# Patent

## Title
Transformation of a plant to promote root and/or leaf growth

## Abstract

The present invention is directed to identify genes involved in the growth of a plant and provide a transformed plant the growth of which is promoted utilizing the genes.

## Background
This application is a National Stage of PCT/JP08/072,595 filed Dec. 5, 2008 and claims the benefit of JP 2007-315953 filed Dec. 6, 2007.
TECHNICAL FIELD
The present invention relates to a transformed plant the growth of which is promoted and a method for producing the plant.
BACKGROUND ART
Roots of a plant plays important roles, such as nutritional absorption and anchoring a plant body upright. However, many of the factors associated with such physiological phenomena remain unknown.
Recently, a method called FOX hunting system has been developed, by which genes whose DNA sequences have been determined but functions have not been understood yet can be comprehensively analyzed (JP Patent Publication No. 2003-018808 A). In this system, full-length cDNA is linked to a high-expression vector and the vector thus obtained is introduced into Arabidopsis thaliana, whereby Arabidopsis thaliana highly-expressing the full-length cDNA is produced. The FOX hunting system described as above is applicable to various genetic analyses.
On the other hand, some patent applications relating to the growth of a plant have been filed, and examples thereof include those relating to a gene-controlling extension of plant roots (JP Patent Publication No. 2004-187564 A), a method for promoting the growth of a plant (JP Patent Publication No. 2004-305051 A), and a method for increasing the growth of a plant and crop yield (JP Patent Publication No. 2002-531083 A). JP Patent Publication No. 2004-187564 A discloses that an Arabidopsis thaliana gene specifically expressed in root tips (AtGCN20-3) was identified utilizing Arabidopsis thaliana T-DNA tag lines. JP Patent Publication No. 2004-305051 A discloses that extension of growing organs such as roots is promoted by over-expression of a plant-derived cyclin B2 gene. JP Patent Publication No. 2002-531083 A discloses that a plant is transformed with a nucleic acid encoding a cyclin protein that is linked to regulatory sequences to produce a transformed plant exhibiting increased growths of roots and shoots.
DISCLOSURE OF THE INVENTION
The present invention is directed to identify genes involved in the growth of a plant, and to provide a transformed plant the growth of which is promoted utilizing the genes and a method for producing the plant.
The present inventors conducted a thorough research to achieve the aforementioned goal. As a result, we have produced Arabidopsis thaliana lines each highly-expressing one rice full-length cDNA (called rice FOX lines), and conducted a root bending assay to isolate Arabidopsis thaliana exhibiting changes in root extension in comparison with the wild-type plant, and sequenced the rice full-length cDNA introduced into the Arabidopsis thaliana thus isolated to identify DNA involved in the growth of a plant, thereby completing the present invention.
The present invention is summarized as follows.
[1] A transformed plant comprising DNA of any of the following (a) to (g) introduced thereinto:
(a) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7;
(b) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 in which one or several nucleotides are deleted, substituted or added, and encoding a protein having an activity to promote the growth of a plant;
(c) DNA comprising a nucleotide sequence having a 90% or more identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, and encoding a protein having an activity to promote the growth of a plant;
(d) DNA which hybridizes to DNA comprising a nucleotide sequence complementary to DNA comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 under stringent conditions, and encodes a protein having an activity to promote the growth of a plant;

(e) DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8;

(f) DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 in which one or several amino acids are deleted, substituted or added, and having an activity to promote the growth of a plant; and

(g) DNA encoding a protein comprising an amino acid sequence having a 90% or more identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, and having an activity to promote the growth of a plant.

[2] The transformed plant according to [1], wherein the activity to promote the growth of a plant is an activity to promote root extension.

[3] The transformed plant according to [1] or [2], wherein the activity to promote the growth of a plant is an activity to increase a leaf area.

[4] The transformed plant according to any of [1] to [3], being a monocotyledon or a dicotyledon.

[5] The transformed plant according to any of [1] to [4], being a plant body, a part of a plant body, a cultured plant cell, or a seed.

[6] A recombinant vector comprising DNA of any of the following (a) to (g):

(a) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7;

(b) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 in which one or several nucleotides are deleted, substituted or added, and encoding a protein having an activity to promote the growth of a plant;

(c) DNA comprising a nucleotide sequence having a 90% or more identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, and encoding a protein having an activity to promote the growth of a plant;

(d) DNA hybridizing to DNA comprising a nucleotide sequence complementary to DNA comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 under stringent conditions, and encoding a protein having an activity to promote the growth of a plant;

(e) DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8; (0 DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 in which one or several amino acids are deleted, substituted or added, and having an activity to promote the growth of a plant; and

(g) DNA encoding a protein comprising an amino acid sequence having a 90% or more identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, and having an activity to promote the growth of a plant.

[7] A method for producing a transformed plant comprising introducing DNA of any of the following (a) to (g) into a plant cell and cultivating the plant:

(a) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7;

(b) DNA comprising a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 in which one or several nucleotides are deleted, substituted or added, and encoding a protein having an activity to promote the growth of a plant;

(c) DNA comprising a nucleotide sequence having a 90% or more identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, and encoding a protein having an activity to promote the growth of a plant;

(d) DNA which hybridizes to DNA comprising a nucleotide sequence complementary to DNA comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 under stringent conditions, and encodes a protein having an activity to promote the growth of a plant;

(e) DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8;

(f) DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 in which one or several amino acids are deleted, substituted or added, and having an activity to promote the growth of a plant; and

(g) DNA encoding a protein comprising an amino acid sequence having a 90% or more identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, and having an activity to promote the growth of a plant.

[8] The method according to [7], wherein the DNA is introduced using the recombinant vector according to [6].

The present specification encompasses the contents described in the specification and/or figures in JP Patent Application No. 2007-315953, based on which the present application claims a priority.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an overview of a root bending assay.
FIG. 2 shows extension of the roots of re-transformed rice FOX lines. Panel A shows rice FOX line 1 (SEQ ID NOs: 1 and 2), and panel B shows rice FOX line 2 (SEQ ID NOs: 3 and 4).
FIG. 3 shows extension of the roots of re-transformed rice FOX lines. Panel C shows rice FOX line 3 (SEQ ID NOs: 5 and 6), and panel D shows rice FOX line 4 (SEQ ID NOs: 7 and 8).
FIG. 4 shows increases in the leaf areas of re-transformed rice FOX lines. Panel A shows rice FOX line 1 (SEQ ID NOs: 1 and 2), and panel B shows rice FOX line 2 (SEQ ID NOs: 3 and 4).



BEST MODE FOR CARRYING OUT THE INVENTION
(1) DNA Involved in Promotion of the Growth of a Plant
DNA involved in promotion of the growth of a plant used in the present invention is DNA of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, and amino acids encoded by the DNA are SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. The sequence information above has been reported to National Center for Biotechnology Information (NCBI). The NCBI accession numbers corresponding to SEQ ID NOs: 1 to 8 are shown below.










TABLE 1







Rice FOX line
Accession No.









1 (SEQ ID NOs: 1, 2)
AK069726



2 (SEQ ID NOs: 3, 4)
AK070346



3 (SEQ ID NOs: 5, 6)
AK067987



4 (SEQ ID NOs: 7, 8)
AK066897










The above-described DNA involved in promotion of the growth of a plant was found as a result of searching through rice genes using the FOX hunting system and the root bending assay.
The FOX hunting system (Full-length cDNA Over-expressor Gene Hunting System) is a method for elucidating the function of DNA based on changes in traits resulted from over-expression of full-length cDNA introduced into a plant (see JP Patent Publication No. 2003-018808 A). In the present invention, rice full-length cDNA was used as full-length cDNA to be introduced into a plant, and Arabidopsis thaliana was used as a plant into which full-length cDNA was to be introduced.
Specifically, approximately 13,000 rice full-length cDNAs are prepared as a pool having an equivalent ratio of the cDNAs (called normalization), and the cDNA is integrated into a T-DNA vector having a regulatory region such as a promoter, an enhancer, and a terminator, and a selection marker such as a drug-resistance gene. The resulted T-DNA vectors are introduced into Agrobacterium, whereby a rice full-length cDNA expression library (called a rice FOX library) is produced. Arabidopsis thaliana is transformed using the Agrobacterium by the floral dipping method to produce Arabidopsis thaliana transformant lines (rice FOX lines). In the FOX hunting system, because only one or two clones are introduced per plant even when plants are infected with a library consisting of hundreds of millions of clones, transformant lines in which each plant has different clone(s) introduced thereinto can be produced. T1 seeds are collected from the aforementioned transformed Arabidopsis thaliana and sown, and then seeds of T2 generation are collected. The T2 seeds are subjected to the root bending assay for screening.
The root bending assay is a method described in the article by Britt et al., (1993), A UV-sensitive mutant of Arabidopsis defective in the repair of pyrimidine-pyrimidinone (6-4) dimers. Science 261:, 1571-1574, which is carried out specifically as follows in the present invention.
T2 seeds of rice FOX lines and wild-type seeds are sown on MS agar media and subjected to a dark treatment and a vernalization treatment. Subsequently, the media are held vertically and the seeds are grown under continuous white light so that roots extend along the surface of the agar medium. Then, the agar medium are placed horizontally and irradiated with UV-B in the dark. Subsequently, the plates are held vertically and rotated by ninety degrees to change the direction of root extension, and the roots are again grown under continuous white light. Then, the lengths of the roots extended after irradiation with UV-B are compared between the wild-type plants and the T2 plants of the rice FOX lines, and the line that has extended the root longer than the wild type is isolated as a candidate UV-B-resistant line.
With regard to the candidate UV-B-resistant rice FOX line thus isolated, the introduced rice full-length cDNA is sequenced, and Arabidopsis thaliana is re-transformed with the rice full-length cDNA, and whether a phenotype exhibiting promoted root extension can be reproduced in the resulted re-transformed plant is verified (see FIG. 1).
The identified DNAs are, specifically, DNAs consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively.
The aforementioned DNA encodes a protein having an activity to promote the growth of a plant. Herein, the “activity to promote the growth of a plant” is not particularly limited as long as it is an activity to promote the growth of a plant in comparison with the wild type, and examples thereof include an activity to promote root extension and/or an activity to increase a leaf area. The degree to which the growth of a plant is promoted is not limited as long as the growth of the plant is statistically significant compared to that of the wild type. For example, the length of the root of the transformed plant of the present invention is preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, and 50% or more longer than the length of the root of the wild type. Also, the leaf area of the transformed plant of the present invention is preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, and 50% or more larger than the leaf area of the wild type.
DNA to be used in the present invention can be DNA consisting of nucleotide sequences of the aforementioned SEQ ID NOs in which one or several nucleotides are deleted, substituted or added as long as the protein encoded by the DNA has an activity to promote the growth of a plant.
In the present specification, “a nucleotide sequence of SEQ ID NO:1 in which one or several nucleotides are deleted, substituted or added” means that, for example, one to 10 nucleotides, preferably one to five nucleotides can be deleted from the nucleotide sequence of SEQ ID NO:1, or one to 10 nucleotides, preferably one to five nucleotides can be added to the nucleotide sequence of SEQ ID NO:1, or one to 10 nucleotides, preferably one to five nucleotides in the nucleotide sequence of SEQ ID NO:1 can be substituted by other nucleotides.
Further, DNA to be used in the present invention can be DNA consisting of a nucleotide sequence having a 90% or more identity to the nucleotide sequences of the aforementioned SEQ ID NOs as long as the protein encoded by the DNA has an activity to promote the growth of a plant.
In the present specification, the “identity” in “a nucleotide sequence having a 90% or more identity to the nucleotide sequence of SEQ ID NO:1” is 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.
The “identity” of the nucleotide sequence as used herein means, in the alignment of two nucleotide sequences, the degree of coincidence between the sequences observed when the two sequences are aligned in such a way that the number of the identical nucleotides is maximized. Specifically, it is expressed as a percentage (%) of the number of the identical nucleotides with respect to the total number of nucleotides. The % identity can be determined using a known algorithm such as BLAST and FASTA. When gaps are introduced, for example, when using FASTA, the number of the gaps is also added on the total number of nucleotides.
Further, DNA to be used in the present invention can be DNA which hybridizes to DNA consisting of a nucleotide sequence complementary to DNA consisting of the nucleotide sequences of the aforementioned SEQ ID NOs under stringent conditions as long as the protein encoded by the DNA has an activity to promote the growth of a plant.
In the present specification, the “stringent conditions” refers to conditions under which a so-called specific hybrid is formed, while a non-specific hybrid is substantially not formed. Examples of such conditions include conditions under which a nucleic acid having a high identity, that is, a complementary strand of DNA consisting of a nucleotide sequence having a 90% or more, preferably a 95% or more, more preferably a 98% or more, and even more preferably a 99% or more identity to the nucleotide sequence of SEQ ID NO:1 hybridizes, while a complementary strand of a nucleic acid less identical than the above does not hybridize. More specifically, such conditions refers to conditions in which the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, and more preferably 300 to 750 mM, a temperature is 25 to 70° C., preferably 50 to 70° C., and more preferably 55 to 65° C., and the formamide concentration is 0 to 50%, preferably 20 to 50%, and more preferably 35 to 45%. Furthermore, under stringent conditions, conditions for washing a filter after hybridization are normally such that the sodium chloride concentration is 15 to 600 mM, preferably 50 to 600 mM, and more preferably 300 to 600 mM, and a temperature is 50 to 70° C., preferably 55 to 70° C., and more preferably 60 to 65° C.
Alternatively, the stringent conditions can be such conditions that hybridization is carried out in 2 to 6× sodium chloride/sodium citrate (SSC, 1×SSC contains 150 mM sodium chloride and 15 mM sodium citrate, and has a pH of 7.0) at room temperature to 40° C., and wash is carried out once or several times with 0.1 to 1×SSC (preferably 0.1 to 0.2×SSC) and 0.1% SDS at 50 to 68° C.
Further, DNA to be used in the present invention can be DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. The protein consisting of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 is encoded by DNA consisting of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, respectively.
Further, DNA to be used in the present invention can be DNA encoding a protein consisting of amino acid sequences of the aforementioned SEQ ID NOs in which one or several amino acids are deleted, substituted or added as long as the protein encoded by the DNA has an activity to promote the growth of a plant.
Also, the amino acid sequence may contain conservative amino acid substitutions. Such substitutions occur, for example, between amino acids sharing a similar structural or electric property. The groups of such amino acids include (1) acidic amino acids: aspartic acid and glutamic acid; (2) basic amino acids: lysine, arginine, and histidine; (3) non-polar amino acids: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; (4) uncharged polar amino acids: glycine, asparagines, glutamine, cysteine, serine, threonine, and tyrosine; and (5) aromatic amino acids: phenylalanine, tyrosine, and tryptophan.
In the present specification, “an amino acid sequence of SEQ ID NO:2 in which one or several amino acids are deleted, substituted or added” means that, for example, one to 10 amino acids, preferably one to five amino acids can be deleted from the amino acid sequence of SEQ ID NO:2, or one to 10 amino acids, preferably one to five amino acids can be added to the amino acid sequence of SEQ ID NO:2, or one to 10 amino acids, preferably one to five amino acids in the amino acid sequence of SEQ ID NO:2 can be substituted by other amino acids.
Further, DNA to be used in the present invention can be DNA encoding a protein consisting of an amino acid sequence having a 90% or more identity to the amino acid sequences of the aforementioned SEQ ID NOs as long as the protein encoded by the DNA has an activity to promote the growth of a plant.
In the present specification, the “identity” in “an amino acid sequence having a 90% or more identity to the amino acid sequence of SEQ ID NO:2” is 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.
The “identity” of the amino acid sequence as used herein means, in the alignment of two amino acid sequences (or nucleotide sequences), the degree of coincidence between the sequences observed when the two sequences are aligned in such a way that the number of the identical amino acid residues is maximized. Specifically, it is expressed as a percentage (%) of the number of the identical amino acid residues with respect to the total number of amino acid residues. The % identity can be determined using a known algorithm such as BLAST and FASTA. When gaps are introduced, for example, when using FASTA, the number of the gaps is also added on the total number of amino acid residues.
DNA to be used in the present invention can be obtained as a nucleic acid fragment by PCR amplification using nucleic acids from a cDNA library or a genome DNA library and the like as a template with primers designed based on the sequence of any of SEQ ID NOs: 1 to 8. Also, the DNA can be obtained as a nucleic acid fragment by carrying out hybridization using nucleic acids from the aforementioned libraries and the like as a template, and employing a DNA fragment, which is a part of the DNA, as a probe. Alternatively, the DNA can be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods such as a chemical synthesis method known in the art.
Deletion, addition, and substitution of the aforementioned DNA or amino acids can be carried out by modifying DNA encoding the aforementioned protein with a technique known in the art. Introduction of mutations in DNA can be achieved by, for example, the Kunkel method or the Gapped duplex method, and mutations can be introduced using a mutation-introducing kit that utilizes a site-specific mutagenesis method (Mutan-K (Takara Bio Inc.) and a LA PCR in vitro mutagenesis kit (Takara Bio Inc.)) and the like.
The conventional technique such as the aforementioned PCR, hybridization, and recombination is described in, for example, Sambrook, J., et al. (1989), Molecular Cloning: Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, and Ausubel et al. (1995), Short Protocols In Molecular Biology, third edition, John Wiley & Sons, Inc.
(2) Recombinant Vector
The recombinant vector of the present invention to be used for transformation of a plant can be constructed by introducing DNA of any of (a) to (g) as described above into an appropriate vector. As the vector, pBI-based, pPZP-based, pSMA-based vectors and the like, which can allow introduction of target DNA into a plant via Agrobacterium, are preferably used. Particularly, a pBI-based binary vector or intermediate vector is preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3, and pBIG2113. A binary vector is a shuttle vector that is replicable in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium containing a binary vector, DNA held between border sequences consisting of a LB sequence and a RB sequence on the vector is integrated into the nuclear DNA of the plant. Also, examples of other vectors include a pUC-based vector that can allow direct introduction of DNA into a plant such as pUC18, pUC19, and pUC9. Further, examples of other vectors include a plant virus vector such as cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV), and tobacco mosaic virus (TMV).
When a binary vector-based plasmid is used, a target DNA is inserted between the border sequences (between LB and RB) of the aforementioned binary vector, and the recombinant vector thus obtained is multiplied in E. coli. Subsequently, the recombinant vectors thus multiplied are introduced into Agrobacterium tumefaciens GV3101, C58, LBA4404, EHA101, and EHA105, or Agrobacterium rhizogenes LBA1334, and the like, by electroporation and the like, and the target DNA is introduced into a plant using the Agrobacterium thus obtained.
In order to insert target DNA into a vector, a method in which purified DNA is cleaved by an appropriate restriction enzyme and then the resulting DNA is linked to a vector by inserting it into a restriction site or a multicloning site of the appropriate vector DNA, and the like, are applied.
Also, it is necessary to integrate target DNA into a vector in such a way that the DNA can exert its function. In this regard, a promoter, an enhancer, a terminator, an origin of replication required when using a binary vector-based plasmid (such as an origin of replication derived from a Ti or Ri plasmid), a selection marker gene, and the like can be linked to upstream, inside, or downstream of the target DNA.
The promoter is not necessary that from a plant as long as it is DNA that can function in a plant cell and induce an expression in a specific tissue or at a specific developmental stage of a plant. Specific examples thereof include cauliflower mosaic virus (CaMV) 35S promoter, a promoter of nopaline-synthase gene, ubiquitin promoter from corn, actin promoter from rice, and PR protein promoter from tobacco.
Examples of the enhancer include an enhancer region containing an upstream sequence of CaMV 35S promoter, a transcription enhancer E12, and an omega sequence, all of which are used to increase expression efficiency of target DNA.
The terminator may be a sequence that can terminate DNA transcription driven by the promoter, and examples thereof include a terminator of nopaline-synthase (NOS) gene, a terminator of octopine-synthase (OCS) gene, and a terminator of CaMV 35S RNA gene.
Examples of the selection marker gene include a hygromycin resistance gene, an ampicillin resistance gene, a neomycin resistance gene, a bialaphos resistance gene, and a dihydrofolate reductase gene.
(3) Transformed Plant and Method for Producing the Plant
The transformed plant of the present invention can be produced by introducing DNA of any of (a) to (g) as described above or the aforementioned recombinant vector into a target plant. In the present invention, “introduction of DNA” means that target DNA is introduced in the aforementioned host plant cell by, for example, a known genetic engineering method in such a way that the DNA can be expressed. The DNA thus introduced can be integrated into a host plant genome DNA or present as being contained within an exogenous vector.
Introduction of DNA or a recombinant vector may be carried out by a known method, and examples thereof include an Agrobacterium method, a PEG-calcium phosphate method, electroporation, a particle gun method, and a microinjection method. The Agrobacterium method includes a method using a protoplast, a method using a piece of tissue, and a method using a whole plant (in planta method). The method using a protoplast can be carried out by a method in which a protoplast is co-cultured with Agrobacterium containing a Ti plasmid or a Ri plasmid (Agrobacterium tumefaciens or Agrobacterium rhizogenes, respectively) and a method in which a protoplast is fused with a spheroplast of Agrobacterium (a spheroplast method). The method using a piece of tissue can be carried out by a method in which steriled cultured leaf discs of a target plant are infected, or by infecting calluses (undifferentiated cultured cells), and the like. Also, the in planta method using a seed or a plant body is feasible by directly treating an imbibed seed, a young plant (young seedling), a potted plant, and the like with Agrobacterium. The aforementioned plant transformation methods can be carried out according to the description in “Shinpan Model shokubutu no jikken protocol, Idengakutekishuho kara genome kaiseki made (literal translation: New edition Experimental protocol for model plant, From genetic technique to genome analysis) (Supervised by SHIMAMOTO, Ko and OKADA, Kiyotaka, Shujunsha Co., Ltd., 2001)” and the like.
Whether the DNA is integrated into the plant body can be confirmed by PCR, Southern hybridization, Northern hybridization, and the like. For example, DNA is prepared from the transformed plant, and primers specific to the target DNA are designed, and PCR is carried out. Subsequently, the amplified product is subjected to agarose electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, and then stained with ethidium bromide, SYBR Green solution, and the like so that the amplified product is detected as a single band, whereby transformation can be confirmed. Also, PCR can be carried out using a primer that is labeled with a fluorescent dye and the like in advance, and then the amplified product can be detected. Further, transformation may also be confirmed by a method in which the amplified product is bound to a solid phase such as a microplate, which is then confirmed by fluorescence or an enzymatic reaction, and the like.
Alternatively, transformation can also be confirmed by producing a vector having various reporter genes, for example, a gene of β-glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), and the like, linked to downstream of the target DNA, and transforming a plant using Agrobacterium having the vector introduced thereinto as described above, and then measuring the expression of the reporter gene.
A plant to be used for transformation in the present invention can be either a monocotyledon or a dicotyledon, and examples thereof include, but are not limited to, a plant belonging to the family Brassicaceae (including Arabidopsis thaliana, cabbage, and rapeseed), the family Poaceae (including rice, corn, barley, and wheat), and the family Solanaceae (including eggplant, tobacco, tomato, and potato).
In the present invention, a plant material to be subjected to transformation may be any of plant organ and tissue such as a stem, a leaf, a seed, an embryo, an ovule, an ovary, a shoot apex, an anther, and pollen, a section of the above plant organ and tissue, an undifferentiated callus, and a cultured plant cell such as a protoplast obtainable by removing a cell wall of the callus by an enzymatic treatment. Also, when the in planta method is used, an imbibed seed and an entire plant body can be used.
When a cultured plant cell is subjected to transformation, in order to regenerate a transformant from the resulted transformed cell, an organ or an organism may be regenerated by a known tissue culture method. A person skilled in the art can easily perform the regeneration as described above using a well-known method for regenerating a plant body from a plant cell. For example, regeneration of a plant body from a plant cell can be performed as follows.
When a plant tissue or a protoplast is used as a plant material to be subjected to transformation, the plant material or the protoplast is cultured in a callus-forming medium, which is prepared by adding inorganic elements, vitamins, carbon sources, sugars as an energy source, plant growth-regulating substances (plant hormones such as auxin, cytokinin, gibberellin, abscisic acid, ethylene, and brassinosteroid), followed by sterilization. The plant tissue or the protoplast is then allowed to form a dedifferentiated callus, which will proliferate into an amorphous mass (hereinafter called “callus induction”). The callus thus formed is transferred to a new medium containing plant growth-regulating substances such as auxin and allowed to further proliferate (subculture).
When the callus induction is performed on a solid medium such as agar and subculture is performed by, for example, liquid culture, each culture can be efficiently carried out in a large amount. Then, the callus proliferated by subculturing is cultured under appropriate conditions to induce re-differentiation of an organ (hereinafter called “re-differentiation induction”), and finally a complete plant body is regenerated. The re-differentiation induction can be carried out by appropriately setting composition of constituents in the medium such as plant growth-regulating substances including auxin and carbon sources, the amounts thereof, light, temperature, and the like. An adventitious embryo, an adventitious root, an adventitious shoot, an adventitious stem, an adventitious leaf, and the like are formed and these adventitious organs are grown into a complete plant body by the aforementioned re-differentiation induction. Alternatively, these adventitious organs can be stored until they grow into a complete plant body (for example, in the form of capsulated artificial seed, dried embryo, and lyophilized cell and tissue).
The transformed plant of the present invention encompasses any of an entire plant body, a part of a plant body (for example, a leaf, a petal, a stem, a root, and pollen), a cultured plant cell (for example, a callus and a protoplast), and a seed, having DNA of any of (a) to (g) as described above introduced thereinto. Further, it also encompasses a progeny plant body obtained by sexual or asexual reproduction of the plant body, a part, a cultured cell, and a seed of the progeny plant body. The transformed plant of the present invention can be mass-produced by obtaining a reproductive material such as a seed and a protoplast from the transformed plant and cultivating or culturing the reproductive material.
The growth of the transformed plant obtained as above, particularly root extension and/or an increase in the leaf area are promoted by overexpression of DNA of any of (a) to (g) as described above. As a result, the root will extend under the ground, whereby lodging resistance and drought resistance can be increased and cultivation of the plant in poor nutrition becomes possible. Further, because the growth of leafs is also promoted and a photosynthetic activity is facilitated, ultimately the growth of an entire plant can be promoted.
EXAMPLE 1
The present invention is further specifically described hereinbelow based on Examples; however, the present invention is not limited to these Examples.
(1) Production of Rice FOX Line by FOX Hunting System
In the present Example, rice full-length cDNA was screened using pBIG2113SF, which was obtained by introducing an SfiI cloning site into a constitutive expression vector, pBIG2113N (Taji, T. et al., Plant J., 2002. 24(4): p.p. 417-426 and Becker, D. et al., Nucleic Acid Res., 1990. 18(1): p. 203).
(i) Production of Normalized Rice Full-Length cDNA Mix
Full-length cDNA was prepared from rice by the CAP trapper method. The resulted cDNA was cloned into a site between SfiI restriction sites in Lambda ZAP or Lambda pLC-1-B (reference: Seki M. et al. Plant J., 15, 707-720 (1998)). Sequences at the 5′ end and 3′ end were sequenced using the vector sequence and cDNAs were grouped, and 20,000 independent clones were identified (reference: Seki M. et al. Plant Physiol. Biochem. 39, 211-220 (2001)). Subsequently, 0.5 μl aliquot from each clone prepared at 50 ng/μl was removed, and all the aliquots were mixed in a single tube. From the mixture, 1 μl aliquot was removed, with which 20 μl of the electric competent cell, DH10B (Gibco BRL), were transformed. Then, approximately 200,000 independent colonies grown on agar media containing Amp were mixed, from which plasmids were collected. The plasmids thus obtained were provided as a normalized rice full-length cDNA mix.
(ii) Production of Rice FOX Library
The normalized rice full-length cDNA mix (2 μg) and 700 μg of pBIG2113SF were mixed and simultaneously completely cleaved by SfiI. After cleavage, the cleaved products were concentrated by isopropanol precipitation. The concentrated products were dissolved in 8 μl of water, into which 1 μl of 10× buffer and 1 μl of T4 ligase were mixed, and a reaction was allowed to proceed for one full day at 16° C. The reaction solution (2 μl) was mixed with 40 μl of the electric competent cell, DH10B, and transformation is performed.
Then, approximately 150,000 independent colonies grown on agar media containing kanamycin (Km) were mixed, from which plasmids were collected. The plasmid solution thus collected (2 μl) was mixed with 40 μl of the electric competent Agrobacterium cell, GV3101, and transformation is performed. Approximately 150,000 independent colonies grown on agar media containing Km were suspended in LB liquid medium, to which glycerol was added to prepare a 15% glycerol solution, which was stored at −80° C. The glycerol solution thus obtained was provided as a rice FOX library.
(iii) Production of Rice FOX Lines
Approximately 200,000 colonies of the aforementioned rice FOX library were grown and suspended in a dipping solution, with which the floral dipping of the wild type Arabidopsis thaliana (ecotype: Colombia) was performed. Seeds (T1 seeds) were harvested and allowed to germinate on nutrient-poor media, BAM, containing hygromycin and then, only plants exhibiting hygromycin resistance (approximately 23,000 lines) were transplanted to soil.
(2) Screening of T2 Generation of Rice FOX Line by Root Bending Assay
Seeds (T2 seeds) were collected from the plants transplanted to soil in (1) (iii) above. The T2 seeds and the wild-type seeds were sterilized and sown on 1.2% agar media (MS medium containing vitamin B5). Then, the seeds were subjected to a dark treatment by wrapping the media with aluminum foils, and then to a vernalization treatment by placing the media at 4° C. for approximately one week. Subsequently, the media were held vertically so that roots would extend along the surface of the agar media, and the seeds were grown at 22° C. for three days under continuous white light. The agar media containing grown roots were placed horizontally, which were then irradiated with approximately 2.3 Wm−2 of UV-B (approximately 8 kJm−2) in the dark. Subsequently, the plates were held vertically and rotated by ninety degrees to change the direction of root extension, and the roots were again grown at 22° C. under continuous white light for three days (FIG. 1).
The lengths of the roots extended for three days after UV-B irradiation were evaluated. The UV-B resistance was judged by quantitatively measuring the root length using an image analysis software (Image J, National Institute for Health, USA). As a result, 49 lines were isolated from 7034 rice FOX lines subjected to the root bending assay as candidate UV-B resistant lines.
(3) Re-Cloning of cDNA and Sequencing of Rice Full-Length cDNA Inserted into Rice FOX Line
Approximately two rosette leaves (approximately 200 mgfw) were collected from each of the candidate rice FOX lines selected in (2) above, from which genomic DNA was extracted. A PCR reaction was carried out on the DNA. The composition of the PCR reaction solution was as shown below, and the reaction conditions were as follows; 40 cycles of 94° C. for 0.5 minute, 55° C. for 0.5 minute, and 72° C. for four minutes.
Composition of the reaction solution:

















Primers (100 pM)
2 × 0.25
μl



dNTP (200 μM)
4
μl



Buffer (×2)
25
μl



Polymerase
0.5
μl



Genomic DNA
10
μl



Distilled water
10
μl



Total
50
μl










The primers used for PCR were as follows:








GTACGTATTTTTACAACAATTACCAACAAC
(SEQ ID NO: 9)


 


GGATTCAATCTTAAGAAACTTTATTGCCAA
(SEQ ID NO: 10)






The PCR products were collected from agarose gels, and then mixed with pBIG2113SF. The resultant was completely cleaved by SfiI and then precipitated by isopropanol, followed by treatment with T4 ligase. Then, E. coli was transformed with the resulted mixture. Plasmids into which the PCR fragments were inserted were selected and the nucleotide sequences of the inserted cDNA fragments were identified using the aforementioned primers.
(4) Confirmation of Phenotype of Promoted Root Extension
T2 seeds of Arabidopsis thaliana transformants into which the rice full-length cDNAs obtained in (3) above were re-introduced and the wild-type seeds, each approximately 20 seeds, were sterilized and sown on 1.2% agar media. Then, the seeds were subjected to a dark treatment by wrapping the media with aluminum foils, and then to a vernalization treatment by placing the media at 4° C. for approximately one week. Subsequently, the media were held vertically so that roots would extend along the surface of the agar media, and the seeds were grown at 22° C. for seven days under continuous white light. Subsequently, images of the plant bodies were taken and the lengths from the boundary between the hypocotyls and the roots to the root tips were measured by the aforementioned image analysis software.
The phenotype of promoted root extension was reproduced in the re-transformants. The images and plots of the root lengths of rice FOX lines 1 to 4 are shown in FIGS. 2 and 3. In panel A of FIG. 2, the root length of the wild type was 15.49 mm (average), while that of rice FOX line 1 was 18.36 mm (average). In panel B of FIG. 2, the root length of the wild type was 13.37 mm (average), while that of rice FOX line 2 was 18.76 mm (average). In panel C of FIG. 3, the root length of the wild type was 16.98 mm (average), while that of rice FOX line 3 was 22.25 mm (average). In panel D of FIG. 3, the root length of the wild type was 13.97 mm (average), while that of rice FOX line 4 was 16.78 mm (average).
The sequences of the rice full-length cDNA introduced into the aforementioned rice FOX lines are of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, and the amino acid sequences are of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
(5) Confirmation of Phenotype of Increased Leaf Area
T2 seeds of Arabidopsis thaliana transformants into which the rice full-length cDNAs obtained in (3) above were re-introduced and the wild-type seeds, each 20 to 30 seeds, were sown in agricultural soil and subjected to a vernalization treatment by being placed at 4° C. for four days. Then, the seeds were cultivated at 22° C. with a 16-hour light period. On day 15 after initiation of cultivation under the light condition, images of the plant bodies were taken and the leaf areas were measured by the aforementioned image analysis software.
The phenotype of increased leaf area was reproduced in the re-transformants. The images and plots of the leaf areas of rice FOX lines 1 and 2 are shown in FIG. 4. In panel A of FIG. 4, the leaf area of the wild type was 3.30 mm2 (average), while that of rice FOX line 1 was 5.22 mm2 (average). In panel B of FIG. 4, the leaf area of the wild type was 3.31 mm2 (average), while that of rice FOX line 2 was 5.11 mm2 (average).
INDUSTRIAL APPLICABILITY
The genes whose function was elucidated according to the present invention are involved in the growth of a plant, particularly root extension. The nutrition-absorption ability of a plant is increased by overexpression of the genes in the plant enabling cultivation of a plant under poor nutritional conditions, and lodging resistance and drought resistance can be increased. Particularly, by enlarging edible parts of agricultural products whose main part is the root, such as root vegetables, a commercial value as an edible crop can be increased in the agricultural products.
Further, the genes are also involved in an increase in the leaf area. Overexpression of the genes in a plant can promote the growth above the ground. Particularly, by increasing edible parts of leaf vegetables by increasing the tissue amount above the ground, a commercial value as edible plants can be increased in the leaf vegetables.
As shown above, overexpression of the genes involved in root extension and an increase in the leaf area by transformation can ultimately produce plants the growth of which is entirely promoted. The present invention is applicable to any plants for which the transformation technique has been established.
All the publications, patents, and patent applications cited in the present specification are incorporated herein by reference in their entirety.

## Claims

1. A method for promoting growth of at least one of a plant root and a plant leaf, the method comprising introducing
DNA comprising SEQ ID NO:3;
DNA having at least 95% identity to the full length nucleotide sequence of SEQ ID NO:3;
DNA encoding a protein comprising SEQ ID NO:4; or
DNA encoding a protein that is at least 95% identical to SEQ ID NO: 4 into a plant cell;
cultivating the plant cell to produce a plant with promoted growth of at least one of the root and the leaf of the plant; and
selecting the plant with promoted growth of at least one of the root and the leaf of the plant.

2. The method according to claim 1, wherein the DNA is introduced using a recombinant vector.

3. The method according to claim 1, wherein the plant is a monocot or a dicot.

4. The method according to claim 1, wherein the DNA comprising SEQ ID NO:3 is introduced into the plant cell.

5. The method according to claim 1, wherein the DNA that is at least 95% identical to SEQ ID NO:3 is introduced into the plant cell.

6. The method according to claim 1, wherein the DNA encoding the protein comprising SEQ ID NO:4 is introduced into the plant cell.

7. The method according to claim 1, wherein the DNA encoding the protein that is at least 95% identical to SEQ ID NO:4 is introduced into the plant cell.

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