U.S. patent number RE39,685 [Application Number 10/265,415] was granted by the patent office on 2007-06-05 for method for shortening internode of inflorescence by introducing gene for petunia transcription factor petspl2.
This patent grant is currently assigned to Director General of National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries, N/A. Invention is credited to Hitoshi Nakagawa, Hiroshi Takatsuji.
United States Patent |
RE39,685 |
Takatsuji , et al. |
June 5, 2007 |
Method for shortening internode of inflorescence by introducing
gene for petunia transcription factor petSPL2
Abstract
A gene encoding DNA which is selected from a) or b): a) DNA
having a nucleotide sequence from the 190th position to the 807th
position of a nucleotide sequence represented in SEQ ID NO: 1 of
the Sequence Listing; or b) DNA which hybridizes to DNA of a) under
stringent conditions, and encodes a transcription factor capable of
altering characters of a plant.
Inventors: |
Takatsuji; Hiroshi (Tsukuba,
JP), Nakagawa; Hitoshi (Kukizaki-machi,
JP) |
Assignee: |
Director General of National
Institute of Agrobiological Resources, Ministry of Agriculture,
Forestry and Fisheries (Ibaragi, JP)
N/A (N/A)
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Family
ID: |
16820182 |
Appl.
No.: |
10/265,415 |
Filed: |
October 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
09156580 |
Sep 18, 1998 |
06215043 |
Apr 10, 2001 |
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Foreign Application Priority Data
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Aug 7, 1998 [JP] |
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10-224852 |
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Current U.S.
Class: |
800/290; 435/468;
800/298; 800/317; 800/278; 536/23.6; 435/69.1; 800/323.1;
435/320.1 |
Current CPC
Class: |
C07K
14/415 (20130101); C12N 15/8261 (20130101); Y02A
40/146 (20180101) |
Current International
Class: |
C12N
15/29 (20060101); A01H 5/00 (20060101); C12N
15/82 (20060101); C12N 15/90 (20060101); C12N
5/04 (20060101) |
Field of
Search: |
;435/69.1,320.1,410,419,430,468 ;536/23.6
;800/278,290,295,298,317,323.1 |
References Cited
[Referenced By]
U.S. Patent Documents
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6297429 |
October 2001 |
Takatsuji et al. |
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Foreign Patent Documents
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11-262390 |
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Sep 1999 |
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JP |
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WO 90/12084 |
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Oct 1990 |
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WO |
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Other References
Sakai et al., Nature, 1995, vol. 378, pp. 199-203. cited by
examiner .
Takatsuji, Hiroshi. "Transcription Factors Controlling Floral Organ
Development." Cell Technology. Plant Technology Series. Shujunsha,
Japan, pp. 96-106. cited by other .
Takatsuji, Hiroshi. The IDEN, vol. 51, No. 4, pp. 34-38 (1997).
cited by other .
Torii, K.U. et al. Plant Cell, vol. 8, pp. 735-746 (1996). cited by
other .
Dehio et al. Plant Mol. Bio., vol. 23, pp. 1199-1210 (1993). cited
by other .
Sakai, Hajime et al. "Role of SUPERMAN in maintaining Arabidopsis
floral whorl boundaries." Nature, vol. 378, pp. 199-203 (1995).
cited by other .
Bancroft, E. et al. (The EU Arabidopsis Genome Project: Mt. Bevan)
Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of
Arabidopsis thaliana. Nature, vol. 391, pp. 485-488 (1998). cited
by other .
Ken-ichi Kubo, et. al.: "Cys.sub.2 /His.sub.2 zinc-finger protein
family of petunia: evolution and general mechanism of
target-sequence recognition" Nucleic Acids Research vol. 26, No. 2
pp. (608-615). cited by other .
Nakagawa et al., "Functional analyses of two SUPERMAN-like
zinc-finger proteins, PetSPL1 and PetSPL2, of petunia", Plant and
Cell Physiology vol. 39, No. Suppl., S63, XP001076675 (1998),
Abstract. cited by other .
Takatsuji et al., "Functional characterization of petunia
TFIIIA-type zinc-finger transcription factor: Involvement in the
transcriptional regulation associated with the control of
floral-organ development", Plant and Cell Physiology vol. 39, No.
Suppl., S3, XP001076676 (1998), Abstract. cited by other .
Takatsuji, Hiroshi et al.; "A New Family of Zinc Finger Proteins in
Petunia: Structure, DNA Sequence Recognition, and Floral
Organ-Specific Expression"; 1994, The Plant Cell, vol. 5, pp.
947-958. cited by other.
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Primary Examiner: Mehta; Ashwin
Attorney, Agent or Firm: Townsend & Townsend and Crew,
LLP
Claims
What is claimed is:
1. An isolated DNA molecule comprising a nucleotide sequence from
the 190th position to the 807th position of the nucleotide sequence
represented in SEQ ID NO:1.
2. An isolated DNA molecule encoding a transcription factor
comprising an amino acid sequence from the 1st position to the
206th position of the amino acid sequence represented in SEQ ID
NO:2.
3. A method for producing a transgenic plant, comprising the steps
of: introducing the DNA molecule of claim 1 into a plant cell; and
regenerating the plant cell into a transgenic plant.
4. A method according to claim 3, wherein the plant is a
dicotyledon.
5. A method according to claim 4, wherein the plant is a member of
the Solanaceae family.
6. A method according to claim 5, wherein the plant is a member of
the Petunia genus.
7. A method according to claim 3, wherein the DNA molecule is
incorporated into a plant expression vector.
8. A transgenic plant produced by the method of claim 3.
9. A method for producing a transgenic plant, comprising the steps
of: introducing the DNA molecule of claim 2 into a plant cell; and
regenerating the plant cell into a transgenic plant.
10. A transgenic plant produced by the method of claim 9.
11. A method for altering characters of a plant, comprising steps
of: introducing the DNA molecule of claim 1 into a plant cell;
regenerating the plant cell into a transgenic plant; and selecting
the plant having .Iadd.an .Iaddend.altered .[.characters.].
.Iadd.character.Iaddend., wherein the .[.characters.].
.Iadd.character .Iaddend.of the plant .[.include one.]. .Iadd.is
.Iaddend.selected from the group consisting of .[.the.].
.Iadd.decreased .Iaddend.height of the plant and .[.the.].
.Iadd.decreased .Iaddend.length of an internode.
12. A method for altering characters of a plant, comprising steps
of: introducing the DNA molecule of claim 2 into a plant cell;
regenerating the plant cell into a transgenic plant; and selecting
the plant having .Iadd.an .Iaddend.altered .[.characters.].
.Iadd.character.Iaddend., wherein the .[.characters.].
.Iadd.character .Iaddend.of the plant .[.include one.]. .Iadd.is
.Iaddend.selected from the group consisting of .[.the.].
.Iadd.decreased .Iaddend.height of the plant and .[.the.].
.Iadd.decreased .Iaddend.length of an internode.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a gene encoding a transcription
factor capable of altering characters of a plant and its use. More
particularly, the present invention relates to the PetSPL2 gene
which is a novel gene derived from Petunia hybrida, genes related
thereto, and the use thereof.
2. Description of the Related Art
In order to clarify regulatory mechanisms controlling the
characters of a plant, for example, morphogenesis of a flower,
molecular biological and molecular genetical studies have been
conducted using Arabidopsis thaliana, Antirrhinum majus, and
Petunia hybrida. In particular, Petunia hybrida is preferably used
as a subject of studies for the following reasons: high value as a
horticultural plant; the presence of various species; ease of
transformation; ease to observe due to its large flower; and
accumulation of genetical findings (H. Takatsuji, "Molecular
mechanism for determining a shape of a plant", Cell Technology,
Plant Cell Technology Series (SHUJUNSHA), pp. 96-106).
Genes which cause mutation have been isolated from mutants in which
floral organs of the above-mentioned plant is altered. As a result,
it is becoming clear that transcription factors play important
roles in differentiation and morphogenesis of a flower. For
example, SUPERMAN of Arabidopsis thaliana is a transcription factor
having a zinc finger motif as a DNA binding domain. It is known
that, in SUPERMAN mutant with its gene mutated, number of stamens
are remarkably increased, and pistils are defective (THE IDEN,
April, 1997 (Vol. 51, No. 4), pp. 34-38).
For understanding the mechanism for the control of characters of a
plant, it is important to identify a novel transcription factor
which is involved in such control. A gene for a transcription
factor which controls morphogenesis of a flower may be introduced
into a plant by using gene engineering procedure. It is possible to
obtain a plant, using gene introduction, having a flower with novel
characters which has not been obtained or is not likely to be
obtained by a conventional breeding. It is considered that a plant
with such novel characters is horticulturally valuable.
SUMMARY OF THE INVENTION
A gene of the present invention has DNA which is selected from a)
or b): a) DNA having a nucleotide sequence from the 190th position
to the 807th position of a nucleotide sequence represented in SEQ
ID NO:1 of the Sequence Listing; or b) DNA which hybridizes to DNA
of a) under stringent conditions, and encodes a transcription
factor capable of altering characters of a plant.
A gene of the present invention encodes a transcription factor
which is selected from i or ii): i) a transcription factor having
an amino acid sequence from the 1st position to the 206th position
of an amino acid sequence represented in SEQ ID NO:2; or ii) a
transcription factor having an amino acid sequence in which one or
more amino acids of i) are subjected to deletion, substitution, or
addition, and being capable of altering characters of a plant.
In one embodiment of the present invention, the characters of a
plant include one selected from the group consisting of the height
of a plant and the length of an internode.
A method for producing a transgenic plant of the present invention
includes the steps of: introducing a plant cell with the
above-mentioned gene; and regenerating a plant body from the plant
cell having the introduced gene.
In one embodiment of the present invention, the plant belongs to
dicotyledon.
In another embodiment of the present invention, the plant belongs
to Solanaceae.
In another embodiment of the present invention, the plant belongs
to Petunia.
In another embodiment of the present invention, the gene is
incorporated into a plant expression vector.
A transgenic plant of the present invention is produced by the
above-mentioned method.
Thus, the invention described herein makes possible the advantages
of (1) providing a gene encoding a transcription factor capable of
altering characters of a plant, in particular, a height of a plant
and a length of an internode; (2) providing a method for producing
a plant with its character altered by introduction of the gene; and
(3) providing a transgenic plant.
These and other advantages of the present invention will become
apparent to those skilled in the art upon reading and understanding
the following detailed description with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of PetSPL2 gene
and its deduced amino acid sequence (SEQ ID NO: 2).
FIG. 2 is a schematic view showing a PetSPL2 high expression vector
(pBIN-35S-PetSPL2).
FIG. 3 shows an autoradiogram of a denatured agarose gel
electrophoresis image detecting mRNA of the PetSPL2 gene in Petunia
hybrida transformed with the PetSPL2 gene.
FIG. 4 shows pictures of morphology of a plant body of wild-type
Petunia hybrida (right) and that of Petunia hybrida transformed
with the PetSPL2 gene (left).
FIG. 5 shows pictures of internodes of wild-type Petunia hybrida
(left) and those of Petunia hybrida transformed with the PetSPL2
gene (right).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be described in detail.
The term "transcription factor" as used herein refers to a protein
which binds to a DNA regulatory region of genes to control the
synthesis of mRNA. Some transcription factors are known to have a
highly conservative amino acid sequence called zinc finger motif in
their DNA binding domains.
A gene of the present invention encodes a transcription factor
capable of altering characters of a plant. This gene may have
either of the following DNAs: a) DNA having a nucleotide sequence
from the 190th position to the 807th position of a nucleotide
sequence represented in SEQ ID NO:1 of the Sequence Listing; or b)
DNA which hybridizes to DNA of a) under stringent conditions, and
encodes a transcription factor capable of altering characters of a
plant.
The gene of the present invention may also have DNA which encodes a
transcription factor capable of altering characters of a plant, and
has a homology of about 60% or more, preferably about 70% or more,
more preferably about 80% or more, and still more preferably about
90% or more, with DNA of a).
Preferably, the gene of the present invention may contain DNA of
a).
The gene of the present invention may also encode either of the
following transcription factors: i) a transcription factor having
an amino acid sequence from the 1st position to the 206th position
of an amino acid sequence represented in SEQ ID NO:2; or ii) a
transcription factor having an amino acid sequence in which one or
more amino acids of i) are subjected to deletion, substitution, or
addition, and being capable of altering characters of a plant.
The number of amino acids subject to deletion, substitution, or
addition may be about 130 or less, preferably about 60 or less,
more preferably about 30 or less, still more preferably about 20 or
less, and still further more preferably 10 or less.
Preferably, the gene of the present invention may encode the
transcription factor of i).
The particularly preferred gene in the present invention is PetSPL2
gene. FIG. 1 shows a cDNA sequence (SEQ ID NO:1) of this gene and
its deduced amino acid sequence (SEQ ID NO:2).
Alterations in "characters of a plant" refer to any changes in at
least one character of a plant. The character of a plant includes,
but is not limited to, at least one of the height of a plant and
the length of an internode of a plant. These changes are evaluated
by comparing the characters of a plant obtained by introducing a
gene of the present invention with the characters of a plant
(wild-type or horticultural type) before introducing the gene.
Example of height change of a plant include, but are not limited
to, a dwarf and a semi-dwarf. The dwarfism is preferably about 1/2
or less, more preferably about 1/3 or less of a standard height of
a plant before introducing the gene.
An example of length change of an internode includes, but is not
limited to, a reduction of an internode. The reduction of an
internode includes any reduction of an internode of a reproductive
branch (i.e., inflorescence) and an internode of a vegetative
branch (i.e., phyllotaxis). The reduction of inflorescence is a
particularly preferable example of a change. The change in length
of an internode preferably acheieves a length of about 1/2 or less,
more preferably about 1/5 or less, and most preferably about 1/10
or less, compared with a standard internode of the plant before
introducing a gene.
An example of changed characters of a plant is a combination of a
drawf and a reduction of an internode, more preferably a
combination of a drawf and a reduction of an internode of an
inflorescence.
The gene of the present invention can be isolated, for example, by
performing polymerase chain reaction (PCR) with genomic DNA of a
plant as a template, using a pair of degenerated primers
corresponding to a conserved region of the amino acid sequence
encoded by a gene of a known transcription factor, and screening a
genomic library of the same plant, using the amplified DNA fragment
thus obtained as a probe. Examples of a pair of primers include a
combination of 5'-CARGCNYTNGGNGGNCAY-3' (SEQ ID NO:3) or
5'-YTNGGNGGNCAYATGAAY-3' (SEQ ID NO:4) with 5'-ARNCKNARYTCNARRTC-3'
(SEQ ID NO:5) in which N is inosine, R is G or A, Y is C or T, and
K is T or G.
PCR can be performed in accordance with the manufacturer's
instructions for a commercially available kit and instruments, or
by a procedure well known to those skilled in the art. A method for
producing a gene library, stringent conditions used for
hybridization with a probe, and a method for cloning a gene are
well known to those skilled in the art. For example, see Maniatis
et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
A nucleotide sequence of the gene thus obtained can be determined
by a nucleotide sequence analysis method known in the art or by a
commercially available automatic sequencer.
The gene of the present invention is not limited to those isolated
from native genome but may include synthetic polynucleotides.
Synthetic polynucleotides can be obtained, for example, by
modifying a sequenced gene as described above using a procedure
well known to those skilled in the art.
The gene of the present invention can be ligated to an appropriate
plant expression vector by a method well known to those skilled in
the art and introduced into a plant cell by a known gene
recombination technique. The introduced gene is incorporated into
the DNA of a plant cell. The DNA of a plant cell includes DNA
contained in various organelles (e.g., mitochondria, chloroplasts,
etc.) of a plant cell, as well as chromosomes.
The "plant" includes both monocotyledon and dicotyledon. The
preferred plant is dicotyledon. The dicotyledon includes both
Archichlamiidae and Sympetalidae. A plant of Sympetalidae is
preferable. Examples of the plants of Sympetalidae include
Gentianales, Solanales, Lamiales, Callitrichales, Plantaginales,
Campanulales, Scrophulariales, Rubiales, Dipsacales, Asterales, and
the like. A plant of Solanales is preferable. Examples of the
plants of Solanales include Solanaceae, Hydrophyllaceae,
Polemoniaceae, Cuscutaceae, Convolvulaceae, and the like.
Solanaceae is preferable. Solanaceae includes Petunia, Datura,
Nicotiana, Solanum, Lycopersicon, Capsicum, Physalis, and Lycium,
etc. Plants of Petunia, Datura, and Nicotiana are preferable.
Petunia is more preferable. Examples of the plants of Petunia
include P. hybrida, P. axillaris, P. inflata, P. violacea, and the
like. A plant of P. hybrida is especially preferable. The "plant"
refers to a plant body having a flower and/or a fruit and a seed
obtained from it, unless otherwise specified.
Examples of the "plant cell" include cells from plant organs such
as leaves and roots, callus, and suspension cultured cells.
The term "plant expression vector" as used herein refers to a
nucleic acid sequence in which various regulatory elements, such as
a promoter, for regulating expression of the gene of the present
invention, are linked to each other so as to be operable in a host
plant cell. Preferably, the plant expression vector may include a
plant promoter, a terminator, a drug resistant gene and an
enhancer. It is well known to those skilled in the art that a type
of the plant expression vector and regulator elements may be varied
depending on the type of host cell. A plant expression vector used
according to the present invention may further contain a T-DNA
region. The T-DNA region allows a gene to be efficiently introduced
to plant genome especially when Agrobacterium is used to transform
a plant.
The term "plant promoter" as used herein refers to a promoter that
functions in a plant. Constitutive promoters as well as
tissue-specific promoters which selectively function in a part of a
plant body, including a flower, are preferable. Examples of plant
promoters include, but are not limited to, Cauliflower mosaic virus
(CaMV) 35S promoter and a promoter of nopaline synthase.
The term "terminator" as used herein refers to a sequence
positioned downstream of a region of a gene encoding a protein,
which is involved in the termination of transcription of mRNA, and
the addition of a poly A sequence. The terminator is known to
contribute to the stability of mRNA, thereby affecting the
expression level of a gene. Examples of such terminators include,
but are not limited to, CaMV 35S terminator and a terminator of a
nopaline synthase gene (Tnos).
A "drug resistant gene" is desirable to facilitate the selection of
transgenic plants. The examples of such drug resistant genes for
use in the invention include, but are not limited to, a neomycin
phosphotransferase II (NPTII) gene for conferring kanamycin
resistance, and a hygromycin phosphotransferase gene for conferring
hygromycin resistance.
An "enhancer" may be used to enhance the expression level of a gene
of interest. As the enhancer, an enhancer region containing a
sequence upstream of the above-mentioned CaMV 35S promoter is
preferable. More than one enhancers may be used in one plant
expression vector.
The plant expression vector according to the present invention may
be produced by using a recombinant DNA technique well known to
those skilled in the art. The examples of preferable vectors for
constructing a plant expression vector include, but are not limited
to pBI-type vectors or pUC-type vectors.
A plant expression vector may be introduced into a plant cell by
using methods well known to those skilled in the art, for example,
a method of infecting a plant cell with Agrobacterium or a method
of directly introducing a vector into a cell. The method using
Agrobacterium may be performed, for example, as described in Nagel
et al., Microbiol. Lett., 67, 325, 1990. According to this method,
Agrobacterium is first transformed with a plant expression vector
by, for example, electroporation, and then the transformed
Agrobacterium is infected to a plant cell by a well-known method
such as a leaf-disk method. Examples of the methods for directly
introducing a plant expression vector into a cell include, but are
not limited to, an electroporation method, a particle gun method, a
calcium phosphate method, and a polyethylene glycol method. These
methods are well known in the art and a method suitable for a
particular plant to be transformed may be suitably selected by
those skilled in the art.
The cells in which plant expression vectors have been introduced
are selected based on their drug resistance such as resistance to
kanamycin. Thereafter, the cells may be regenerated to a plant body
by using a conventional method.
Expression of the introduced gene of the present invention in the
regenerated plant body can be confirmed by using a procedure well
known to those skilled in the art. This confirmation can be
performed by northern blot analysis, for example. More
specifically, the total RNAs may be extracted from leaves of a
resultant plant, and may be subjected to denatured agarose gel
electrophoresis, and then, RNAs may be blotted onto an appropriate
membrane. The blot can be hybridized with a labelled RNA probe
complementary to a part of the introduced gene to detect mRNA from
the gene of the present invention.
The plant of the present invention is a transgenic plant produced
by the above-mentioned procedure. It is preferable that the altered
characters of the transgenic plant (i.e., a height of a plant
and/or a length of an internode) include that which is not found in
a known wild-type or horticultural type. It is also preferable that
the altered characters of a plant are horticulturally valuable.
Furthermore, it is preferable that altered characters of a plant
are stably conserved over subsequent generations.
EXAMPLES
Hereinafter, the present invention will be described by way of the
following illustrative examples. Restriction enzymes, plasmids and
the like used in the following examples are available from
commercial sources.
Example 1
Isolation of PetSPL2 Gene
The protein encoded by the SUPERMAN gene of Arabidopsis thaliana
was compared with the protein encoded by the GmN479 gene (Kouchi et
al., personal communication) expressed specifically in soy bean
root nodules. Three different degenerate primers for use in PCR
were synthesized based on the amino acid sequences commonly present
in both proteins. The nucleotide sequences of two primers oriented
5' to 3' in the genes are 5'-CARGCNYTNGGNGGNCAY-3' (primer 1,
corresponding to an amino acid sequence QALGGH; SEQ ID NO:6) and
5'-YTNGGNGGNCAYATGAAY-3'(SEQ ID NO:4). (Primer 2, corresponding to
an amino acid sequence LGGHMN; SEQ ID NO:7), respectively, and a
nucleotide sequence of a primer oriented 3' to 5' in the genes is
5'-ARNCKNARYTCNARRTC-3' (SEQ ID NO:5) (primer 3, corresponding to
an amino acid sequence DLELRL; SEQ ID NO:8), wherein N is inosine,
Y is either C or T, R is either G or A, and K is either T or G.
A first set of PCR was conducted with primer 1 and primer 3 under
the following conditions: 94.degree. C. for 10 minute, followed by
30 cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds and 72.degree. C. for 60 seconds, and subsequently
72.degree. C. for 7 minutes, using as a template a genomic DNA of a
petunia (Petunia hybrida var. Mitchell) extracted according to the
method described in Boutry, M. and Chua N. H. (1985) EMBO J. 4,
2159-2165. In addition, a second PCR was conducted with primer 2
and primer 3, while using as a template a portion of the product
from the first PCR. The reaction conditions were the same as those
used in the first PCR. Amplified DNA fragments were inserted into
the TA cloning vector (produced by Invitrogen), which were then
introduced into E. coli according to a conventional method.
Plasmids were extracted from the transformed E. coil and the
nucleotide sequences of the DNA fragment was determined. The
results revealed that a part of zinc finger motif contained in
common in SUPERMAN and GmN479 was encoded within the resulting DNA
fragment. The gene from which this DNA fragment was derived was
designated as PetSPL2 gene. In the same series of experiments, the
presence of 3 other DNAs (PetSPL1, 3 and 4 genes) containing a
nucleotide sequence similar to that of PetSPL2 was demonstrated.
For details regarding the PetSPL3 gene, see Japanese Patent
Application No. 10-65921.
To clone cDNA of the PetSPL2 gene, the DNA fragment described above
and a GENETRAP cDNA selection kit (produced by BRL) were used to
screen a cDNA library of petunia floral buds (Petunia hybrida var.
Mitchell) which had been created, using the BRL kit, within a
pSPORT plasmid vector (produced by BRL). Several clones for the
PetSPL2 gene were obtained by screening this cDNA library. Thus,
cDNA for the PetSPL2 gene derived from Petunia was isolated.
Example 2
Analysis of the Nucleotide Sequence and Amino Acid Sequence of
PetSPL2 gene
The longest clone out of the clones obtained in Example 1 contained
a PetSPL2 gene cDNA fragment of about 1.0 kb. The DNA nucleotide
sequence of this cDNA fragment was determined (SEQ ID NO:1). From
an open reading frame contained in the resulting DNA nucleotide
sequence, an amino acid sequence of the protein was deduced (SEQ ID
NO:2).
The comparison of the nucleotide sequences indicated that the
PetSPL2 gene showed 58%, 67% and 51% nucleotide sequence homology
to the SUPERMAN, PetSPL1 and PetSPL4 genes, respectively. The
PetSPL2 gene showed 52% nucleotide sequence homology to PetSPL3
gene. This comparison of the nucleotide sequences was conducted
only within the coding region of each gene.
The deduced amino acid sequence of PetSPL2 contained a single
TFIIIA-type zinc finger motif similar to that of SUPERMAN. On this
basis, it was presumed that PetSPL2 was a transcription factor.
PetSPL2 showed about 37% and 23% homology to SUPERMAN and PetSPL3,
respectively, in the full-length amino acid sequence.
Table 1 compares the amino acid sequence of SUPERMAN with that of
each PetSPL in the zinc finger motif. Amino acid homology (about
100%) of PetSPL2 to SUPERMAN in the zinc finger motif was shown to
be the same as the corresponding homology (about 100%) of PetSPL1
to SUPERMAN and to be higher compared with that (about 76%) of
PetSPL3 to SUPERMAN (wherein the amino acid sequence homology was
calculated assuming that the zinc finger motif extends from the 4th
C to the 24th H). Table 1 also shows a comparison of C terminal
hydrophobic region of SUPERMAN with that of each PetSPL.
TABLE-US-00001 TABLE 1 Zinc-finger ##STR00001## C-terminal
hydrophobic region ##STR00002##
From the results described above, it was presumed that PetSPL2 is a
novel transcription factor with a closer relationship to SUPERMAN
than to PetSPL3, and belongs to a class different from that of
PetSPL3.
Example 3
Construction of a Plant Expression Vector Containing a
Polynucleotide Encoding PetSPL2
A DNA fragment (HindIII-XbaI fragment) containing a CaMV 35S
promoter in a plasmid pBI221 (purchased from Clontech) and a DNA
fragment (SacI-EcoRI fragment) containing a NOS terminator were
sequentially inserted into a multicloning site of plasmid PUCAP
(van Engelen, F. A. et al., Transgenic Res. 4:288-290 (1995)) to
produce pUCAP35S. On the other hand, pSPORT/PetSPL2 plasmid
containing PetSPL2 was cleaved at KpnI and SacI sites (which are
the sites within this vector), and inserted between KpnI and SacI
sites of the pUCAP35. This recombinant plasmid was further cleaved
with AscI and PacI, and the resultant DNA fragment encoding PetSPL2
was introduced into AscI and PacI sites of a binary vector pBINPLUS
(van Engelen, F. A. et al., (1995), supra).
The constructed PetSPL2 gene high expression vector
(pBIN-35S-PetSPL2) includes, as shown in FIG. 2a, a CaMV 35S
promoter region (P35S; 0.9 kb), a polynucleotide of the present
invention encoding PetSPL2 (PetSPL2; 1.0 kb) and a terminator
region of nopaline synthase (Tnos: 0.3 kb). In FIG. 2, Pnos and
NPTII indicate a promoter region of nopaline synthase and neomycin
phosphotransferase II gene, respectively. LB and RB indicate T-DNA
left border and T-DNA right border, respectively.
Example 4
Introduction of the PetSPL2 Gene into PETUNIA Cells
(1) (Transformation of Agrobacterium tumefaciens)
Agrobacterium tumefaciens LBA4404 line (purchased from Clontech)
was cultured in an L medium containing 250 .mu.g/ml streptomycin
and 50 .mu.g/ml rifampicin at 28.degree. C. According to the method
of Nagel et al. (1990) (supra), a cell suspension of this strain
was prepared. The PetSPL2 gene high expression vector constructed
in Example 3 was introduced into the above described strain by
electroporation.
(2) (Introduction of a polynucleotide encoding PetSPL2 into Petunia
cell)
The Agrobacterium tumefaciens LBA4404 line obtained in (1) was
cultured (at 28.degree. C., 200 rpm) with agitation in YEB medium
(D. M. Glover ed. DNA Cloning, IPL PRESS, second edition, p.78),
followed by a 20-fold dilution with sterilized water. Leaf sections
of petunia (Petunia hybrida var. Mitchell) were cultured in this
diluted solution. After 2-3 days, the Agrobacterium was removed
using a medium containing carbenicillin, and thereafter these leaf
sections were subcultured in a selection medium by transferring to
new media every 2 weeks. The Kanamycin resistance trait conferred
by the expression of the NPTII gene derived from pBINPLUS,
introduced together with the above-mentioned PetSPL2 gene, was used
as an indicator to select transformed petunia cells. Callus was
induced from the transformed cells using a conventional method, and
then re-differentiated into a plant body.
Example 5
Expression of the PetSPL2 Gene in a PetSPL2 Transformed Plant
Total RNAs were extracted from leaves of 14 PetSPL2 transformed
petunias obtained in Example 4. 10 .mu.g each of the extracts was
subjected to denatured agarose gel electrophoresis, and blotted
onto a Genescreen plus filter (produced by DuPont) in accordance
with a conventional method. A PetSPL2 antisense RNA was labelled
using DIG RNA labelling kit (produced by Boeringer Mannheim).
Hybridization and filter washing were performed with the labelled
RNA according to the instructions of the kit. After the washing,
the filter was exposed to an XAR film (produced by Kodak) for 1
hour at room temperature. FIG. 3 shows an autoradiogram of an image
of denatured agarose gel electrophoresis which detected PetSPL2
gene mRNAs from 13 petunias. These results indicated that 4 out of
13 individual transformant petunias expressed PetSPL2 mRNA at a
high level under the control of a high expression promoter.
Example 6
Phenotype of a Transformant Petunia Expressing PetSPL2 Gene at High
Level
Phenotypes as described below were commonly observed in 3 petunias
out of 4 individual transformant petunias expressing the PetSPL2
gene at high levels. The remaining one petunia expressed the
PetSPL2 gene at relatively low level compared with the above three
petunias. The most significant change observed in the plant bodies
was shortening of the internode length of their inflorescences
(i.e., suppression of internode elongation) and dwarfism associated
therewith (FIG. 4; left panel shows a PetSPL2-transformant petunia
and right panel shows a wild type petunia). This change was
observed more extensively in the reproductive stage, namely
inflorescence, than in the vegetative stage. The internode length
of the inflorescence was shown to be less than one tenth of the
wild type (FIG. 5; left panel shows internodes of a wild type
petunia and right panel shows those of a PetSPL3-transformant
petunia). Other changes were rounding of leaves, a moderate
decrease in size of flowers and the like (FIG. 4).
For a gene involved in controlling the internode elongation of
inflorescence, an ERECTA gene of Arabidopsis thaliana has been
reported (Torii et al., 1996, Plant Cell, 8:735). However, no
significant homology between the ERECTA gene and the PetSPL2 gene
of the present invention is found at either a nucleotide sequence
level or an amino acid sequence level. Some of the genes which are
involved in plant hormone synthesis and control thereof (rolA,
etc.) are also known to shorten the internode length. However,
these plant hormone-related genes are known for exhibiting multiple
effects as well as controlling the internode elongation (Dehio et
al., 1993, Plant Mol. Bio., 23:1199).
From the results described above, it has been shown that the
PetSPL2-transformant petunia becomes more dwarfish with shortening
of the internode length such that the appearance of the flowers
change significantly as compared to the wild types. Accordingly, it
is understood that introduction of the PetSPL2 gene is useful,
especially for ornamental flowers or horticultural types whose
internode length is prone to elongation. A significant change in
the appearance of flowers may confer a new value for appreciation
on plants. In addition, suppression of the height of a plant may
have a significant horticultural value with respect to making the
plant resistant to dislodging. Furthermore, fruit trees having the
PetSPL gene introduced therein are expected to become compact in
their shapes. This is meaningful because it may make
fruit-harvesting work more efficient.
According to the present invention, a gene encoding a transcription
factor capable of altering morphology, and the like of a plant is
provided. By utilizing the present gene, a plant with altered
character can be produced. The generated plant is horticulturally
useful because it is provided with the character which is not found
or rarely found in a wild-type and horticultural type.
Various other modifications will be apparent to and can be readily
made by those skilled in the art without departing from the scope
and spirit of this invention. Accordingly, it is not intended that
the scope of the claims appended hereto be limited to the
description as set forth herein, but rather that the claims be
broadly construed.
SEQUENCE LISTINGS
1
181997DNAPetunia hybrida var. MitchellCDS(190)..(810)PetSPL2
transcription factor 1cccagtgcca ttttttctct ctagtcaagc tctctatatc
atcatcacta ttcccttggc 60tgcagtaaca ctcctattta accctcacaa aaaaattacc
agagggcagc aaaaaatgct 120tgaacataat tattatactt actattaagc
tagatttcct cttgatcttg ctaggtttga 180ctggagaaa atg gca ggc atg gat
aga aac agt ttc aac agt aag tac ttc 231 Met Ala Gly Met Asp Arg Asn
Ser Phe Asn Ser Lys Tyr Phe 1 5 10aaa aac aaa agc atc atg gca aga
cag atg gag tac ttg aat aac aac 279Lys Asn Lys Ser Ile Met Ala Arg
Gln Met Glu Tyr Leu Asn Asn Asn 15 20 25 30aat ggc gac aat aac aac
aac aat aat gtt aca agc tca tta cga gat 327Asn Gly Asp Asn Asn Asn
Asn Asn Asn Val Thr Ser Ser Leu Arg Asp 35 40 45aat tat gga aat gaa
gat cat tta ctt ggt gga cta ttc tct tgg cct 375Asn Tyr Gly Asn Glu
Asp His Leu Leu Gly Gly Leu Phe Ser Trp Pro 50 55 60cca aga tct tat
aca tgt agc ttt tgt aaa agg gaa ttt aga tct gct 423Pro Arg Ser Tyr
Thr Cys Ser Phe Cys Lys Arg Glu Phe Arg Ser Ala 65 70 75caa gct ctt
ggt gga cac atg aat gtt cat aga aga gat aga gcc att 471Gln Ala Leu
Gly Gly His Met Asn Val His Arg Arg Asp Arg Ala Ile 80 85 90ttg aga
caa tca cca cct aga gat att aat agg tat tct ctt cta aac 519Leu Arg
Gln Ser Pro Pro Arg Asp Ile Asn Arg Tyr Ser Leu Leu Asn 95 100 105
110ctt aat ctt gaa cca aac cct aac ttt tac cct agt cat aac cct agt
567Leu Asn Leu Glu Pro Asn Pro Asn Phe Tyr Pro Ser His Asn Pro Ser
115 120 125ttt tca aga aaa ttc cca cct ttt gaa atg agg aaa tta gga
aaa gga 615Phe Ser Arg Lys Phe Pro Pro Phe Glu Met Arg Lys Leu Gly
Lys Gly 130 135 140gtt gtt cca aac aat cac ttg aaa agt gcc aga ggg
cgt ttt gga gtt 663Val Val Pro Asn Asn His Leu Lys Ser Ala Arg Gly
Arg Phe Gly Val 145 150 155gag aaa att gac tct ttc atg caa gaa aaa
gaa tgt act act aca gtg 711Glu Lys Ile Asp Ser Phe Met Gln Glu Lys
Glu Cys Thr Thr Thr Val 160 165 170atc aag aag tcc gag ttt cta aga
ttg gac ttg gga att ggg ttg atc 759Ile Lys Lys Ser Glu Phe Leu Arg
Leu Asp Leu Gly Ile Gly Leu Ile175 180 185 190agt gaa tca aag gaa
gat tta gat ctt gaa ctt cga ctg gga tcc act 807Ser Glu Ser Lys Glu
Asp Leu Asp Leu Glu Leu Arg Leu Gly Ser Thr 195 200 205taactatatc
taatttttac ggcattaagg tttgtaaatt gagtcgacag cttagtcaaa
867actacttatg cactttaata tggcttcttg tgctatattt atttatttta
catggctgta 927tctaggtttg cattttaaga tttagtacct tgtcagatta
aaagaaaacg aaagttaaat 987taaaaaaaaa 9972206PRTPetunia sp. 2Met Ala
Gly Met Asp Arg Asn Ser Phe Asn Ser Lys Tyr Phe Lys Asn 1 5 10
15Lys Ser Ile Met Ala Arg Gln Met Glu Tyr Leu Asn Asn Asn Asn Gly
20 25 30Asp Asn Asn Asn Asn Asn Asn Val Thr Ser Ser Leu Arg Asp Asn
Tyr 35 40 45Gly Asn Glu Asp His Leu Leu Gly Gly Leu Phe Ser Trp Pro
Pro Arg 50 55 60Ser Tyr Thr Cys Ser Phe Cys Lys Arg Glu Phe Arg Ser
Ala Gln Ala 65 70 75 80Leu Gly Gly His Met Asn Val His Arg Arg Asp
Arg Ala Ile Leu Arg 85 90 95Gln Ser Pro Pro Arg Asp Ile Asn Arg Tyr
Ser Leu Leu Asn Leu Asn 100 105 110Leu Glu Pro Asn Pro Asn Phe Tyr
Pro Ser His Asn Pro Ser Phe Ser 115 120 125Arg Lys Phe Pro Pro Phe
Glu Met Arg Lys Leu Gly Lys Gly Val Val 130 135 140Pro Asn Asn His
Leu Lys Ser Ala Arg Gly Arg Phe Gly Val Glu Lys145 150 155 160Ile
Asp Ser Phe Met Gln Glu Lys Glu Cys Thr Thr Thr Val Ile Lys 165 170
175Lys Ser Glu Phe Leu Arg Leu Asp Leu Gly Ile Gly Leu Ile Ser Glu
180 185 190Ser Lys Glu Asp Leu Asp Leu Glu Leu Arg Leu Gly Ser Thr
195 200 205318DNAArtificial SequenceDescription of Artificial
Sequencedegenerate primer 1 3cargcnytng gnggncay 18418DNAArtificial
SequenceDescription of Artificial Sequencedegenerate primer 2
4ytnggnggnc ayatgaay 18517DNAArtificial SequenceDescription of
Artificial Sequencedegenerate primer 3 5arncknaryt cnarrtc
1766PRTArtificial SequenceDescription of Artificial Sequenceamino
acids present in both SUPERMAN gene of Arabidopsis thaliana and
GmN479 gene of soy bean root nodules 6Gln Ala Leu Gly Gly His 1
576PRTArtificial SequenceDescription of Artificial Sequenceamino
acids present in both SUPERMAN gene of Arabidopsis thaliana and
GmN479 gene of soy bean root nodules 7Leu Gly Gly His Met Asn 1
586PRTArtificial SequenceDescription of Artificial Sequenceamino
acids present in both SUPERMAN gene of Arabidopsis thaliana and
GmN479 gene of soy bean root nodules 8Asp Leu Glu Leu Arg Leu 1
5943PRTArtificial SequenceDescription of Artificial
SequenceSUPERMAN zinc finger motif 9Ser Tyr Thr Cys Ser Phe Cys Lys
Arg Glu Phe Arg Ser Ala Gln Ala 1 5 10 15Leu Gly Gly His Met Asn
Val His Arg Arg Asp Arg Ala Arg Leu Arg 20 25 30Leu Gln Gln Ser Pro
Ser Ser Ser Ser Thr Pro 35 401042PRTArtificial SequenceDescription
of Artificial SequencePetSPL1 zinc finger motif 10Ser Tyr Thr Cys
Ser Phe Cys Lys Arg Glu Phe Arg Ser Ala Gln Ala 1 5 10 15Leu Gly
Gly His Met Asn Val His Arg Arg Asp Arg Ala Arg Leu Arg 20 25 30Leu
Gln Ser Pro Pro Arg Glu Asn Gly Thr 35 401141PRTArtificial
SequenceDescription of Artificial SequencePetSPL2 zinc finger motif
11Ser Tyr Thr Cys Ser Phe Cys Lys Arg Glu Phe Arg Ser Ala Gln Ala 1
5 10 15Leu Gly Gly His Met Asn Val His Arg Arg Asp Arg Ala Ile Leu
Arg 20 25 30Gln Ser Pro Pro Arg Asp Ile Asn Arg 35
401243PRTArtificial SequenceDescription of Artificial
SequencePetSPL3 zinc finger motif 12Ser Tyr Glu Cys Asn Phe Cys Lys
Arg Gly Phe Ser Asn Ala Gln Ala 1 5 10 15Leu Gly Gly His Met Asn
Ile His Arg Lys Asp Lys Ala Lys Leu Lys 20 25 30Lys Gln Lys Gln His
Gln Arg Gln Gln Lys Pro 35 401343PRTArtificial SequenceDescription
of Artificial SequencePetSPL4 zinc finger motif 13Phe Tyr Arg Cys
Ser Phe Cys Lys Arg Gly Phe Ser Asn Ala Gln Ala 1 5 10 15Leu Gly
Gly His Met Asn Ile His Arg Lys Asp Arg Ala Lys Leu Arg 20 25 30Glu
Ile Ser Thr Asp Asn Leu Asn Ile Asp Gln 35 401433PRTArtificial
SequenceDescription of Artificial SequenceSUPERMAN C-terminal
hydrophobic region 14Ile Leu Arg Asn Asp Glu Ile Ile Ser Leu Glu
Leu Glu Ile Gly Leu 1 5 10 15Ile Asn Glu Ser Glu Gln Asp Leu Asp
Leu Glu Leu Arg Leu Gly Phe 20 25 30Ala1533PRTArtificial
SequenceDescription of Artificial SequencePetSPL1 C-terminal
hydrophobic region 15Leu Met Lys Arg Ser Glu Phe Leu Arg Leu Glu
Leu Gly Ile Gly Met 1 5 10 15Ile Asn Glu Ser Lys Glu Asp Leu Asp
Leu Glu Leu Arg Leu Gly Tyr 20 25 30Thr1633PRTArtificial
SequenceDescription of Artificial SequencePet SPL2 C-terminal
hydrophobic region 16Val Ile Lys Lys Ser Glu Phe Leu Arg Leu Asp
Leu Gly Ile Gly Leu 1 5 10 15Ile Ser Glu Ser Lys Glu Asp Leu Asp
Leu Glu Leu Arg Leu Gly Ser 20 25 30Thr1733PRTArtificial
SequenceDescription of Artificial SequencePetSPL3 C-terminal
hydrophobic region 17Gly Ser Val Asp Ser Arg Glu Asn Arg Leu Pro
Ala Arg Asn Gln Glu 1 5 10 15Thr Thr Pro Phe Tyr Ala Glu Leu Asp
Leu Glu Leu Arg Leu Gly His 20 25 30Glu1833PRTArtificial
SequenceDescription of Artificial SequencePetSPL4 C-terminal
hydrophobic region 18Cys Gly Thr Leu Asp Glu Lys Pro Lys Arg Gln
Ala Glu Asn Asn Asp 1 5 10 15Met Gln Gln Asp Asp Ser Lys Leu Asp
Leu Glu Leu Arg Leu Gly Pro 20 25 30Asp
* * * * *