# Patent

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

## Abstract

A technique by which the production of plant biomass can be significantly increased is provided. A protein phosphatase 2C gene having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is over-expressed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a divisional patent application of U.S. patent application Ser. No. 12/922,432 filed on Dec. 3, 2010, which is a National Stage of International Application No. PCT/JP2009/054953 filed Mar. 13, 2009, claiming priority based on Japanese Patent Application No. JP 2008-066460 filed Mar. 14, 2008, the contents of all of which are incorporated herein by reference in their entirety.


TECHNICAL FIELD
The present invention relates to: a plant in which a given gene is over-expressed; a method for increasing the production of biomass and/or seeds through overexpression of a given gene; and a method for producing such plant capable of producing an increased amount of biomass and/or seeds.
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 production. 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 introduction takes place, desirable traits cannot always be acquired.
Various gene introduction techniques are known as techniques for increasing the production of plant biomass, as disclosed in Patent Documents 1-7, for example. However, none of these techniques can be said to exert sufficient effects of increasing the production of biomass.
PATENT DOCUMENTS


    Patent Document 1: JP Patent Publication (Kohyo) No. 2001-505410 A
    Patent Document 2: JP Patent Publication (Kohyo) No. 2001-519659 A
    Patent Document 3: JP Patent Publication (Kohyo) No. 2007-530063 A
    Patent Document 4: JP Patent Publication (Kokai) No. 2005-130770 A
    Patent Document 5: JP Patent Publication (Kohyo) No. 2000-515020 A
    Patent Document 6: JP Patent Publication (Kohyo) No. 9-503389 A
    Patent Document 7: JP Patent Publication (Kokai) No. 2005-52114 A


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 plant biomass 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 increased by causing overexpression of a gene encoding protein phosphatase 2C having characteristic consensus sequences. Thus, they have completed the present invention.
Specifically, the plant according to the present invention is a plant in which a gene encoding protein phosphatase 2C having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is over-expressed.
Also, the method for increasing the production of biomass according to the present invention comprises causing the overexpression of a gene encoding protein phosphatase 2C having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order.
Furthermore, the method for producing a plant according to the present invention comprises the steps of: preparing a transformed plant in which a gene encoding protein phosphatase 2C having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is over-expressed; and measuring the amount of biomass of a progeny plant 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 encoding protein phosphatase 2C can be at least one type of gene selected from the group consisting of At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), At5g27930-AtPP2C6-7 (SEQ ID NO:36), At2g20050 (SEQ ID NO:66), and At3g06270 (SEQ ID NO:67), or a gene functionally equivalent thereto.
In the present invention, the gene encoding protein phosphatase 2C preferably encodes any one of the following proteins (a) to (c):
(a) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and 48;
(b) a 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 an amino acid sequence selected from the group consisting of SEQ ID NOS: 5, 7, 36, 42, and 48 and having protein phosphatase 2C activity; and
(c) a protein that is encoded by a polynucleotide hybridizing under stringent conditions to a polynucleotide comprising a nucleotide sequence complementary to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 4, 6, 35, 41, and 47 and has protein phosphatase 2C activity.
Also, in the present invention, an example of the above functionally equivalent gene is a protein phosphatase 2C gene from an organism other than Arabidopsis thaliana. Another example of an organism other than Arabidopsis thaliana is an organism selected from the group consisting of rice (Oryza sativa), black cottonwood (Populus trichocarpa), european grape (Vitis vinifera), Medicago truncatula (Medicago truncatula), alfalfa (Medicago sativa), Physcomitrella patens (Physcomitrella patens), ice plant (Mesembryanthemum crystallinum), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), corn (Zea mays), rapeseed (Brassica rapa), tomato (Solanum lycopersicum), monkey flower (Mimulus guttatus), and monocellular red alga (Cyanidioschyzon merolae).
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.
Effect of the Invention
The plant according to the present invention is a plant capable of producing significantly improved amount of biomass and/or seeds compared with wild-type plants. Also, the method for increasing the production of biomass and/or seeds according to the present invention can realize the significantly increased production of biomass and/or seeds 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 and/or seeds 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. Also, through application of the present invention, for example, the productivity can be improved when seeds are directly products or ingredients contained in seeds are directly products and this can be achieved at lower cost.
This description hereby incorporates the entire content of the description and/or the drawings of Japanese Patent Application No. 2008-066460, which is the basis of the priority claim of this application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-1, 1-2 and 1-3 are characteristic diagrams showing the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program for amino acid sequences encoded by At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), At5g27930-AtPP2C6-7 (SEQ ID NO:36), At2g20050 (SEQ ID NO:66), and At3g06270 (SEQ ID NO:67).
FIGS. 2-1, 2-2 and 2-3 are characteristic diagrams showing the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program for amino acid sequences encoded by At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), and At5g27930-AtPP2C6-7 (SEQ ID NO:36).
FIG. 3 is a photo showing the above-ground parts of wild-type plants and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was introduced.
FIG. 4 is a characteristic diagram showing the results of measuring the amounts of biomass of the above-ground parts of wild-type plants and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was introduced. The result for the wild-type plants is the average value for 12 individual wild-type plants and each result for the transformed plants is the average value for 5 individual transformed plants.
FIG. 5 is a characteristic diagram showing the results of measuring the amounts of seeds of wild-type plants and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was introduced. The result for wild-type plants is the average value for 12 individual wild-type plants and each result for the transformed plants is the average value for 5 individual transformed plants.
FIG. 6 shows photos showing the above-ground parts of a rice control plant into which a plant expression vector (constructed by ligating 3 multiple cloning sites of a pST-Bluel vector to pBI-sGFP-R4R3) was introduced and a transformed rice plant into which the coding region of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) was introduced.
FIG. 7 shows photos showing the above-ground parts of a control rice plant into which no gene was introduced and a transformed rice plant into which the coding region of rice-derived PP2C (protein phosphatase 2C) (Os05g0358500) was introduced.
FIG. 8 is a photo showing the above-ground parts of a wild type plant and a transformed plant into which a fragment containing ORF of a PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) was introduced.
FIG. 9 is a photo showing the above-ground parts of a wild-type plant and a transformed plant into which a fragment containing ORF of a PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) was introduced.
FIG. 10 is a photo showing the above-ground parts of a wild-type plant and transformed plants into which a fragment containing ORF of a PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)) was introduced.
FIG. 11 is a photo showing the above-ground parts of a wild-type plant and transformed plants into which a fragment containing ORF of a PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)) was introduced.
FIG. 12 is a photo showing the above-ground parts of a wild-type plant and transformed plants into which the coding region of a rice-derived PP2C (protein phosphatase 2C) gene (Os05g0358500) was introduced.



BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be described in detail as follows.
The plant according to the present invention is a plant in which: a gene encoding protein phosphatase 2C having characteristic consensus sequences is over-expressed; and the amount of biomass is significantly improved compared with wild-type plants. The plant according to the present invention may be a plant in which the protein phosphatase 2C gene is over-expressed in all plant tissues or at least some plant tissues. Here, the term “plant tissue(s)” refers to plant organ(s) such as leaves, stems, seeds, roots, and flowers.
Here, the term “overexpression” refers to an expression level that can be confirmed as a transcript as a result of transcription of the protein phosphatase 2C gene introduced into a plant.
Protein Phosphatase 2C Gene
The protein phosphatase 2C gene to be over-expressed in a plant encodes protein phosphatase 2C that has 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side. In addition, a gene group classified as Group E as in FIG. 1 of Topographic cladogram (on page 237 of Reference: TRENDS in Plant Science Vol. 9 No. 5 May 2004 pages 236-243) encodes protein phosphatase 2C having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side. In addition, the reference predicts the presence of 76 protein phosphatase 2C genes in Arabidopsis thaliana and discloses the results of producing a phylogenetic tree of these genes using T-Coffee software (reference; Notredame, C. et al. 2000 T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205-247) as in FIGS. 1-1, 1-2 and 1-3. In this phylogenetic tree, protein phosphatase 2C genes classified as members of Group E encode protein phosphatase 2C that has 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side. The 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 are characteristic sequences in Group E in the above-mentioned classification and serve as a basis for clear differentiation from other groups.
Group E in the above classification includes protein phosphatase 2C genes specified by Arabidopsis thaliana-derived At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), At5g27930-AtPP2C6-7 (SEQ ID NO:36), At2g20050 (SEQ ID NO:66), and At3g06270 (SEQ ID NO:67). FIGS. 1-1, 1-2 and 1-3 show the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program (which can be used with the DDBJ of the National Institute of Genetics (on world wide web at clustalw.ddbj.nig.ac.jp/top-j)) for the amino acid sequences encoded by these Arabidopsis thaliana-derived protein phosphatase 2C genes, At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), At5g27930-AtPP2C6-7 (SEQ ID NO:36), At2g20050 (SEQ ID NO:66), and At3g06270 (SEQ ID NO:67) (with the amino acid (sequence) substitution matrix used herein being a default matrix known as BLOSUM (Blocks of Amino Acid Substitution Matrix)). As shown in FIGS. 1-1, 1-2 and 1-3, these protein phosphatase 2C genes classified as members of Group E have consensus sequences characteristic in the regions denoted as I to III. These regions denoted as I to III are subjected with a rice-derived protein phosphatase 2C gene (described later) to alignment analysis, so that the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 can be defined.
Herein, in the amino acid sequence shown in SEQ ID NO: 1, which is an amino acid residue denoted as “Xaa,” may be any amino acid, and it is not limited to any particular amino acid. However, the 1st amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably leucine (three character code: Leu and single character code: L; the same applies to the following) or phenylalanine (Phe, F). The 4th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably valine (Val, V), isoleucine (Ile, I), or methionine (Met, M). The 16th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably serine (Ser, S) or alanine (Ala, A). The 17th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 1 is preferably lysine (Lys, K), arginine (Arg, R), glutamine (Gln, Q), or asparagine (Asn, N). More specifically, a consensus sequence comprising the amino acid sequence shown in SEQ ID NO: 1 is preferably (L/F)XG(V/I/M)FDGHGXXGXXX(S/A)(K/R/Q/N)XV. In such amino acid sequence, pluralities of amino acids in parentheses represent possible variations of amino acid residues at the relevant positions. Also, in the following amino acid sequences, “X” means that any amino acid residue may be present at the relevant position.
Also, such a consensus sequence may be a sequence containing the following 3 amino acid residues on the N-terminal side of Region I in FIGS. 1-1, 1-2 and 1-3: (D/E/N)XX.
Here, in the amino acid sequence shown in SEQ ID NO: 2, an amino acid residue denoted as “Xaa,” may be any amino acid, and it is not limited to any particular amino acid. However, the 5th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G), alanine (Ala, A), or serine (Ser, S). The 6th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably valine (Val, V), leucine (Leu, L), or isoleucine (Ile, I). The 9th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), methionine (Met, M), or leucine (Leu, L). The 12th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G) or alanine (Ala, A). The 15th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably leucine (Leu, L), valine (Val, V), or isoleucine (Ile, I). The 17th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably isoleucine (Ile, I), valine (Val, V), or methionine (Met, M). The 18th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably glycine (Gly, G) or alanine (Ala, A). The 22nd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably aspartic acid (Asp, D) or histidine (His, H). The 26th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably valine (Val, V) or isoleucine (Ile, I). The 27th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 2 is preferably leucine (Leu, L), methionine (Met, M), or isoleucine (Ile, I). More specifically, a consensus sequence comprising the amino acid sequence shown in SEQ ID NO: 2 is preferably SGXT(G/A/S)(V/L/I)XX(I/V/F/M/L)XX(G/A)XX(L/V/I)X(I/V/M)(A/G)NXG(D/H)SRA(V/I)(L/M/I). In such amino acid sequence, pluralities of amino acids in parentheses represent possible variations of amino acid residues at the relevant positions. Also, in the following amino acid sequences, “X” means that any amino acid residue may be present at the relevant position.
Here, the amino acid sequence shown in SEQ ID NO: 3, an amino acid residue denoted as “Xaa,” may be any amino acid, and it is not limited to any particular amino acid. However, the 4th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably methionine (Met, M), valine (Val, V), or phenylalanine (Phe, F). The 5th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably serine (Ser, S), alanine (Ala, A), or threonine (Thr, T). The 7th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably alanine (Ala, A) or serine (Ser, S). The 8th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably phenylalanine (Phe, F), isoleucine (Ile, I), or valine (Val, V). The 14th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably lysine (Lys, K) or glutamic acid (Glu, E). The 18th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V) or leucine (Leu, L). The 19th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine (Ile, I) or valine (Val, V). The 23rd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably glutamic acid (Glu, E), glutamine (Gln, Q), or aspartic acid (Asp, D). The 24th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine (Ile, I), valine (Val, V), or phenylalanine (Phe, F). The 29th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine (Ile, I), leucine (Leu, L), or valine (Val, V). The 30th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably serine (Ser, S), threonine (Thr, T), or asparagine (Asn, N). The 33rd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably aspartic acid (Asp, D), asparagine (Asn, N), or histidine (His, H). The 35th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably phenylalanine (Phe, F) or tyrosine (Tyr, Y). The 36th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), phenylalanine (Phe, F), or methionine (Met, M). The 37th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V), leucine (Leu, L), or isoleucine (Ile, I). The 38th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L) or valine (Val, V). The 40th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably threonine (Thr, T) or serine (Ser, S). The 43rd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V), isoleucine (Ile, I), or methionine (Met, M). The 44th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably tryptophan (Trp, W) or phenylalanine (Phe, F). The 45th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably aspartic acid (Asp, D) or glutamic acid (Glu, E). The 47th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L), isoleucine (Ile, I), or methionine (Met, M). The 48th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably serine (Ser, S), threonine (Thr, T), or proline (Pro, P). The 49th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably asparagine (Asn, N) or serine (Ser, S). The 52nd amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably valine (Val, V) or alanine (Ala, A). The 55th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably leucine (Leu, L), valine (Val, V), isoleucine (Ile, I), or methionine (Met, M). The 56th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is preferably isoleucine (Ile, I) or valine (Val, V). Preferably, an example of the consensus sequence comprising the amino acid sequence shown in SEQ ID NO: 3 is more specifically GXA(M/V/F)(S/A/T)R(A/S)(F/I/V)GDXXX(K/E)XXG(V/L)(I/V)XXP(E/Q/D)(I/V/F)XXXX(I/L/V)(T/S)XX(D/N/H)X(F/Y)(L/I/V/F)(V/L/I)(L/V)A(T/S)DG(V/I/M)(W/F)(D/E)X(L/I/M)(S/T/P)(N/S)XX(V/A)XX(L/V/I/M)(I/V). In such amino acid sequence, pluralities of amino acids in parentheses represent possible variations of amino acid residues at the relevant positions. Also, in the following amino acid sequences, “X” means that any amino acid residue may be present at the relevant position.
Here, the 20th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is more preferably alanine (Ala, A), serine (Ser, S), or cysteine (Cys, C). Also, the 50th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 3 is more preferably aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), glutamine (Gln, Q), or asparagine (Asn, N).
Variations of amino acid residues that can be present at given positions are determined based on the following reasons. As described in Reference (1) (“McKee Biochemistry,” 3rd ed., Chapter 5 Amino Acid•Peptide•Protein 5.1 Amino Acid; editorial supervisor: Atsushi Ichikawa; translation supervisor: Shinichi Fukuoka; publisher: Ryosuke Sone; publishing office: Kagaku-Dojin Publishing Company, INC, ISBN4-7598-0944-9), it is well known that amino acids are classified based on side chains having similar properties (e.g., chemical properties and physical sizes). Also, it is well known that molecular evolutionary substitution frequently takes place among amino acid residues classified in a given group, while retaining protein activity. Based on these concepts, a substitution (mutation) score matrix for amino acid residues (BLOSUM: Blocks of Amino Acid Substitution Matrix) is proposed in FIGS. 2-1, 2-2 and 2-3 of Reference (2): Henikoff S., Henikoff J. G., Amino-acid substitution matrices from protein blocks, Proc. Natl. Acad. Sci. U.S.A., 89, 10915-10919 (1992) and is broadly used. Reference (2) is based on a finding that amino acid substitutions that take place among amino acids with side chains having similar chemical properties result in less structural or functional changes in the entire protein. According to References (1) and (2) above, amino acid side chain groups to be used in multiple alignment can be considered based on indices such as chemical properties and physical sizes. They are shown as amino acid groups with a score of 0 or higher and preferably as amino acid groups with a score of 1 or higher through the use of the score matrix (BLOSUM) disclosed in Reference (2). Typical groups are the following 8 groups. Further precisely grouped amino acid groups may be amino acid groups with a score of 0 or higher, preferably a score of 1 or higher, and further preferably a score of 2 or higher.
1) Aliphatic Hydrophobic Amino Acid Group (ILMV Group)
This group is a group of amino acids having aliphatic hydrophobic side chains, among neutral nonpolar amino acids disclosed in Reference (1) above, which is composed of V (Val, valine), L (Leu, leucine), I (Ile, isoleucine), and M (Met, methionine). Among amino acids classified as neutral nonpolar amino acids according to Reference (1), FGACWP is not included in this “aliphatic hydrophobic amino acid group” because of the following reasons: G (Gly, glycine) and A (Ala, alanine) are the same size as that of or smaller in size than a methyl group and have weak non polar effects; C (Cys, cysteine) may play an important role in S—S bonds and has a property of forming a hydrogen bond with an oxygen atom or a nitrogen atom; F (Phe, phenylalanine) and W (Trp, tryptophan) have side chains with significantly large molecular weights and have strong aromatic effects; P (Pro, proline) has strong imino acid effects, so as to fix the angle of the main chain of the polypeptide.
2) Group Having Hydroxymethylene Group (ST Group)
This group is a group of amino acids (from among neutral polar amino acids) having hydroxymethylene groups in side chains, which is composed of S (Ser, serine) and T (Thr, threonine). Hydroxy groups existing in the side chains of S and T constitute sugar-binding sites, so that these sites are often important for a polypeptide (protein) to have specific activity.
3) Acidic Amino Acid (DE Group)
This group is a group of amino acids having acidic carboxyl groups in side chains, which is composed of D (Asp, aspartic acid) and E (Glu, glutamic acid).
4) Basic Amino Acid (KR Group)
This group is a group of basic amino acids, which is composed of K (Lys, lysine) and R (Arg, arginine). These K and R are positively charged within a wide pH range and have basic properties. On the other hand, H (His, histidine) classified in basic amino acids is almost never ionized at pH 7, so that H is not classified in this group.
5) Methylene Group=Polar Group (DHN Group)
This group is characterized in that: in all cases, a methylene group as a side chain binds to an α-carbon element beyond which a polar group is present; and the physical sizes of methylene groups (nonpolar groups) closely resemble from each other. This group is composed of N (Asn, asparagine; polar group is an amide group), D (Asp, aspartic acid; polar groups are carboxyl groups), and H (His, histidine; polar groups are imidazole groups).
6) Dimethylene Group=Polar Group (EKQR Group)
This group is characterized in that: in all cases, linear hydrocarbon having a length longer than that of a dimethylene group binds as a side chain to an α-carbon element, beyond which a polar group is present; and the physical sizes of dimethylene groups that are nonpolar groups closely resemble from each other. This group is composed of E (Glu, glutamic acid, polar group is a carboxyl group), K (Lys, lysine; polar groups are amino groups), Q (Gln, glutamine; polar groups are amide groups), and R (Arg, arginine; polar groups are imino groups and amino groups).
7) Aromatic Series (FYW Group)
This group is a group of aromatic amino acids having benzene nuclei in the side chains and characterized by having chemical properties unique in aromatic series. This group is composed of F (Phe, phenylalanine), Y (Tyr, tyrosine), and W (Trp, tryptophan).
8) Ring & Polar (HY Group)
This group is a group of amino acids having both ring structures in the side chains and polarity, which is composed of H (H, histidine; Both ring structures and polar groups are imidazole groups), and Y (Tyr, tyrosine; Ring structures are benzene nuclei and polar groups are hydroxy groups).
As described above, it is understood that: in the given amino acid sequences shown in SEQ ID NOS: 1-3, an amino acid residue denoted as Xaa may be any amino acid; or amino acid residues denoted as Xaa may be substituted with each other within the above groups 1)-8). Hence, in the present invention, the protein phosphatase 2C gene to be over-expressed in a plant may be a protein phosphatase 2C gene from any plant, as long as it has the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side.
More specifically, examples of an Arabidopsis thaliana protein phosphatase 2C-coding gene having the 3 consensus sequences (comprising the amino acid sequences shown in SEQ ID NOS: 1-3) in such order from the N-terminal side include At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), At5g27930-AtPP2C6-7 (SEQ ID NO:36), At2g20050 (SEQ ID NO:66), and At3g06270 (SEQ ID NO:67). In the present invention, at least one type of gene selected from the gene group is over-expressed. Particularly in the present invention, it is preferable to cause overexpression of at least one type of gene selected from among At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), and At5g27930-AtPP2C6-7 (SEQ ID NO:36). Particularly, in the present invention, it is more preferable to cause overexpression of at least one type of gene selected from among At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), and At5g27930-AtPP2C6-7 (SEQ ID NO:36) and it is most preferable to cause overexpression of a gene specified by At3g05640 (SEQ ID NO:5).
In addition, FIGS. 2-1, 2-2 and 2-3 show the results of alignment analysis using a CLUSTAL W (1.83) multiple sequence alignment program (that can be used with the DDBJ of the National Institute of Genetics (on world wide web at clustalw.ddbj.nig.ac.jp/top-j)) for amino acid sequences encoded by At1g03590 (SEQ ID NO:63)-AtPP2C6-6, At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), and At5g27930 (SEQ ID NO:36)-AtPP2C6-7 (amino acid (sequence) substitution matrix used herein is default matrix, BLOSUM (Blocks of Amino Acid Substitution Matrix)).
That is, FIGS. 2-1, 2-2 and 2-3 show the 3 consensus sequences in protein phosphatase 2C encoded by At1g03590-AtPP2C6-6 (SEQ ID NO:63), At1g16220 (SEQ ID NO:61), At1g79630 (SEQ ID NO:62), At5g01700 (SEQ ID NO:65), At3g02750 (SEQ ID NO:42), At5g36250 (SEQ ID NO:64), At5g26010 (SEQ ID NO:59), At4g32950 (SEQ ID NO:60), At3g16800 (SEQ ID NO:48), At3g05640 (SEQ ID NO:5), and At5g27930-AtPP2C6-7 (SEQ ID NO:36). Regions denoted as I-III in FIGS. 2-1, 2-2 and 2-3 are subjected with an ortholog of a rice-derived protein phosphatase 2C gene (described later) to alignment analysis, so that the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 above can be defined as the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 53-55, respectively.
The consensus sequence shown in SEQ ID NO: 53 is more specifically (L/F)CG(V/I/M)FDGHGXXGXX(V/I)(S/A)(K/R)XV. The consensus sequence shown in SEQ ID NO: 54 is more specifically SGXT(G/A/S)(V/L)XX(I/V/F/L)XX(G/A)XX(L/V/I)X(I/V/M)(A/G)NXG(D/H)SRA(V/I)(L/M/I). The consensus sequence shown in SEQ ID NO: 55 is more specifically GLA(M/V)(S/A)R(A/S)(F/L)GDXX(L/I/V)KX(Y/F/H)G(V/L)(I/V)XXP(E/Q/D)(I/V/F)XXXX(I/L/V)(T/S)XXDX(F/Y)(L/I/V/M)(V/L/I)LA(T/S)DG(V/I/M)WDX(L/I/M/V)(S/T)NX(E/D)(V/A)XX(L/V/I)(I/V).
In addition, in such amino acid sequences, pluralities of amino acids in parentheses represent possible variations of amino acid residues at the relevant positions. Also, in these amino acid sequences, “X” means that any amino acid residue may be present at the relevant position.
Here, the 9th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 54 is more preferably isoleucine (Ile, I), valine (Val, V), or phenylalanine (Phe, F). Also, the 11th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 54 is more preferably glutamine (Gln, Q) or histidine (His, H). Moreover, the 13th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 54 is more preferably lysine (Lys, K), glutamic acid (Glu, E), serine (Ser, S), glutamine (Gln, Q), aspartic acid (Asp, D), or asparagine (Asn, N).
Here, the 7th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably alanine (Ala, A). Also, the 8th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably phenylalanine (Phe, F). Moreover, the 11th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably phenylalanine (Phe, F) or tyrosine (Tyr, Y). Furthermore, the 13th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably leucine (Leu, L) or isoleucine (Ile, I). Moreover, the 15th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably aspartic acid (Asp, D), serine (Ser, S), or glutamic acid (Glu, E). Furthermore, the 20th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably serine (Ser, S), alanine (Ala, A), or cysteine (Cys, C). Moreover, the 27th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably histidine (His, H) or arginine (Arg, R). Furthermore, the 34th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably glutamine (Gln, Q), glutamic acid (Glu, E), or histidine (His, H). Furthermore, the 36th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably leucine (Leu, L), isoleucine (Ile, I), or valine (Val, V). Furthermore, the 47th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably leucine (Leu, L), isoleucine (Ile, I), or valine (Val, V). Furthermore, the 50th amino acid residue from the N-terminal side in the amino acid sequence shown in SEQ ID NO: 55 is more preferably lysine (Lys, K), glutamic acid (Glu, E), glutamine (Gln, Q), aspartic acid (Asp, D), or asparagine (Asn, N).
As examples, the nucleotide sequence of the coding region in the gene specified by At3g05640 (SEQ ID NO:5) is shown in SEQ ID NO: 4 and the amino acid sequence of protein phosphatase 2C encoded by the gene specified by At3g05640 is shown in SEQ ID NO: 5. Also, the nucleotide sequence of the coding region in the gene specified by At5g27930 (SEQ ID NO:36) is shown in SEQ ID NO: 35 and the amino acid sequence of protein phosphatase 2C encoded by the gene specified by At5g27930 is shown in SEQ ID NO: 36. Moreover, the nucleotide sequence of the coding region in the gene specified by At3g02750 (SEQ ID NO:42) is shown in SEQ ID NO: 41 and the amino acid sequence of protein phosphatase 2C encoded by the gene specified by At3g02750 is shown in SEQ ID NO: 42. Furthermore, the nucleotide sequence of the coding region in the gene specified by At3g16800 (SEQ ID NO:48) is shown in SEQ ID NO: 47 and the amino acid sequence of protein phosphatase 2C encoded by the gene specified by At3g16800 is shown in SEQ ID NO: 48.
Also, in the present invention, genes functionally equivalent to genes listed above may also be over-expressed. Here, the term “functionally equivalent gene” refers to, for example, a gene (from an organism other than Arabidopsis thaliana) that: has the 3 consensus sequences (preferably, the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 53-55. The same applies to the following) comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side; and encodes protein phosphatase 2C. Also, the term “functionally equivalent gene” refers to a gene that encodes a protein having protein phosphatase 2C activity. The term “protein phosphatase 2C activity” refers to Mg2+- or Mn2+-dependent serine/threonine phosphatase (Ser/Thr phosphatase) activity. Therefore, whether or not a gene encodes a protein having protein phosphatase 2C activity can be confirmed by examining whether or not the gene product has serine/threonine phosphatase activity in the presence of Mg2+ or Mn2+. Conventionally known techniques can be appropriately employed for determining serine/threonine phosphatase activity. For example, a commercially available activity determination kit ProFluor (registered trademark) Ser/Thr Phosphatase Assay (Promega) can be used.
Here, example of organisms is not limited to Arabidopsis thaliana. For example, rice (Oryza sativa) is included. Specifically, an example of a functionally equivalent gene is a rice Os05g0358500 gene. The nucleotide sequence of a coding region of the Os05g0358500 gene is shown in SEQ ID NO: 6 and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 7. Also, examples of the above-mentioned rice-derived functionally equivalent gene include Os11g0109000 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 8 and 9, respectively), Os12g0108600 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 10 and 11, respectively), Os02g0471500 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 12 and 13, respectively), Os04g0321800 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 14 and 15, respectively), Os11g0417400 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 16 and 17, respectively), Os07g0566200 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 18 and 19, respectively), Os08g0500300 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 20 and 21, respectively), Os02g0224100 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 22 and 23, respectively), and Os02g0281000 (the nucleotide sequence and the amino acid sequence are shown in SEQ ID NOS: 56 and 57, respectively).
Moreover, examples of the above-mentioned functionally equivalent genes from plants other than Arabidopsis thaliana and rice include genes (UniProt data base Accession Nos. A9P973, A9PFS0, and A9P7U4) from black cottonwood (Populus trichocarpa), genes (UniProt data base Accession Nos. A7PRZ8, A7Q8H4, A7PV59, A5C3B0, A5BF43, A7QFG6, A7P4H7, A5C0C9, A5AP53, A7QQF9, and A5BDP5) from european grape (Vitis vinifera), genes (UniProt data base Accession Nos. Q2HW33 and Q4L0F8) from Medicago truncatula (Medicago truncatula), a gene (GenBank data base Accession No. AY651248) from alfalfa (Medicago sativa), genes (UniProt data base Accession Nos. A9SE70, A9SE69, and A9RFU1) from Physcomitrella patens (Physcomitrella patens), a gene (UniProt data base Accession No. 2511453C) from ice plant (Mesembryanthemum crystallinum), a gene (UniProt data base Accession No. A8HQG8) from Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), genes (GenBank data base Accession Nos. BT024031, BT017414, and BT024134) from corn (Zea mays), genes (GenBank data base Accession Nos. AC189312 and AC189579) from rapeseed (Brassica rapa), genes (GenBank data base Accession Nos. AP009550, AP009302, and AP009278) from tomato (Solanum lycopersicum), a gene (GenBank data base Accession No. AC182571) from monkey flower (Mimulus guttatus), and a gene (GenBank data base Accession No. AP006489) from monocellular red alga (Cyanidioschyzon merolae).
In these plants other than Arabidopsis thaliana, which are represented by the above examples, a gene encoding protein phosphatase 2C that has the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side can be easily searched for and/or identified from a known database such as GenBank based on the above-listed nucleotide sequence of Arabidopsis thaliana-derived protein phosphatase 2C gene or amino acid sequence of protein phosphatase 2C.
In addition, a protein phosphatase 2C gene to be over-expressed in the present invention is not limited to the above described protein phosphatase 2C genes comprising the nucleotide sequences and the amino acid sequences shown in SEQ ID NOS: 4-23, 35, 36, 41, 42, 47, and 48. Hence, the protein phosphatase 2C 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 shown in odd numbers of SEQ ID NOS: 4-23 or the amino acid sequence shown in SEQ ID NO: 36, 42, or 48, and, has protein phosphatase 2C activity. 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 altering a nucleotide sequence encoding the above protein phosphatase 2C 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, protein phosphatase 2C genes to be over-expressed herein may be genes homologous to the protein phosphatase 2C genes comprising the nucleotide sequences and the amino acid sequences shown in SEQ ID NOS: 4-23. 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 protein phosphatase 2C genes comprising the nucleotide sequences and the amino acid sequences shown in SEQ ID NOS: 4-23, 35, 36, 41, 42, 47, and 48 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; the 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 in such order from the N-terminal side; and protein phosphatase 2C 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 protein phosphatase 2C genes comprising the nucleotide sequences and the amino acid sequences shown in SEQ ID NOS: 4-23, 35, 36, 41, 42, 47, and 48 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 protein phosphatase 2C genes comprising the nucleotide sequences shown in even numbers of SEQ ID NOS: 4-23 or the nucleotide sequence shown in 35, 41, or 47. 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 and/or seeds compared with wild-type plants, as a result of overexpression of a protein phosphatase 2C gene having the above described 3 consensus sequences that comprise the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order. Examples of a technique for causing the overexpression of such protein phosphatase 2C gene include a technique for modifying a promoter of an endogenous protein phosphatase 2C gene in a target plant, a technique for introducing an expression vector in which an exogenous protein phosphatase 2C gene is arranged under control of a promoter that enables overexpression, and a technique by which the two above techniques are performed simultaneously.
A preferred example is a technique for introducing an expression vector in which the above protein phosphatase 2C gene is arranged under control of a promoter that enables overexpression into a target plant.
Expression Vector
An expression vector is constructed to contain a promoter that enables overexpression and the above described protein phosphatase 2C 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 overexpression of a protein phosphatase 2C gene within 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 overexpression in a plant can also be used herein. As such promoter, any conventionally known promoter can be used. When the above described protein phosphatase 2C gene is site-specifically over-expressed using such promoter, a plant organ in which the gene is over-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 protein phosphatase 2C 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 and the above protein phosphatase 2C gene, and if necessary, the above other DNA segments may be introduced in an predetermined order. For example, the above protein phosphatase 2C 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 protein phosphatase 2C 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 transfer 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 overexpression of the above-described protein phosphatase 2C 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), rapeseed (Brassica rapa, Brassica napus, Brassica campestris), cabbage (Brassica oleracea var. capitata), napa (Brassica rapa var. pekinensis), ging-geng-cai (Brassica rapa var. chinensis), turnip (Brassica rapa var. rapa), turnip greens (Brassica rapa var. hakabura), potherb mustard (Brassica rapa var. lancinifolia), komatsuna (Brassica rapa var. peruviridis), pak choi (Brassica rapa var. chinensis), daikon (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, protein phosphatase 2C genes that can be used in the present invention can be isolated from various plants and used. Such protein phosphatase 2C 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, a protein phosphatase 2C gene that is isolated from a monocotyledon is preferably over-expressed. In particular, when a plant required to increase the biomass production is rice, the rice-derived protein phosphatase 2C gene (SEQ ID NO: 6) is preferably over-expressed.
In addition, in the present invention, even when a plant required to increase the biomass production is a monocotyledon, a dicotyledon-derived protein phosphatase 2C gene may be over-expressed. Specifically, for example, the Arabidopsis thaliana-derived protein phosphatase 2C gene (SEQ ID NO: 4) may be introduced into not only dicotyledons, but also a variety of plants that are classified as monocotyledons, so that the gene is over-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 such that the protein phosphatase 2C gene is over-expressed 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 exerting the significantly increased production of biomass and/or seeds per plant compared with wild-type plants can be provided through overexpression of the above described protein phosphatase 2C 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. Also, the term “significantly increased production of seeds” refers to a situation in which the total amount and/or total number of seeds harvested from a plant is statistically significantly high compared with wild-type plants. That is, the term “significantly increased production of seeds” may refer to any of: a case in which the size of each seed is improved; a case where the size per seed is equivalent but the number of seeds is improved; or a case in which the size per seed is improved and the number of seeds is also improved.
According to the present invention, the production of biomass and/or seeds by plants 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 over-expressing the above protein phosphatase 2C gene.
EXAMPLES
The present invention will be specifically described in the following reference examples and examples. However, the examples are not intended to limit the technical scope of the present invention.
Example 1
Preparation of Transformants (Arabidopsis thaliana) Through Introduction of the PP2C (Protein Phosphatase 2C) Gene (At3g05640 (SEQ ID NO:5))
1. Materials and Methods
1-1. Experimental Materials
As experimental materials, seeds of Arabidopsis thaliana mutants (Activation-tag T-DNA lines: Weigel T-DNS 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/1 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 4 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, 0. 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 crushed under 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 reaction 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; Liu, Y. G. and Whttier, R. F., 1995, Genomics 25, 674-681; Liu, Y. G. et al., 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.









TL1:



(SEQ ID NO: 24)



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



 



TL2:



(SEQ ID NO: 25)



5′-CGC TGC GGA CAT CTA CAT TTT TG-3′



 



TL3:



(SEQ ID NO: 26)



5′-TCC CGG ACA TGA AGC CAT TTA C-3′



 



P1:



(SEQ ID NO: 27)



5′-NGT CGA SWG ANA WGA A-3′






In addition, in SEQ ID NO: 25, “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 reaction 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: 24)
0.5
pmol



Arbitrary primer 1 (SEQ ID NO: 27)
100
pmol



TaKaRa Ex Taq (Takara Bio)
1.0
unit



Total
20
μl

















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 reaction 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: 25)
5
pmol


Arbitrary primer 1 (SEQ ID NO: 27)
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 reaction 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
μl


2.5 mM dNTPs (Takara Bio)
0.5
μl


3rd specific primer (TL3: SEQ ID NO: 26)
10
pmol


Arbitrary primer 1 (SEQ ID NO: 27)
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 2 nd 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, 498-bp sequence information was obtained (SEQ ID NO: 28).
The Arabidopsis Information Resource (TAIR on world wide web at arabidopsis.org) was subjected to a BLAST search for the thus obtained sequence. Thus, the insertion site was found to be the gene of [AGI (Arabidopsis Genome Initiative gene code) code: At3g05630] of Arabidopsis thaliana chromosome 3.
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 the T-DNA insertion site (At3g05630) revealed in 1-2-3.
1-2-5. Obtainment of Predicted Genes
For amplification of a fragment containing the ORF region of PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) predicted to be activated in 1-2-4, PCR primers 5640PF1 and 5640PR1 were designed and synthesized based on the sequence information disclosed at the TAIR (on world wide web at arabidopsis.org/home). In addition, these primers were designed, so that a restriction enzyme site (BsrG I or Sal I) required for introduction into expression vectors was added to the terminus of each primer.







5640PF1 (SEQ ID NO: 29):


5′-ACG CGT CGA CAT GGG ACA TTT CTC TTC CAT GTT


 


CAA CGG-3′


 


5640PR1 (SEQ ID NO: 30):


5′-TGT ACA TGT ACA CTA TAG AGA TGG CGA CGA CGA


 


TGA AGA ATG G-3′






According to the method described in 1-2-2, a template DNA was prepared from wild-type Arabidopsis thaliana (ecotype Col-0). Phusion High-Fidelity DNA Polymerase (New England BioLabs: NEB) was used as an enzyme and the above 5640PF1 and 5640PR1 were used as primers. The relevant PCR reaction solution composition and reaction conditions are shown in Table 7 and Table 8, respectively.










TABLE 7








Template (genomic DNA)
60
ng



10 × HF buffer (NEB)
5
μl



10 mM dNTPs (NEB)
1.0
μl



Each primer
20
pmol



Phusion High-Fidelity
1.0
unit



DNA Polymerase





Total
50
μl

















TABLE 8







#1:
98° C. (30 seconds)


#2:
15 cycles of 98° C. (10 seconds)/55° C.



(30 seconds)/72° C. (30 seconds)


#5:
72° C. (10 minutes)









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 GFX PCR DNA and a 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 PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was cloned into a vector, followed by determination of the nucleotide sequence and sequence analysis.
1-2-6. Construction of Plant Expression Vector
A fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was inserted into a plant expression vector pBI121 containing an omega sequence from tobacco mosaic virus. Thus, a construct was prepared.
First, the pCR2.1 vector, in which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) 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 2700 by containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) and pBI121 containing the omega sequence were each fractioned and purified from the gel using GFX PCR DNA and a GEL Band Purification Kit (Amersham).
For introduction of a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) 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 DH5α, 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 PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) was subcloned into an expression vector, followed by determination of the nucleotide sequence 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 Agrobacterium tumefaciens C58C1 strain by electroporation (Plant Molecular Biology Mannal, Second Edition, B. G. Stanton and A. S. Robbert, Kluwer Acdemic Publishers 1994). Subsequently, Agrobacterium tumefaciens in 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. (Steven J. Clough and Andrew F. Bent, 1998, The Plant Journal 16, 735-743).
Transformants were selected using kanamycin-containing medium. T1 generation plants were produced by self-pollination from the transformants, so that T2 seeds were obtained.
1-2-8. Confirmation of the Phenotype of Transformant
T2 seeds produced in 1-2-7 were aseptically sowed and then the resulting plants were transplanted into pots (each with a diameter of 50 mm) containing vermiculite mixed soil. As control plants for comparison, Arabidopsis plants that had not undergone recombination were transplanted. They were cultivated under conditions of 22° C. and 16 hours in the light/8 hours in the dark, and with a light intensity ranging from about 30 to 45 μmol/m−2/s−1, for a total of 11 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 and seeds were weighed using an electronic balance.
1-3. Results
Regarding the results of 1-2-8, 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 PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) had been introduced. Also, FIG. 4 and FIG. 5 show the results of measuring the total amounts of biomass and seeds of the above-ground parts of the plants.
As shown in FIGS. 3, 4, and 5, it was revealed that in the case of transformed plants into which the fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g05640 (SEQ ID NO:5)) had been introduced, the total amounts of biomass of the above-ground parts were much higher (about 1.9 to 2.1 times) than the amounts of the same in the cases of wild-type plants. In addition, the amounts of seeds were much greater (by about 1.7 to 1.8 times) than the same in the cases of wild-type plants.
Example 2
Preparation of Transformants (Rice) Through Introduction of the PP2C (Protein Phosphatase 2C) cDNA (At3g05640 (SEQ ID NO:5))
2. Materials and Methods
2-1. Experimental Materials
As experimental materials, Arabidopsis transformants into which a fragment containing ORF of the PP2C gene (At3g05640 (SEQ ID NO:5)) prepared in 1 was introduced into Arabidopsis thaliana and rice (Oryza sativa L. ssp. japonica (cv. Nipponbare)) to make transformants as experimental materials.
2-2. Methods
2-2-1. Obtainment of PP2C (Protein Phosphatase 2C) cDNA (At3g05640 (SEQ ID NO:5))
Arabidopsis transformants prepared by introduction of a fragment containing ORF of the PP2C gene (At3g05640 (SEQ ID NO:5)) prepared in 1 were grown until the plants reached 4 weeks of age. Total RNA was isolated from the above-ground parts, then RT-PCR was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems), so that cDNA was prepared.
PCR was performed using the following primers that had been designed based on the nucleotide sequence (SEQ ID NO: 4) of the coding region of PP2C (At3g05640 (SEQ ID NO:5)) and PrimeSTAR HS DNA Polymerease (Takara Bio). The thus amplified fragment was TA-cloned into a pCR-Blunt II-TOPO vector (Invitrogen®).









AP041-F:



(SEQ ID NO: 31)



5′-AGGATCCATGGGACATTTCTCTTCCATGT-3′



 



AP041-R:



(SEQ ID NO: 32)



5′-AGAGCTCCTATAGAGATGGCGACGACG-3′






2-2-2. Construction of Plant Expression Vector

A GUS (β-Glucuronidase) portion of pIG121-Hm (Ohat, S. et al., 1990, Plant Cell Physiol. 31, 805-813) was substituted with sGFP (S65T) having an intron fragment of Ricinus communis-derived catalase, so that a plant expression vector, pBIsGFP, was constructed. Furthermore, a sequence containing pDEST R4-R3 recombination sites (attR4 and attR3) included in a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen®) was inserted, so that a destination vector, pBI-sGFP-R4R3, was constructed.
A corn-derived ubiquitin gene promoter (SEQ ID NO: 33: Christensen, A. H. and Quail, P. H., Transgenic Research 1996, 5, 213-218), PP2C cDNA (At3g05640 (SEQ ID NO:5)) obtained in 2-2-1, and an Agrobacterium tumefaciens Ti plasmid-derived nopaline synthase gene (NOS) terminator (obtained from SEQ ID NO: 34: pIG121-Hm) were cloned by BP reaction to result in donor clones, pDONR P4-P1R, pDONR 221, and DONR P2R-P3, respectively, included in a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen), so that entry clones were prepared.
An LR reaction was performed for each of the thus prepared entry clones and a destination vector, pBI-sGFP-R4R3, so that a plant expression vector containing the corn-derived ubiquitin gene promoter, PP2C cDNA (At3g05640 (SEQ ID NO:5)), and the nopaline synthase gene (NOS) terminator, in such order, was constructed. The nucleotide sequences of the thus obtained expression vectors were determined and sequence analysis was conducted.
2-2-3. Gene Introduction into Rice Using Agrobacterium Method
The plant expression vectors constructed in 2-2-2 were introduced into an Agrobacterium tumefaciens EHA101 strain. Then Agrobacterium tumefaciens in which the plant expression vector had been introduced was introduced into rice (Oryza sativa L. ssp. japonica (cv. Nipponbare)). Specifically, the experiment was conducted under conditions in accordance with the method disclosed in JP Patent No. 3141084.
Transformed rice plants that had grown in hygromycin-containing medium were selected and then T1 seedlings (about 12 cm) were aseptically prepared.
2-2-4. Confirmation of Phenotype of Transformant
T1 plants prepared in 2-2-3 were transplanted in pots with a diameter of about 10 cm containing Kumiai Hitetsu culture soil No. 2 (JA Aichi Keizairen (economic federation)). After acclimatization, the plants were transplanted into 1/5000a Wagner pots containing the same culture soil and then cultivated under conditions of 30° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 100 μmol m−2s−1.
As control plants, T1 plants into which a plant expression vector (constructed by ligating 3 multiple cloning sites of a pST-Bluel vector (Novagen) to a destination vector pBI-sGFP-R4R3) had been introduced were similarly cultivated.
2-3. Results
Regarding the results of 2-2-4 above, FIG. 6 shows photos showing a control rice plant and a transformed rice plant prepared by introduction of the coding region of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)). As shown in FIG. 6, in the above-ground parts of the transformed rice plant into which the coding region of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) had been introduced, the total amount of biomass was improved compared with the control rice plant. It was revealed through the above results that when the Arabidopsis thaliana-derived PP2C gene is expressed at a high level in a plant other than Arabidopsis thaliana, the production of plant biomass can be increased.
Example 3
Preparation of Transformant (Rice) Through Introduction of PP2C (Protein Phosphatase 2C) cDNA (Os05g0358500)
3. Materials and Methods
3-1. Experimental Materials
As experimental materials, rice (Oryza sativa L. ssp. japonica (cv. Nipponbare)) was used.
3-2. Methods
3-2-1. Obtainment of Rice PP2C (Protein Phosphatase 2C) cDNA (Os05g0358500)
In this Example, a rice homologous gene (PP2C gene (Os05g0358500)) homologous to PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) used in Examples 1 and 2 was used. The entire sequence was chemically synthesized based on the nucleotide sequence (SEQ ID NO: 6) of the coding region of rice PP2C (Os05g0358500). A fragment of the chemically synthesized entire sequence was cloned into pDONR 221 that was a donor clone of a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen®).
3-2-2. Construction of Plant Expression Vector
A corn-derived ubiquitin gene promoter (SEQ ID NO: 33: Christensen, A. H. and Quail, P. H., Transgenic Research 1996, 5, 213-218) and an Agrobacterium tumefaciens Ti plasmid-derived nopaline synthase gene (NOS) terminator (SEQ ID NO: 34: obtained from pIG121-Hm) were cloned by BP reaction to result in donor clones, pDONR P4-P1R and DONR P2R-P3, respectively, included in a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen®), so that entry clones were prepared.
An LR reaction was performed for pDONR 221 into which the rice PP2C (Os05g0358500) cDNA sequence prepared in 3-2-1 had been cloned, pDONR P4-P1R into which the above prepared corn-derived ubiquitin gene promoter sequence had been cloned, DONR P2R-P3 into which the nopaline synthase gene (NOS) terminator sequence had been cloned, and the destination vector pBI-sGFP-R4R3 constructed in 2-2-2. Thus, a plant expression vector containing the corn-derived ubiquitin gene promoter, the rice PP2C cDNA (Os05g0358500), and the nopaline synthase gene (NOS) terminator, in such order, was constructed. The nucleotide sequence of the thus obtained expression vector was determined and then sequence analysis was conducted.
3-2-3. Gene Introduction into Rice Using Agrobacterium Method
The plant expression vector constructed in 2-2-2 was introduced into an Agrobacterium tumefaciens EHA101 strain. Then Agrobacterium tumefaciens in which the plant expression vector had been introduced was introduced into rice (Oryza sativa L. ssp. japonica (cv. Nipponbare)). Specifically, the experiment was conducted under conditions according to the method disclosed in JP Patent No. 3141084.
Transformed rice plants that had grown in hygromycin-containing medium were selected and then T1 seedlings (about 12 cm) were aseptically prepared.
3-2-4. Confirmation of the Phenotype of Transformant
T1 plants prepared in 3-2-3 were transplanted in pots with a diameter of about 10 cm containing Kumiai Hitetsu culture soil No. 2 (JA Aichi Keizairen (economic federation)). After acclimatization, the plants were transplanted into 1/5000a Wagner pots containing the same culture soil and then cultivated under conditions of 30° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 100 μmol m−2s−1.
As control plants, rice plants, into which no gene had been introduced, were regulated to be at almost the same growth stage as that of transformants, transplanted simultaneously with the transplantation of transformants, and then cultivated similarly.
3-3. Results
As the results of 3-2-4 above, FIG. 7 shows photos showing control rice plants and transformed rice plants into which the coding region of rice-derived plant PP2C (protein phosphatase 2C) (Os05g0358500) had been introduced. As shown in FIG. 7, in the above-ground parts of the transformed rice plants into which the coding region of the rice-derived PP2C (protein phosphatase 2C) (Os05g0358500) had been introduced, the total amount of biomass was improved compared with the control rice plants. It was revealed by the above results that when the rice-derived PP2C gene is expressed at a high level in a rice plant, the production of rice plant biomass can be increased.
Example 4
Preparation of Transformant (Arabidopsis thaliana) Through Introduction of PP2C (Protein Phosphatase 2C) Gene (At5g27930 (SEQ ID NO:36))
4. Materials and Methods
4-1. Experimental Materials
As experimental materials, wild-type Arabidopsis thaliana (ecotype Col-0) was used.
4-2. Methods
4-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein Phosphatase 2C) Gene (At5g27930 (SEQ ID NO:36))
In this Example, a PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order was used, instead of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) used in Examples 1 and 2. For amplification of a fragment containing an ORF region of Arabidopsis PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)), PCR primers, APO42-F5 and APO42-R, were designed and synthesized based on the sequence information disclosed in TAIR (on world wide web at arabidopsis.org/home). Also, PCR primers SalI-APO42-F2 and APO42-BsrGI-R2 were also designed and synthesized so as to add a sequence (restriction enzyme site: BsrG I or Sal I) on the vector side required upon cloning of the thus amplified fragment into the vector using an In-Fusion cloning system (Clontech). The nucleotide sequence of the coding region in the PP2C gene (At5g27930 (SEQ ID NO: 36)) is shown in SEQ ID NO: 35 and the amino acid sequence of the protein encoded by the PP2C gene (At5g27930) is shown in SEQ ID NO: 36.







AP042-F5:


(SEQ ID NO: 37)


5′-ATGGGACATTTCTCATCGATGTTC-3′ 


 


AP042-R:


(SEQ ID NO: 38)


5′-TTACTTTAAAATCGTCATGGCATGATG-3′ 


 


SalI-AP042-F2


(SEQ ID NO: 39)


5′-AATTACTATTTACAATTACAGTCGACATGGGACATTTCTCATCGA


 


TGTTCAATGGA-3′ 


 


AP042-BsrGI-R2


(SEQ ID NO: 40)


5′-AGCCGGGCGGCCGCTTTACTTGTACATTACTTTAAAATCGTCATGG


 


CATGATGATGTTG-3′ 






PCR was performed using template DNA prepared from wild-type Arabidopsis thaliana (ecotype Col-0) according to the method of 1-2-2, the above primers, APO42-F5 and APO42-R, and PrimeSTAR HS DNA Polymerase (Takara Bio), so that a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) was obtained.
4-2-2. Construction of Plant Expression Vector
A fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) was inserted into a plant expression vector pBI121 containing a tobacco mosaic virus-derived omega sequence, so that a construct was prepared.
A fragment containing the PP2C gene (At5g27930 (SEQ ID NO:36)) obtained in 4-2-1 was cloned into a vector using an In-Fusion cloning system (Clontech), so that a construct was prepared. The nucleotide sequence of the thus obtained expression vector in which the fragment containing the PP2C gene (At5g27930 (SEQ ID NO:36)) had been subcloned was determined and sequence analysis was conducted.
4-2-3. Gene Introduction into Arabidopsis thaliana Using Agrobacterium Method
The plant expression vector constructed in 4-2-2 was introduced by electroporation (Plant Molecular Biology Mannal, Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain. 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. (1998, The Plant Journal 16: 735-743). T1 seeds were obtained by self-pollination.
4-2-4. Confirmation of the Phenotype of Transformant
T1 seeds obtained in 4-2-3 were aseptically sowed in kanamycin-containing medium, so that T1 plants were prepared. Seedlings selected using kanamycin-containing medium were transplanted in pots with a diameter of 50 mm containing vermiculite-mixed soil. As control plants, non-recombinant Arabidopsis plants were transplanted. They were cultivated under conditions of 22° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 30-45 μmol m−2s−1.
4-3. Results
As the results of 4-2-4 above, FIGS. 8 and 9 show photos showing the above-ground parts of wild-type plants and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At5g27930 (SEQ ID NO:36)) had been introduced. As shown in FIGS. 8 and 9, in the above-ground parts of the transformed plants into which the fragment containing ORF of the PP2C gene (At5g27930 (SEQ ID NO:36)) had been introduced, the total amount of biomass was improved compared with the wild-type plants. It was revealed by the above results that when the PP2C (protein phosphatase 2C) gene having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is expressed at a high level, the production of the plant biomass can be increased.
Example 5
Preparation of Transformant (Arabidopsis thaliana) Through Introduction of PP2C (Protein Phosphatase 2C) Gene (At3g02750 (SEQ ID NO:42))
5. Materials and Methods
5-1. Experimental Materials
As experimental materials, wild-type Arabidopsis thaliana (ecotype Col-0) was used.
5-2. Methods
5-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein Phosphatase 2C) Gene (At3g02750 (SEQ ID NO:42))
In this Example, a PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)) having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order was used, instead of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) used in Examples 1 and 2. For amplification of a fragment containing an ORF region of the PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)), PCR primers, AP036-F4 and AP036-R, were designed and synthesized based on the sequence information disclosed in TAIR (on world wide web at arabidopsis.org/home).
Also, PCR primers, SalI-AP036-F2 and AP036-BsrGI-R2, were designed and synthesized so as to add a sequence (restriction enzyme site: BsrG I or Sal I) on the vector side required upon cloning of the thus amplified fragment into the vector using an In-Fusion cloning system (Clontech). The nucleotide sequence of the coding region in the PP2C gene (At3g02750 (SEQ ID NO:42)) is shown in SEQ ID NO: 41 and the amino acid sequence of the protein encoded by the PP2C gene (At3g02750) is shown in SEQ ID NO: 42.







AP036-F4:


(SEQ ID NO: 43)


5′-ATGGGGTCCTGTTTATCTGCAG-3′ 


 


AP036-R:


(SEQ ID NO: 44)


5′-TCACTTTCCAGGCACAAATCTTG-3′ 


 


SalI-AP036-F2


(SEQ ID NO: 45)


5′-AATTACTATTTACAATTACAGTCGACATGGGGTCCTGTTTATCTG


 


CAGAGAGCAGG-3′ 


 


AP036-BsrGI-R2


(SEQ ID NO: 46)


5′-AGCCGGGCGGCCGCTTTACTTGTACATCACTTTCCAGGCACAAAT


 


CTTGGTAAGTT-3′ 






PCR was performed using template DNA prepared from wild-type Arabidopsis thaliana (ecotype Col-0) according to the method of 1-2-2, the above primers, and PrimeSTAR HS DNA Polymerase (Takara Bio), so that a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)) was obtained.
5-2-2. Construction of Plant Expression Vector
A fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)) was inserted into a plant expression vector pBI121 containing a tobacco mosaic virus-derived omega sequence, so that a construct was prepared.
A fragment containing the PP2C gene (At3g02750 (SEQ ID NO:42)) obtained in 5-2-1 was cloned into a vector using an In-Fusion cloning system (Clontech), so that a construct was prepared. The nucleotide sequence of the thus obtained expression vector in which the fragment containing the PP2C gene (At3g02750 (SEQ ID NO:42)) had been subcloned was determined and sequence analysis was conducted.
5-2-3. Gene Introduction into Arabidopsis thaliana Using Agrobacterium Method
The plant expression vector constructed in 5-2-2 was introduced by electroporation (Plant Molecular Biology Mannal, Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain. 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. (1998, The Plant Journal 16: 735-743). T1 seeds were obtained by self-pollination.
5-2-4. Confirmation of the Phenotype of Transformant
T1 seeds obtained in 5-2-3 were aseptically sowed in kanamycin-containing medium, so that T1 plants were prepared. Seedlings selected using kanamycin-containing medium were transplanted in pots with a diameter of 50 mm containing vermiculite-mixed soil. As control plants, non-recombinant Arabidopsis plants were transplanted. They were cultivated under conditions of 22° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 30-45 μmol m−2s−1.
5-3. Results
As the results of 5-2-4 above, FIG. 10 shows a photo showing the above-ground parts of wild-type plants and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g02750 (SEQ ID NO:42)) had been introduced. As shown in FIG. 10, in the above-ground parts of the transformed plants into which the fragment containing ORF of the PP2C gene (At3g02750 (SEQ ID NO:42)) had been introduced, the total amount of biomass was improved compared with the wild-type plants. It was revealed by the above results that when the PP2C (protein phosphatase 2C) gene having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is expressed at a high level, the production of the plant biomass can be increased.
Example 6
Preparation of Transformants (Arabidopsis thaliana) Through Introduction of PP2C (Protein Phosphatase 2C) Gene (At3g16800 (SEQ ID NO:48))
6. Materials and Methods
6-1. Experimental Materials
As experimental materials, wild-type Arabidopsis thaliana (ecotype Col-0) was used.
6-2. Methods
6-2-1. Obtainment of Arabidopsis thaliana PP2C (Protein Phosphatase 2C) Gene (At3g16800 (SEQ ID NO:48))
In this Example, a PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)) having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order was used, instead of PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) used in Examples 1 and 2. For amplification of a fragment containing an ORF region of the PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)), PCR primers, APO40-F4 and APO40-R, were designed and synthesized based on the sequence information disclosed in TAIR (on world wide web at arabidopsis.org/home). Also, PCR primers, SalI-APO40-F2 and APO40-BsrGI-R2, were synthesized and designed so as to add a sequence (restriction enzyme site: BsrG I or Sal I) on the vector side required upon cloning of the thus amplified fragment into the vector using an In-Fusion cloning system (Clontech). The nucleotide sequence of the coding region in the PP2C gene (At3g16800 (SEQ ID NO:48)) is shown in SEQ ID NO: 47 and the amino acid sequence of the protein encoded by the PP2C gene (At3g16800) is shown in SEQ ID NO: 48.







AP040-F4:


(SEQ ID NO: 49)


5′-ATGGTGCTTTTACCAGCGTTTTTG-3′ 


 


AP040-R: 


(SEQ ID NO: 50)


5′-CTAAGAAGGACGAAAGAAGAGAC-3′ 


 


SalI-AP040-F2


(SEQ ID NO: 51)


5′-AATTACTATTTACAATTACAGTCGACATGGTGCTTTTACCAGCGT


 


TTTTGGACGGATTAG-3′ 


 


AP040-BsrGI-R2:


(SEQ ID NO: 52)


5′-AGCCGGGCGGCCGCTTTACTTGTACACTAAGAAGGACGAAAGAAG


 


AGACAGAGAAC-3′ 






PCR was performed using template DNA prepared from wild-type Arabidopsis thaliana (ecotype Col-0) according to the method of 1-2-2, the above primers, and PrimeSTAR HS DNA Polymerase (Takara Bio), so that a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)) was obtained.
6-2-2. Construction of Plant Expression Vector
The fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)) was inserted to a plant expression vector pBI121 containing a tobacco mosaic virus-derived omega sequence, so that a construct was prepared.
The fragment containing the PP2C gene (At3g16800 (SEQ ID NO:48)) obtained in 6-2-1 was cloned into a vector using an In-Fusion cloning system (Clontech), so that a construct was prepared. The nucleotide sequence of the thus obtained expression vector into which the fragment containing the PP2C gene (At3g16800 (SEQ ID NO:48)) had been subcloned was determined and sequence analysis was conducted.
6-2-3. Gene Introduction into Arabidopsis thaliana Using Agrobacterium Method
The plant expression vector constructed in 6-2-2 was introduced by electroporation (Plant Molecular Biology Mannal, Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain. 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. (1998, The Plant Journal 16: 735-743). T1 seeds were obtained by self-pollination.
6-2-4. Confirmation of the Phenotype of Transformant
T1 seeds obtained in 6-2-3 were aseptically sowed in kanamycin-containing medium, so that T1 plants were prepared. Seedlings selected using kanamycin-containing medium were transplanted in pots with a diameter of 50 mm containing vermiculite-mixed soil. As control plants, non-recombinant Arabidopsis plants were transplanted. They were cultivated under conditions of 22° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 30-45 μmol m−2s−1.
6-3. Results
As the results of 6-2-4 above, FIG. 11 shows a photo showing the above-ground parts of a wild-type plant and transformed plants into which a fragment containing ORF of the PP2C (protein phosphatase 2C) gene (At3g16800 (SEQ ID NO:48)) had been introduced. As shown in FIG. 11, in the above-ground parts of the transformed plants into which the fragment containing ORF of the PP2C gene (At3g16800 (SEQ ID NO:48)) had been introduced, the total amount of biomass was improved compared with the wild-type plant. It was revealed by the above results that when the PP2C (protein phosphatase 2C) gene having 3 consensus sequences comprising the amino acid sequences shown in SEQ ID NOS: 1-3 from the N-terminal side in such order is expressed at a high level, the production of the plant biomass can be increased.
Example 7
Preparation of Transformant (Arabidopsis thaliana) Through Introduction of PP2C (Protein Phosphatase 2C) cDNA (Os05g0358500)
7. Materials and Methods
7-1. Experimental Materials
An experimental material, wild-type Arabidopsis thaliana (ecotype Col-0) was used.
7-2. Methods
7-2-1. Obtainment of Rice PP2C (Protein Phosphatase 2C) cDNA (Os05g0358500)
In this Example, a rice homologous gene (PP2C gene (Os05g0358500)) homologous to PP2C (protein phosphatase 2C) (At3g05640 (SEQ ID NO:5)) used in Examples 1 and 2 was used. The entire sequence was chemically synthesized based on the nucleotide sequence (SEQ ID NO: 6) of the coding region of rice PP2C (Os05g0358500). A fragment of the chemically synthesized entire sequence was cloned into pDONR 221 that was a donor clone of a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen®).
7-2-2. Construction of Plant Expression Vector
A cauliflower mosaic virus-derived 35S (CaMV35S Ω) promoter (SEQ ID NOS: 58) containing a tobacco mosaic virus-derived omega sequence and an Agrobacterium tumefaciens Ti plasmid-derived nopaline synthase gene (NOS) terminator (SEQ ID NOS: 34: obtained from pIG121-Hm) were cloned by BP reaction to result in donor clones, pDONR P4-P1R and DONR P2R-P3, respectively, included in a MultiSite Gateway Three-Fragement Vector Construction Kit (Invitrogen®), so that entry clones were prepared.
An LR reaction was performed for pDONR 221 into which the rice PP2C (Os05g0358500) cDNA sequence prepared in 7-2-1 had been cloned, pDONR P4-P1R into which the above prepared CaMV35S Ω promoter sequence had been cloned, DONR P2R-P3 into which the nopaline synthase gene (NOS) terminator sequence had been cloned, and the destination vector pBI-sGFP-R4R3 constructed in 2-2-2. Thus, a plant expression vector containing the CaMV35S Ω promoter, the rice PP2C cDNA (Os05g0358500), and the nopaline synthase gene (NOS) terminator in such order was constructed. The nucleotide sequence of the thus obtained expression vector was determined and then sequence analysis was conducted.
7-2-3. Gene Introduction into Arabidopsis thaliana Using Agrobacterium Method
The plant expression vector constructed in 7-2-2 was introduced by electroporation (Plant Molecular Biology Mannal, Second Edition, B. G. Stanton A. S. Robbert, Kluwer Acdemic Publishers 1994) into an Agrobacterium tumefaciens C58C1 strain. 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. (1998, The Plant Journal 16: 735-743). T1 seeds were obtained by self-pollination.
7-2-4. Confirmation of the Phenotype of Transformant
T1 seeds obtained in 7-2-3 were aseptically sowed in kanamycin-containing medium, so that T1 plants were prepared. Seedlings selected using kanamycin-containing medium were transplanted in pots with a diameter of 50 mm containing vermiculite-mixed soil. As control plants, non-recombinant Arabidopsis plants were transplanted. They were cultivated under conditions of 22° C., 16 hours in the light/8 hours in the dark, and a light intensity of about 30-45 μmol m−2s−1.
7-3. Results
As the results of 7-2-4 above, FIG. 12 shows a photo showing the above-ground parts of a wild-type plant and transformed plants into which the coding region of the rice-derived PP2C (protein phosphatase 2C) gene (Os05g0358500) had been introduced. As shown in FIG. 12, in the above-ground parts of the transformed plants into which the coding region of the rice-derived PP2C gene (Os05g0358500) had been introduced, the total amount of biomass was improved compared with the wild-type plant. It was revealed by the above results that when the rice-derived PP2C gene is expressed at a high level in Arabidopsis thaliana, the production of Arabidopsis biomass can be increased.
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 comprising overexpressing in a plant a gene encoding protein phosphatase 2C comprising a protein selected from the group consisting of (a) and (b):
(a) a protein comprising SEQ ID NO:42; and
(b) a protein comprising an amino acid sequence that has a deletion of one to five amino acids, a substitution of one to five amino acids, an addition of one to five amino acids, or an insertion of one to five amino acids with respect to SEQ ID NO:42 and having protein phosphatase 2C activity; and
thereby increasing the production of biomass as compared to a wild-type plant.

2. A method for producing a transformed plant comprising the step of introducing into a plant a gene encoding protein phosphatase 2C comprising a protein selected from the group consisting of (a) and (b):
(a) a protein comprising a SEQ ID NO:42; and
(b) a protein comprising an amino acid sequence that has a deletion of one to five amino acids, a substitution of one to five amino acids, an addition of one to five amino acids, or an insertion of one to five amino acids with respect to SEQ ID NO:42 and having protein phosphatase 2C activity;
thereby producing a transformed plant.

3. The method according to claim 1, wherein the plant is a dicotyledon.

4. The method according to claim 1, wherein the plant is a plant of the family Brassicaceae.

5. The method according to claim 1, wherein the plant is Arabidopsis thaliana.

6. The method according to claim 1, wherein the plant is Brassica rapa.

7. The method according to claim 1, wherein the plant is a monocotyledon.

8. The method according to claim 1, wherein the plant is a plant of the family Gramineae.

9. The method according to claim 1, wherein the plant is rice (Oryza sativa).

10. The method according to claim 1, wherein the plant is sugarcane (Saccharum officinarum).

11. The method according to claim 2, wherein the plant is a dicotyledon.

12. The method according to claim 2, wherein the plant is a plant of the family Brassicaceae.

13. The method according to claim 2, wherein the plant is Arabidopsis thaliana.

14. The method according to claim 2, wherein the plant is Brassica rapa.

15. The method according to claim 2, wherein the plant is a monocotyledon.

16. The method according to claim 2, wherein the plant is a plant of the family Gramineae.

17. The method according to claim 2, wherein the plant is rice (Oryza sativa).

18. The method according to claim 2, wherein the plant is sugarcane (Saccharum officinarum).