Note: Descriptions are shown in the official language in which they were submitted.
CA 02519997 2005-09-22
DESCRIPTION
STRESS-INDUCIBLE PROMOTER AND METHOD FOR USING THE SAME
Field of the invention
The present invention relates to a stress-inducible promoter derived
from rice and a method for using the same.
Prior Art
Plants possess tolerance mechanisms to cope with various types of
environmental stresses in nature, such as dehydration, high temperature,
freezing, or salt stress. As the stress tolerance mechanism has been
elucidated at a molecular level in recent years, stress tolerant plants have
been
produced via biotechnological techniques. For example, it has been shown
that stress proteins such as LEA proteins, water channel proteins, or
synthases
for compatible solutes are induced in cells when they are exposed to stress,
thereby protecting the cells from such stress. Thus, research has been
attempted in which genes of LEA proteins of barley or detoxification enzymes
of tobacco, genes of synthases for osmoregulatory substances (e.g., sugar,
proline, or glycinebetaine), or the like are introduced into host plants.
Research using genes encoding w-3 fatty acid desaturase of Arabidopsis
thaliana, the D9-desaturase of blue-green algae, or the like, which are
modification enzymes of the cellular membrane lipid, has also been attempted.
In such research, a gene was bound to the 35S promoter of the cauliflower
mosaic virus and introduced into a plant. The level of stress tolerance of the
recombinant plant was, however, unstable, and the expression level of the
introduced gene was low. Thus, none of these was put to practical use.
On the other hand, a stress tolerance mechanism is found to be
intricately associated with several genes (Shinozaki K, Yamaguchi-Shinozaki
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K. Plant Physiol., 1997, Oct; 115(2), pp. 327-334). Accordingly, research
whereby a gene that encodes a transcription factor and that also
simultaneously activates the expression of the aforementioned several genes is
ligated to a constitutive promoter and introduced into a plant, thereby
enhancing the plant's stress tolerance, has been attempted (Liu et al., The
Plant Cell, 1998, 10: 1391-1406). When expressions of several genes are
simultaneously activated, however, the energy of the host plant becomes
directed towards the synthesis of the gene product or intracellular metabolism
resulting from the gene product. Accordingly, the growth of the plant itself
becomes retarded or results in a dwarf.
In contrast, the present inventors isolated from Arabidopsis thaliana
the DREBIA, DREB1 B, DREB1 C, DREB2A, and DREB2B genes encoding the
transcription factors that bind to a stress-responsive element and
specifically
activate the transcription of genes located downstream of such element (JP
Patent Publication (Unexamined Application) No. 2000-60558). They
reported that the introduction of the genes into a plant by ligating them to a
stress-inducible rd29A promoter enabled production of a stress-tolerant plant
without retarding plant growth (JP Patent Publication (Unexamined
Application) No. 2000-116260).
The rd29A promoter is derived from Arabidopsis thaliana, which is a
dicotyledonous plant. It is able to function in monocotyledonous plants,
although its activity level is low. Accordingly, a stress-inducible promoter
capable of a high level of activity in monocotyledonous plants has been
awaited.
Disclosure of the Invention
The present invention is directed to discovering a stress-inducible
promoter that can effectively function in monocotyledonous plants such as rice
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and to providing a novel environmental stress-tolerant plant using such
promoter.
The present inventors have conducted concentrated studies in order to
attain the above object. As a result, they have succeeded in isolating a
potent
stress-inducible promoter from the rice genome. They have also found that
the environmental stress tolerance of the monocotyledonous plant could be
significantly improved with the use of such promoter. This has led to the
completion of the present invention.
Specifically, the present invention relates to a stress-inducible
promoter derived from rice. More specifically, the promoter consists of the
following DNA (a) or (b):
(a) DNA consisting of the nucleotide sequence as shown in SEQ ID NO:
1 or 10; or
(b) DNA hybridizing under stringent conditions with DNA consisting
of a nucleotide sequence that is complementary to the DNA consisting of a
nucleotide sequence as shown in SEQ ID NO: 1 or 10 and expressing
stress-inducible promoter activity.
The term "stress" used herein refers to dehydration stress, low
temperature stress, or salt stress.
The present invention provides a recombinant vector comprising the
aforementioned promoter. The vector may comprise other structural genes or
regulatory genes under the control of the promoter according to the present
invention. It is particularly preferable that the vector comprises structural
genes and/or regulatory genes for enhancing stress tolerance.
Examples of preferable structural genes for enhancing stress tolerance
include the P5CS gene, which is a key enzyme for proline synthesis (Yoshiba
Y. et al., 1999, BBRC 261), and the AtGo1S3 gene for galactinol synthesis
(Taji T. et al., 2002, Plant J. 29: 417-426).
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Examples of preferable regulatory genes for enhancing stress tolerance
include the Arabidopsis thaliana-derived DREB transcription factor genes (JP
Patent Publication (Unexamined Application) No. 2000-60558), the
rice-derived OsDREB transcription factor genes (Japanese Patent Application
No. 2001-358268, Dubouzet et al., Plant J. in press), and the NCED gene,
which is a key enzyme for the biosynthesis of the plant hormone ABA (luchi S.
et al., 2001, Plant J. 27: 325-333).
The Arabidopsis thaliana-derived DREB transcription factor genes and
the rice-derived OsDREB transcription factor genes are particularly
preferable.
The rice-derived OsDREB transcription factor genes are most preferable.
The present invention provides a transgenic plant that is obtained by
introducing the vector of the present invention into a suitable host.
According to one embodiment of the present invention, the transgenic plant is
obtained by introducing the vector of the present invention into a host plant.
In such a case, the host plant is preferably a monocotyledonous plant, and
such
monocotyledonous plant is preferably rice.
By introducing the promoter of the present invention into plants, the
present invention can further provide a method for enhancing stress tolerance
in plants. The promoter of the present invention exhibits potent
stress-inducible promoter activity that has never been observed in
monocotyledonous plants, and thus, the promoter of the present invention is
more suitable for enhancing the stress tolerance of monocotyledonous plants.
Brief Description of Drawings
Fig. 1 shows the results of Northern analysis on a0022(LIP9) when
each type of stress is applied.
Fig. 2 shows the nucleotide sequence of a0022(LIP9) in its promoter
region.
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Fig. 3 shows the structure of a GUS expressing construct, wherein Tg7
represents a g7 terminator, HPT represents hygromycin phosphotransferase,
Pnos represents an Nos promoter, and Tnos represents a Nos terminator.
Fig. 4 is a graph showing the GUS activities of transgenic tobacco or
rice prepared by introducing various promoters ligated to GUS genes when
dehydration stress is applied.
Fig. 5 is a photograph showing the results of GUS staining on
transgenic rice prepared by introducing an LIPS promoter ligated to GUS
genes when salt stress is applied.
Fig. 6 shows the results of analyzing the expression levels of the
introduced genes and the target genes (LIP9 (a0022), WS1724 (a0066), and
salT (;a2660)) in the transgenic rice and in the wild-type rice by the
Northern
method, wherein "a," "b," and "c" each independently represent a transgenic
plant line.
Fig. 7 shows the nucleotide sequence of a0066(WS1724) in its promoter
region.
Fig. 8 is a graph showing the GUS activities of transgenic rice prepared
by introducing a WS1724 promoter ligated to GUS genes when dehydration
stress is applied, wherein right bars represent GUS activities when
dehydration
stress is applied and left bars represent the controls.
Fig. 9 is a photograph showing the results of GUS staining on
transgenic rice prepared by introducing a WS1724 promoter ligated to GUS
genes when dehydration stress is applied.
Embodiments of the Invention
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The promoter of the present invention is a rice-derived promoter, which
is induced specifically by environmental stress such as low temperature,
dehydration, or salt stress.
1. Identification of the promoter of the present invention
The promoter of the present invention can be identified as follows.
Plants that were given stress are compared with plants that were not given
stress, and genes that are expressed at significantly different levels
(stress-inducible genes) are first screened for. Based on the genome
information, a sequence that is considered to be a promoter of the gene is
then
screened for.
A process for identifying the promoter of the present invention is
hereafter described.
1.1 Preparation of mRNA
At the outset, mRNA for screening for the stress-inducible genes is
prepared.
A source of mRNA may be a part of a plant such as a leaf, stem, root, or
flower or a plant as a whole. Alternatively, a plant obtained by sowing seeds
on a solid medium such as GM medium, MS medium, or #3 medium and
growing them aseptically may be used. The source may be a callus or a
cultured cell of the plant that was aseptically grown.
In this screening process, differences in gene expression levels are
observed between plants that were given stress and plants that were not given
stress. Thus, it is necessary to prepare mRNAs for each of the plants. A
method for applying stress is suitably determined depending on the types of
plants to be used. In general, dehydration stress can be applied by growing
plants without water for 2 to 4 weeks. Low temperature and freezing stresses
can be applied by growing plants at 15 to -10 C for 1 to 10 days. Salt stress
can be applied by growing plants in 100 to 600 mM NaCl for 1 hour to 7 days.
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In the case of rice, for example, hydroponically grown rice is exposed to low
temperature stress (10 to -4 C), salt stress (150 to 250 mM NaCl), and
dehydration stress (desiccated state).
Plants that were given stress and plants that were not given stress are
frozen with liquid nitrogen and ground in a mortar, etc. From the resulting
ground material, a crude RNA fraction is extracted by the glyoxal method, the
guanidine thiocyanate and cesium chloride method, the lithium chloride and
urea method, the proteinase K and deoxyribonuclease method, or the like.
From this crude RNA fraction, poly(A)+ RNA (mRNA) can be then obtained
by the affinity column method using oligo dT-cellulose, poly U-Sepharose*
carried on Sepharose*2B, or the like or by the batch method. The resulting
mRNA may further be fractionated by sucrose density gradient centrifugation
or the like, if necessary.
1.2 Screening for stress-inducible gene
The stress-inducible genes are screened for based on a comparison of
differences in gene expression levels between plants that were given stress
and
plants that were not given stress. Methods for comparing the gene
expression levels are not particularly limited, and examples thereof include
conventional methods such as RT-PCR, real time PCR, subtraction,
differential display, differential hybridization, and cross hybridization.
A method using solid phase samples such as gene chips and cDNA
microarrays is especially suitable for implementing the screening procedure
because such method can simultaneously detect the expression of several
thousands to several tens of thousands of genes qualitatively and
quantitatively.
(1) Preparation of cDNA microarray
The cDNA microarray used in the screening procedure is not
particularly limited as long as the cDNA of the monocotyledonous plant (e.g.,
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rice), i.e., a detection target of the promoter, is spotted thereon. An
existing
array may be used, or an array may be prepared based on conventional methods
(e.g., The Plant Cell (2001) 13: 61-72 Seki et al.).
When preparing the cDNA microarray, the cDNA library of the plant of
interest should be prepared first. The cDNA library can be produced by
conventional methods using mRNA prepared in accordance with the method in
(1) as a template. The cDNA to be spotted is not particularly limited as long
as it is derived from monocotyledonous plants. From the viewpoint of ease
in later analyses of genome databases, cDNA derived from monocotyledonous
plants such as rice with advanced genome analysis is preferable. Plants may
be in a normal state (without treatment). However, plants are preferably
exposed to stress such as dehydration, salt, or low temperature.
When producing the cDNA library, a commercially available kit (e.g.
ZAP*-cDNA Synthesis Kit, Stratagene) is first used for reverse transcription
of
mRNA and single-stranded cDNA synthesis. Then, double-stranded cDNA is
synthesized using the resulting single-stranded cDNA as a template.
Subsequently, an adaptor containing a suitable restriction site is added to
the
resulting double-stranded cDNA, which is then inserted into a cloning site of
a
lambda phage vector. The resulting DNA is packaged in vitro using a
commercially available kit (e.g., Gigapack* III Gold packaging extract
(Stratagene)), caused to infect an E. coli host, and then amplified. Thus, the
cDNA library of interest can be obtained.
Once the cDNA library is produced, this cDNA or a region with a high
specificity in such cDNA (e.g., the UTR region containing no repeating
sequence on the 3' side) is amplified by PCR to produce a probe to be
immobilized on the array. When probes for all the genes of interest are
produced by repeating this procedure, these probes are spotted on a slide
glass
using a commercially available spotter (e.g., one manufactured by Amersham).
Thus, the cDNA microarray of interest is obtained.
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(2) Detection of gene expression level
Gene expression levels can be detected by the cDNA microarray as
signal intensity obtained when sample mRNA (or cDNA) labeled with a
suitable reagent is hybridized with the cDNA probe on the microarray. In
general, the expression level of the gene is preferably determined as a
comparative value with a suitable control or the ratio of expression levels
between two samples to be compared, with respect to the differences in the
amount of cDNA probes spotted on the array. In the case of the present
screening procedure, mRNA derived from plants that were not given stress
(without treatment) is employed as a control, and relative expression levels
of
mRNA derived from plants that were given stress may be detected in relation
thereto.
Detection is carried out as follows. mRNAs of the control and the
sample (or cDNA thereof) are labeled with different fluorescent dyes (e.g.,
Cy3 and Cy5) and hybridized with the cDNA probe on the array. For example,
mRNA is extracted from the plants that were given stress and subjected to
reverse transcription in the presence of Cy5-labeled dCTP to prepare
Cy5-labeled cDNA. Subsequently, mRNA is extracted from plants that were
not given stress (without treatment), and Cy3-labeled cDNA is prepared in the
same manner. Cy5-labeled cDNA (sample) is mixed with an equivalent
amount of Cy3-labeled cDNA (control), and the resultant is hybridized with
cDNA on the array. Cy3 may be used for labeling the sample, and Cy5 may
be used for labeling the control. Alternatively, other suitable label reagents
may also be used.
The obtained fluorescence intensity is read using a fluorescent signal
detector and then converted into a numerical value. This numerical value is
equivalent to the ratio of the gene expression levels of the sample relative
to
the control. The fluorescence intensity read using a scanner is optionally
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subjected to error adjustment or normalization of variances for each sample.
Normalization can be carried out based on the genes that are commonly
expressed in each sample, such as house keeping genes. Further, a threshold
line for reliability may be determined to remove data with low correlation.
(3) Selection of stress-inducible genes
Based on the analytical results by the array, stress-inducible genes are
specified as genes that are expressed at significantly different levels
between
plants that were given stress and plants that were not given stress. The term
"significantly different" used herein refers to, for example, an intensity
level
of 1,000 or higher, and a difference between two plants of three times or
more.
(4) Analysis of expression by Northern blotting
The thus selected genes are further subjected to Northern analysis and
the like. Thus, the expression levels of the genes are confirmed to be
enhanced with respect to stress tolerance levels. For example, plants are
exposed to various levels of stress such as salt, dehydration, or low
temperature stress in the manner described above. RNA is then extracted
from the plant and separated by electrophoresis. The separated RNA is
transferred to a nitrocellulose membrane and hybridized with a labeled cDNA
probe that is specific for the gene. Thus, the expression level thereof can be
detected.
If the expression level of the selected gene is enhanced in a
stress-dependent manner, it can be confirmed that the gene is stress-
inducible.
Examples of stress-inducible genes selected from the rice cDNA library
include a0022 (LIP9: SEQ ID NO: 2) and a0066 (WS1724: SEQ ID NO: 8) of
the present invention. a0022 and a0066 are identification numbers (ID No.)
of cDNA immobilized on the microarray.
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1.3 Screening for promoter sequence
(1) Screening of gene database
Subsequently, detection software (e.g., Blast) is used to search existing
gene databases (e.g., the DDBJ database) for promoter sequences of the
stress-inducible genes. Regarding a plant such as rice, the genome of which
has been mostly decoded, all promoter sequences controlling specified
stress-inducible genes can be searched for by using existing databases.
Promoter sequences are selected as regions that are considered to be promoters
from among the upstream regions in genome genes that are highly genomically
identical to the stress-inducible gene (cDNA). Based on the genome
information of stress-inducible genes, for example, the region approximately 1
to 2 kb upstream of the site that is presumed to be an initiation codon for
these
genes is deduced to be a promoter region.
Some of the conventional stress-inducible promoters have in their
sequences cis elements involved with promoter activities, such as dehydration
responsive elements (DRE), abscisic acid responsive elements (ABRE), and
low temperature responsive elements. When a stress-inducible transcription
factor is bound to the cis element, the aforementioned promoter is activated,
and the stress-tolerance-imparting genes that are under the control of the
promoter are allowed to express. If the cis element is contained in the
upstream region that has been screened, accordingly, this region is highly
likely to be a stress-inducible promoter.
Thus, the genome information of a gene highly homologous to the
aforementioned a0022 (LIP9: SEQ ID NO: 2) was obtained, and a deduced
LIP9 promoter sequence (SEQ ID NO: 1) was screened for from the region 1.1
kb upstream thereof. Similarly, a deduced WS1724 promoter sequence (SEQ
ID NO: 10) was screened for from the upstream region of a gene highly
homologous to a0066 (WS1724: SEQ ID NO: 8).
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(2) Confirmation of functionality of stress-inducible promoter
Subsequently, the functionality of the deduced promoter sequence is
confirmed by changes in promoter activity when stress is applied.
At the outset, a primer is produced based on the promoter sequence
deduced in the section above. PCR is carried out using genome DNA as a
template, and the promoter is cloned. Subsequently, a reporter gene is
ligated downstream of the promoter to produce a reporter plasmid. The
produced reporter plasmid is then introduced into a plant, thereby
investigating the expression of the reporter gene when stress is applied to
the
plant (preferably its T2 generation). Examples of reporter genes include
3-glucuronidase (e.g., GUS: pBI12l, Clontech), luciferase gene, and green
fluorescent protein gene. GUS is preferable because its activity can be
indicated by numerical values and its expression can be visually observed via
staining.
1.4 Promoter of the present invention
Based on the above, the rice genome-derived LIP9 promoter sequence
(SEQ ID NO: 1) was found to be a stress-inducible promoter, which was
expressed highly in a dehydration-, low temperature-, or salt stress-dependent
manner.
As mentioned above, the LIP9 promoter is induced specifically by
every type of stress. The structural and functional features thereof are as
follows.
1) The LIP9 promoter comprises in its structure 2 DRE cis elements
associated with dehydration stress induction (Fig. 2).
2) The expression level of the LIP9 is high in a plant that allows
overexpression of the OsDREB 1 gene (Japanese Patent Application No.
2001-358268), which is a rice-derived transcription factor that binds to a DRE
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cis element and activates the transcription of the gene located downstream
thereof.
3) The LIP9 promoter comprises the DRE sequence to which the
OsDREB1 protein binds. Accordingly, the LIP9 promoter is deduced to be
optimal for overexpression of the OsDREB gene.
Also, the WS1724 gene is a target of the OsDREB gene. Such
deduction is made based on the fact that the WS1724 promoter comprises in its
structure 2 DRE sequences, and it is made based also on the expression pattern
of a0066 when stress is applied (the expression pattern is inducible by
dehydration, salt, and low temperature stresses and the rate of induction by
low temperature is slower than that by dehydration and salt).
The promoter of the present invention is not limited to DNA consisting
of the nucleotide sequence as shown in SEQ ID NO: 1 or 10. The
stress-inducible promoter of the present invention includes DNA that
hybridizes under stringent conditions with DNA consisting of a nucleotide
sequence that is complementary to the DNA consisting of the nucleotide
sequence as shown in SEQ ID NO: 1 or 10 as long as such DNA has
stress-inducible promoter activity. Under the "stringent conditions,"
hybridization is carried out in 30%-50% formamide at 37 C to 50 C in 6 x SSC,
and preferably in 50% formamide at 42 C in 6 x SSC.
2. Recombinant vector
The recombinant vector of the present invention comprises the
promoter of the present invention. The vector may comprise other functional
structural genes or regulatory genes downstream of the promoter of the present
invention. Examples of preferable genes include structural genes and/or
regulatory genes for enhancing stress tolerance. The term "functional" refers
to a state in which other structural genes or regulatory genes are suitably
expressed under the control of the promoter of the present invention.
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Structural genes for enhancing stress tolerance encode a protein that
plays roles in enhancing plants' tolerance to environmental stress such as
dehydration, low temperature, or salt stress. Examples thereof include: LEA
proteins; water channel proteins; synthases for compatible solutes;
detoxification enzyme of tobacco; synthases for osmoregulatory substances
(e.g., sugar, proline, or glycinebetaine); genes encoding w-3 fatty acid
desaturase of Arabidopsis thaliana and the D9-desaturase of blue-green algae,
which are modification enzymes of the cellular membrane lipid; P5CS, which
is a key enzyme of proline synthesis; and the AtGolS3 gene for galactinol
synthesis.
A regulatory gene for enhancing stress tolerance regulates the activity
of a stress-inducible promoter and the expression of genes for imparting
stress
tolerance, thereby enhancing stress tolerance in plants. Examples thereof
include: Arabidopsis thaliana-derived transcription factors such as DREBIA,
DREB2A, DREBIB, and DREBIC genes (JP Patent Publication (Unexamined
Application) No. 2000-60558); rice-derived transcription factors such as
OsDREB I A, OsDREB 1 B, OsDREB 1 C, OsDREB 1 D, and OsDREB2A genes
(Japanese Patent Application No. 2001-358268); and NCED genes, which are
key enzymes for the biosynthesis of the plant hormone ABA.
When the promoter of the present invention comprises a specific cis
element, the gene of the transcription factor that binds to the cis element
and
enhances its promoter activity is particularly preferably ligated downstream
of
the promoter.
As described above, the LIP9 promoter according to the present
invention comprises in its structure 2 DRE sequences. Thus, the DREB or
OsDREB gene (for example, OsDREB 1 A, OsDREB 1 B, OsDREB 1 C,
OsDREB1D, OsDREB2A, or OsDREB2B gene) is preferably ligated
downstream of the LIP9 promoter. The OsDREB gene is most preferable.
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Since the WS1724 promoter also comprises 2 DRE sequences and it is
deduced to be the target of OsDREB, the DREB or OsDREB gene (for example,
OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, OsDREB2A, or
OsDREB2B gene) is preferably ligated downstream of the WS1724 promoter.
The OsDREB gene is most preferable.
The vector of the present invention is constructed so as to be
functional by ligating (inserting) the promoter of the present invention or
the
promoter and another regulatory gene or structural gene to (into) an
appropriate vector. The vector into which the promoter is to be inserted is
not particularly limited as long as it is capable of replicating genes of
interest
in a host. For example, plasmid DNA, phage DNA, or the like may be used.
Plasmid DNA includes: plasmids for E. coli hosts such as pBR322, pBR325,
pUC 118, and pUC 119; plasmids for Bacillus subtilis hosts such as pUB 110 and
pTP5; plasmids for yeast hosts such as YEp13, YEp24, and YCp50; and
plasmids for plant cell hosts such as pBI221 and pBI121. Phage DNA
includes 2 phage DNA and the like. Further, an animal virus vector such as a
retrovirus or vaccinia virus vector, or an insect virus vector such as a
baculovirus vector, may also be used.
The promoter of the present invention is inserted into a vector by
cleaving the purified DNA with an appropriate restriction enzyme and then
inserted into the restriction site or the multi-cloning site of an appropriate
vector for ligation.
The recombinant vector of the present invention may comprise a
splicing signal, poly(A) addition signal, selection marker, ribosome binding
sequence (SD sequence) or the like, if so desired. Examples of selection
markers are dihydrofolate reductase genes, ampicillin tolerance genes,
neomycin tolerance genes, and the like.
3. Transgenic plant
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The transgenic plant of the present invention can be produced by
introducing the recombinant vector of the present invention into a host so
that
promoter activity can be expressed. Hosts are not particularly limited as
long as the promoter of the present invention can function therein. Hosts are
preferably plants, and the monocotyledonous plants such as rice are
particularly preferable.
When plants or plant cells are used as hosts, for example, cells
established from rice, maize, wheat, Arabidopsis thaliana, tobacco, or carrot
or protoplasts prepared from these plants are used. Methods for introducing
recombinant vectors into plants include a method of Abel et al., which
utilizes
polyethylene glycol (Abel, H. et al. Plant J. 5:421-427, 1994), and
electroporation.
4. Stress tolerant transgenic plant
(1) Production of transgenic plant
Structural genes and/or regulatory genes for enhancing stress tolerance
are introduced into plants so as to be under the control of the promoter of
the
present invention. Thus, functional transgenic plants with enhanced
tolerance to environmental stress such as low temperature, freezing, or
dehydration stress can be produced. An example of particularly preferable
host plants are monocotyledonous plants.
A method for introducing the promoter of the present invention, etc.
into a host plant includes indirect introduction such as the Agrobacterium
infection method and direct introduction such as the particle gun method, the
polyethylene glycol method, the liposome method, and the microinjection
method. Up to the present, it had been difficult to carry out the
Agrobacterium infection method to produce transgenic plants from
monocotyledonous plants such as rice. However, the addition of
acetosyringon enabled Agrobacterium to infect rice. Thus, the
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Agrobacterium infection method became applicable for monocotyledonous
plants.
Production of transgenic plants using Agrobacterium is hereafter
described.
A recombinant vector to be introduced into a plant can be prepared by
cleaving with an appropriate restriction enzyme DNA comprising the promoter
of the present invention and a structural gene and/or regulatory gene for
enhancing stress tolerance, ligating an appropriate linker to the resulting
DNA
if necessary, and inserting the DNA into a cloning vector for the plant cell
host.
A binary vector type plasmid such as pBI2113Not, pBI2113, pBIlOl, pBIl21,
pGA482, pGAH, or pBIG, or an intermediate vector type plasmid such as
pLGV23Neo, pNCAT, or pMON200, may be used as cloning vectors.
When a binary vector type plasmid is used, the gene of interest is
inserted between the border sequences (LB, RB) of the binary vector. The
resulting recombinant vector is amplified in E. coli. The amplified
recombinant vector is then introduced into Agrobacterium tumefaciens C58,
LBA4404, EHA 101, C58C 1 RifR, EHA 105, etc., by freeze-thawing,
electroporation, or the like. The resulting Agrobacterium is used to
transform the plant.
In the present invention, the three-member conjugation method
(Nucleic Acids Research, 12:8711, 1984) may also be used in addition to the
method described above to prepare an Agrobacterium to be introduced into
plants. Specifically, plasmid-containing E. coli comprising the gene of
interest, helper plasmid-containing E. coli (e.g. pRK2013), and an
Agrobacterium are mixed and cultured on a medium containing rifampicin and
kanamycin. Thus, a zygote Agrobacterium to be allowed to infect plants can
be obtained.
For the expression of a foreign gene, etc., in plant bodies, a terminator
for plants, etc., should be located downstream of the structural gene.
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Specific examples of terminator sequences that may be utilized in the present
invention include cauliflower mosaic virus-derived and nopaline synthase
gene-derived terminators. Terminators are not limited to the aforementioned,
as long as they are known to be functional in plant bodies.
In order to efficiently select transgenic cells of interest, use of an
effective selection marker gene is preferable. As such a selection marker,
one or more genes selected from the kanamycin tolerance (NPTII) gene, the
hygromycin phosphotransferase (htp) gene that confers tolerance to the
antibiotic hygromycin on plants, the phosphinothricin acetyl transferase (bar)
gene that confers tolerance on bialaphos, and the like, can be used. The
promoter of the present invention and the selection marker gene may be
incorporated together into a single vector. Alternatively, they may each be
incorporated into separate vectors.
If the plant is infected with the thus prepared Agrobacterium, a
transgenic plant of interest can be produced.
The transgenic plant is sowed onto a medium containing an adequate
antibiotic, and plants containing promoters and genes of interest are
selected.
The selected plants are transferred to pots containing Bonsol No. 1, black
soil,
or the like and are further grown. Generally, the genes are introduced into
the
genome of the host plant in a similar manner. Due to differences in the
locations on the genome into which the genes have been introduced, however,
the expression of the introduced genes varies. This phenomenon is called a
"position effect." By analyzing transgenic plants with DNA fragments from
the introduced gene as a probe by Northern blotting, it is possible to select
those transgenic plants in which the introduced gene is expressed more highly.
(2) Confirmation of stress tolerance
Whether or not the promoter of the present invention, or a structural
gene and/or regulatory gene for enhancing stress tolerance, is integrated in
the
18
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transgenic plant and in the subsequent generation thereof can be confirmed by
extracting DNA from cells and tissues of those plants and detecting the
introduced gene by PCR or Southern analysis, which are conventional in the
art.
The expression level and the expression organ of a gene in a
transgenic plant can be analyzed by extracting RNA from cells and tissues of
the plant and detecting the mRNA of the introduced gene by RT-PCR or
Northern analysis, which are conventional in the art. Alternatively, the
transcription product of the introduced gene can be analyzed directly by
Western blotting using an antibody against the above product or the like.
The tolerance to environmental stresses of the transgenic plant into
which the promoter of the present invention has been introduced can be
evaluated by setting the transgenic plant in a pot containing a soil
comprising
vermiculite, perlite, Bonsol, and the like or hydroponically growing plants,
exposing the plants to various types of environmental stresses, and examining
the survival of the plants. Environmental stresses include low temperature,
dehydration, and salt stresses. For example, tolerance to dehydration stress
can be evaluated by leaving the plant without water for 2 to 4 weeks and then
examining the survival thereof. Tolerance to low temperature and freezing
stresses can be evaluated by leaving the plant at 15 to -10 C for 1 to 10
days,
growing it at 20 to 35 C for 2 days to 3 weeks, and then examining its
survival
ratio. Tolerance to salt stress can be evaluated by leaving the plant in 100
to
600 mM NaCl for 1 hour to 7 days, growing it at 20 to 35 C for 1 to 3 weeks,
and then examining its survival ratio. Thus, use of the promoter of the
present invention can significantly enhance stress tolerance without retarding
the growth of plants (particularly monocotyledonous plants).
(3) An example of a preferable transgenic plant
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An example of a preferable transgenic plant according to the present
invention is one prepared by introducing a vector comprising the functional
OsDREB gene ligated downstream of the LIPS or WS1724 promoter in a
monocotyledonous plant, such as rice or wheat. Since the LIP9 promoter
comprises 2 DRE regions, the OsDREB gene can effectively exhibit the effects
of stress tolerance by binding to the cis elements. Similarly, the WS1724
promoter comprises 2 DRE regions, and thus, the expression level of the
OsDREB gene can be enhanced and the stress tolerance of the plant can be
improved.
Examples
The present invention is described in greater detail with reference to
the following examples, although the technical scope of the present invention
is not limited thereto.
[Example 1] Identification of stress-inducible rice gene
Stress-inducible rice genes were searched for using cDNA microarrays
and Northern analysis.
1. Production of rice cDNA microarray
Rice seeds (Nihonbare) that were grown hydroponically for 2 to 3
weeks were subjected to dehydration, salt, or low temperature stress.
Dehydration stress was applied by air-drying at room temperature, salt stress
was applied by culturing in a 250 mM NaCl solution, and low temperature
stress was applied by cultivation at 4 C. The rice that had been subjected to
each type of stress was frozen with liquid nitrogen. Total RNA was extracted
from the frozen sample by the guanidine thiocyanate and cesium chloride
method, and mRNA was prepared using the Oligo(dt)-cellulose column.
cDNA was synthesized using the resulting mRNA as a template and using a
HybriZAP-2.1 two-hybrid cDNA Gigapack cloning kit (Stratagene), and the
cDNA was inserted and cloned at the EcoRI-Xhol cleavage site of
CA 02519997 2005-09-22
HybriZAP-2.1 phagemid vector. This phagemid DNA was packaged using
Gigapack III Gold packaging extract (Stratagene). The obtained lambda
phage particles containing cDNA were used to infect host E. coli, which were
then amplified, and these particles were subsequently recovered in the form of
a phage suspension.
The nucleotide sequences of the cDNA clones were sequenced to select
about 1,500 independent clones. The selected clones were amplified by PCR
and stamped onto a poly-L-lysine-coated microslide glass (model S7444,
Matsunami) using the GTMASS System (Nippon Laser and Electronic
Laboratory). Thereafter, the clones were immobilized by UV cross-linking to
produce the rice cDNA microarray (The Plant Cell, 2001, 13: 61-72 Seki et
al.).
2. Microarray analysis
mRNAs were purified from rice plants that had been subjected to
dehydration, salt, or low temperature stress or treated with 100 M abscisic
acid (5 hours or 10 hours) in the same manner as in the section above and from
rice plants that had not been subjected to stress (without treatment). mRNA
derived from rice plants without treatment was employed as a control, and
mRNA derived from rice plants that had been subjected to each type of stress
or treated with abscisic acid was employed as a sample. cDNA microarray
analysis was carried out by dual-fluorescent labeling using Cy3 and Cy5. As
a result of the microarray analysis, the genes with intensities of 1,000 or
higher and the genes with expression levels as high as 3 times that of the
control, were selected as candidate stress-inducible genes. Thus, a0022
(LIP9: SEQ ID NO: 2) and a0066 (WS1724: SEQ ID NO: 8) were selected as
stress-inducible genes.
3. Expression analysis via Northern hybridization
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The characteristic expression of the genes selected in the section above
was analyzed via Northern hybridization. Rice plants were first exposed to
abscisic acid, dehydration, low temperature, salt, or water stress, and
sampling
was accomplished regarding the rice that had been subjected to stress every 0,
1, 2, 5, and 10 hours. The abscisic acid, dehydration, low temperature, or
salt stress was applied in the same manner as in 1., and water stress was
applied by immersing the plants in pure water. Total RNA was prepared from
each sample, electrophoresis was carried out, and the expression of each gene
was observed by the Northern method. The results are shown in Fig. 1.
As is apparent from Fig. 1, the expression of the a0022 gene was
induced by the abscisic acid, dehydration, low temperature, or salt stress. In
particular, the expression thereof was rapidly induced by abscisic acid,
dehydration, or salt stress. In contrast, the expression thereof was slowly
induced by low temperature stress. The a0066 gene was a target of OsDREB,
based on the expression pattern when stress is applied (the expression pattern
is inducible by dehydration, salt, and low temperature stresses and the rate
of
induction by low temperature is slower than that by dehydration and salt).
[Example 2] Screening of promoter sequence
1. Screening of rice genome database
Using BLAST, the rice genome database of DDBJ was searched for a
identical site of cDNA:a0022 (LIP9: SEQ ID NO: 2), which was selected as a
stress-inducible gene in Example 1. As a result, in the gene in which identity
was observed, the sequence located 1.1 kb upstream of the initiation codon
toward the 5' side of the gene was selected as a promoter sequence (SEQ ID
NO: 1). A similar search was conducted concerning a0066 (WS1724: SEQ ID
NO: 8), and the promoter sequence thereof (SEQ ID NO: 10) was selected.
Fig. 2 shows the structure of the LIP9 promoter region. As is
apparent from Fig. 2, LIP9 comprises in its structure 2 DRE cis elements
22
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((A/G)CCGAC). Fig. 7 shows the structure of the WS1724 promoter region.
The WS1724 promoter was also found to comprise in its structure 2 DRE cis
elements ((A/G)CCGAC).
2. Cloning
Based on the selected promoter sequences, primers were designed, PCR
was carried out using rice genome DNA as a template, and cloning was carried
out. The primer sequences and the conditions for PCR used are as follows.
Primer sequences for LIP9 promoter:
Forward primer: 5'-CACGAAGCTTTCATCAGCTATTCATCAA-3' (SEQ ID NO: 3)
Reverse primer: 5'-CCGGATCCTCGATCGATGGATTCAGCTA-3' (SEQ ID NO: 4)
Primer sequences for WS1724 promoter:
Forward primer: 5'-CCATTGGATCCAGCCGTGGAAGTCCAAC-3' (SEQ ID NO: 11)
Reverse primer: 5'-GCCGGGGATCCTTGGCGCCTCTCTCTCT-3' (SEQ ID NO: 12)
PCR conditions: 30 cycles of 95 C for 1 minute, 55 C for 1 minute, and 68 C
for 2 minutes
[Example 3] Activity of LIP9 promoter against stress
(1) Preparation of transgenic plant
The promoter site of pBIG29APHSNot was substituted with an
ubiquitin promoter of maize to produce G-ubi plasmid. The G-ubi plasmid
was cleaved with BamHI-HindIII and ligated to a fragment of a similarly
cleaved LIP9 promoter. The plasmid into which the LIP9 promoter had been
incorporated was cleaved with BamHI-EcoRI and ligated to the Gus gene,
which was similarly cleaved out from pBI221 (Clontech) with BamHI-EcoRI,
to produce a GUS-expressing construct G-LIP9:GUS (Fig. 3). The plasmid
G-LIP9:GUS was introduced by electroporation into Agrobacterium EHA105,
which was washed with 10% glycerol after culturing, thereby preparing
Agrobacterium EHA105 (G-LIP9:GUS). Rice was infected with this
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Agrobacterium EHA105 (G-LIP9:GUS) in the following manner to prepare a
transgenic plant of interest.
Rice seeds were immersed in 70% ethanol for 1 minute and sterilized
by immersion in 2% sodium hypochlorite for 1 hour. The sterilized seeds
were then washed with sterilized water, and 9 grains each of the seeds were
sowed onto a plate of N6D solid medium (3.98 g of CHU[N6] Basal Salt
Mixture (Sigma), 30 g of sucrose, 100 mg of myo-inositol, 300 mg of casamino
acid, 2,878 mg of L-proline, 2 mg of glycine, 0.5 mg of nicotinic acid, 0.5 mg
of pyridoxine hydrochloride, 1 mg of thiamine hydrochloride, 2 mg of 2,4-D,
and 4 g of Gellite per liter; pH 5.8), followed by culturing for 24 days.
Thus,
callus formation was induced. The callus formed from approximately 20
grains of the seeds was transferred to new N6D solid medium, followed by
culturing for an additional three days.
Separately, Agrobacterium EHA105 (G-LIP9:GUS) was cultured in 5
ml of YEP medium containing 100 mg/l rifampicilin and 20 mg/l kanamycin
(10 g of Bacto peptone, 10 g of Bacto yeast extract, 5 g of NaCl, and 406 mg
of
MgC12.6H20 per liter; pH 7.2) at 28 C for 24 hours. This Agrobacterium was
diluted with AAM medium containing 20 mg/l acetosyringon (10 mg of
MnSO4.5H2O, 3 mg of H3BO3, 2 mg of ZnSO4.7H2O, 250 pg of Na2MoO4.2H2O,
25 g of CuSO4.5H2O, 25 g of CoCl2.6H2O, 750 g of KI, 150 mg of
CaCl2.2H2O, 250 mg of MgSO4.7H20, 40 mg of Fe-EDTA, 150 mg of
NaH2PO4.2H2O, 1 mg of nicotinic acid, 10 mg of thiamine hydrochloride, 1 mg
of pyridoxine hydrochloride, 100 mg of myo-inositol, 176.7 mg of L-arginine,
7.5 mg of glycine, 900 mg of L-glutamine, 300 mg of aspartic acid, and 3 g of
KC1 per liter; pH 5.2) to bring O.D.660 to 0.1. Thus, 20 ml of Agrobacterium
suspension was prepared.
Subsequently, the Agrobacterium suspension was added to and then
mixed with the callus, which was cultured for 3 days, for 1 minute.
Thereafter, this callus was placed on a sterilized paper towel to remove
excess
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Agrobacterium suspension and then cultured on 2N6-AS solid medium, on
which the sterilized filter paper was placed, (3.98 g of CHU[N6] Basal Salt
Mixture, 30 g of sucrose, 10 g of glucose, 100 mg of myo-inositol, 300 mg of
casamino acid, 2 mg of glycine, 0.5 mg of nicotinic acid, 0.5 mg of pyridoxine
hydrochloride, 1 mg of thiamine hydrochloride, 2 mg of 2,4-D, 10 mg of
acetosyringon, and 4 g of Gellite per liter; pH 5.2) at 25 C for 3 days in the
dark. After culturing for 3 days, the culture product was thoroughly washed
with an aqueous solution of 3% sucrose containing 500 mg/l carbenicillin until
the solution did not become clouded. The washed culture product was further
cultured on N6D solid medium containing 500 mg/l carbenicillin and 10 mg/1
hygromycin for 1 week. Thereafter, the resulting culture product was
transferred onto a N6D solid medium containing 500 mg/l carbenicillin and 50
mg/l hygromycin and cultured for 18 days. Furthermore, the callus was
transferred to a redifferentiation medium (4.6 g of Murashige and Skoog Plant
Salt Mixture (Nihon Pharmaceutical Co., Ltd), 30 g of sucrose, 30 g of
sorbitol,
2 g of casamino acid, 100 mg of myo-inositol, 2 mg of glycine, 0.5 mg of
nicotinic acid, 0.5 mg of pyridoxine hydrochloride, 0.1 mg of thiamine
hydrochloride, 0.2 mg of NAA, 2 mg of kinetin, 250 mg of carbenicillin, 50 mg
of hygromycin, and 8 g of agarose per liter; pH 5.8). The product was
transferred to a new medium every week and redifferentiated. Those having
buds that had grown to approximately 1 cm were transferred to a hormone-free
medium (4.6 g of Murashige and Skoog Plant Salt Mixture (Nihon
Pharmaceutical Co., Ltd), 30 g of sucrose, 2 mg of glycine, 0.5 mg of
nicotinic
acid, 0.5 mg of pyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride,
50 mg of hygromycin, and 2.5 g of Gellite per liter; pH 5.8). Plant bodies,
which have grown to approximately 8 cm on the hormone-free medium, were
transferred to a pot containing synthetic particulate potting soil (Bonsol No.
1,
Sumitomo Chemical Co., Ltd.) to allow the transgenic plants to produce seeds.
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Similarly, the rd29A promoter (Nature Biotechnology, 1999, 17:
287-291), the 35S promoter, or the salT promoter (SEQ ID NO: 5) was ligated
upstream of the GUS gene to produce constructs. The obtained constructs
were introduced into rice and/or tobacco.
The salT promoter is a stress-inducible promoter isolated from the rice
genome via screening conducted in the same manner as with the case of the
LIP9 promoter. The ID No. of cDNA of the salT promoter immobilized on
the microarray is a2660. Although the salT promoter does not comprise a
special cis sequence in its structure, it is confirmed that expression thereof
is
induced by the abscisic acid, dehydration, low temperature, or salt stress.
(2) Promoter activity against dehydration stress
The T2 generation of the obtained GUS-expressing transgenic rice was
grown hydroponically for 2 weeks and exposed to dehydration stress in the
same manner as in Example 1.
In the case of GUS-expressing transgenic tobacco, a plant, which was
regenerated from a T1 generation plant, was grown in a plant cone for 3 to 5
weeks, and a grown leaf was bisected. One part thereof was designated as a
control, and the other was exposed to dehydration stress by being air dried at
room temperature.
The GUS activities of both transgenic rice and tobacco were assayed
based on changes in fluorescence intensities resulting from the decomposition
of 4-methylumbelliferyl-(3-D-glucuronide. Fig. 4 shows the GUS activities
of the transgenic plants to which various promoters were introduced at the
time of the application of dehydration stress.
As is apparent from Fig. 4, the activity level of the stress-inducible
salT or LIP9 promoter in monocotyledonous plants, i.e., rice, is higher than
that of the rd29A promoter. In particular, the activity of the LIP9 promoter
26
CA 02519997 2005-09-22
was as high as approximately two times that of the salT promoter. While the
LIP9 promoter also exhibited stress-inducible promoter activities in tobacco,
which is a dicotyledonous plant, its activity was weaker than that in rice.
(3) Promoter activity against salt stress
Subsequently, the entire body of the rice to which the LIP9
promoter-GUS construct has been introduced was immersed in salt water and
then subjected to GUS staining. As a result, the entire plant was stained
(Fig.
5). Based on this, the LIP9 promoter was found to function in all parts of the
plant that had been subjected to salt stress.
[Example 4] WS1724 promoter activity against stress
In the same manner as in Example 3, transgenic rice was produced
using the WS1724 promoter, and stress response thereof was examined.
(1) Production of transgenic plants
The promoter site of pBIG29APHSNot was substituted with an
ubiquitin promoter of maize to produce G-ubi plasmid. The G-ubi plasmid
was cleaved with BamHI-HindIll and ligated to a fragment of the similarly
cleaved WS1724 promoter. PCR fragment of the WS1724 promoter were cut
with BamHI and blunt-ended, and ligated to a site of the pBIG vector, which
was cleaved with Smal to produce a GUS-expressing construct (WS1724:GUS).
Subsequently, WS1724:GUS plasmid was introduced by electroporation into
Agrobacterium EHA105, which was washed with 10% glycerol after culturing,
thereby preparing Agrobacterium EHA105 (WS1724:GUS). Rice was
infected with this Agrobacterium EHA105 (WS1724:GUS) to prepare a
transgenic plant of interest.
(2) Promoter activity against dehydration stress
27
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The GUS-expressing transgenic rice was exposed to dehydration stress
in the same manner as in Example 3 to assay GUS activities thereof based on
changes in fluorescence intensities resulting from the decomposition of
4-methylumbelliferyl-(3-D-glucuronide. As a result, GUS activities observed
in the leaves of the transgenic rice to which dehydration stress had been
applied (where the leaves had been cut and allowed to stand for 24 hours) were
higher than those observed in the leaves of the controls (where such leaves
had
been cut and then frozen immediately). The transgenic rice to which
dehydration stress had been applied (for 24 hours) was subjected to GUS
staining, and GUS activities were observed both in the roots and in the
leaves.
[Example 5] Expression of genes introduced into transgenic rice, LIP9 genes,
and WS1724 genes
A transgenic plant was prepared in the same manner as in Example 3.
This transgenic plant was prepared by introducing the OsDREB1A gene (SEQ
ID NO: 6) or the DREBIC gene (SEQ ID NO: 8) that is under the control of an
ubiquitin promoter of maize or 35S promoter into rice. The mRNA level of
the OsDREB1A and DREBIC genes that had been introduced into the
transgenic plant and the mRNA level of the LIP9 (a0022), WS1724 (a0066),
and salT (a2660), the expression of which was considered to be altered by the
introduced genes, were analyzed by the Northern method.
The OsDREB 1 A gene (SEQ ID NO: 6), the DREB 1 C gene (SEQ ID NO:
7), the LIP9 gene (a0022, SEQ ID NO: 2), the WS1724 gene (a0066, SEQ ID
NO: 8), and the salT gene (a2660, SEQ ID NO: 9) were employed as probes
(the sequences of the coding regions were employed as probes concerning SEQ
ID NOs: 6 and 7 in the sequence listings). As a control, rice in which only
the vector had been transformed was similarly analyzed.
The transgenic rice was selected in a 0.1% Benlate solution containing
mg/ml hygromycin for 5 days. Thereafter, the plant was transferred to a
28
CA 02519997 2005-09-22
pot containing Bonsol No. 1 and was grown for 12 days. The wild-type rice
was grown similarly. Total RNA was extracted from each plant and
electrophoresed. Expression of each gene was analyzed by the Northern
method in the same manner as in Example 1. The results are shown in Fig. 6,
wherein "a," "b," and "c" each independently represent a transgenic plant
line.
In the transgenic rice into which the OsDREB 1 A and DREB 1 C genes
had been introduced, the expression of the LIP9 gene having the DRE sequence
in the promoter region was found to be induced. In contrast, the expression
of the salT gene having no DRE sequence in the promoter region was found to
be inconsistent with the expression of the introduced gene (OsDREB1A or
DREBIC). Also, the expression of the WS1724 gene, which comprises the
DRE sequence in the promoter region and is deduced to be a target of OsDREB,
as with the LIP9 was induced by these transgenic plants.
The LIP9 or WS1724 promoter comprises the DRE sequence, and the
expression level of the LIP9 or WS1724 gene is high in transgenic plants
wherein overexpression of the OsDREB lA genes is observed. LIP9 and
WS1724 was considered to be the target gene of the OsDREB genes, including
OsDREB 1 A. Accordingly, the LIP9 or WS1724 promoter was deduced to be
optimal for overexpression of the OsDREB gene.
[Reference Example 1] Production of pBE35S:OsDREB1A, G-ubi:OsDREBIA,
and G35S- ShA:OsDREB1A
G-ubi and G35S-Sh0 were prepared as follows. At the outset, pBIG
plasmid (Nucleic Acids Research 18: 203, 1990) was cleaved with BamHI,
blunt-ended, and ligated to delete the BamHI cleavage site. Thereafter, the
plasmid was cleaved with Hindlll and EcoRl. The resulting fragment was
ligated to a fragment of approximately 1.2 kb, which was obtained by cleavage
of pBE2113Not plasmid in the same manner, thereby preparing a pBIG2113Not
plasmid.
29
CA 02519997 2005-09-22
Subsequently, pBIG2113Not was cleaved with HindIll and BamHI and
ligated to a fragment of rd29A promoter (approximately 0.9 kb, Nature
Biotechnology 17: 287-291, 1999), which was cleaved in the same manner,
thereby preparing pBIG29APHSNot plasmid. Further, this pBIG29APHSNot
plasmid was cleaved with HindIll and Sall and then ligated to a fragment of
the ubiquitin gene (Ubi-1) promoter (approximately 2.0 kb, Plant Molecular
Biology 18: 675-689, 1992) of maize or a fragment (approximately 1.6 kb,
Proceeding National Academy of Science USA 96: 15348-15353, 1999)
containing CaMV 35S promoter of p35S-shA-stop and a part of the intron of a
sucrose synthase gene (Shl) of maize, which was cleaved in the same manner.
Thus, G-ubi plasmid or G35S-shA plasmid was prepared.
pBE2l13Not, G-ubi, and G35S-shA described above were each cleaved
with BamHI, and ligated to a fragment of the similarly cleaved OsDREB1A
gene encoding a transcription factor of rice using Ligation High (Toyobo Co.,
Ltd.). E. coli DH5a was transformed using the thus obtained ligation
product. After the transgenic E.coli was cultured, pBE35S:OsDREB1A,
G-ubi: OsDREB1A, and G35S-ShA: OsDREB1A plasmids were purified
therefrom. Subsequently, the nucleotide sequences thereof were determined,
and those having the OsDREB1A gene bound in the sense direction were
selected.
The plasmid pBE35S: OsDREB1A-containing E. coli DH5a, helper
plasmid pRK2013-containing E. coli HB101, and Agrobacterium C58 were
mixed and cultured on LB agar medium at 28 C for 24 hours. Generated
colonies were scraped off and suspended in 1 ml of LB medium. This
suspension (10 l) was applied to LB agar medium containing 100 mg/l
rifampicilin and 20 mg/l kanamycin and cultured at 28 C for 2 days, thereby
obtaining zygote Agrobacterium C58 (pBE35S: OsDREB1A). By
electroporation, the G-ubi: OsDREB1A plasmid and the G35S-ShA:
OsDREB 1 A plasmid were separately introduced into Agrobacterium EHA 105,
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and washed with 10% glycerol after culturing. Thus, Agrobacterium
EHA105 (G-ubi:OsDREB1A) and Agrobacterium EHA105 (G35S-shA:
OsDREB 1 A) were obtained.
c
Industrial Applicability
The present invention provides a stress-inducible promoter that is
effectively functional in monocotyledonous plants. Such promoter comprises
the DRE sequence. If the OsDREB gene or the like is ligated so as to be
under the control of the promoter and introduced into plants, accordingly,
transgenic monocotyledonous plants, such as rice, having potent stress
tolerance can be produced.
Free Text of Sequence Listing
SEQ ID NO: 3 - description of artificial sequence: primer
SEQ ID NO: 4 - description of artificial sequence: primer
SEQ ID NO: 11 - description of artificial sequence: primer
SEQ ID NO: 12 - description of artificial sequence: primer
31
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SEQUENCE LISTING
<110> Japan International Research Center for Agricultural Sciences
Incorporated Administrative Agency, National Agriculture and Bio-
oriented Research Organization
<120> Stress Inducible Promoter and Its Uses
<130> PH-2004-PCT
<140>
<141>
<150> JP 2003-80847
<151> 2003-03-24
<160> 12
<170> Patentln Ver. 2.1
<210> 1
<211> 1066
<212> DNA
<213> Oryza sativa
<220>
<223> Inventor: Shinozaki, Kazuko; Katsura, Koji; Ito, Yusuke
<400> 1
tcatcagcta tcatcaaagc gaaggaaaga aagaaaaata aaaggaaaag aactggctgg 60
aaattagaga agccccggac gactcgatct gggggtggca aattaatcag tgtgatcaac 120
agggataact tatcccgtcc gaccaaatcc accaaccaaa ccaagacccg atttgttagg 180
ctgtgaaaga cggatcagtg ggaccctgat ctacggaccc catatgtcac cgtccaggtc 240
tctggatctc tcccgtcgtc ctaatcagac accgcgcgcg cggtgccgtc gctctcgagc 300
cgtgtcccgc tcccaactcg tcacaaaagc gatcacagac tcttccttcc tctgctggga 360
gagaagaaaa attggccgcg atgatgccga taaagaggaa aaagggatga gaatccgatg 420
gaaaaaaact gatgttaatc tatcgctact gctgcgcact aagacgaatc gtatccgaac 480
aagaaacgct tacgttactg ttcctaaatg gatcgctccg ctcatcactt aaccaaaaat 540
cgattaggaa attgacggac agcgacgccc gaagccaagt gtctcgtcgc gtaggcgtcg 600
aggcctcgaa gcagagggag cggagaggcg gacgcgccgc ccacgcctcc tctccctcgg 660
tgacacggcc gtctggctcc acatggcgcc gacctctccc gatgcgtcca cccgtcccga 720
ggcaccgcca cgtcggaacc agccggccgc cccacgcgat tgccgacacg cgtcgcggcg 780
ccactggctc acccgctgcc tgcctctgcc tgccccccat ctcgtcgcca tttcccgccc 840
acgcttcttg tcctcgcgtc gcctacgcgt acgtacgata caaacgccgc acctttcgat 900
cccctccgct atataaggag ggcatctgcc tcgccacctt cttcatccga aagcaaaagc 960
gactcgtcac agctcaaaca agtcaagagc gaatagttct tgctgatctg ttgtttgatt 1020
actttagttc tcgagaggct ttagctgaat ccatcgatcg aggatg 1066
<210> 2
<211> 1245
<212> DNA
<213> Oryza sativa
<400> 2
gctagcagag ctcgtcacag ctcaaacaag tcaagagcga atagttcttg ctgatctgtt 60
gtttgattac tttagttctc gagaggcttt agctgaatcc atcgatcgat catggaggat 120
gagaggaaca cggagagcca ccagggtggc gaggctgcag agcaggtgga ggtgaaggac 180
aggggcctct tcgacaacct ccttggcagg aagaaggacg atcagccgga ggagaagaag 240
catgaggagg agcttgtcac cggcatggag aaggtctccg tggaagagcc aaagaaggag 300
gagcaccacg ccgagggcga gaagaaggag agcctcctct ccaagctgca ccgatccagc 360
tccagctcca gctcgtcgag tgatgaggaa gaggaggtga tcgatgacaa cggcgaggtg 420
1
CA 02519997 2005-11-02
gtcaagagga agaagaagaa ggggctcaag gagaagatca aggagaagct gcccggccac 480
aaggaccatg ccggtgagca tgctcctccg cccgcggcga cgggcttccc gcgccggctc 540
cgctgcatcc gtggtgacgg ccgcgcccac gccactcctg ctcccgtggt gactcacggc 600
gatcaccacc acgacaccgc cgtccccgtg gaaaagatcg agggtgatca cgccagacgg 660
aggcgaccct gccacgtgca cccgaggagg aaaaaagggc ttcctcgaca agatcaagga 720
gaagctgccc ggcggccaca agaagccgga agacgcaact gctgtgccgc cgccggccgc 780
ctcaccggct gctcctgcca ctactccggc gccagcacac ccaccgccgg ctacagagga 840
agtgagcagc ccggatggga aggagaagaa gggtatactg ggcaagatca tggagaaact 900
gcccggttac cacaagggct ccggcgagga agacaagacc gccgccgccg ccaccggcga 960
gcacaagagc agcgcttaat tggggcgtgt gtgagaccag gccatggttg gaatttggaa 1020
gtgtttggcg tgtgttagtt tggtgctttt tctgcactgc agctttgtta agttcgtgtc 1080
aagattggtc aaggcctggt cagcgaagcc cgatcagtga tcgaagtttg tgtttcgtgt 1140
ggggtacggg cttcagtttg ctatagtcaa gtactagatg ttgagtttgt ttaattatta 1200
ttggcactct tgtattggtt ttgggctggg cattctgcct tggta 1245
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer
<400> 3
cacgaagctt tcatcagcta ttcatcaa 28
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer
<400> 4
ccggatcctc gatcgatgga ttcagcta 28
<210> 5
<211> 1608
<212> DNA
<213> Oryza sativa
<400> 5
caacaaccac tactgaacac ggctaagtgt gtttcctctc ctcgaagatg tcgttattgc 60
gttcttttct gctattccat acatatcaat ctctagagga acaccttact ctagctttca 120
gacaagggac ggtggtaaat cacgtcgtat cctccatggg gtgtgctccg aaaaaccttc 180
cctcatgcat tagagatcat gggtggaatt tagcgatggc acaccttatt tataatttag 240
ttactctccg gcggtaccat ctgcttccgt ttgttgatcg atgctggcga tgatgtgtgt 300
gagtatcgat caacagaatg atcggacgct atttttgggg tcgttttttt tcagcattga 360
ggagggatga ggattgcttg caacatgcag gtgctgctca aaacaacggt taagcagata 420
tccgtcaatt tgatagtaag atctgtaacg cgtggtcttt cgagctgaaa actatggact 480
ctttgaaaca aagataatat tatattaaat tctattattc aaagatatct aaatatttag 540
aaagatatta ataatgttat taaactttga cttacttaaa acaagtccaa aactgcatgt 600
ccctaaatcg ccagaagata aggaacacct gtacccgtga taacagaggg gtatgaaatt 660
tggacacgag gcttctttgg cagacgtggc gctgagtgag cttggctcgc ttggtcaaac 720
tccgtgcagg gacattcagt tagctagcta gcagcattgt cgacaataag atagccttta 780
aatgttagca ctcaccagct tgtcaaaaac caaggcttgg tgacggcggc ttcagaatga 840
aggatagatg gataaatgtc tagaatatta taaagtccaa caaaagatgg agcacatgca 900
tgaaagatta cgtacacgaa tgcagttgat acagtggatg ttaggcataa gaagcactat 960
aaatagaggg tgcaatcccc attgccctac acaactacac aagtcgacta tcattacaag 1020
gaaatttaag cgaccacgaa ggtatgaaag catagcagta ctctgcattt tttttttttg 1080
2
CA 02519997 2005-11-02
atgttgttct agctagctct gcttaaggtt ttcctttctt tcgttctttg tttttttttt 1140
gtaagctcaa ctagttgcat gcaatttaga ttttatcctt ttacagttgg aaaaacatcc 1200
ctataaatat taccatgaat gcatagagat tcgaggaagc tacaaattgg acgactgatt 1260
ccaaaaaaaa aaaaaaaatc agatggtcac atcattgcta ttgttttgtg aaagtacaaa 1320
agcactcgtt cggattcaaa ttacttgtgc aaattaatta aaaaccatag aaatgatcat 1380
gttaccccta cacatttcgg aaacaatacc atatatgtta gtgtgcgatc attcaaattg 1440
atttatatct gaacaaaact gagtgggaat acggtgagca aacttgacga ttccaaaata 1500
atttatattt aggcaaaatt ttacaacttc aaagttcaaa caagctaacc tgaaaaatca 1560
tgtttgaatt tactaagatg tgcttttgta tttactaaac agagtatg 1608
<210> 6
<211> 927
<212> DNA
<213> Oryza sativa
<220>
<221> CDS
<222> (69)..(782)
<400> 6
cacactcgag cagagcaaat acagttcagg aatcaggagc aagcagaaac acacacacaa 60
atccgaag atg tgc ggg atc aag cag gag atg agc ggc gag tcg tcg ggg 110
Met Cys Gly Ile Lys Gln Glu Met Ser Gly Glu Ser Ser Gly
1 5 10
tcg ccg tgc agc tcg gcg tcg gcg gag cgg cag cac cag acg gtg tgg 158
Ser Pro Cys Ser Ser Ala Ser Ala Glu Arg Gln His Gln Thr Val Trp
15 20 25 30
acg gcg ccg ccg aag agg ccg gcg ggg cgg acc aag ttc agg gag acg 206
Thr Ala Pro Pro Lys Arg Pro Ala Gly Arg Thr Lys Phe Arg Glu Thr
35 40 45
agg cac ccg gtg ttc cgc ggc gtg cgg cgg agg ggc aat gcc ggg agg 254
Arg His Pro Val Phe Arg Gly Val Arg Arg Arg Gly Asn Ala Gly Arg
50 55 60
tgg gtg tgc gag gtg cgg gtg ccc ggg cgg cgc ggc tgc agg ctc tgg 302
Trp Val Cys Glu Val Arg Val Pro Gly Arg Arg Gly Cys Arg Leu Trp
65 70 75
ctc ggc acg ttc gac acc gcc gag ggc gcg gcg cgc gcg cac gac gcc 350
Leu Gly Thr Phe Asp Thr Ala Glu Gly Ala Ala Arg Ala His Asp Ala
80 85 90
gcc atg ctc gcc atc aac gcc ggc ggc ggc ggc ggc ggg gga gca tgc 398
Ala Met Leu Ala Ile Asn Ala Gly Gly Gly Gly Gly Gly Gly Ala Cys
95 100 105 110
tgc ctc aac ttc gcc gac tcc gcg tgg ctc ctc gcc gtg ccg cgc tcc 446
Cys Leu Asn Phe Ala Asp Ser Ala Trp Leu Leu Ala Val Pro Arg Ser
115 120 125
tac cgc acc ctt cgc cga cgt ccg cca cgc cgt gcc gag gcc gtc gag 494
Tyr Arg Thr Leu Arg Arg Arg Pro Pro Arg Arg Ala Glu Ala Val Glu
130 135 140
gac ttc ttc cgg cgc cgc ctc gcc gac gac gcg ctg tcc gcc acg tcg 542
Asp Phe Phe Arg Arg Arg Leu Ala Asp Asp Ala Leu Ser Ala Thr Ser
145 150 155
3
CA 02519997 2005-11-02
tcg tcc tcg acg acg ccg tcc acc cca cgc acc gac gac gac gag gag 590
Ser Ser Ser Thr Thr Pro Ser Thr Pro Arg Thr Asp Asp Asp Glu Glu
160 165 170
tcc gcc gcc acc gac ggc gac gag tcc tcc tcc ccg gcc agc gac ctg 638
Ser Ala Ala Thr Asp Gly Asp Glu Ser Ser Ser Pro Ala Ser Asp Leu
175 180 185 190
gcg ttc gaa ctg gac gtc ctg agt gac atg ggc tgg gac ctg tac tac 686
Ala Phe Glu Leu Asp Val Leu Ser Asp Met Gly Trp Asp Leu Tyr Tyr
195 200 205
gcg agc ttg gcg cag ggg atg ctc atg gag cca cca tcg gcg gcg ctc 734
Ala Ser Leu Ala Gln Gly Met Leu Met Glu Pro Pro Ser Ala Ala Leu
210 215 220
ggc gac gac ggt gac gcc atc ctc gcc gac gtc cca ctc tgg agc tac 782
Gly Asp Asp Gly Asp Ala Ile Leu Ala Asp Val Pro Leu Trp Ser Tyr
225 230 235
tagagctcaa tcaactgtac aattttgcct cttttttctc tcttttctgg cttccgatgc 842
caaaattttg gtactgtacg gacactactt tcggtaatgt gatggaacaa gttgcaaaac 902
aaaaaaaaaa aaaaaaaaaa aaaaa 927
<210> 7
<211> 944
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (135)..(785)
<400> 7
cctgaattag aaaagaaaga tagatagaga aataaatatt ttatcatacc atacaaaaaa 60
agacagagat cttctactta ctctactctc ataaacctta tccagtttct tgaaacagag 120
tactcttctg atca atg aac tca ttt tct gcc ttt tct gaa atg ttt ggc 170
Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly
1 5 10
tcc gat tac gag tct ccg gtt tcc tca ggc ggt gat tac agt ccg aag 218
Ser Asp Tyr Glu Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys
15 20 25
ctt gcc acg agc tgc ccc aag aaa cca gcg gga agg aag aag ttt cgt 266
Leu Ala Thr Ser Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg
30 35 40
gag act cgt cac cca att tac aga gga gtt cgt caa aga aac tcc ggt 314
Glu Thr Arg His Pro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly
45 50 55 60
aag tgg gtg tgt gag ttg aga gag cca aac aag aaa acg agg att tgg 362
Lys Trp Val Cys Glu Leu Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp
65 70 75
ctc ggg act ttc caa acc get gag atg gca get cgt get cac gac gtc 410
Leu Gly Thr Phe Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val
80 85 90
gcc gcc ata get ctc cgt ggc aga tct gcc tgt ctc aat ttc get gac 458
Ala Ala Ile Ala Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp
95 100 105
4
CA 02519997 2005-11-02
tcg get tgg cgg cta cga atc ccg gaa tca acc tgt gcc aag gaa atc 506
Ser Ala Trp Arg Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Glu Ile
110 115 120
caa aag gcg gcg get gaa gcc gcg ttg aat ttt caa gat gag atg tgt 554
Gln Lys Ala Ala Ala Glu Ala Ala Leu Asn Phe Gln Asp Glu Met Cys
125 130 135 140
cat atg acg acg gat get cat ggt ctt gac atg gag gag acc ttg gtg 602
His Met Thr Thr Asp Ala His Gly Leu Asp Met Glu Glu Thr Leu Val
145 150 155
gag get att tat acg ccg gaa cag agc caa gat gcg ttt tat atg gat 650
Glu Ala Ile Tyr Thr Pro Glu Gln Ser Gln Asp Ala Phe Tyr Met Asp
160 165 170
gaa gag gcg atg ttg ggg atg tct agt ttg ttg gat aac atg gcc gaa 698
Glu Glu Ala Met Leu Gly Met Ser Ser Leu Leu Asp Asn Met Ala Glu
175 180 185
ggg atg ctt tta ccg tcg ccg tcg gtt caa tgg aac tat aat ttt gat 746
Gly Met Leu Leu Pro Ser Pro Ser Val Gln Trp Asn Tyr Asn Phe Asp
190 195 200
gtc gag gga gat gat gac gtg tcc tta tgg agc tat taaaattcga 792
Val Glu Gly Asp Asp Asp Val Ser Leu Trp Ser Tyr
205 210 215
tttttatttc catttttggt attatagctt tttatacatt tgatcctttt ttagaatgga 852
tcttcttctt tttttggttg tgagaaacga atgtaaatgg taaaagttgt tgtcaaatgc 912
aaatgttttt gagtgcagaa tatataatct tt 944
<210> 8
<211> 353
<212> DNA
<213> Oryza sativa
<400> 8
gctagcagag tagcaatcca ttccgatcca tcaaatttct cttgagaccg tagagagaga 60
gagaggcgcc aaccatggcc ggcatcatcc acaagatcga ggagaagctc cacatgggcg 120
gaggcgagca caagaaggaa gacgagcaca agaaggaggg ggagcaccac aagaaggacg 180
gggagcacaa ggaaggcgtg gtggagaaga tcaaggacaa gatcaccggc gaccacggcg 240
acggcggcga gcacaaggag aagaaggaca agaagaagaa gaaggagaag aagcacggcg 300
aggagggcca ccaccacgac ggccacagca gcagcagcag cgacagcgac tgg 353
<210> 9
<211> 545
<212> DNA
<213> Oryza sativa
<400> 9
cgactatcat tacaaggaaa tttaagcgac cacgaagagt atgacgctgg tgaagattgg 60
tccgtggggc ggaaatggag ggtcagctca ggacatcagt gtgccaccca agaagctgtt 120
aggcgtgaca atctacagct cagatgcaat cagatccatt gccttcaact acatcggtgt 180
ggatggacag gaatatgcca ttggtccatg gggtgggggc gaaggcacct ctacagagat 240
taaactgggc tcctctgagc agatcaagga gatttctgga acccatggcc cagtctatga 300
tctggctgac attgtcacct atcttaagat tgtgacaagt gctaataata catacgaggc 360
tggagtccca aatggaaagg aattcagcat tccactgcaa gactctggcc atgtcgttgg 420
attctttgga aggtctggaa cgcttatcga cgcaattggc atctacgtcc acccttgatt 480
cccagtggtc aaagaattac tacctactac catatctacg aaataatgtt ccatggtgtt 540
gttgt 545
CA 02519997 2005-11-02
<210> 10
<211> 1357
<212> DNA
<213> Oryza sativa
<400> 10
agccgtggaa gtccaacctg caggctcagg ctgcagatcg cccaaggcgc acttgcctcc 60
acgatggctt gtcctcaacc gctcggaagg cgagatccaa ttggcaattt gttcaacgca 120
gggagagagg aggagactgg aacgggatca ttggacattg gttgatgaat tgcaatttgg 180
atgacgaggc cgcgagggtc agaccgtcgg agagtgagat gatggttata caagtgtact 240
agtaggacgg acggtggcac cggccagaag cagcagattt tgtgcaaacg ttgagcccgc 300
aacacgtggc cggcatcgac ccgctacacg gacgcagcgc cccccccccc cccccccccg 360
cggacccacg cgggccggcc gcgctgtcgc cgtgctgccg actacgccgt cgaaatcaac 420
gcgtccgcct cgatcctccc ctgccgacgc tgtacaagtg gcgaccagaa aacaccatgt 480
agtatttgat ctcgtctaag agcaagttta atactatagt ccactattag ctccaattta 540
tttataactg atctaatagc caattcacac aataattgct tactatacta ttaatatatg 600
gtctcacatg tcatacacat attccgtctt ggagttcgtg ctgcagctgg ctacagatct 660
gtagcccgct gctcttctct ctcagagcga gtataatagt acaaactgga ctggcgatag 720
gagaaacacg tcagctacag tgttgagctg gatgagtgag aagaggagag agagtgagag 780
tgggcgacaa ttttatcgcc ggctctagca ccagcttcga gagaaaagtg gtgagcgcag 840
aggttgtgag ctgcatgtgt gagacgaagc ttaagttatt ttattatgat gtgaagttga 900
tgggtccagc gttgcaggtc atttattgta ttcacaagat gcaaagagag ctactagctg 960
agttggatgg aattaacgcc ggctgtctac gctactatta accttgctct catcttttat 1020
ctcatcaaaa tatatttata gctggctaat agtctgctat cgtacctgct ctaatgcata 1080
cgttttttct ctctgtggca aaacggttgg tgcgttacac ggggtgcacg aagccatgca 1140
tcaccctgct caacccgtct ccttttttag cctaatcttt tcctccttat ccgatgggcc 1200
ttccgtttct caagacaccc ccacaccgcc ccggccctct ataaatacca accacgacga 1260
gccaagcgaa catcaccaca gctagatcat tagcaatcca ttccgatcca tcaaatttct 1320
cttgagaccg tagagagaga gagaggcgcc aaccatg 1357
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer
<400> 11
ccattggatc cagccgtgga agtccaac 28
<210> 12
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer
<400> 12
gccggggatc cttggcgcct ctctctct 28
6
