comprises a nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 95% identity to SEQ ID NO: 3.

15. The method according to any one of claims 12 to 14, wherein the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 8.

16. The variant Myb-related gene according to any one of claims 1 to 6 or the expression vector according to claim 7 or the host or progeny according to any one of claims 8 to 10 or the method according to any one of claims 11 to 15 substantially as herein before described in the Examples and/or Drawings. 42