# Patent

## Title
Gene for increasing the production of plant biomass and method of use thereof

## Abstract

A technique by which the production of plant biomass can be significantly increased is provided. The AT3G05660 gene encoding a receptor-like protein having a leucine-rich repeat structure or a gene functionally equivalent to such a gene is introduced, or an expression control region of an endogenous gene corresponding to the gene is modified.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2009/069155 filed Nov. 11, 2009, claiming priority based on Japanese Patent Application No. 2008-288869, filed Nov. 11, 2008, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to: a plant into which a given gene is introduced or the expression control region of the given endogenous gene corresponding to the gene is modified; a method for increasing the production of biomass through introduction of a given gene or modification of an expression control region of the given endogenous gene; and a method for producing a plant capable of producing an increased amount of biomass.
BACKGROUND ART
The term “biomass” generally refers to the total amount of organisms that inhabit or exist in a given area. When such term is used with regard to plants, in particular, it refers to dry weight per unit area. Biomass units are quantified in terms of mass or energy. The expression “biomass” is synonymous with “Seibutsutairyo” or “Seibutsuryo.” In the case of plant biomass, the term “standing crop” is occasionally used for “biomass.” Since plant biomass is generated by fixing atmospheric carbon dioxide with the use of solar energy, it can be regarded as so-called “carbon-neutral energy.” Accordingly, an increase of plant biomass is effective for global environmental preservation, the prevention of global warming, and mitigation of greenhouse gas emissions. Thus, technologies for increasing the production of plant biomass have been industrially significant.
Plants are cultivated for the purpose of using some tissues thereof (e.g., seeds, roots, leaves, or stems) or for the purpose of producing various materials, such as fats and oils. Examples of fats and oils produced from plants that have been heretofore known include soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn oil, safflower oil, palm oil, and rapeseed oil. Such fats and oils are extensively used for household and industrial applications. Also, fats and oils produced from plants are used as raw materials for biodiesel fuel or bioplastic, and the applicability thereof is increasing for alternative energy to petroleum.
In particular, an energy crop such as sugar cane can be used as a raw material for biofuel. Hence, the increased production of the total mass of a plant itself (the amount of plant biomass) is expected. Under such circumstances, improvement in productivity per unit of cultivation area is required in order to increase the production of the amount of plant biomass. It has been found that if the number of cultivated plants is assumed to be constant per unit of cultivation area, improvement in the amount of biomass per plant would be necessary.
However, it is thought that since many genes are involved in the amount of plant biomass (a so-called “kind of quantitative trait”), individual gene introduction or individual genetic modification cannot lead to an effective increase in the production of plant biomass. Meanwhile, a great deal of difficulties are associated with introduction of many genes in a desired state into a plant. Such gene introduction is also problematic in that if successful gene introduction takes place, desirable traits cannot always be acquired.
Various gene transfer techniques are known as techniques for increasing the production of plant biomass, as disclosed in JP Patent Publication (Kohyo) No. 2001-505410 A; JP Patent Publication (Kohyo) No. 2001-519659 A; JP Patent Publication (Kohyo) No. 2007-530063 A; JP Patent Publication (Kokai) No. 2005-130770 A; JP Patent Publication (Kohyo) No. 2000-515020 A; JP Patent Publication (Kohyo) No. 9-503389 A (1997); and JP Patent Publication (Kokai) No. 2005-52114 A, for example. However, according to techniques disclosed in Patent Documents (JP Patent Publication (Kohyo) No. 2001-505410 A; JP Patent Publication (Kohyo) No. 2001-519659 A; JP Patent Publication (Kohyo) No. 2007-530063 A; JP Patent Publication (Kokai) No. 2005-130770 A; and JP Patent Publication (Kohyo) No. 2000-515020 A), only the effects of increasing the production of partial plant biomass, such as plant seeds, tubers, roots, and leaves, can be obtained. Also, techniques disclosed in JP Patent Publication (Kohyo) No. 9-503389 A (1997) and JP Patent Publication (Kokai) No. 2005-52114 A disclose only the effects of increasing the production of biomass under nitrate-unlimited growth conditions or by delaying the flowering time so as to lengthen the growth period. Hence, Patent Documents (JP Patent Publication (Kohyo) No. 2001-505410 A; JP Patent Publication (Kohyo) No. 2001-519659 A; JP Patent Publication (Kohyo) No. 2007-530063 A; JP Patent Publication (Kokai) No. 2005-130770 A; JP Patent Publication (Kohyo) No. 2000-515020 A; JP Patent Publication (Kohyo) No. 9-503389 A (1997); and JP Patent Publication (Kokai) No. 2005-52114 A) disclose no techniques for achieving the effects of increasing the production of the biomass of an entire plant through application of general culture and/or growth conditions.
DISCLOSURE OF THE INVENTION
Object to be Achieved by the Invention
In view of the above circumstances, an object of the present invention is to search for genes having novel functions of drastically improving the amount of the biomass of an entire plant even under general culture and/or growth conditions and thus to provide a technique with which the production of plant biomass can be drastically increased.
Means to Achieve the Object
As a result of intensive studies to achieve the above object, the present inventors have made the novel finding that the production of plant biomass can be drastically improved by: introducing a gene encoding a receptor-like protein that has a leucine-rich repeat structure and a characteristic domain in a molecule thereof; or modifying an expression control region of an endogenous gene corresponding to the gene. Thus, they have completed the present invention.
Specifically, the plant according to the present invention is a plant into which the AT3G05660 gene encoding a receptor-like protein having a leucine-rich repeat structure or a gene functionally equivalent to such a gene is introduced, or in which an expression control region of an endogenous gene corresponding to the gene is modified.
Also, the method for increasing the production of biomass according to the present invention comprises introducing the AT3G05660 gene encoding a receptor-like protein having a leucine-rich repeat structure or a gene functionally equivalent to the gene, or modifying an expression control region of an endogenous gene corresponding to the gene.
Furthermore, the method for producing a plant according to the present invention comprises the steps of: preparing a transformed plant into which the AT3G05660 gene encoding a receptor-like protein having a leucine-rich repeat structure or a gene functionally equivalent to such a gene is introduced, or in which an expression control region of an endogenous gene corresponding to the gene is modified; and measuring the amounts of biomass of progeny plants of the transformed plant and then selecting a line in which the amount of biomass is significantly improved.
In the present invention, the above gene preferably encodes any one of the following proteins (a) to (c):
(a) a protein comprising the amino acid sequence of SEQ ID NO: 3;
(b) a receptor-like protein comprising an amino acid sequence that has a deletion, a substitution, an addition, or an insertion of one or a plurality of amino acids with respect to the amino acid sequence of SEQ ID NO: 3 and having a leucine-rich repeat structure; and

(c) a receptor-like protein that is encoded by a polynucleotide hybridizing under stringent conditions to a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 and has a leucine-rich repeat structure.

Also, in the present invention, an example of the above functionally equivalent gene is a gene encoding a receptor-like protein, which is derived from an organism other than Arabidopsis thaliana and has a leucine-rich repeat structure.
Examples of plants to be subjected to the present invention include dicotyledons such as plants of the family Brassicaceae. Examples of plants of the family Brassicaceae include Arabidopsis thaliana and rapeseed. Other examples of plants to be subjected to the present invention include monocotyledons such as plants of the family Gramineae. Examples of plants of the family Gramineae include rice and sugarcane.
This description hereby incorporates the entire content of the description and/or the drawings of Japanese Patent Application No. 2008-288869, which is the basis of the priority claim of this application.
Effect of the Invention
The plant according to the present invention is a plant capable of producing significantly improved amount of biomass compared with wild-type plants. Also, the method for increasing the production of biomass according to the present invention can realize the significantly increased production of biomass compared with target wild-type plants. Furthermore, the method for producing a plant according to the present invention makes it possible to produce a plant capable of producing significantly improved amount of biomass compared with wild-type plants. Therefore, through application of the present invention, for example, productivity can be improved when the plant itself is a product and this can be achieved at lower cost.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a characteristic diagram showing the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program for the amino acid sequences encoded by AT3G05660 (SEQ ID NO: 3) and AT3G05650 (SEQ ID NO: 22).
FIG. 2-1 is a characteristic diagram showing the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program for the amino acid sequences encoded by AT3G05660 (SEQ ID NO: 3), AT2G33080 (SEQ ID NO: 23), and AT3G05650 (SEQ ID NO: 22).
FIG. 2-2 is a characteristic diagram showing the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program for the amino acid sequences encoded by AT3G05660 (SEQ ID NO: 3), AT2G33080 (SEQ ID NO: 23), and AT3G05650 (SEQ ID NO: 22).
FIG. 3 is a photo showing the aerial parts of wild-type plants and transformed plants into which a fragment containing ORF of AT3G05660 was introduced.
FIG. 4 is a characteristic diagram showing the results of measuring the total amounts of the biomass of the aerial parts of: a wild-type plant; a transformed plant into which the LRR-RLK protein gene (AT1G69990) was introduced; a transformed plant into which the LRR-RLK protein gene (AT5G39390) was introduced; a transformed plant into which the LRR protein gene (AT3G05650) was introduced; a transformed plant into which the LRR protein gene (AT2G33080) was introduced; and a transformed plant into which the protein gene (AT3G05660) was introduced.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be explained in detail as follows.
The plant according to the present invention is a plant into which the AT3G05660 gene encoding a receptor-like protein (hereinafter, abbreviated as LRR-RLP) having a leucine-rich repeat structure or a gene functionally equivalent to such a gene is introduced, or in which an expression control region of an endogenous gene corresponding to the AT3G05660 gene is modified; and the amount of biomass is significantly improved compared with wild-type plants. The expression level of a target gene can be significantly increased compared with that in a wild-type plant by exogenously introducing the target gene or modifying an expression control region of an endogenous gene corresponding to the AT3G05660 gene. The plant according to the present invention may be a plant in which the LRR-RLP gene is expressed in all plant tissues or at least some plant tissues. Here, the term “plant tissue(s)” is meant to include plant organ(s) such as leaves, stems, seeds, roots, and flowers.
Also, the term “expression control region” refers to a promoter region to which RNA polymerase binds and a region to which another transcription factor binds. A transcriptional regulatory region is preferably modified by substituting a promoter region, for example, among endogenous transcriptional regulatory regions with a promoter region that enables a higher expression level.
LRR-RLP Gene
In the present invention, an LRR-RLP gene is the AT3G05660 gene or a gene functionally equivalent to such a gene. In addition, as described in Reference 1 (Plant Physiology, June 2003, Vol. 132, pp. 530-543), LRR-RLP is involved in receptor-like kinase (RLK)-signals and is a protein analogous to an extracellular domain of RLK. LRR-RLP encoded by the AT3G05660 gene comprises a characteristic domain consisting of the amino acid sequence of SEQ ID NO: 1. Hence, when LRR-RLP (AT2G33080 or AT3G05650) having high homology with LRR-RLP encoded by the AT3G05660 gene is compared with AT3G05660 at the amino acid sequence level, it is understood that the amino acid sequence of SEQ ID NO: 1 is specific in AT3G05660.
FIG. 1 shows the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program (that can be used for DDBJ of the National Institute of Genetics (clustalw.ddbj.nig.ac.jp/top-j.html)) for AT3G05660 and AT3G05650. Also, FIG. 2 shows the results of similar alignment analysis for AT3G05660, AT2G33080, and AT3G05650.
As shown in FIG. 1 and FIG. 2, it is understood that the amino acid sequence of SEQ ID NO: 1 (in FIGS. 1 and 2, denoted as Region A) is a specific domain capable of distinguishing AT3G05660 from AT2G33080 and AT3G05650. Regarding AT3G05660 having a domain consisting of the amino acid sequence of SEQ ID NO: 1, it was revealed in the Examples described later that AT3G05660 has effects of significantly improving the production of biomass. The nucleotide sequence of the coding region of the AT3G05660 gene is of SEQ ID NO: 2. The amino acid sequence of LRR-RLP encoded by the AT3G05660 gene is of SEQ ID NO: 3.
Also, in the present invention, a gene functionally equivalent to the AT3G05660 gene may also be introduced into a plant. Here, the term “functionally equivalent gene” refers to, for example, a gene from an organism other than Arabidopsis thaliana, corresponding to the AT3G05660 gene encoding LRR-RLP.
The above genes, which are from organisms other than Arabidopsis thaliana and are functionally equivalent to the AT3G05660 gene, are not particularly limited and can be specified by searching for a database containing gene sequences of various organisms. Specifically, DDBJ/EMBL/GenBank International Nucleotide Sequence Database or SWISS-PROT database is searched, for example, using the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 3 as a query sequence, so that the sequence can be easily searched for and/or identified from such a known database.
In addition, LRR-RLP genes in the present invention are not limited to the above described LRR-RLP genes comprising the nucleotide sequences and the amino acid sequences specified by SEQ ID NOS. Hence, the LRR-RLP gene may be a gene that contains an amino acid sequence having a deletion, a substitution, an addition, or an insertion of one or a plurality of amino acid sequences with respect to the amino acid sequences specified by the above-described SEQ ID NOS, and has activity of functioning as LRR-RLP. Here the term “a plurality of amino acids” refers to 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1 to 3 amino acids, for example. In addition, amino acid deletion, substitution, or addition can be performed by modifying a nucleotide sequence encoding the above LRR-RLP gene by a technique known in the art. Mutation can be introduced into a nucleotide sequence by a known technique such as the Kunkel method or the Gapped duplex method or a method based thereof. For example, mutation is introduced with a mutagenesis kit using site-directed mutagenesis (e.g., Mutant-K or Mutant-G (both are trade names of Takara Bio)) or the like, or a LA PCR in vitro Mutagenesis series kit (trade name, Takara Bio). Also, a mutagenesis method may be: a method using a chemical mutation agent represented by EMS (ethyl methanesulfonate), 5-bromouracil, 2-aminopurine, hydroxylamine, N-methyl-N′-nitro-N nitrosoguanidine, or other carcinogenic compounds; or a method that involves radiation treatment or ultraviolet [UV] treatment typically using X-rays, alpha rays, beta rays, gamma rays, an ion beam, or the like.
Also, LRR-RLP genes may be genes homologous to the LRR-RLP genes comprising the nucleotide sequences and the amino acid sequences specified by the above-described SEQ ID NOS. Here, the term “homologous gene” generally refers to a gene that has evolutionarily branched off from a common ancestor gene, including a homologous gene (ortholog) of 2 types of species and a homologous gene (paralog) generated by overlapping branching that takes place within the same species. In other words, the above term “functionally equivalent gene” refers to a homologous gene such as an ortholog or a paralog. Furthermore, the above term “functionally equivalent gene” may also refer to a gene that does not evolve from a common gene, but simply has analogous functions.
Examples of genes analogous to the LRR-RLP genes comprising the nucleotide sequences and the amino acid sequences specified by the above described SEQ ID NOS include genes encoding proteins: having amino acid sequences that have 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more similarity to these amino acid sequences; having a common sequence comprising the amino acid sequence of SEQ ID NO: 1; and having LRR-RLP activity. Here, the value of similarity refers to a value that can be found based on default setting using a computer mounted with a BLAST (Basic Local Alignment Search Tool) program and a database containing gene sequence information.
Also, genes analogous to LRR-RLP genes comprising the nucleotide sequences and the amino acid sequences specified by the above SEQ ID NOS can be identified by, when the plant genome information remains unclarified, extracting the genome from a target plant or constructing a cDNA library for a target plant and then isolating a genomic region or cDNA hybridizing under stringent conditions to at least a portion of the LRR-RLP genes comprising the nucleotide sequences and amino acid sequences specified by the above SEQ ID NOS. Here, the term “stringent conditions” refers to conditions under which namely a specific hybrid is formed, but a non-specific hybrid is never formed. For example, such conditions comprise hybridization at 45° C. with 6×SSC (sodium chloride/sodium citrate), followed by washing at 50° C. to 65° C. with 0.2-1×SSC and 0.1% SDS. Alternatively, such conditions comprise hybridization at 65° C. to 70° C. with 1×SSC, followed by washing at 65° C. to 70° C. with 0.3×SSC. Hybridization can be performed by a conventionally known method such as a method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
The plant according to the present invention exerts significantly improved production of biomass compared with wild-type plants, as a result of introduction of the AT3G05660 gene or a gene functionally equivalent to the gene into a plant or modification of an expression control region of an endogenous gene corresponding to the gene. Examples of a technique for introducing the LRR-RLP gene into a plant include a technique for introducing an expression vector in which an exogenous LRR-RLP gene has been arranged under control of a promoter that enables expression within the plant. Examples of a technique for modifying an expression control region of an endogenous gene corresponding to the gene include a technique for modifying a promoter of an endogenous LRR-RLP gene in a target plant.
A preferred example is a technique for introducing an expression vector in which the above LRR-RLP gene is arranged under control of a promoter that enables expression in a target plant.
Expression Vector
An expression vector is constructed to contain a promoter that enables expression within plants and the above described LRR-RLP gene. As a vector serving as a mother body for an expression vector, various conventionally known vectors can be used. For example, plasmids, phages, cosmids, or the like can be used and such vector can be appropriately selected depending on plant cells into which it is introduced and introduction methods. Specific examples of such vector include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, and pBI vectors. Particularly, when a method for introduction of a vector into a plant uses Agrobacterium, a pBI binary vector is preferably used. Specific examples of such pBI binary vector include pBIG, pBIN19, pBI101, pBI121, and pBI221.
A promoter to be used herein is not particularly limited, as long as it enables expression of the LRR-RLP gene in a plant. Any known promoter can be appropriately used. Examples of such promoter include a cauliflower mosaic virus 35S promoter (CaMV35S), various actin gene promoters, various ubiquitin gene promoters, a nopaline synthase gene promoter, a tobacco PR1a gene promoter, a tomato ribulose 1,5-bisphosphate carboxylase×oxidase small subunit gene promoter, a napin gene promoter, and an oleosin gene promoter. Of these, a cauliflower mosaic virus 35S promoter, an actin gene promoter, or a ubiquitin gene promoter can be more preferably used. The use of each of the above promoters enables strong expression of any gene when it is introduced into plant cells.
Also, a promoter having functions of causing site-specific expression in a plant can also be used herein. As such a promoter, any conventionally known promoter can be used. When the above described LRR-RLP gene is site-specifically expressed using such a promoter, a plant organ in which the gene is expressed can be more increased than wild-type plant organs.
In addition, an expression vector may further contain other DNA segments in addition to a promoter and the above LRR-RLP gene. Such other DNA segments are not particularly limited and examples thereof include a terminator, a selection marker, an enhancer, and a nucleotide sequence for enhancing translation efficiency. Also, the above recombinant expression vector may further have a T-DNA region. A T-DNA region can enhance efficiency for gene introduction particularly when the above recombinant expression vector is introduced into a plant using Agrobacterium. 
A transcription terminator is not particularly limited, as long as it has functions as a transcription termination site and may be any known transcription terminator. For example, specifically, a transcription termination region (Nos terminator) of a nopaline synthase gene, a transcription termination region (CaMV35S terminator) of cauliflower mosaic virus 35S, or the like can be preferably used. Of them, the Nos terminator can be more preferably used. In the case of the above recombinant vector, a phenomenon such that an unnecessarily long transcript is synthesized and that a strong promoter decreases the number of copies of a plasmid after introduction into plant cells can be prevented by arranging a transcription terminator at an appropriate position.
As a transformant selection marker, a drug resistance gene can be used, for example. Specific examples of such drug resistance gene include drug resistance genes against hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol, and the like. Transformed plants can be easily selected by selecting plants that can grow in medium containing the above antibiotics.
An example of a nucleotide sequence for increasing translation efficiency is an omega sequence from tobacco mosaic virus. This omega sequence is arranged in an untranslated region (5′UTR) of a promoter, so that the translation efficiency of the fusion gene can be increased. As such, the recombinant expression vector may contain various DNA segments depending on purposes.
A method for constructing a recombinant expression vector is not particularly limited. To an appropriately selected vector serving as a mother body, the above promoter, the above LRR-RLP gene, a transcription repressor converting polynucleotide, and if necessary, the above other DNA segments may be introduced in an predetermined order. For example, the above LRR-RLP gene and a promoter (and, if necessary, a transcription terminator or the like) are linked to construct an expression cassette and then the cassette may be introduced into a vector. In construction of an expression cassette, for example, cleavage sites of DNA segments are prepared to have protruding ends complementary to each other and then performing a reaction with a ligation enzyme, making it possible to specify the order of the DNA segments. In addition, when an expression cassette contains a terminator, DNA segments may be arranged in the following order from upstream: a promoter, the above LRR-RLP gene, and a terminator. Also, reagents for construction of an expression vector (that is, types of restriction enzymes, ligation enzymes, and the like) are also not particularly limited. Hence, commercially available reagents can be appropriately selected and used.
Also, a method for replicating (a method for producing) the above expression vector is not particularly limited and conventionally known replication methods can be used herein. In general, such expression vector may be replicated within Escherichia coli as a host. At this time, preferred types of Escherichia coli may be selected depending on the types of vector.
Transformation
The above-described expression vector is introduced into a target plant by a general transformation method. A method for introducing an expression vector into plant cells (transformation method) is not particularly limited. Conventionally known appropriate introduction methods can be used depending on plant cells. Specifically, a method using Agrobacterium or a method that involves direct introduction into plant cells can be used, for example. As a method using Agrobacterium, a method described in Bechtold, E., Ellis, J. and Pelletier, G. (1993) In Planta Agrobacterium-mediated gene introduction by infiltration of adult Arabidopsis plants. C.R. Acad. Sci. Paris Sci. Vie, 316, 1194-1199., or a method described in Zyprian E, Kado Cl, Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid. Plant Molecular Biology, 1990, 15(2), 245-256. can be employed, for example.
As a method for directly introducing an expression vector into plant cells, microinjection, electroporation, a polyethylene glycol method, a particle gun method, protoplast fusion, a calcium phosphate method, or the like can be employed.
Also, when a method for directly introducing DNA into plant cells is employed, DNA that can be used herein contains transcriptional units required for the expression of a target gene, such as a promoter and a transcription terminator, and a target gene. Vector functions are not essential in such case. Moreover, a DNA that contains a protein coding region alone of a target gene having no transcriptional unit may be used herein, as long as it is integrated into a host's transcriptional unit and then the target gene can be expressed.
Examples of plant cells into which the above expression vector or an expression cassette containing no expression vector, but a target gene is introduced include cells of each tissue of plant organs such as flowers, leaves, and roots, calluses, and suspension-cultured cells. At this time, an appropriate expression vector may be constructed according to the types of plant to be produced or a versatile expression vector may be constructed in advance and then introduced into plant cells.
Plants into which an expression vector is introduced or in other words, plants required to increase the production of biomass are not particularly limited. Specifically, through expression of the above-described LRR-RLP gene, effects of increasing the production of biomass can be expected for all plants. Examples of target plants include, but are not limited to, dicotyledons and monocotyledons, such as plants (see below) belonging to the families Brassicaceae, Gramineae, Solanaceae, Leguminosae, Salicaceae, and the like.
Family Brassicaceae: Arabidopsis thaliana (Arabidopsis thaliana), Aburana (rapeseed) (Brassica rapa, Brassica napus), cabbage (Brassica oleracea var. capitata), rapeseed (Brassica rapa, Brassica napus), Natane (rapeseed) (Brassica rapa, Brassica napus), napa (Brassica rapa var. pekinensis), ging-geng-cai (Brassica rapa var. chinensis), turnip (Brassica rapa var. rapa), coleseed greens (Brassica rapa var. hakabura), potherb mustard (Brassica rapa var. lancinifolia), komatsuna (Brassica rapa var. peruviridis), pak choi (Brassica rapa var. chinensis), daikon (Brassica Raphanus sativus), Japanese horseradish (Wasabia japonica), and the like.

Family Solanaceae: tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solaneum tuberosum), tomato (Lycopersicon lycopersicum), chile pepper (Capsicum annuum), petunia, and the like.

Family Leguminosae: soy (Glycine max), pea (Pisum sativum), broad bean (Vicia faba), Wisteria (Wisteria floribunda), peanuts (Arachis hypogaea), bird's foot trefoil (Lotus corniculatus var. japonicus), common bean (Phaseolus vulgaris), azuki bean (Vigna angularis), Acacia, and the like.

Family Asteraceae: florists' daisy (Chrysanthemum morifolium), sunflower (Helianthus annuus), and the like.

Family Arecaceae: oil palm (Elaeis guineensis, Elaeis oleifera), coconut (Cocos nucifera), date palm (Phoenix dactylifera), copernicia, and the like.

Family Anacardiaceae: wax tree (Rhus succedanea), cashew nut (Anacardium occidentale), lacquer tree (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera), and the like.

Family Cucurbitaceae: pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), snake gourd (Trichosanthes cucumeroides), gourd (Lagenaria siceraria var. gourda), and the like.

Family Rosaceae: almond (Amygdalus communis), rose (Rosa), strawberry (Fragaria), cherry (Prunus), apple (Malus pumila var. domestica), and the like.

Family Caryophyllaceae: carnation (Dianthus caryophyllus) and the like.

Family Salicaceae: poplar (Populus trichocarpa, Populus nigra, or Populus tremula) and the like.

Family Gramineae: corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), bamboo (Phyllostachys), sugarcane (Saccharum officinarum), napier grass (Pennisetum pupureum), erianthus (Erianthus ravenae), miscanthus (Japanese silver grass) (Miscanthus virgatum), sorghum (Sorghum) and switch grass (Panicum), and the like.

Family Liliaceae: tulip (Tulipa), lily (Lilium), and the like.

Of these examples, energy crops such as sugarcane, corn, rapeseed, and sunflower, which can serve as raw materials for biofuel, may be preferable targets. This is because the costs for biofuel such as bioethanol, biodiesel, biomethanol, bioDME, bioGTL (BTL), and biobutanol can be reduced by increasing the production of biomass using energy crops.
Also, as described above, LRR-RLP genes that can be used in the present invention can be isolated from various plants and used. Such LRR-RLP genes can be appropriately selected and used, depending on the types of target plant required to increase the biomass production. Specifically, when a plant required to increase the biomass production is a monocotyledon, an LRR-RLP gene isolated from a monocotyledon is preferably expressed.
In addition, in the present invention, even when a plant required to increase the biomass production is a monocotyledon, a dicotyledon-derived LRR-RLP gene may be introduced. Specifically, for example, the Arabidopsis thaliana-derived LRR-RLP gene (SEQ ID NO: 2) may be introduced into not only dicotyledons, but also a variety of plants that are classified as monocotyledons, so that the gene is expressed.
Other Steps and Methods
After the above transformation, a step of selecting proper transformants from plants can be performed by a conventionally known method. Such selection method is not particularly limited. For example, selection can be made based on drug resistance such as hygromycin resistance. Alternatively, after the growth of transformants, plants are directly weighed or the any organs or tissues thereof are weighed, the weights are compared with those of wild-type plants, and then plants with significantly increased weights thereof may be selected.
Also, progeny plants can be obtained from transformed plants obtained by transformation according to a conventional method. Progeny plants retaining a trait resulting from the introduction of the LRR-RLP gene or a trait resulting from modification of an expression control region of a relevant endogenous LRR-RLP gene are selected based on the amount of biomass. Therefore, a stable plant line capable of producing an increased amount of biomass because of having the above trait can be produced. Also, plant cells or reproductive materials, such as seeds, fruits, stocks, calluses, tubers, cut ears, or lumps, may be obtained from a transformed plant or an offspring plant thereof. A stable plant line capable of producing an increased amount of biomass because of having the above trait can be mass-produced therefrom based on such materials.
In addition, examples of the term “plant(s)” in the present invention include at least any of grown plants, plant cells, plant tissues, calluses, and seeds. Specifically, in the present invention, any forms of plants that can be finally grown to mature plants are regarded as “plants.” Also, examples of such plant cells include various forms of plant cells, such as suspended culture cells, protoplasts, and leaf sections. Plants can be obtained through the growth and differentiation of these plant cells. In addition, regeneration of plants from plant cells can be performed using a conventionally known method depending on the type of plant cells.
As explained above, according to the present invention, plants capable of achieving the significantly increased production of biomass per plant compared with wild-type plants can be provided through introduction of the above LRR-RLP gene having a specific domain into a plant or modification of an expression control region of the endogenous LRR-RLP gene. Here, the term “significantly increased production of biomass” refers to a situation in which the total weight of each plant is statistically significantly increased compared with the same of a wild-type plant. In this case, even when some plant tissues become specifically large and the sizes of the other tissues are equivalent to those of a wild-type plant, it is concluded that the production of biomass is increased if the total weight of the entire plant is large.
According to the present invention, the production of biomass is increased. Hence, improvement in productivity can be achieved in both of the following cases: a case in which a purpose is to produce the whole plant; and a case in which a purpose is to produce some plant tissues (e.g., seeds) or components contained in plants. For example, when a purpose is to produce fats and oils contained in plant seeds, the amounts of fats and oils that can be harvested per area under cultivation can be greatly improved. Here, examples of fats and oils include, but are not particularly limited to, plant-derived fats and oils such as soybean oil, sesame oil, olive oil, coconut oil, rice oil, cottonseed oil, sunflower oil, corn oil, safflower oil, and rapeseed oil. Also, the thus produced fats and oils can be broadly used for household uses or industrial uses and can be further used as raw materials for biodiesel fuel. Hence, according to the present invention, the above fats and oils for household uses or industrial uses, biodiesel fuel, and the like can be produced at low cost with the use of plants in which the LPR-RLP gene has been introduced or an expression control region of the endogenous LPR-RLP gene has been modified.
EXAMPLES
The present invention will be specifically described in the following examples. However, the examples are not intended to limit the technical scope of the present invention.
Example 1
1. Materials and Methods
1-1. Experimental Materials
As experimental materials, seeds of Arabidopsis thaliana mutants (Activation-tag lines: Weigel T-DNA lines, Total of 20072 lines) were used. In addition, the seeds were purchased from the Nottingham Arabidopsis Stock Centre (NASC). Regarding the seeds used as experimental materials, Weigel, D. et al., 2000, Plant Physiol. 122, 1003-1013 can be referred to.
1-2. Methods
1-2-1. Selection of Salt-Resistant Mutants
Seeds of Weigel T-DNA lines were aseptically sowed on 125 mM or 150 mM NaCl-containing modified MS agar (1%) medium [vitamins in B5 medium, 10 g/l sucrose, and 8 g/L agar (for bacterial medium; Wako Pure Chemical Industries, Ltd.)] and then cultured at 22° C. under 30-100 μmol/m2/sec illumination (a cycle of 16 hours in the light/8 hours in the dark). Two to four weeks after sowing, salt-resistant mutant candidates were selected. In addition, regarding MS medium, see Murashige, T. et al., 1962, Physiol. Plant. 15, 473-497. Also, regarding the B5 medium, see Gamborg, O. L. et al., 1968, Experimental Cell Research 50, 151-158.
1-2-2. DNA Preparation
A site for insertion of T-DNA into the genome of the thus selected salt-resistant Arabidopsis thaliana line was determined by a TAIL-PCR method. First, young leaves were harvested from the cultivated Arabidopsis thaliana plants, and then they were crushed while undergoing liquid nitrogen freezing. DNA was prepared using a DNA preparation kit (DNeasy Plant Mini Kit, QIAGEN) according to the standard protocols included with the kit.
1-2-3. TAIL-PCR Method and Presumption of T-DNA Insertion Site
Three (3) types of specific primer, TL1, TL2, and TL3, were determined to be located near the left T-DNA sequence (T-DNA left border) of an activation-tagging vector (pSKI015: GenBank accession No. AF187951) used in Weigel T-DNA lines. With the use of an arbitrary primer P1 and the following PCR solutions and reaction conditions, TAIL-PCR (supervisors, Isao Shimamoto and Takuji Sasaki, New Edition, Plant PCR Experimental Protocols, 2000, pp. 83-89, Shujunsha, Tokyo, Japan; Genomics, 25, 674-681, 1995, Plant J., 8, 457-463, 1995) was performed, so that genomic DNA adjacent to T-DNA was amplified.
The specific sequences of the primers TL1, TL2, TL3, and P1 are as follows.









(SEQ ID NO: 4)



TL1: 5′-TGC TTT CGC CAT TAA ATA GCG ACG G-3′



 



(SEQ ID NO: 5)



TL2: 5′-CGC TGC GGA CAT CTA CAT TTT TG-3′



 



(SEQ ID NO: 6)



TL3: 5′-TCC CGG ACA TGA AGC CAT TTA C-3′



 



(SEQ ID NO: 7)



P1: 5′-NGT CGA SWG ANA WGA A-3′






In addition, in SEQ ID NO: 7, “n” represents “a,” “g,” “c,” or “t” (location: 1 and 11), “s” represents “g” or “c” (location: 7), and “w” represents “a” or “t” (location: 8 and 13).
The 1st PCR solution composition and reaction conditions are shown in Table 1 and Table 2, respectively.











TABLE 1









Template (genomic DNA)
10
ng



10 × PCR buffer (Takara Bio)
2
μl



2.5 mM dNTPs (Takara Bio)
1.6
μl



1st specific primer (TL1: SEQ ID NO: 4)
0.5
pmol



Arbitrary primer P1 (SEQ ID NO: 7)
100
pmol



TaKaRa Ex Taq (Takara Bio)
1.0
unit



Total
20
μ1


















TABLE 2







#1:
94° C. (30 seconds)/95° C. (30 seconds)


#2:
5 cycles of 94° C. (30 seconds)/65° C. (30 seconds)/72° C.



(1 minute)


#3:
1 cycle of 94° C. (30 seconds)/25° C. (1 minute)→raised to 72° C.



within 3 minutes/72° C. (3 minutes)


#4:
94° C. (15 seconds)/65° C. (30 seconds)/72° C. (1 minute),



94° C. (15 seconds)/68° C. (30 seconds)/72° C. (1 minute), and



15 cycles of 94° C. (15 seconds)/44° C. (30 seconds)/72° C.



(1 minute)


#5:
72° C. (3 minutes)









The 2nd PCR solution composition and reaction conditions are shown in Table 3 and Table 4, respectively.









TABLE 3 







Template (50-fold dilution of the 1st PCR product)
1
μl


10 × PCR buffer (Takara Bio)
2
μl


2.5 mM dNTPs (Takara Bio)
1.5
μl


2nd specific primer (TL2: SEQ ID NO: 5)
5
pmol


Arbitrary primer P1 (SEQ ID NO: 7)
100
pmol


TaKaRa Ex Taq (Takara Bio)
0.8
unit


Total
20
μl

















TABLE 4







#6:
94° C. (15 seconds)/64° C. (30 seconds)/72° C. (1 minute),



94° C. (15 seconds)/64° C. (30 seconds)/72° C. (1 minute), and



12 cycles of 94° C. (15 seconds)/44° C. (30 seconds)/72° C.



(1 minute)


#5:
72° C. (5 minutes)









The 3rd PCR solution composition and reaction conditions are shown in Table 5 and Table 6, respectively.









TABLE 5 







Template (50-fold dilution of the 2nd PCR product)
1
μl


10 × PCR buffer (Takara Bio)
5
μ1


2.5 mM dNTPs (Takara Bio)
0.5
μl


3rd specific primer (TL3: SEQ ID NO: 6)
10
pmol


Arbitrary primer P1 (SEQ ID NO: 7)
100
pmol


TaKaRa Ex Taq (Takara Bio)
1.5
unit


Total
50
μl



















TABLE 6









#7:
20 cycles of




94° C. (30 seconds)/44° C. (30 seconds)/72° C. (1 minute)



#5:
72° C. (3 minutes)










Subsequently, the 2nd and the 3rd reaction products were subjected to agarose gel electrophoresis and then the presence or the absence of amplification and the specificity of reaction products were confirmed. Also, the 3rd amplification products were subjected to a sequencing reaction directly using a BigDye Terminator Cycle Sequencing Kit Ver. 3. 1 (Applied Biosystems) and the specific primer TL3. Thus, a nucleotide sequence was determined using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
As a result, 5 types of nucleotide sequence were determined. Specifically, the sequence information of 538 bp, the sequence information of 311 bp, the sequence information of 498 bp, and the sequence information of 633 bp were obtained. The thus obtained types of sequence information are of SEQ ID NOS: 8-11, respectively.
Using the thus obtained sequence information, The Arabidopsis Information Resource (TAIR: arabidopsis.org/) was subjected to a BLAST search. Thus, the T-DNA insertion sites were found to be: a portion between the gene of Arabidopsis thaliana chromosome 1 [AGI (The Arabidopsis Genome Initiative gene code) code: At1g69990] and the gene [AGI (The Arabidopsis Genome Initiative gene code) code: At1g70000]; the gene of Arabidopsis thaliana chromosome 5 [AGI (The Arabidopsis Genome Initiative gene code) code: At5g39400]; the gene of Arabidopsis thaliana chromosome 3 [AGI (The Arabidopsis Genome Initiative gene code) code: At3g05630]; and the gene of Arabidopsis thaliana chromosome 2 [AGI (The Arabidopsis Genome Initiative gene code) code: At2g33110].
1-2-4. Prediction of Activated Genes
Activated genes were predicted from the sequence of a presumed open reading frame (ORF) gene existing within a 10-Kb range near each T-DNA insertion site (the portion between At1g69990 and At1g70000, At5g39400, At3g05630, and At2g33110) revealed in 1-2-3.
1-2-5. Preparation of Mutant Containing Predicted Gene Via Introduction
For amplification of fragments containing the ORF regions of the LRR-RLK (leucine-rich repeat receptor-like protein kinase) gene (AT1G69990), the LRR-RLK (leucine-rich repeat receptor-like protein kinase) gene (AT5G39390), the LRR (leucine-rich repeat) protein gene (AT3G05650), the LRR (leucine-rich repeat) protein gene (AT2G33080), and the LRR (leucine-rich repeat) protein gene (AT3G05660), which were predicted as being activated in 1-2-4, a pair of primers was designed and synthesized for each gene based on the sequence information disclosed in TAIR (arabidopsis.org/home.html) (Table 7). In addition, these pairs of primers were designed so that restriction enzyme sites required upon introduction into expression vectors were added to the primers (Table 7).










TABLE 7








Restriction


Gene
Forward
Reverse
enzyme site



















AT1G69990
5′-ACG CGT CGA CCC 
5′-TGT ACA TGT 
Sal I
BsrG I



ATC ATG AAA ACG
ACA AGT GAG AAC





ATC TCA ATC TTC 
GGT AGA TAA GTA 





TTC GTC-3′
AGT GG-3′





(SEQ ID NO: 12)
(SEQ ID NO: 13)




 


AT5G39390
5′-ACG CGT CGA CCA 
5′-TGT ACA TGT ACA 
Sal I
BsrG I



AAC GAC GTA TCT
GGA GAA CTT TGA





CAT AAG TCG ACG 
AGA TCA TCG AGA 





CA-3′
GG-3′





(SEQ ID NO: 14)
(SEQ ID NO: 15)




 


AT3G05650
5′-ACG CGT CGA CCC 
5′-TGT ACA TGT ACA 
Sal I
BsrG I



ATC ACA CAC ACA
CAG CGT AAA TGA 





TAC ACA CAC-3′
AGA ACA CCC CAA 





(SEQ ID NO: 16)
ACT GAA C-3′ 






(SEQ ID NO: 17)




 


AT2G33080
5′-ACG CGT CGA CAT 
5′-TGT ACA TGT ACA 
Sal I
BsrG I



GTC AGG ATC ACA 
TCA GCA CTT GCT 





TCT GCG TTT GC-3′
CCT GTT CTT CG-3′





(SEQ ID NO: 18)
(SEQ ID NO: 19)




 


AT3G05660
5′-ACG CGT CGA CGG 
5′-AAG GAA AAA AGC 
Sal I
Not I



AGA AGC AAA ACC 
GGC CGC CCG TAC 





TCA TAG AAG TCA 
AAC GTT GCT TTC 





ATG AGT CTC ATT 
TTC GCC GAC GTC-3′





CC-3′
(SEQ ID NO: 21)





(SEQ ID NO: 20)









According to the method described in 1-2-2, a template DNA was prepared from wild-type Arabidopsis thaliana (Col-0 ecotype). Takara Ex Taq (Takara Bio Inc.), Platinum Pfx DNA Polymerase (Invitrogen), or Phusion High-Fidelity DNA Polymerase (NEW ENGLAND BioLabs: NEB) was used as an enzyme. The primer pairs listed in Table 7 were used as primers. The relevant PCR solution composition and reaction conditions were used according to protocols included with each enzyme. PCR amplification products were subjected to electrophoresis with 2% agarose gel (TAE buffer) and then fragments were stained with ethidium bromide. A gel containing target fragments was excised using a scalpel. Target DNA fragments were eluted and purified using a GFX PCR DNA and GEL Band Purification Kit (Amersham). Adenin was added to the thus obtained DNA fragment using an A-Addition Kit (QIAGEN). The amplified DNA to which adenine had been added was ligated to a TA-Cloning pCR2.1 vector using a TOPO TA Cloning Kit (Invitrogen) and then transformed into competent cells (E. coli TOP 10) included with the kit. After transformation, cells were cultured in LB medium supplemented with 50 μl/ml kanamycin and then transformants were selected. Colonies that had appeared were subjected to liquid culture in LB medium supplemented with 50 μl/ml kanamycin. Plasmid DNA was prepared from the thus obtained microorganisms using a Plasmid Mini Kit (QIAGEN).
The thus obtained fragment containing ORF of the LRR-RLK gene (AT1G69990) was cloned into a vector, the thus obtained fragment containing ORF of the LRR-RLK gene (AT5G39390) was cloned into a vector, the thus obtained fragment containing ORF of the LRR (protein gene (AT3G05650) was cloned into a vector, the thus obtained fragment containing ORF of the LRR protein gene (AT2G33080) was cloned into a vector, and the thus obtained fragment containing ORF of the LRR protein gene (AT3G05660) was cloned into a vector, followed by determination of the nucleotide sequences and sequence analysis.
1-2-6. Construction of Plant Expression Vector
A fragment containing ORF of the LRR-RLK gene (AT1G69990), the LRR-RLK gene (AT5G39390), the LRR protein gene (AT3G05650), the LRR protein gene (AT2G33080), or the protein gene (AT3G05660) was inserted into a plant expression vector pBI121 containing an omega sequence from tobacco mosaic virus. Thus, constructs were prepared.
First, the pCR2.1 vector, in which a fragment containing ORF of the LRR-RLK gene (AT1G69990) had been cloned in 1-2-5, was treated with restriction enzymes Sal I and BsrG I.
Next, similarly pBI121 containing an omega sequence was treated with restriction enzymes Sal I and BsrG I. The products digested with these restriction enzymes were subjected to 0.8% agarose gel electrophoresis. A fragment of about 1850 bp containing ORF of the LRR-RLK gene (AT1G69990) and pBI121 containing the omega sequence were each fractioned and purified from the gel using a GFX PCR DNA and GEL Band Purification Kit (Amersham).
For introduction of a fragment containing ORF of the LRR-RLK gene (AT1G69990) using a pBI121 fragment containing the omega sequence as a vector, the vector and the insert were mixed at a ratio of 1:10, followed by an overnight ligation reaction at 16° C. using an equivalent amount of a TaKaRa Ligation kit ver. 2 (Takara Bio Inc.).
The total amount of the reaction solution was added to 100 μl of competent cells (E. coli strain DH5a, TOYOBO), so that transformation was performed according to protocols included with the kit. Cells were applied to LB agar medium containing 50 μg/ml kanamycin and then cultured overnight. Colonies that had appeared were subjected to liquid culture in LB medium supplemented with 50 μg/ml kanamycin. Plasmid DNA was prepared from the thus obtained microorganisms using a Plasmid Mini Kit (QIAGEN).
The thus obtained fragment containing ORF of the LRR-RLK gene (AT1G69990) was subcloned into an expression vector, followed by determination of the nucleotide sequences and sequence analysis.
In addition, the LRR-RLK gene (AT5G39390) and the LRR protein gene (AT2G33080) were incorporated into expression vectors according to the above method, except that primers listed in Table 7 were used, followed by determination of the nucleotide sequences and sequence analysis. The LRR protein gene (AT3G05650) was cloned into a TA-Cloning pCR2.1 vector. Then the vector was treated with an restriction enzyme EcoR I, blunt-ended using a DNA Blunting Kit (Takara Bio Inc.), treated with phenol chloroform, and then treated with a restriction enzyme BsrG I. pBI121 containing an omega sequence was treated with a restriction enzyme Sal I, blunt-ended using a DNA Blunting Kit (Takara Bio Inc.), treated with phenol chloroform, and then treated with a restriction enzyme BsrG I. The resultant was incorporated into an expression vector according to the above method, followed by determination of the nucleotide sequence and sequence analysis. The protein gene (AT3G05660) was treated with a restriction enzyme Not I, blunt-ended using a DNA Blunting Kit (Takara Bio Inc.), treated with phenol chloroform, and then treated with a restriction enzyme Sal I. pBI121 containing an omega sequence was treated with a restriction enzyme BsrG I, blunt-ended using a DNA Blunting Kit (Takara Bio Inc.), treated with phenol chloroform, and then treated with a restriction enzyme Sal I. The resultant was incorporated into an expression vector according to the above method, followed by determination of the nucleotide sequences and sequence analysis.
1-2-7. Gene Introduction into Arabidopsis thaliana Using Agrobacterium Method
The plant expression vector constructed in 1-2-6 was introduced into the Agrobacterium tumefaciens C58C1 strain by electroporation (Plant Molecular Biology Manual, Second Edition, B. G. Stanton and A. S. Robbert, Kluwer Acdemic Publishers 1994). Subsequently, Agrobacterium tumefaciens into which the plant expression vector had been introduced was introduced into wild-type Arabidopsis thaliana (ecotype Col-0) by an infiltration method described by Clough et al. (The Plant Journal 16, 735-743, 1998).
Transformants were selected using kanamycin-containing medium. T2 generation plants were produced by self-pollination.
1-2-8. Confirmation of the Phenotype of Transformant
Measurement of Amount of Biomass:
T2 seeds produced in 1-2-7 were each aseptically sowed in MS agar medium containing 50 mg/L kanamycin and 0.5% sucrose. After 2 weeks, the resulting plants were transplanted into pots (each with a diameter of 50 mm) containing vermiculite mixed soil. As control plants, Arabidopsis plants that had not undergone recombination and had been aseptically sowed in MS agar medium containing 0.5% sucrose were transplanted. They were cultivated under conditions of 23° C. and 8 hours in the light/16 hours in the dark (short-day conditions) and with a light intensity of about 160 μE/cm2 for a total of 6 weeks after transplantation. After cultivation, above-ground parts of the plants were placed in paper bags and dried under conditions of 22° C. and humidity of 60% for 2 weeks. The total amounts of biomass were weighed using an electronic balance.
2. Results
Regarding the results of 1-2-8 (Measurement of amount of biomass), FIG. 3 shows a photo of the above-ground parts of wild-type plants and transformed plants into which a fragment containing ORF of the protein gene (AT3G05660) had been introduced. Also, FIG. 4 shows the results of measuring the total amounts of biomass of the above-ground parts of a wild-type plant, a transformed plant into which the LRR-RLK protein gene (AT1G69990) had been introduced, a transformed plant into which the LRR-RLK protein gene (AT5G39390) had been introduced, a transformed plant into which the LRR protein gene (AT3G05650) had been introduced, a transformed plant into which the LRR protein gene (AT2G33080) had been introduced, and a transformed plant into which the protein gene (AT3G05660) had been introduced.
As shown in FIGS. 3 and 4, it was revealed that in the case of transformed plants into which the fragment containing ORF of the protein gene (AT3G05660) had been introduced, the total amount of biomass of the above-ground parts was much greater than that of a wild-type plant (by about 1.5 times). Meanwhile, it was revealed that in the case of a transformed plant into which the LRR-RLK gene (AT1G69990), the LRR-RLK gene (AT5G39390), the LRR protein gene (AT3G05650), or the LRR protein gene (AT2G33080) had been introduced, the amount of biomass was almost equivalent to that of a wild-type plant.
Based on the above results, it was revealed that the amounts of biomass would be drastically improved in plants into which the AT3G05660 gene had been introduced.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A method for increasing the production of biomass in a plant as compared to a wild-type plant, wherein said method comprises step (i) or (ii):
(i) introducing, into a plant, a polynucleotide encoding a protein selected from the group consisting of (a) to (b); or (ii) activating an expression control region of an endogenous gene encoding a protein selected from the group consisting of (a) to (b), wherein proteins (a) to (b) are:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 3; and
(b) a protein comprising the amino acid sequence of SEQ ID NO: 3 but in which 1-10 amino acids have been deleted, substituted, and/or added in the sequence of SEQ ID NO: 3.

2. The method according to claim 1, wherein said plant is a dicotyledon.

3. The method according to claim 1, wherein said plant is of the family Brassicaceae.

4. The method according to claim 1, wherein said plant is Arabidopsis thaliana.

5. A method for selecting a plant with increased production of biomass, wherein said method comprises steps (1) to (3):
(1) the step of (i) introducing, into a plant, a polynucleotide encoding a protein selected from the group consisting of (a) to (b); or (ii) activating, in a plant, an expression control region of an endogenous gene encoding a protein selected from the group consisting of (a) to (b), wherein proteins (a) to (b) are:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 3; and
(b) a protein comprising the amino acid sequence of SEQ ID NO: 3 but in which 1-10 amino acids have been deleted, substituted, and/or added in the sequence of SEQ ID NO: 3;

(2) measuring the amount of biomass produced by the plant resulting from step (1); and
(3) selecting the plant resulting from step (1) when the plant exhibits increased production of biomass in comparison with a wild-type plant.

6. The method according to claim 5, wherein said plant is a dicotyledon.

7. The method according to claim 5, wherein said plant is of the family Brassicaceae.

8. The method according to claim 5, wherein said plant is Arabidopsis thaliana.