Title of Invention

"METHOD FOR INCREASING EXPRESSION OF AT LEAST TWO STRESS DEFENSE GENES IN A PLANT"

Abstract The present invention provide methods of imparting stress tolerance, characterized in that an expression amount of at least one stress defense gene is increased compared with a non-transformant by transforming the plant with an exogenous spermidine synthase (SPDS) gene, an exogenous S-adenosylmethionine decarboxylase (SAMDC) gene, an exogenous arginine decarboxylase (ADC) gene, an ornithine decarboxylase (ODC) gene and/or a spermine synthase (SPMS) gene under the control of a promoter capable of functioning in the plant.
Full Text DESCRIPTION
Method for increasing expression of stress defense genes
Technical Field
The present invention relates to a method of increasing
expression amounts of stress defense genes involved in stress
tolerance or stress resistance in plants, and a method of
imparting various stress defense effects by increasing the
expression amounts of the stress defense genes involved in the
stress tolerance or the stress resistance.
BACKGROUND ART
Plants adapt to various types of environmental stress such
as the temperature and salt of their habitats. However, in terms
of temperature stress, for example, plants are susceptible to hot
or cold temperatures when exposed to environments over or under
the maximum or minimum optimum growth temperature, leading to
impairment upon the gradual or sudden loss of the physiological
functions of cells. Elf forts have been made to expand the
temperature adaptability of plants by breeding means such as
selection or cross breeding in order to make use of wild plants
adapted to various temperature environments for food crops,
horticultural plants, and the like. The planting period in which
vegetables, flowers and ornamental plants, fruit trees, and the
like can be cultivated has been expanded by such breeding means
as well as by protected horticulture. However, Japan in
particular extends a considerable length to the north and south,
with extreme variation in climate and considerable change in
seasons from area to area, resulting in a greater risk of crop
exposure to temperature environments that are not conducive to
growth, depending on the area and season. Rice, for example,
which originates in tropical regions, can now be cultivated in
cooler areas such as the Tohoku district and Hokkaido as a result
of improvements in varieties since the Meiji period, and are now
cultivated as staples of these regions, but unseasonably low
temperatures in early summer in these areas recently have
resulted in cold-weather damage, leading to the problem of severe
shortages even now. Recently, abnormal atmospheric phenomena
attributed to global warming or El Nino have resulted in major
crop damage, and the rice shortages caused by severe cold-weather
damage in 1993 are still remembered. Culinary plants include many
crops of tropical origin among fruits and vegetables such as
tomatoes, cucumbers, melons, and water melon. Such crops are in
high demand and are extremely important in terms of agriculture,
and they have long been involved in greenhouse culture. However,
since the oil shock of 1974, the conservation of resources and
lowering warming costs have become a problem. The conservation of
resources in protected horticulture has been studied from a
variety of perspectives, from the structural concerns of green
houses to cultivation techniques, but the most basic
consideration is increasing the cold tolerance of crops.
Hot; temperature can be a major form of stress for plants,
and, in particular, the growth and yield of crops is tremendously
affected by heat during summer.
In regard to salt stress, it is said that about 10% of all
land surface area is salt damaged, and the spread of saline soil,
primarily in arid areas such as Southeast Asia and Africa is
becoming a serious agricultural problem.
Drought stress can be a major form of stress for plants, and
is significantly affected by the amount and distribution of
precipitation when temperature is not a limiting factor. The
growth and yield of crops is tremendously dependent upon drought
stress Ln semi-arid regions and the like which are important
areas of crop cultivation.
Osmotic stress or water stress can be a major form of stress
for plants, arid is significantly affected by the amount and
distribution of precipitation when temperature is not a limiting
factor. The growth and yield of crops is tremendously dependent
upon osmotic: stress or water stress in semi-arid regions and the
like which are important areas of crop cultivation.
Cross breeding, breeding making use of recent genetic
engineering techniques, methods making use of the action of plant
hormones and plant regulators, and the like have been employed to
improve tolerance against these various types of stress.
Stress-tolerant plants have thus far been produced using
genetic engineering techniques. Genes reported to have been used
in the improvement of cold tolerance include fatty acid
desaturase genes of membrane lipids (co-3 desaturase gene,
glycerol-3-phosphate acyltransferase gene, and stearoyl-ACPdesaturase
gene), pyruvic-phosphate dikinase genes involved in
photosynthesis, and genes coding for proteins with
cryoprotection/preverition activity (COR15, COR85, and kinl) .
Genes reported to have been used in the improvement of
tolerance against salt, drought, and water stress include glycine
betaine synthetase genes of osmotic regulators (choline
monooxygenase gene and betaine aldehyde hydrogenase gene) and
proline synthetase genes (l-pyroline-5-carboxylate synthetase).
As the method of improving the stress tolerance by
increasing the expression amount of the stress defense gene
involved in the stress tolerance or the stress resistance, the
method of utilizing a gene encoding a transcription factor (DREB
gene) has been reported (Non-patent document 1: Nature
Biotechnology, 17, 287-291, 1999; Patent document 2: The Plant
Cell, 10, 1391-1406, 1998) . In both reports, it has been
described that the expression amount of the stress defense gene
group such as rd29A, kinl and P5CS are increased to enhance the
tolerance against drought stress, salt stress and freeze stress
by excessively expressing the DREB gene constantly in plants, but
remarkable inhibition of growth is observed, individuals which
stop the growth and development and are withered are observed,
and adverse effects on the growth and development are shown.
There are the cases in which the gene involved in the
stress tolerance or the stress resistance other than DREB gene
has been introduced. It has been known that a cold regulated
protein/LEA protein is a late embryogenesis abundant (LEA)
protein and is induced by stress, and it has been reported that
the tolerance against drought stress and salt stress is enhanced
by introducing HV1 which is the LEA protein into rice plant
(Plant Physiology, 110, 249-257, 1996). It has been reported that
a pathogen related PR-1 protein is a protein induced by pathogen
infection and that the tolerance against heavy metal stress and
pathogen infection stress is enhanced by introducing a CABPR1
gene which is one of PR-1 (pathogenesis-related protein I) into
tobacco (Plant; Cell Rep., Feb 18, 2005). Peroxidase is known to
be one (EC 1.11.1.7) of cell wall enzymes in the plants and to be
induced by disease and stress. It has been reported that the
tolerance against oxidative stress and pest stress is enhanced by
introducing it into the plants(Plant Physiology, 132, 1177-1185,
2003; J. Econ. Entomol., 95(1), 81-88, 2002). However, for these
stress tolerance and stress resistance, at most only two types of
stress tolerances are imparted by introducing one type of the
gene involved in the stress tolerance and the stress resistance
into the plant. In the natural world, the plants suffer multiple
stresses simultaneously, and thus, it is necessary to impart
defense effects on the multiple stresses simultaneously in order
to increase productivity of crops. Also, in many of the plants
transformed with the forgoing genes, actually the sufficient
effect to an industrially applicable extent has not been obtained,
and actually these plants have not come into practical use.
There are genes suggested to be deeply involved in stress
although they have not been introduced into the plant. It has
been reported that an old regulated protein/corlS is a gene
induced by low temperature stress and is deeply involved in
freeze stress tolerance (Pro. Natl. Acad. Sci. USA, 93, 13404-
13409, 1996; Pro. Natl. Acad. Sci. USA, 95, 14570-14575, 1998).
It. has been reported that an early response dehydration
prote.in/ERD15 is a gene induced by drought stress and is deeply
involved in drought stress tolerance (Plant Physiology, 106, 1707,
1994). It has been reported that a salt stress induced tonoplast
intrinsic protein/aquaporin and a water channel protein/aquaporin
of Gene Number 7 are water channel proteins induced by stress,
and are deeply involved in the tolerance against salt stress,
osmotic stress and low temperature stress (Mol. Cells., 9(1), 84-
90, L999; Foods Food Ingredients J. Jpn., 176, 40-45). It has
been reported that a dehydration induced protein/RD22 is a
protein induced by drought stress, and is deeply involved in
drought: stress tolerance (Plant Cell., 15(1), 63-78, 2003). It
has been reported that a senescence associated protein senl is a
protein induced by aging stress, salt stress, osmotic stress and
low temperature stress, and is deeply involved in the tolerance
against aging stress, salt stress, osmotic stress and low
temperature stress (Plant Physiology, 130, 2129-2141, 2002).
Additionally, there are genes shown to be involved in the stress
tolerance and the stress resistance. If these genes are
introduced into the plant, the stress resistance corresponding to
each gene is enhanced, but it is difficult to increase the
expression amounts of the multiple stress defense genes
simultaneously.
Owing to environmental problems and food problems, it is a
very important subject to impart the stress defense effects to
the plants, and several attempts to improve the stress defense
have been performed by gene recombination technology, but
actually the sufficient effect to an industrially applicable
extent has not been obtained, and actually no plants has come
into practical use. Furthermore, the expression levels of the
stress defense gene group have been increased to improve the
stress tolerance by excessively expressing the gene encoding DREB
which is one of transcription factors in the plants. However, the
remarkable inhibitory effect on the growth and development is
observed, and it is problematic in that seeds can not be
collected. Therefore, it is an object of the present invention to
provide a method of imparting various stress defense effects by
simply increasing expression amounts of multiple stress defense
genes w.i thin the range in which no adverse effect is given to the
growth and development of plants before and/or during
Disclosure of the Invention
As a result of an extensive study for accomplishing the
above object, the present inventors have found that a polyamine
amount before and during encountering stress is increased by
isolating a spermidine synthase (SPDS) gene, introducing the gene
into a plant and excessively expressing the gene under promoter
control, thereby increasing expression levels of multiple genes
involved in stresses and improving parameters for various stress
tolerances within the range in which no adverse effect is given
to the growth and development of plants. Furthermore, the present
inventors have found that various stress tolerance parameters are
improved by similarly increasing the expression levels of the
multiple genes involved in stresses to impart the stress defense
effects using not only the spermidine synthase (SPDS) gene but
also an S-adenosylmethionine decarboxylase (SAMDC) gene, an
arginine decarboxylase (ADC) gene, an ornithine decarboxylase
(ODC) gene and a spermine synthase (SPMS) gene capable of
controlling an amount of contained spermidine or spermine. In
addition, the present inventors have found that it can be
important to excessively express in a form containing 5'-nontranslated
region concerning the ADC gene, the ODC gene and the
SAMDC gene having 5'-non-translated region (e.g., uORF: small
upstream open reading frame) which has been described to control
an expression amount and a translation amount. That is, the
present invention relates to imparting the stress defense effects
to the plants.
1. A method of inducing an expression of at least two stress
defense genes in a plant, characterized in that an expression
amount of at least one stress defense gene is increased compared
with a non-transformant by transforming the plant with an
exogenous opermidine synthase (SPDS) gene, an exogenous
adenosylmethionine decarboxylase (SAMDC) gene, an exogenous
arginine: decarboxylase (ADC) gene, an exogenous ornithine
decarboxylase (ODC) gene and/or an exogenous spermine synthase
(SPMS) qene under the control of a promoter capable of
functioning in the plant.
16. A method of imparting stress defense effects to a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) gene, an exogenous arginine (ADC) decarboxylase gene, an
ornithine decarboxylase (ODC) gene and/or a spermine synthase
(SPMS) gene under the control of a promoter capable of
functioning in the plant.
17. A method of imparting stress defense effects to a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) gene, an exogenous arginine (ADC) decarboxylase gene, an
exogenous ornithine decarboxylase (ODC) gene and/or an exogenous
spermine synthase (SPMS) gene under the control of a promoter
capable of functioning in the plant, and a transformed plant in
which expression levels of the stress defense genes have been
increased compared with a non-transformed plant (wild type) is
selected
18 . A method of enhancing productivity of a plant characterized
in that; expression amounts of at least two stress defense genes
are increased compared with a non-transformant by transforming
the plant with an exogenous spermidine synthase (SPDS) gene, an
exogenous S-adenosylmethionine decarboxylase (SAMDC) gene, an
exogenous arginine (ADC) decarboxylase gene, an exogenous
ornithine decarboxylase (ODC) gene and/or an exogenous spermine
synthase (SPMS) gene under the control of a promoter capable of
functioning in the plant.
19. A method of enhancing stress tolerance in a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) qene, an exogenous arginine (ADC) decarboxylase gene, an
exogenous ornithine decarboxylase (ODC) gene and/or an exogenous
spermine synthase (SPMS) gene under the control of a promoter
capable of functioning in the plant.
According to the present invention, disorders due to
various stresses encountered in the growth, development and
cultivation processes can be avoided, the growth inhibition and
yield reduction can be reduced, as well as the stabilization of
the cultivation, enhancement of productivity, enlargement of
cultivation regions and expansion of cultivation periods can be
anticipated. It becomes possible to cultivate the plants in
barren lands and salt; accumulated soils, and it can be
anticipated to contribute to the global warming and the food
problem.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a view showing results of Northern blotting of
transformants (TSP, OSP) in which a polyamine synthase gene has
been introduced and a wild type (WT).
Fig. 2 is a view comparing with respect to the tolerance
against osmotic stress between transformants (TSP) in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 3 is a view comparing with respect to the tolerance
against drought; stress between transformants (TSP) in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 4 is a view comparing with respect to the tolerance
against drought, stress between transformants (OSP) in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 5 is a view comparing with respect to the tolerance
against: cold stress between transformants (TSP) in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 6 is a view comparing with respect to the tolerance
against salt stress between transformants (TSP) in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 7 is a view comparing with respect to the tolerance
against salt stress between transformed sweet potato in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 8 is a view comparing with respect to the formation
rate of the root tubers between transformed sweet potato in which
a polyamine synthase gene has been introduced and a wild type
(WT) .
Fig. 9 is a photograph comparing with respect to the root
sight between transformed sweet potato in which a polyamine
synthase gene has been introduced and a wild type (WT).
Fig. 10 is a view comparing with respect to the yield of
the root tubers between transformed sweet potato in which a
polyamine synthase gene has been introduced and a wild type (WT).
Fig. 11 is a photograph comparing with respect to the root
sight between transformed sweet potato in which a polyamine
synthase gene has been introduced and a wild type (WT).
Fig. 12 is a view comparing with respect to the free
polyamine content in the leaf and the root tuber between
transformed sweet potato in which a polyamine synthase gene has
been introduced and a wild type (WT).
Detailed Description of the Invention
As used in the present invention, "stress" refers to stress
received from the environment, such as high temperatures, low
temperatures, low pH, low oxygen, oxidation, osmotic pressure,
drought, water, weak light, cadmium, ozone, air pollution, UV
rays, pathogens, salt, herbicides, intense light, flooding, aging,
heavy metals and pests. As used in the present invention, "nontrans
formants" or "untransformed plants" mean any plants to which
at least one gene selected from exogenous spermidine synthase
(SPDS) genes, S-adenosylmethionine decarboxylase (SAMDC) genes,
arginine decarboxylase (ADC) genes, ornithine decarboxylase (ODC)
genes and spermine synthase (SPMS) genes has not been introduced.
As such, wild species, as well as cultivated varieties
established through common cross breeding, natural or artificial
variants thereof, transgenic plants incorporating exogenous genes
other than sperrnidine synthase genes, and the like are all
included. Documents cited in this specification are incorporated
herein by reference.
Polyamine synthase gene
In the present invention, unexpectedly it has been found
that the expression amounts of multiple genes (stress defense
genes) involved in stress defense are increased by introducing a
polyamine synthase gene into the plant. Furthermore, the present
inventors have found the method in which the expression amounts
of the multiple stress defense genes are increased within the
range in which no adverse effect is given to the growth and
development of the plants to impart various stress defense
effects by transforming the plants particularly with the
polyamine synthase gene such as spermidine synthase (SPDS) gene,
S-adenosylmethionine decarboxylase (SAMDC) gene, arginine
decarboxylase (ADC) gene, ornithine decarboxylase (ODC) gene and
spermine synthase (SPMS) gene capable of increasing the amount of
contained spermidine or spermine among the polyamine synthase
genes. Herein, the SPDS gene, the SAMDC gene, the ADC gene, the
ODC gene and the SPMS gene are sometimes collectively referred to
as the "polyamine synthase gene". Therefore, the "polyamine
synthase gene" includes the SAMDC gene, the ADC gene, the ODC
gene and the SPMS gene.
As used in the present invention, "spermidine synthase"
(SPDS) genes" are genes cording for amino acids of enzymes
involved in spermidine or spermine biosynthesis. The SPDS is a
rate-.limi.ting enzyme which produces spermidine. Spermidine
synthase: (SPDS: EC2.5.1.16) is an enzyme catalyzing the reaction
producing sperrnidine and methylthioadenosine from putrescine and
adenosy]methylthiopropylamine. "Arginine decarboxylase (ADC)
genes" are genes coding for amino acids of enzymes involved in
spermidine or spermine biosynthesis. The ADC is a rate-limiting
enzyme which produces spermidine. Arginine decarboxylase (ADC:
EC4.1.1. 19.) is an enzyme catalyzing the reaction producing
agrnatine and carbon dioxide from L-arginine.
"S-adenosylmethionine decarboxylase (SAMDC) genes" are genes
coding tor amino acids of enzymes involved in spermidine or
spermine biosynthesis. S-adenosylmethionine decarboxylase (SAMDC:
EC4.1.1.50.) is an enzyme catalyzing the reaction producing
adenosylmethylthiopropylamine and carbon dioxide from
S-adenosylmethionine. "Ornithine decarboxylase (ODC) genes" are
genes coding for amino acids of enzymes involved in spermidine or
spermine biosynthesis. Ornithine decarboxylase (ODC: EC4.1.1.17.)
is an enzyme catalyzing the reaction producing putrescine and
carbon dioxide from L-ornithine. "Spermine synthase (SPMS) genes"
are genes coding for amino acids of enzymes involved in spermine
biosynthesis. The SPMS is a rate-limiting enzyme which produces
spermidine. For carrying out the present invention, the most
preferable "polyamine synthase genes" are SPDS genes.
These polyamine synthase genes may be derived from any,
such as plants, microbes and animals, as long as the gene
expression for stress defense is enhanced without causing adverse
effects on growth. Genes which are already isolated can also be
used. For example, spermidine synthase (SPDS) genes have been
isolated from plants such as Arabidopsis thaliana and tabacco
(Plant Cell Physiol., 39(1), 73-79, (1998)), tomatoes (Plant
Physiol., 120, 935, (1999)), coffee (Plant Science., 140, 161-168,
(1999)), peas (Plant Molecular Biology, 39, 933-943, (1999)),
apples (Mol. Gen. Genomics, 268, 799-807, (2003)), Cucurbita
ficifclA.a Bouche (W002/23974, W003/84314) . Furthermore, it has
been attempted that the spermidine synthase (SPDS) gene is
introduced into tobacco which is a model plant, and the change of
contained polyamine has been examined, but the expression level
of the stress defense genes and the improvement of environmental
stress tolerances have not been examined (Journal of Plant
Physiology, 162: 989-1001, 2004). Arginine decarboxylase (ADC)
genes have been isolated from oats (Mol. Gen. Genet., 224, 431-
436 (1990)), tomatoes (Plant Physiol., 103, 829-834 (1993)),
Arabidopsis thaliana (Plant Physiol., Ill, 1077-1083 (1996)),
peas (Plant Mol. Biol., 28, 997-1009 (1995)), and Cucurbita
ficifolia Bouche (W002/23974, W003/84314); S-adenosylmethionine
decarboxylase (SAMDC) genes have been isolated from potatoes
(Plant Hoi. Biol., 26, 327-338 (1994)), spinach (Plant Physiol.,
107, 1461-1462 (1995)), and Cucurbita ficifolia Bouche
(W002/23974, W003/84314); ornithine decarboxylase (ODC) genes
have been isolated from datura (Biochem. J., 314, 241-248
(1996)); spermine synthase (SPMS) genes have been isolated from
Arabidopsis thaliana (EMBO J., 19, 4248-4256, (2000))
In one preferable embodiment of the present invention, it
is important that the ADC gene, the ODC gene or the SAMDC gene is
excessively expressed in a form of containing the 5'-nontranslated
region (e.g., uORF) which affects the expression and
the translation under the control of an inducible type promoter.
The "uORF" of the present invention indicates an upstream
open reading frame, and is present 5' upstream (5'-non-translated
region) of the ORF which encodes amino acids. The uORF is present
in the 5'-non-translated region of the polyamine synthase genes
(ADC gene, ODC gene and SAMDC gene), and is believed to control
the expression arid the translation of the polyamine synthase
genes.
According to the present invention, the polyamine synthase
gene isolated from microorganisms and animals can express the
stress defense genes such as DREB, CBF1, LEA and COR about 1.1 to
10 times, preferably about 1.3 to 10 times, more preferably about
1.4 to 8 times and particularly about 1.5 to 5 times more highly
compared with non-transformants. More preferably, the polyamine
synthase gene isolated from the microorganisms and the animals
can express the stress defense genes such as DREB, CBF1, LEA and
COR in the range of about 1.5 to 4 times more highly compared
with the non-transformants.
In addition, polyamine synthase genes can also be isolated
from various plants. Specific examples include dicotyledons such
as Cucurbitaceae; Solanaceae; Brassicaceae such as Arabidopsis
thaliand; Papilionaceae such as alfalfa and Vigna unguiculata;
Malvaceae; and Asteraceae; or monocotyledons such as Gramineae,
including rice, wheat, barley, and corn. Cucurbitaceae,
Brassicaceae and Gramineae are preferred, and Cucurbita ficifolia
Bouche, Arabidopsis thaliana, rice, corn, wheat, cotton, soybeans
and rapeseed are more preferred.
In the invention, the most suitable conditions for
obtaining polyamine synthase genes are also found. That is, plant
tissue in which the plant-derived polyamine synthase genes of the
invention are isolated may be in the form of seeds or in the
process of growing. The genes may be isolated from part or all of
the tissue of growing plants. Any part can be used to isolate
genes, but whole plants, buds, flowers, ovaries, fruit, leaves,
stems, roots, and the like are preferred. Roots and leaves are
more preferred.
Preferred examples of polyamine metabolism-related enzyme
genes used in the present invention include the spermidine
synthase gene. Specific examples include:
* DNA having the base sequence represented by base numbers 77
through 1060 in the base sequence given in SEQ ID NO. I
(Cucurbita ficifolia Bouche);
* DNA having the base sequence represented by base numbers 118
through 1281 in the base sequence given in SEQ ID NO. 3 (rice) ;
and
* DNA having the base sequence represented by base numbers 456
through 1547 in the base sequence given in SEQ ID NO. 5 (Cucurbita
ficifoUa Bouche) .
* DNA having the base sequence represented by base numbers 541
through 2661 in the base sequence given in SEQ ID NO. 7 (Cucurbita
ficifolia Bouche) .
* DNA having the base sequence represented by base numbers 1
through 1020 in the base sequence given in SEQ ID NO. 9
(Arabidopsis thaliana) .
Further examples include:
* DNA having a base sequence capable of hybridizing under
stringent conditions with DNA or their complementary chains
having any of the above sequences, and coding for a polypeptide
with spermidine synthase activity equivalent to those sequences.
Still further examples include:
* DNA comprising any of the above amino acid sequences with 1 or
more bases deleted, substituted, inserted, or added, and coding
for a polypeptide with spermidine synthase activity equivalent to
those sequences.
"Polyamine synthase genes" include known genes as well as
DNA having a base sequence capable of hybridizing under stringent
conditions with the genes or their complementary chains, and
coding for a polypeptide with polyamine synthase activity
equivalent to those sequences. Furthermore, DNA comprising amino
acid sequences encoded by any of the above DNA, in which 1 or
more bases deleted, substituted, inserted, or added, and coding
for a polypeptide with polyamine synthase activity equivalent to
those sequences are included.
The "stringent conditions" referred to here mean conditions
under which base sequences coding for a polypeptide with enzyme
activity equivalent to the polyamine synthase (e.g. spermidine
synthase) encoded by a specific polyamine synthase gene (e.g.
polyamine) synthase genes) sequence form hybrids with the specific
sequence (referred to as specific hybrids), and base sequences
coding for polypeptides with no such equivalent activity do not
form hybrids with the specific sequence (referred to as nonspecific
hybrids). One with ordinary skill in the art can readily
select such conditions by varying the temperature during the
hybridization reaction and washing process, the salt
concentration (luring the hybridization reaction and washing
process, and so forth. Specific examples include, but are not
limited to, conditions under which hybridization is brought; about
at 42°C in 6 x SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 x
33PE (3M NaCl, 0, 2 M NaH2P04, 20 mM EDTA-2Na, pH 7.4), and the
product, is washed with 0.5 x SSC at 42°C. Preferably, the
condition where the hybridization is performed in 50%
formaldehyde, 6 x SCC (0.9 M NaCl, 0.09M trisodium citrate) or 6
x SSPE (3 M NaCl, 0.2 M NaH2P04, 20 mM EDTA2Na, pH 7.4) at 42°C
and washing with 0.1 x SCC at 42°C is further performed is
included.
The "base sequences with 1 or more bases deleted,
substituted, inserted, or added" referred to here are widely
known by those having ordinary skill in the art to sometimes
retain physiological activity even when the amino acid sequence
of a protein generally having that physiological activity has one
or more amino acids substituted, deleted, inserted, or added.
Genes that have such modifications and that code for a polyamine
synthase (e.g. spermidine synthase) can also be used in the
present invention. For example, the poly A tail or 5', 3' end
nontranslation regions may be "deleted," and bases may be
"deleted" to the extent that amino acids are deleted. Bases may
also be "substituted," as long as no frame shift results. Bases
may also be "added" to the extent that amino acids are added.
However, it is essential that such modifications do not result in
the loss of polyamine synthase (e.g. spermidine synthase)
activity. "Genes with one or more bases deleted, substituted, or
added" are preferred. Such modified DNA can be obtained by
modifying the DNA base sequences of the invention so that amino
acids at specific sites are substituted, deleted, inserted, or
added by site-specific mutagenesis, for example (Nucleic Acid
Research, Vol. 10, No. 20, 6487-6500 (1982)).
Polyandries
Polyamines, the general term for aliphatic hydrocarbons
with 2 or more primary amino groups, are ubiquitous natural
substances in organisms, with more than 20 types discovered so
far. Typical polyamines include putrescine, spermidine, and
spermine. The known primary physiological action of polyamines
includes (1) nucleic acid stabilization and structural
modification through interaction with nucleic acids; (2)
promotion of various nucleic acid synthesis systems; (3)
activation of protein synthesis systems; and (4) stabilization of
cell membranes and enhancement of membrane permeability of
substances. Reports on the role of polyamines in plants include
cell protection and promotion of nucleic acid or protein
biosynthesis during cellular growth or division. As used in the
invention, "spermidine" , one of the typical polyamines, is an
ubiquitous natural substance in organisms, and is an aliphatic
hydrocarbon with three primary amino group.
The involvement of polyamines in various types of
environmental stress has recently been reported. They have been
implicated in cold stress (J. Japan Soc. Hortic. Sci., 68, 780-
787 (1999); J. Japan Soc. Hortic. Sci., 68, 967-973 (1999); Plant
Physiol. 124, 431-439 (2000)); salt stress (Plant Physiol. 91,
500-504 (1984)); acid stress (Plant Cell Physiol. 38(10), 1156-
1166 (1997)); osmotic stress (Plant Physiol. 75, 102-109 (1984));
pathogen infection stress (New Phytol., 135, 467-473 (1997)); and
herbicide stress (Plant Cell Physiol. 39(9), 987-992 (1998)), but
all of these reports assume the involvement of polyamines based
on the correlation between growth reaction or stress tolerance
and changes in polyamine concentration, yet their involvement at
the genetic level between environmental stress tolerance and
polyamine synthase genes was not well studied.
There are other cases in which the polyamine synthase gene
has been introduced into the plant, but regulation of the
expression amount of the stress defense gene has not been studied.
For example, the plant transformed with the spermidine synthase
(SPDS) gene has been reported in tobacco (Non-patent document
Journal of Plant Physiology 161, 989-1001, 2004). The SPDS gene
derived from Datura stramonium has been excessively expressed
constantly in tobacco, and the change of contained polyamine
amounts has been examined. However, the change of the expression
amount of the stress defense genes and the stress tolerance are
not shown at all. The method of increasing the expression amounts
of the stress defense gene group by transforming the plant with
the spermidine synthase (SPDS) gene thereby imparting the stress
defense effects has not been reported until now. Also, the method
of increasing the expression amounts of the stress defense gene
group by transforming the plant with the S-adenosylmethionine
decarboxylase (SAMDC) gene, the arginine decarboxylase (ADC) gene,
the ornithine decarboxylase (ODC) gene and/or the spermine
synthase (SPMS) gene thereby imparting the stress defense effects
has not been reported until now. In addition, it has been
attempted that the polyamine synthase gene is introduced into the
plant, and the change of the contained polyamine amounts has been
examined, but the relationship between the expression level of
the stress defense gene and the improvement of various stress
tolerances has not been examined at all. In the present invention,
the relationship between the expression levels of the stress
defense genes and stress defense genes and the improvement of
various stress tolerances has been disclosed for the first time.
As a result of an extensive study for imparting the stress
defense effects to the plants, the present inventors have found
that it is very important for imparting or improving various
stress tolerances to increase the amount of contained spermidine
or spermine before or during encountering stresses by
transforming the plant with the spermidine synthase (SPDS) gene
and increase the expression levels of the stress defense genes by
the action of increased spermidine or spermine thereby imparting
the stress defense effects. Without wishing to be bound to any
theory, the present inventors believe that (1) spermidine or
spermine increased by transforming the plant with the SPDS gene
acts as a second messenger (signal transducing substance) and
activates tyrosine kinase involved in signal transduction thereby
inducing the expression of the stress defense genes, and (2)
spermidine or spermine increased by transforming the plant with
the SPDS gene is metabolized by polyamine oxidase (PAO) resulting
in increased levels of hydrogen peroxide which activate the
signai transduction thereby inducing the expression of the stress
defense qenes. Since the increase of contained spermidine or
spermine and the action thereof are important for the induction
of the expression of the stress defense genes and the increase of
the expression level thereof, likewise the effect of increasing
the expression levels of the stress defense genes is obtained
also using the S-adenosylmethionine decarboxylase (SAMDC) gene,
the arginine decarboxylase (ADC) gene, the ornithine
decarboxylase (ODC) gene or the spermine synthase (SPMS) which
can increase the amount of contained spermidine or spermine. A
time period that the expression level of the stress defense gene
is enhanced may be any of constantly, under the condition of nonstress,
before encountering stress and under the condition of
stress. However, the present inventors have found that it is
important to increase the expression amounts of the stress
defense genes within the range in which no effect is given to the
growth and development of the plant constantly or under the
condition of non-stress and impart the stress defense effects to
the plant previously (preliminarily) before encountering stress
(vaccine effect), thereby exhibiting the excellent tolerance and
resistance against various stresses when encountering stress. In
addition, the present inventors have found that the expression
levels of the stress defense genes such as DREB, CBF1 and COR are
increased within the range in which no adverse effect is given to
the growth and development to impart the stress defense effects
by introducing the gene such as SPDS, SAMDC, ADC, ODC and SPMS
into the plant and excessively expressing it under the control of
the promoter, thereby improving the parameters of various stress
tolerances and enhancing the productivity (e.g., yield) and
characters, and have completed the present invention.
Stress defense genes
In the present invention, the "stress defense gene" is a
gene whose expression/induction or expression amount is increased
when the plant encounters stress, and the gene involved in or
ssociated with the stress tolerance. In one preferable
embodiment of the invention, the stress defense genes are the
following 49 genes or the genes having 60% or more, preferably
70% or more, more preferably 80% or more, still more preferably
85% or more and particularly 90% or more homology to these genes
with specific Accession Number.
(Table Removed)
number
In another preferable embodiment, the stress defense genes
can belong to the following 1 to 11 categories:
I. CBF1/DREBlB
For example, CBF1, DREB1B of Gene Numbers I, 25 and 42 are
the genes encoding the transcription factors expressed and
induced by stresses (The Plant Cell, 10, 1391-1406, 1998, Proc.
Natl. Acad. Sci. U.S.A., 94, 1035-1040, 1997, Plant Physiol., 130,
639-648, 2002), and are shown to have an ERF/AP2 DNA binding
domain arid act as a factor to activate the transcription (Biochem.
Biophys. Res. Commun., 290, 998-1009, 2002, Physiol. Plant, 112,
171-175, 2001). It has been reported that the tolerance against
environmental stresses such as drought, freeze and low
temperature is enhanced by introducing CBF1, DREBlB genes into
the plant (Science, 280, 104-106, 1998, Plant Physiol., 127, 910-
917, 2001, Plant Physiol., 130, 618-626, 2002).
II. CBF3/DREB1A
For example, DREB1A, CBF3 of Gene Numbers 27 and 28 are the
genes encoding the transcription factors expressed and induced by
stresses (The Plant Cell, 10, 1391-1406, 1998, Plant J., 16, 433-
442, 1998, Biochem. Biophys. Res. Commun., 250, 161-170, 1998,
Plant Physiol., 130, 639-648, 2002), and are shown to have an
ERF/AP2 DNA binding domain and act as a factor to activate the
transcription (Plant Cell, 13, 61-72, 2001, Biochem. Biophys. Res.
Commun., 290, 998-1009, 2002, The Plant Journal, 38, 982-993,
2004) . It has been reported that the tolerance against
environmental stresses such as drought, salt, freeze and low
temperature is enhanced by introducing CBF1, DREBlB genes into
the plant (The Plant Cell, 10, 1391-1406, 1998, Nat. Biotech., 17,
287-291, 1999, Plant Physiol., 124, 1854-1865, 2000) .
III. DREB2B
For example, DREB2B of Gene Number 32 is the gene encoding
the transcription factors expressed and induced by drought and
salt stress (The Plant Cell, 10, 1391-1406, 1998, Plant Mol.
Biol., 42, 657-665, 2000) , and are shown to have an ERF/AP2 DNA
binding domain and act as a factor to activate the transcription
(Biochem. Biophys. Res. Commun., 290, 998-1009, 2002) .
IV. LTI78, COR78, rd29A
For example, it has been reported that LTI78(low
temperature induced protein 78), COR78 and rd29A of Gene Number
38 are the genes encoding the proteins expressed and induced by
low temperature stress, drought stress and salt stress and are
deeply involved in the tolerance against low temperature stress,
drought stress and salt stress (Plant Physiol., 103, 1047-1053,
1993, Plant Mol. Biol., 21, 641-653, 1993, Plant Cell, 6,251-264,
1994, Journal of Experimental Botany, 47, 291-305, 1996, Plant
Physiol, 130, 2129-2141, 2002). It has been reported that the
genes (DREB1A, DREB2A) encoding the transcription factors which
bind to the promoter of rd29A are isolated and that the
expression amount of rd29A is increased by introducing the DREB1A
gene into the plant thereby enhancing the tolerance against
environmental stresses such as drought, salt, freeze and low
temperature (The Plant Cell, 10, 1391-1406, 1998, Nat. Biotech.,
17, 287-291, 1999, Plant Physiol., 124, 1854-1865, 2000).
V. RD22/rd22
For example, it has been reported that RD22 and rd22 of
Gene Number 8 are the genes encoding the proteins expressed and
induced by drought stress and are deeply involved in the
tolerance against drought stress (The Plant Cell, 5, 1529-1539,
1993, Mol. Gen. Genet., 247, 391-398, 1995, The Plant Cell, 9,
1859-1868, 1997, Plant Cell., 15(1), 63-78, 2003) .
VI. Corlb
For example, it has been reported that cor15 of Gene Number
3 is the gene encoding the protein expressed and induced by low
temperature stress and drought stress and is deeply involved in
the freeze stress tolerance (Plant Mol. Biol., 23, 1073-1077,
1993, Journal of Experimental Botany, 47, 291-305, 1996). It has
been reported that the freeze stress tolerance is enhanced by
introducing cor!5 gene into the plant (Pro. Natl. Acad. Sci. USA,
93, 13404-13409, 1996, Journal of Plant Physiology, 163, 213-219,
2006), and that the freeze stress tolerance of a chloroplast was
enhanced when the freeze stress tolerance of the chloroplast
isolated from the transformed plant was examined (Pro. Natl. Acad.
Sci. USA, 95, 14570-14575, 1998).
VII. ERD15
For example, it has been reported that ERD15 of Gene Number
5, 30 and 37 are the genes encoding the proteins induced by
drought stress and are deeply involved in the tolerance against
drought stress (Plant Physiology, 106, 1707, 1994) .
VIII. LEA protein
For example, LEA protein of Gene Number 2 is known to be
the gene encoding late embryogenesis abundant(LEA)protein and to
be expressed and induced by various stresses, and it has been
reported that the tolerance against drought stress and salt
stress is enhanced by introducing HVA1 gene which is the LEA
protein gene derived from barley into rice plant, and that the
salt stress tolerance is enhanced in yeast in which the LEA
protein gene derived from tomato has been highly expressed (Plant
Physiology, 110, 249-257, 1996, Gene, 170, 243-248, 1996).
IX. PR-1
For example, PR-1 of Gene Number 4 is known to be the gene
encoding the protein induced by pathogen infection, and to have
an antibacterial activity (Physiol. Mol. Plant Pathol., 55, 85-97,
1999). It has been reported that the tolerance against heavy
metal stress and pathogen infection stress is enhanced by
introducing CABPR1 (Capsicum annuum basic pathogenesis-related
protein 1) gene which is one of PR-1(pathogenesis-related protein
1) into tobacco (Plant Cell Rep., Feb 18, 2005).
X. Peroxidase
For example, peroxidase of Gene Number 19 or 44 is known to
be one (EC 1.11.1.7) of cell wall enzymes in the plant and to be
induced by diseases and stresses, and it has been reported that
the tolerance against oxidative stressoxidative stress and pest
stress ir> enhanced by introducing it into the plant (Plant
Physiology, 132, 1177-1185, 2003, J. Econ. Entomol., 95(1), 81-88,
XI. Hslpro-1
For example, it has been reported that Hslpro-1 (nematode
resistance protein) gene of Gene Number 22 is isolated as a
tolerance gene of nematode and is deeply involved in nematode
tolerance (Science, 275, 832-834, 1997, Plant Mol. Biol., 52,
643-660, 2003) .
In the above 49 genes or genes having 60% or more homology
to the genes and further the genes belonging to I to XI, the
description of the gene of each Gene Number is only an
exemplification, and the stress defense gene can be changed
depending on the type of the plant. Therefore, when the
expression amounts of two or more of 49 genes, genes having 60%
or more homology to the genes and the stress defense genes
belonging to I to XI are increased in the transformant obtained
by introducing the specific polyamine synthase gene into the
plant, this case is included in the method of the present
invention.
For the above stress defense genes, the expression amounts
of two or more of 49 genes (including the genes having 60% or
more homology) and distinct genes of I to XI may be increased,
and the expression amounts of two or more of the genes belonging
to the same group may be increased.
Imparting of stress defense effects
As noted above, in the present invention, "stress" includes
stress received from the environment, such as high temperatures,
low temperatures, low pH, low oxygen, oxidation, salt, osmotic,
drought, water, flooding, cadmium, copper ozone, air pollution,
UV rays, intense light, weak light, pathogens, disease pests,
herbicides and aging . Of these, "heat stress" is stress on
plants due to exposure of the plants to environments over the
upper Limit of optimal growth temperature of the plant. Plants
subject to heat stress are damaged as a result of gradual or
sudden Joss of cellular physiological function. "Cold stress" is
stress on plants due to exposure of the plants to environments
below the minimum optimal growth temperature of the plant. Plants
subject to cold stress are damaged as a result of gradual or
sudden loss of cellular physiological function. "Salt stress" is
stress on plants due to exposure of the plants to environments
over the maximum optimal growth salt concentration of the plant.
Plants subject to salt stress are damaged as a result of gradual
or sudden loss of cellular physiological function due to
intracellular infiltration of excess salt. "Osmotic stress" is
stress on plants due to exposure of the plants to environments
over or under the maximum or minimum optimal growth osmotic of
the plant. Plants subject to osmotic stress are damaged as a
result of gradual or sudden loss of cellular physiological
function. "Drought stress" is stress on plants due to exposure of
the plants to environments under the minimum optimal growth
moisture concentration of the plant. Plants subject to drought
stress are damaged as a result of gradual or sudden loss of
cellular physiological function. "Water stress" is stress on
plants due to exposure of the plants to environments under the
minimum optimal growth moisture concentration of the plant.
Plants subject to water stress are damaged as a result of gradual
or sudden loss of cellular physiological function. Stress under
weak light is stress on plants due to exposure of the plants to
environments under the minimum optimal growth light intensity of
the plant. Plants subject to stress under weak light are damaged
as a result of gradual or sudden loss of cellular physiological
function. "Herbicide stress" is stress on plants due to exposure
of the plants to environments over the maximum optimal growth
herbicide concentration of the plant. Plants subject to herbicide
stress are damaged as a result of gradual or sudden loss of
cellular physiological function. "Pathogen stress" is stress
which the plant receives by being infected or diseased with a
pathogen which is unsuitable for plant growth, and the plant
which has received pathogen stress damages physiological
functions gradually or rapidly to cause the disease. "Pest
stress" is stress which the plant receives by insect damage or
being infected with or encountering a pest which is unsuitable
for the plant growth, and the plant which has received pest
stress damages physiological functions gradually or rapidly to
cause the disease. "Heavy metal stress" is stress on plants due
to exposure of the plants to environments under the minimum
optimal growth heavy metal concentration of the plant. Plants
subject to drought stress are damaged as a result of gradual or
sudden loss of cellular physiological function.
In the present invention, the "method of imparting the
stress defense effects" refers to the method of imparting the
stress defense effects in comparison with the period before
introduction by introducing the exogenous polyamine synthase gene
into the plant. Specifically, the "method of imparting low
temperature stress defense effects" is the method capable of
avoiding or reducing the growth inhibition, the disease and the
productivity decrease due to low temperature stress which the
plant encounters in the growth process by imparting the stress
defense effects to the plant. The "method of imparting high
temperature stress defense effects" is the method capable of
avoiding or reducing the growth inhibition, the disease and the
productivity decrease due to high temperature stress which the
plant: encounters in the growth process by imparting the stress
defense effects to the plant. The "method of imparting salt
stress defense effects" is the method capable of avoiding or
reducing the growth inhibition, the disease and the productivity
decrease-: due to salt stress which the plant encounters in the
growth process by imparting the stress defense effects to the
plant. The "method of imparting osmotic stress defense effects"
is the method capable of avoiding or reducing the growth
inhibition, the disease and the productivity decrease due to
osmotic stress which the plant encounters in the growth process
by imparting the stress defense effects to the plant. The "method
of imparting oxidative stressoxidative stress defense effects" is
the method capable of avoiding or reducing the growth inhibition,
the disease and the productivity decrease due to oxidative
stressoxidative stress which the plant encounters in the growth
process by imparting the stress defense effects to the plant. The
"method of imparting herbicide stress defense effects" is the
method capable of avoiding or reducing the growth inhibition, the
disease and the productivity decrease due to herbicide stress
which the plant encounters in the growth process by imparting the
stress defense effects to the plant. The "method of imparting
freeze stress defense effects" is the method capable of avoiding
or reducing the growth inhibition, the disease and the
productivity decrease due to freeze stress which the plant
encounters in the growth process by imparting the stress defense
effects to the plant. The "method of imparting drought stress
defense effects" is the method capable of avoiding or reducing
the growth inhibition, the disease and the productivity decrease
due to drought stress which the plant encounters in the growth
process by imparting the stress defense effects to the plant. The
"method of imparting pathogen infection stress defense effects"
is the method capable of avoiding or reducing the growth
inhibition, the disease and the productivity decrease due to
pathogen infection stress which the plant encounters in the
growth process by imparting the stress defense effects to the
plant. The "method of imparting pest stress defense effects" is
the method capable of avoiding or reducing the growth inhibition,
the disease arid the productivity decrease due to pest stress
which the plant encounters in the growth process by imparting the
stress defense effects to the plant. The "method of imparting
disease stress defense effects" is the method capable of avoiding
or reducing the growth inhibition, the disease and the
productivity decrease due to disease stress which the plant
encounters in the growth process by imparting the stress defense
effects to the plant. The "method of imparting aging stress
defense effects" is the method capable of avoiding or reducing
the growth inhibition, the disease and the productivity decrease
due to aging stress which the plant encounters in the growth
process by imparting the stress defense effects to the plant. The
"method of imparting heavy metal stress defense effects" is the
method capable of avoiding or reducing the growth inhibition, the
disease and the productivity decrease due to heavy metal stress
which the plant encounters in the growth process by imparting the
stress defense effects to the plant. The stabilization of
cultivation, the enhancement of productivity and yield, and the
effective utilization of the cultivation, the environment, the
period, the region and the area can be anticipated by using the
expression amounts of the stress defense genes as indicators or
controlling them. In addition, it is possible to anticipate the
enhancement of productivity of various useful substances (e.g.,
starch, protein) obtained from the plants by increasing the
productivity and the yield of the plants. The expression amount
of the stress defense gene can be utilized as the indicator for
the method of selecting and diagnosing cultivation effective
crops.
A method of the invention can be carried out by introducing
an exogenous polyamine synthase gene to plants having no
exogenous polyamine synthase gene through genetic engineering
means arid making it retained in a stable manner. As used herein,
"retained in a stable manner" means that the polyamine synthase
gene is expressed in the plant at least in which the polyamine
synthase gene has been introduced, and is retained in the plant
cells long enough to result in the improvement of stress
tolerance. The polyamine synthase gene is, therefore, preferably
incorporated on the chromosomes of the host plant. The polyamine
synthase gene or genes should even more preferably be retained in
subsequent generations.
As used herein, "exogenous" means not intrinsic to the plant,
but externally introduced. Accordingly, an "exogenous polyamine
metabolism-related enzyme gene" may be a polyamine synthase gene
homologous to the host plant (that is, derived from the host
plant:) , which is externally introduced by genetic manipulation.
The use of a host-derived polyamine synthase gene is preferred in
consideration of the identity of the codon usage.
The exogenous polyamine synthase gene may be introduced into
plants by any method of genetic engineering. Examples include
protoplast fusion with heterologous plant cells having the
polyamine synthase gene, infection with a plant virus having a
viral genome genetically manipulated to express the polyamine
synthase gene, or transformation of host plant cells using an
expression vector containing the polyamine synthase gene.
The;: plants of the invention are preferably transgenic plants
which are obtained by the transformation of cells of plants
lacking the exogenous polyamine synthase gene in an expression
vector containing the exogenous polyamine synthase under the
control of a promoter capable of functioning in plants.
Examples of promoters capable of functioning in plants
include the 35S promoter of the cauliflower mosaic virus (CaMV)
which functions in plant cells, the nopaline synthase gene (NOS)
promoter, octopine synthase gene (DCS) promoter, phenylalanine
ammonia lyase (PAL) gene promoter, and chalcone synthase (CHS)
gene promoter, ubiquitin (Ubi-1) promoter, peroxidase gene
promoter. Other well-known plant promoters not limited to these
are also available.
Constitutive promoters include a CaMV35S promoter, an actin
promoter (Plant Cell, 2, 163-171, 1990), an ubiquitin promoter
(Plant Mol. Biol., 18, 675-689, 1992) and a rice cyclophilin
promoter (Plant Mol. Biol., 25, 837-843, 1994). If not only the
promoter to constantly or constitutively express in entire organs
but also the promoter specific for the organ or tissue is used,
it: is possible to express the objective gene only in the
particular organ or tissue, and impart the stress defense effects
only to the particular organ or tissue. As the promoter specific
for leaf tissues, an aldP promoter(Mol. Gen. Genet., 248, 668-674,
1995) and a rbcs promoter(Plant Cell Physiol., 35, 773-778, 1994)
can be utilized. As the promoter specific for flower tissues, a
chsA chalcone synthase promoter (Plant Mol. Biol., 15, 95-109,
1990) and an LAT52 promoter (Mol. Gen. Genet., 217, 240-245,
1989) can be utilized. As the promoter specific for roots, root
tubers and stem tubers, an SbPRPl promoter (Plant Mol. Biol., 21,
109-119, 1993) and a sporamin promoter (Mol. Gen. Genet., 225,
369, 199L) can be utilized. For example, the stress defense
effects can be imparted only to the root tuber by using the
polyamine synthase gene and the sporamin promoter which works
specifically for the root tuber.
As inducible promoters, a stress inducible promoter, a
temperature inducible promoter, a light inducible promoter, a
period inducible promoter, a pathogen inducible promoter, and a
disease inducible promoter and the like can be utilized. For
example, by the use of the polyamine synthase gene and the
promoter (e.g., BN115 promoter: Plant physiol.,106, 917-928,
1999) which can induce the transcription only when the plant
encounters the low temperature, it is possible to control
polyamine metabolism of the plant only at low temperature to
impart the low temperature stress defense effect. By the use of
the polyamine synthase gene and the promoter (e.g., Atmyb2
promoter: The Plant Cell, 5, 1529-1539, 1993) which can induce
the transcription only when the plant encounters the drought, it
is possible to control polyamine metabolism of the plant only at
drought to impart the drought stress defense effect. By the use
of peroxidase promoter (Patent No. 3571639, Patent No. 3259178)
induced by various stresses, it is possible to control polyamine
metabolism of the plant only at various stresses to impart the
various stress defense effect. Furthermore, by the use of the
polyamine synthase gene and the promoter which works in a
vegetative stage, it is possible to impart the stress defense
effects only in the vegetative stage.
Preferably, from a notion that it is particularly important
for the stress defense to previously (preliminarily) impart the
stress defense effects to the plant (vaccine-like effect) by
increasing the expression amounts of the stress defense genes
within the range where no adverse effect is given to the growth
and development of the plant before encountering stress,
preferably the steady or constitutive promoter, the promoter
specific tor the organ or the tissue, and the promoter specific
for the period depending on the growth and development can be
used. In particular, the steady or constitutive promoter is
preferable.
The exogenous polyamine synthase gene in the expression
vector of the present invention is located downstream of the
promoter so that transcription is controlled by the promoter
capable of functioning in plants. A transcription termination
signal (terminator region) capable of functioning in plants
should also be added downstream of the polyamine synthase gene.
An example is the terminator NOS (nopaline synthase) gene.
The expression vector of the present invention may also
contain a cis-regulatory element such as an enhancer sequence.
The expression vector may also contain a marker gene for
selecting transformants such as a drug-resistance gene marker,
examples of which include the neomycin phosphotransferase II
(NPTII) gene, the phosphinothricin acetyl transferase (PAT) gene,
and the glyophosate resistance gene. Because the incorporated
gene is sometimes dropped in the absence of selection pressure,
it is advantageous to ensure that a herbicide resistance gene is
also present on the vector so that the use of a herbicide during
cultivation will always result in conditions involving selection
pressure.
To facilitate mass production and purification, the
expression vector should also contain a selection marker gene
(such as ampicillin resistance gene or tetracycline resistance
gene) in E. coli and a replication origin capable of autonomous
replication in E. coli. The expression vector of the present
invention can be constructed in a simple manner by inserting the
selection marker gene as needed and an expression cassette of the
polyamine synthase gene at the cloning site of an E. coli vector
(pUC or pBR series).
When the exogenous polyamine metabolism-related enzyme gene
is introduced by infection with Agrobacterium tumefaciens or
Aqrobacterium rhizogenes, the polyamine synthase gene expression
cassette can be inserted in the T-DNA region (region transferred
to plant chromosome) on a Ti or Ri plasmid of the cells. At
present, binary vector systems are used in standard methods of
transformation with Agrobacterium. The necessary functions for T--
DNA transfer are independently provided by both the T-DNA itself
and the Ti (or Ri) plasmid, these structural elements being
divided on separate vectors. The binary plasmid has 25 bp border
sequences at both ends necessary for cleaving and combining the
T-DNA, and the plant hormone gene inducing crown gall (or hairy
root) is removed, simultaneously providing room for inserting the
exogenous gene. Examples of commercially available binary vectors
include pBHOl and pBI121 (by Clontech) . The Vir region involved
in the incorporation of the T-DNA has trans action on the
separate Ti (or Ri) plasmid referred to as the helper plasmid.
Various conventionally known methods can be used for the
transformation of the plants. Examples include the PEG method in
which protoplasts are isolated from plant cells by treatment with
a cell wall-degrading enzyme such as cellulase or hemicellulase,
and polyethylene glycol is added to a suspension of the
protoplasts and an expression vector containing the
aforementioned polyamine synthase gene expression cassette to
incorporate the expression vector into the protoplasts by a
process such as endocytosis; the liposome method in which an
expression vector is introduced by ultrasonic treatment or the
like into lipid membrane vesicles such as phosphatidylcholine,
and the vesicles are fused with protoplasts in the presence of
PEG; methods of fusion in a similar process using micelles; and
electroporation in which electrical pulses are applied to a
suspension of protoplasts and an expression vector to incorporate
the vectors in the external solution into the protoplasts.
However, these methods are complicated in that they require a
culturing technique for the redifferentiation of the protoplasts
into plants. Processes for introducing the gene into intact cells
with cell walls include direct injection such as microinjection
in which a micropipette is inserted into cells to inject the
vector DNA in the pipettes under hydraulic or gas pressure into
the cells, or the particle gun method in which metal
microparticles coated with DNA are accelerated through the
detonation of an explosive or gas pressure and thus introduced
into the cells, and methods involving the use of infection with
Agrobacterium. Drawbacks of microinjection are the need for
considerable training and the small number of cells that are
handled. It is therefore more desirable to transform plants with
more convenient methods such as the Agrobacterium method and the
particle gun method. The particle gun method is useful in that
genes can be directly introduced into the apical meristem of
plants while cultivated. In the Agrobacterium method, the genomic
DNA of a plant virus such as the tomato golden mosaic virus
(TGMV) or another gemirii virus is simultaneously inserted between
the border sequences into the binary vector, so that the viral
infection can spread throughout the entire plant and the target
gene can be simultaneously introduced into the entire plant
simply by inoculating cells at any location of the cultivated
plant with the viral cell suspension. These methods are known in
the art, and the ordinary skilled person can choose a suitable
method for a plant being transformed.
Transformed plants in accordance with the invention can be
evaluated, for example, for their cold stress tolerance by low
temperature treatment for 1 to 10 days at 0 to 20°C, followed by
growth at 25 to 30°C to study the state of growth, low temperature
damage, or the like. In the transformed plants produced in the
present invention, for example, the low temperature stress
tolerance can be evaluated by low temperature treatment at 0 to
20°C for 1 to 10 days followed by growing at 25 to 30°C to examine
the state; of growth, low temperature damages, and the like. The
low temperature stress tolerance can be evaluated by growing
maize at 10 to 18°C for the whole growth period and examining the
state of growth and the wet weight (yield). Heat stress tolerance
can be evaluated by low temperature treatment for 1 to 10 days at
35 to 50°C, followed by growth at 25 to 30°C to study the state of
growth, high temperature damage, or the like. The high
temperature stress tolerance can be evaluated by growing maize at
35 to 45°C for the whole growth period and examining the state of
growth and the wet weight (yield). Salt stress tolerance can be
evaluated by studying the state of growth, salt stress damage, or
the like following growth at 25 to 30°C on medium containing 10 to
300 mM NaCl. The salt stress tolerance can be evaluated by
growing the maize in the potting compost containing 10 to 15 mM
NaCl for the whole growth period and examining the state of
growth and the wet weight (yield). Drought and water stress
tolerance can be evaluated by studying the state of growth and
the extent of damage after the supply of water has been
terminated. The tolerance against drought and water stress can be
evaluated by growing the maize in the potting compost where
watering is limited and examining the state of growth and the wet
weight (yield).
Examples of plants which may be transformed in the
invention include, but are not limited to, dicotyledons,
monocotyledons, herbaceous plants, and shrubs. Examples include
sweet potatoes, tomatoes, cucumbers, squash, melons, watermelon,
tobacco, Arabidopsis thaliana, bell peppers, eggplant, beans,
taro, spinach, carrots, strawberries, white potatoes, rice, corn,
alfalfa, wheat, barley, soybeans, rapeseed, sorghum, Eucalyptus,
poplar, kenaf, Eucormia ulmoides, sugarcane, sugar beet, cassava,
betterave, sago palm, Chenopodium album, lilies, orchids,
carnations, roses, chrysanthemum, petunias, Torenia fournieri,
antirrhinum, cyclamen, gypsohila, geranium, sunflowers, Zoisia
japonica, cotton, matsutake mushrooms, shiitake mushrooms,
mushrooms, ginseng, citrus fruits, bananas, and kiwi fruit. Sweet
potatoes, tomatoes, cucumbers, rice, corn, soybeans, wheat,
petunias, Torenia fournieri, Eucalyptus, and cotton are preferred.
According to the present invention, it became possible to
increase the polyamine amount before or during encountering
stress by introducing the polyamine synthase gene into the plant
and to impart multiple stress defense effects by increasing the
expressxon amounts of multiple genes involved in the stress
tolerance or the stress resistance. Furthermore, it is possible
to anticipate the stabilization of the cultivation, the
enhancement of the productivity and the yield, and the
enlargement of the cultivation regions and the areas. Furthermore,
it is also possible to anticipate the enhancement of the
productivity of useful substances (e.g., starch, natural dyes)
obtained from the plants by increasing the productivity and the
yield of the plants.
The plants of the present invention are not limited to
entire plants (whole plants) and include callus thereof, seeds,
all plant tissues, leaves, stems, vines, roots, root tubers or
stem tubers, flowers and the like. In addition, progenies thereof
are also included in the plants of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
The invention is illustrated in further detail by the
following examples, but they are provided only as examples and do
not in any way limit the scope of the invention.
Method of analyzing polyamine
A wild type (non-recombinant plant) and transformants (gene
recombinant plants) can be simultaneously cultivated, and the
amounts of polyamine contained in the leaf, stem, root, seed,
fruit and the like can be examined. Since the amount of contained
polyamine is changed depending on the state of growth, it is
important to examine the tissues whose growth periods and
cultivation periods are the same (e.g., in the case of leaves,
the same leaf age) by cultivating under the same condition.
Polyamine includes free polyamine, combined polyamine and bound
polyamine, extraction methods thereof are different, but all can
be analyzed (Plant Cell Physiol., 43(2), 196-206, 2002). As a
specific example, the method of analyzing free polyamine in the
leaf will be described in detail. The leaves (about 0.1 to 1.0 g)
(young leaves at the same leaf age) are sampled and frozen/stored.
Dilution internal standard solution (1,6-hexanediarnine, internal
standard content = 7.5 or 12 nrnol) and 5% perchloric acid aqueous
solution (5 to 10 mL per 1.0 g specimen fresh weight) were added
to the sampled specimen, and was thoroughly ground down and
extracted using an ornnimixer at room temperature. The ground
solution was centrifuged at 4° C, 35,000 x g for 20 minutes, and
the supernatant: was collected and was taken as the free polyamine
solution. Four hundred microliters of free polyamine solution,
2 0 0 o f saturated sodium carbonate aqueous solution, and 200 [XL
of dansyl chloride/acetone solution (10 mg/mL) were added into a
microtube with a screw cap, and lightly mixed. After firmly
closing with a tube stopper and covering with aluminum foil,
dansylation was conducted by heating for 1 hour in a 60°C water
bath. After allowing the tube to cool, 200 of proline aqueous
solution (100 mg/mL) was added and mixed. The tube was covered
with aluminum foil and heated again for 30 minutes in a water
bath. After standing to cool, the acetone was removed by spraying
nitrogen gas, and then 600 of toluene was added and vigorously
mixed. After allowing the tube to stand quietly and separate into
2 phases, 300 to 4 0 0 o f toluene in the upper layer was
separated into a microtube. The toluene was completely removed by
spraying nitrogen gas. 100 to 200 ^L of methanol was added to the
tube and the dansylated free polyamine was dissolved. The free
polyamine content of putrescine, spermidine and spermine was
assayed by the internal standard method using high performance
liquid chromatography connected to a fluorescence detector
(exitataon wavelength is 365 run, emission wavelength is 510 run) .
A piBondapak C18 (manufactured by Waters, Co.: 027324, 3.9x300 mm,
particle size 10 [4m) was used for the HPLC column. The polyamine
content in the specimens was calculated by deriving the peak
areas of the polyamine and internal standard from the HPLC charts
of the standard solution and specimens. For example, from the
results of the polyamine analysis, cell lines in which the amount
of contained spermidine or spermine has been increased 1.1 to
times compared with non-transformed plant (wild type) which has
not been transformed with the exogenous polyamine synthase gene
are selected or screened in the cell lines of transformants
transformed with the exogenous polyamine synthase gene.
Method of analyzing stress defense gene using microarray
T3 homozygous cell lines in which the amount of contained
spermidine or spermine has been increased 1.1 to 3.0 times
compared with the wild type are selected in the transformants.
The seeds from the wild type (WT) and the T3 homozygous cell
lines (TSP-16, OSP-2) are seeded in plastic pots containing the
potting compost (Metromix 250 supplied from Hyponex Japan).
Sufficient water is given to the soil, which is then covered with
Saran wrap to perform the cold temperature treatment for 2 days
(synchronization). The pots after the cold temperature treatment
are transferred to a cultivation room, and acclimation for about
one week is performed under a long day condition (22°C, 16 hours'
light, 50 fimol m~2 sec"1 PPFD) . After one week, the Saran wrap is
removed, and the cultivation is started under the above long day
condition. On the 50th day (just before internode elongation)
after the start of cultivation, an overground part and a root
part are separately sampled. Their fresh weights are measured,
and immediately they are frozen in liquid nitrogen and stored at
-80°C. Total RNA is extracted using TRIZOL reagent(supplied from
GIBCO-BRL) in accordance with its protocol. Furthermore, the
total RN7i is purified using RNeasy column (supplied from Qiagen)
in accordance with its protocol. Probes are prepared from 40 (xg
of 3 kinds of total RNA (WT, TSP-16, OSP-2). The probes are
prepared using LabelStar Array labeling kit (supplied from
Qiagen) by Cyanine 3-dUTP and Cyanine 5-dUTP in accordance with
its protocol. cDNA array chips (donated by Professor Takayuki
Kawauchi of Nara Institute of Science and Technology Graduate
University) and/or DNA array chips (Arabidopsis supplied from
Agilent Technologies) are used for the array analysis. The array
chips are prehybridized in prehybridization buffer (4*SSC, 1% BSA,
0.1% SDS) at 37°C for one hour. The array chips are washed with
highly purified Milli Q water. This manipulation is repeated
twice. Contained water is removed using a plate centrifuge (1500
rpm, 5 minutes) . The array chips are dried in an incubator set at
65°C for one or more hours. Hybridization is performed using the
probe produced by LabelStar Array labeling kit. The array chips
are hybridized with hybridization buffer (4 x SSC, 10 x Denhart
solution, 1% BSA, 0.2% SDS, 1 of poly A, 0.03 of
yeast tRNA) containing 35 of the probe at 60 to 65°C for 17
hours. After the hybridization, the array chips are washed with
washing solutions starting from 1 x SSC and 0.2% SDS at 65°C to
finally 0.2 x SSC at room temperature. Scanning and data analysis
are performed using Scan ArraySOOO and QuantArray software
(supplied from GSI Lumonics) or ScanArray4000XL (supplied from
Packard Biochip Technologies). A fluorescence value of a negative
control is used for background, and the background is subtracted
from a fluorescence value of each spot. Either a median
normalization method or a global normalization method is used for
the normalization. In order to increase reliability of the
microarray analysis, the analysis is repeated several times for
each array chip.
Example 1: Cloning spermidine synthase gene derived from plant
A spermidine synthase gene derived from Cucurbita ficifolia
Bouch was acquired in accordance with the description in Example
2 in W002/23974 (FSPD1, SEQ ID NOS:1 and 2). A spermidine
synthase gene (OSPD2, SEQ ID NOS:3 and 4) derived from the rice
plant was acquired by the method shown below. In accordance with
the description in Example 2 in W002/23974, the spermidine
synthase gene(FSPDl, SEQ ID NOS:1 and 2) derived from Cucurbita
ficifolia Bouch, an S-adenosylmethionine decarboxylase gene
(FSAM24, SEQ ID NOS:5 and 6) and an arginine decarboxylase gene
(FADC76, SEQ ID NOS:7 and 8) were obtained. A spermidine synthase
gene (FSPM5, SEQ ID NOS:9 and 10) derived from Arabidopsis
tha Liana was obtained in accordance with the description in
Example 1 in ,JP 2002-351750 A. The spermidine synthase gene
(OSPD2, SEQ ID NOS:3 and 4) derived from the rice plant was
acquired by the method shown below.
(1) Preparation of poly (A) + RNA
After removing rice chaff from fully matured seeds of a
rice plant cultivar ("Yukihikari"), the seeds were immersed in
70% ethanol for 5 minutes, and subsequently sterilized by
immersing them in a sterilization solution (5% sodium
hypochlorite, 0.02% Triton X-100) for 20 minutes in a beaker
similarly sterilized. The sterilized seeds were washed three
times with sterilized water in the sterilized beaker. After
washing, the seeds were placed on a growth medium (MS inorganic
salts, MS vitamins, 30 g/L of sucrose, 8 g/L of Phytagar, pH 5.8),
and cultured in a plant incubator (MLR-350HT, supplied from
Sanyo) at 26°C under a light place condition (45 mol nf2 s"1, a
light phase for 16 hours and a dark phase for 8 hours,
hereinafter this light condition is referred to as the light
place). On about 10th day, the low temperature treatment was
started by lowering the temperature in the incubator to 12°C at
day and night. On 3 days after the start of the treatment,
sampling was performed. The samples were stored at -80°C until
use for RNA extraction.
Young seedlings (about 3 g) were immediately frozen in
liquid nitrogen, and finely pulverized in a mortar in the
presence of liquid nitrogen. Total RNA was extracted using TRIZOL
reagent(supplied from GIBCO-BRL) in accordance with its protocol.
A total RNA solution was incubated at 65°C for 5 minutes, and then
rapidly cooled on ice. An equivalent amount of 2 x binding buffer
(10 mM Tris-HCl, 5 mM EDTA2Na, I M NaCl, 0.5% SDS, pH 7.5) was
added to this total RNA solution. This mixture was overlaid in an
oligo dT cellulose column (supplied from Clontech) previously
equilibrated with equilibration buffer (10 mM Tris-HCl, 5 mM
EDTA2Na, 0.5 M NaCl, 0.5% SDS, pH 7.5). Then, the column was
washed with about 10 time amount of the foregoing equilibration
buffer, and subsequently, the poly (A) + RNA was eluted with
elution buffer (10 mM Tris-HCl, 5 mM EDTA2Na, pH 7.5). The
foregoing aqueous solution of 3 M sodium acetate at 1/10 time
amount and ethanol at 2.5 time amount were added to the resulting
elution, and the mixture was then left stand at -80°C. Thereafter,
the centrifugation at 1.0,000 x g was performed, and the resulting
precipitate was washed with 70% ethanol and dried under reduced
pressure?. This dried preparation was dissolved again in 500 [iL of
TE buffer, and repeatedly purified on the oligo dT cellulose
column. The obtained poly (A) + RNA derived from the young
seedlings of the rice plant to which the cold temperature
treatment had been given was used for making a cDNA library.
(2) Preparation of cDNA library
The cDNA library was prepared using a Marathon cDNA
Amplification Kit (by Clontech) according to protocol. The poly
(A) +RNA was used as template, and reverse transcriptase and
modified lock-docking oligo(dT) primer with two degenerate
nucleotide positions at the 3' end were used to synthesize cDNA.
A Marathon cDNA adapter (the 5' end phosphorylated to facilitate
binding to both ends of the ds cDNA with T4 DNA ligase) was
ligated to both ends of the synthesized cDNA. The resulting
adapter-linked cDNA was used as a cDNA library.
(3) Design of PCR primers
The? base sequences of spermidine synthase genes already
isolated from plants or mammals were compared. Regions with
extremely highly conserved homology were selected to synthesize
DNA oligomers (sequence primers I 'II).
SPDS primer I (SEQ ID NO. 11): 5' -GTTTTGGATGGAGTGATTCA-3'
SPDS primer II (SEQ ID NO. 12): 5'-GTGAATCTCAGCGTTGTA-3'
(4) Amplification by PCR
The cDNA library obtained in (2) was used as template, and
the sequence primers designed in (3) were used for PCR. The PCR
steps involved 5 cycles of 30 seconds at 94°C, 1 minute at 45°C,
and 2 minutes at 72°C, followed by 30 cycles of 30 seconds at 94°C,
1 minute at 55°C, and 2 minutes at 72°C.
(5) Agaiose GeJ Electrophoresis
The PCR amplified products were separated by
electrophoresis with 1.5% agarose, and the electrophoresed gel
was stained with ethidium bromide to detect amplified bands on a
UV transi1lumiriator.
(6) Verification and Recovery of PCR Amplified Products
The detected amplified bands were verified and were cut out
of the agarose gel with a razor. The pieces of gel were
transferred to 1.5 mL microtubes, and the DNA fragments were
isolated and purified from the gel using a QIAEX II Gel
Extraction Kit (by QIAGEN). The recovered DNA fragments were
subcloned to the pGEMT cloning vector (by Promega), transformed
with E. coli, and then used to prepare plasmid DNA in the usual
manner.
(7) Sequencing
The sequencing of the sequences inserted into the plasmids
were determined by the dideoxy method (Messing, Methods in
Enzymol., 101, 20-78 (1983)).
(8) Detection of Homology
A homology search of the base sequences of these genes
against a database of known gene base sequences revealed that the
obtained genes had 70 to 100% homology with known plant-derived
spermidine synthase (SPDS) genes
(9) Isolation of full length gene
The full length gene was isolated by 5' -RACE (rapid
amplification of cDNA ends) using Marathon cDNA Amplification Kit
(supplied from Clontech) and the method of integrating 3'-RACE
(Chenchik et al., 1995). 5'-RACE was performed by PCR with the
cDNA library as a template using API primer (5'-
CCATCCTAATACGACTCACTATAGGGC-3' ) and a primer (5'-
TCCCTCGCGTAGCTGTCGGGTTTGA-3') specific for the gene. The PCR was
performed with 35 cycles of 94°C for 30 seconds, 60°C for 45
seconds and 72°C for 2 minutes and then one cycle of 94°C for 30
seconds, 60°C for 45 seconds and 72°C for 7 minutes. 3'-RACE was
performed by PCR with the cDNA library as a template using API
primer -CCATCCTAATACGACTCACTATAGGGC-3' ) and a primer (5'-
ACACAACGCCTCCTGGTCGAAGAGC-3') specific for the gene. The PCR was
performed with 35 cycles of 94°C for 30 seconds, 60°C for 45
seconds and 72°C for 2 minutes and then one cycle of 94°C for 30
seconds, 60°C for 45 seconds and 72°C for 7 minutes. Gene
fragments obtained in 5'-RACE and 3'RACE were subcloned into
pGEM-T cloning vector (supplied from Promega), respectively.
Furthermore, all base sequences were determined in accordance
with the foregoing method, and analyzed by DINASIS-Mac version
3.6 software package (supplied from Hitachi Software Engineering)
The full length spermidine synthase gene derived from
Cucurbita fid folia Bouch was designated as FSPD1 (SEQ ID NOS: 1
and 2), the full length spermidine synthase gene derived from the
rice plant was designated as OSPD2 (SEQ ID NOS:3 and 4), the full
length S-adenosylmethionine decarboxylase gene was designated as
FSAM24 (SEQ ID NOS:5 and 6), the full length arginine
decarboxylase gene was designated as FADC76 (SEQ ID NOS:7 and 8),
and the full length spermidine synthase gene derived from
Arabidopsis thaliana was designated as FSPM5 (SEQ ID NOS:9 and
10) .
The obtained FSPDl and OSPD2 were compared with known
spermidine synthase genes derived from the plants at amino acid
level, and consequently, FSPDl was observed to have about 80%
homology to the spermidine synthase gene derived from the other
plant. OSPD2 was observed to have 100% homology to OsSPDS2
(Journal of Plant Physiology, 161, 883-886, 2004) which was the
spermidine synthase gene derived from the rice plant. FSAM24 was
compared with known S-adenosylmethionine decarboxylase genes
(SAMDC genes) derived from the plants at amino acid level, and
consequently FSAM24 was observed to have 63 to 66% homology.
FADC76 was compared with known arginine decarboxylase genes
derived from the plants, and consequently, FADC76 was observed to
have 71 to 77% homology. FSPM5 was compared with spermidine
synthase gene (ACL5: GeriBarik Accession Number AF184093) derived
from Arabidopsis thaliana at amino acid level, complete
concordance of the amino acids was observed.
Example 2: Preparation and analysis of transgenic Arabidopsis
thaliana
(I) Preparation of Expression Construct
The FSPD1 polyamine synthase gene given in SEQ ID NO.l was
cleaved with Xhol in such a way that the entire reading frame of
the base sequence was included, and the fragment was purified by
the glass milk method. pGEM-7Zf (Promega) was then cleaved with
XhoJ, and the FSPD1 fragments were subcloned in the sense and
antisense directions. The FSPD1 fragments were again cleaved with
the Xbal and Kpril restriction enzymes at the multicloning site of
pGEM-7Zi, and were each subcloned to the binary vector pBI101-Hm2
to which the 35S promoter (or horseradish peroxidase C2 promoter
[Patent No. 3259178] which was a stress/disease inducible
promoter') had been ligated. The resulting plasmid was designated
pBI35S-FSPDl4-/~, pBIC2-FSPDl + /-. Transformed E. coli JM109 was
designated Escherichia coli JMl09/pBl35S-FSPDl+/-, Escherichia
col] JM109/PB1C2-FSPD1+/-.
The OSPD2 polyamine synthase gene given in SEQ ID NO. 3 was
cleaved with Xhol in such a way that the entire reading frame of
the base; sequence was included, and the fragment was purified by
the glass milk method. pGEM-7Zf (Promega) was then cleaved with
Xhol, and the FSPD1 fragments were subcloned in the sense and
antisense directions. The FSPD1 fragments were again cleaved with
the Xba'I and Kpnl restriction enzymes at the multicloning site of
pGEM-7Zf, and were each subcloned to the binary vector pBI101-Hm2
to which the 35S piomoter (or horseradish peroxidase C2 promoter
[Patent No. 3259178] which was a stress/disease inducible
promoter) had been ligated. The resulting plasmid was designated
pBI35S-03PD2+ pB.I.C2-OSPD2 Transformed E. coli JM109 was
designated EsoherLchia coli JM109/pBI35S-OSPD2+, Escherichia
col i JM1 09/pBIC2-OSPD24/- .
The FSATC-M polyamine synthase gene given in SEQ ID MO. 5 was
cleaved with Not I in such a way that the 5' nontranslation region
(uORF sequence) and the entire reading frame of the base sequence
were included, and the ends were blunted. The fragments were
subcioned in the sense and antisense directions to the binary
vector pBI101-Hm2 to which the (blunted) 35S promoter (or
horseradish peroxidase C2 promoter [Patent No. 3259178] which was
a stress/disease inducible promoter) had been ligated. The
resulting plasmid was designated pBI35S-FSAM24+/, pBIC2-
FSAM24+/-. Transformed E. coli JM109 was designated Escherichia
coli JM109/pBl35S-FSAM24+/-, Escherichia coli JM109/pBIC2-
FSAM24+/-.
The FADC76 polyamine synthase gene given in SEQ ID NO. 7 was
cleaved with NotI in such a way that the 5' nontranslation region
(uORF sequence) and the entire reading frame of the base sequence
were included, and the ends were blunted. The fragments were
subcloned in the sense and antisense directions to the binary
vector pBI101-Hm2 to which the (blunted) 35S promoter (or
horseradish peroxidase C2 promoter [Patent No. 3259178] which was
a stress/disease inducible promoter) had been ligated. The
resulting plasmid was designated pBI35S-FADC76+/-, pBIC2-
FADC76+/-. Transformed E. coli JM109 was designated Escherichia
coli JM109/pBI35S- FADC76+/-, Escherichia coli JM109/pBIC2-
FADC76+/-.
The FSPM5 polyamine synthase gene given in SEQ ID NO. 9 was
cleaved with Xhol in such a way that the entire reading frame of
the base sequence was included. The fragments were subcloned in
the sense and antisense directions to the binary vector pBHOl-
Hm2 to which the (blunted) 35S promoter (or horseradish
peroxidase C2 promoter [Patent No. 3259178] which was a
stress/disease inducible promoter) had been ligated. The
resulting plasmid was designated pBI35S-FSPM5+/-, pBIC2-FSPM5+/-.
Transformed E. coli JM109 was designated Escherichia coli
JM109/pBI35S-FSPM5+/-, Escherichia coli JTM109/pBIC2-FSPM5+/-.
(2) Introduction of plasmids to Agrobacterium
The E. coli pBI35S-FSPDl+/-, E. coli pBIC2-FSPDl+/-, E. coli
pBI35S-FSAM24+/-, E. coli pBIC2-FSAM24+/-, E. coli pBI35SFADC76+/-,
E. coli pBIC2-FADC76+/-, E. coli pBI35S-OSPD2+/-, E.
coli pBIC2-OSPD2+/-, E. coli pB!35S-FSPM5+/- or E. coli pBIC2-
FSPM5+/- obtained in (1) and the E. coli strain HB101 with the
pRK2013 helper plasmid were cultured for 1 night at 37°C on LB
medium containing 50 mg/L kanamycin, and the Agrobacterium C58
strain was cultured for 2 nights at 37°C on LB medium containing
50 mg/L kanamycin. Cells were harvested from 1.5 mL of each
culture in Eppendorf tubes and then washed with LB medium. The
cells were suspended in 1 mL of LB medium, 100 \iL each of the
three types of cells were mixed to inoculate LB agar medium and
cultured at 28°C to allow the plasmids to be conjugated with the
Agrobacterium (tripartite conjugation). After 1 or 2 days,
portions were scraped with a platinum loop and smeared on LB agar
medium containing 50 mg/L kanamycin, 20 mg/L hygromycin, and 25
mg/L chloramphenicol. After 2 days of culture at 28°C, a variety
of single colonies were selected. The resulting transformants
were designated C58/pBl35S-FSPDl+/-, C58/pBIC2-FSPDl+/-,
C58/pBl35S-F3AM24+/-, C58/pBIC2-FSAM24+/-, C58/pBI35S-FADC76+/-,
C58/pBIC2-FADC76+/-, C58/pBl35S-OSPD2+/-, C58/pBIC2-OSPD2+/-,
C58/pB!35S-FSPM5+/- or C58/pBIC2-FSPM5+/-. Transgenic Arabidopsis
thaliana was prepared by reduced pressure infiltration ((3)
through (6) below).
(3) Cultivation of Arabidopsis thaliana
Potting compost Metromix (Hyponex Japan) was placed in
plastic pots, the surfaces were covered with netting mesh, and 2
to 5 seeds (donated by Professor Takayuki Kawauchi of Nara
Institute of Science and Technology Graduate University) of
Arabidopsis thaliana (referred to below as the "Columbia strain"
or "wild type") were inoculated through the interstices of the
mesh. The pots were placed for 2 days at 4°C in a low temperature
chamber to germinate, and were then transferred for cultivation
under 22°C long-day conditions (16 hour long day/8 hour night) .
After about 4 to 6 weeks, lateral shoots were induced by top
pruning plants in which the main axis flower stalk was extended
to between 5 and 10 cm. After about 1 to 2 weeks of top pruning,
the plants were infected with Agrobacterium.
( 4 ) Preparation of Agrobacterium suspension
Two days before infection, the Agrobacterium prepared in
(2) above was used to inoculate 10 mL LB medium containing
antibiotics (50 mL kanamycin, 20 mL hygromycin) for 24
hours of shaking culture at 28°C. Portions of the culture were
transferred to 1000 ml LB medium containing antibiotics (50 jxg/mL
kanamycin, 20 /mL hygromycin) for about another 24 hours of
shaking culture at 28°C (to an OD60o of between 1.2 and 1.5). Cells
were harvested from the culture at ambient temperature and were
resuspended in suspension medium for infiltration (0.5 x MS salt,
0.5 x Gamborg B5 vitamin, 1% sucrose, 0.5 g/L MES, 0.44 ^M
benzylaminopurine, 0.02% Silwet-77) to an ODeoo of between 0.8 and
1.
(5) Agrobacterium Infection
The potting soil in the pots of Arabidopsis thaliana
prepared in (3) above was watered to prevent the potting soil
from absorbing the Agrobacterium suspension prepared in (4) above.
Approximately 200 to 300 mL of the Agrobacterium suspension was
placed in 1000 mL beakers, and the potted Arabidopsis thaliana
was turned upside down to dip the plants in the suspension. The
beakers in which the pots had been placed were put into a
dessicator, which was suctioned with a vacuum pump to about -
0.053 MPa (400 mmHg) , and the plants were then allowed to stand
for about 10 minutes. The negative pressure was gradually
released, the plants were then taken out of the Agrobacterium
suspension, the excess Agrobacterium suspension was wiped off
with a Kimtowel, and the pots were placed on their sides in deepbottomed
trays. A small amount of water was introduced, and the
plants were covered with saran wrap. The plants were allowed to
stand in this manner for about 1 day. The saran wrap was then
removed, and the pots were placed upright and irrigation was
stopped for about I week. The potting compost was then gradually
watered, and seeds were harvested from matured pods for about 3
to 5 weeks. The harvested seeds were strained through a tea
strainer to eliminate debris and husks, and the seeds were placed
in a dessicator and thoroughly dried.
(6) Obtaining Transformed Plants
100 \iL (about 2000) seeds obtained in (5) above were
transferred to 1.5 mL Eppendorf tubes and soaked for 2 minutes in
70% ethanol and 15 minutes in 5% sodium hypochlorite solution,
and the seeds were finally washed five times with sterile water
to disinfect the seeds. The disinfected seeds were transferred to
15 mL falcon tubes, about 9 mL of 0.1% aseptic agar solution was
added, and the contents were vigorously mixed. A 0.1% agar
mixture of seeds was evenly spread on selection medium (1 x MS
salt, 1 x Gamborg B5 vitamin, 1% sucrose, 0.5 g/L MES, 0.8% agar,
100 mg/L carbenicillin, 50 mg/L kanamycin, 40 mg/L hygromycin, 8
g/L Phytagar, pH 5.7) like plating the phages. The plates were
dried for about 30 minutes in a clean bench, a 4°C low temperature
treatment was performed for 2 days, the plates were transferred
to a 22°C growth chamber, and transformants with antibiotic
resistance were selected. Plants with about 3 to 5 true leaves
were again transferred to fresh selection medium and cultivated
until 4 to 6 true leaves had grown. Transformants with antibiotic
resistance (Tl) were planted in pots filled with compost and
acclimated under humid conditions for about 5 to 7 days. After
acclimation, the plants were cultivated at 23°C under long day
conditions (16 hour long days/8 hour nights). The resulting
transformed plants (Tl) and plants T2 grown from seeds (T2)
obtained from the transformed plants were analyzed for genes
introduced by PCR or Southern hybridization and their levels of
expression by Northern hybridization were analyzed, and
transformants which are confirmed that the target spermidine
synthase genes had been incorporated in a stable manner and
expressed was selected. Seeds T3 were also harvested from the
plants T2, and antibiotic resistance tests (segregation analysis)
were conducted to obtain homozygotes (T2) based on the proportion
in which transformants appeared. Seeds T2 and seeds T3 obtained
from the homozygotes (T3 homozygous cell line) were used in the
following tests.
(7)Northern Blotting Analysis
In order to confirm expression levels of FSPD1 and OSPD2 in
T2 transformants obtained in (6), Northern blotting was performed.
Total RNA was extracted from untransformed wild type (WT) and T2
transfonnant (FSPD1 introduced cell lines: TSP-14, 15, 16, 17,
19; OSPD2 introduced cell lines: OSP-1, 2) rosette leaves. The
RNA extraction was performed according to an ordinary method. 10
Hg of the resulting total RNA was electrophoresed on 1.5%
formaldehyde agarose gel and blotted over night on HyBond N nylon
membranes. The RNA was fixed with a UV crosslinker and then prehybridized
for 2 hours at 42°C in pre-hybridization buffer (50%
formamide, 5 x SSPE, 5 x Denhardt's, 0.1% SDS, 80 [iq/mL salmon
sperm DNA, pH 7.0). Probes were prepared with the use of 32P-dCTP
and a random label kit (by Amersham) from the cDNA of the rice
SPDS gene fracment arid Cucurbits ficifolia Bouche SPDS gene
fragment obtained in (6) of Example 1. The probe was added to the
pre-hybridization mixture for hybridization over night at 42°C.
After the hybridization, the membranes were washed twice for 30
minutes at 55°C, beginning with a washing solution containing 2 x
SSC and 0.1% SDS, for 30 minutes at 50°C with a washing solution
containing 0.5 x SSC and 0.1% SDS, and ending with a washing
solution containing 0.1 x SSC and 0.1% SDS. Autoradiographs of
the membranes were taken using X-ray film (Kodak). Part of the
results of Northern blotting are given in Figure l.The results in
Figure 1 show that no expression of the exogenous Cucurbita
ficifolia Bouche SPDS gene (FSPD1) or rice SPDS gene (OSPD2) was
detected in the wild type (WT), but that signals were detected in
all the cell lines at high levels, and expression of FSPD1 and
OSPD2 was confirmed.
(8) Polyamine analysis
Cell lines were selected from the results of PCR (or
Southern analysis), Northern analysis or Western analysis.
Polyamine analysis was performed for the cell lines in which the
polyamine metabolism-relating enzyme gene had been absolutely
introduced and the gene was stably expressed. The cell lines,
TSP-14, TSP-15, TSP-16, TSP-17, TSP-19 and TSP-101, in which
F3PD1 had been introduced in a sense direction, the cell lines,
TSA-1 and TSA-4 in which FSAM24 had been introduced in the sense
direction, the cell lines, TAD-3 and TAD-5, in which FADC76 had
been introduced in the sense direction, and the cell lines, TSM-3
and TSM-7, in which FSPM5 had been introduced in the sense
direction were selected. The cell lines, OSP-1, OSP-2, OSP-5 and
OSP-7, in which OSPD2 had been introduced in the sense direction
were selected. About 0.1 to 0.5 g of rosette leaves were sampled
from wild type (WT) and transformants (TSP, OSP), and stored
frozen. Diluted internal standards (1,6-hexanediamine, internal
standard amount = 7.5 or 12 nmol) as well as 5% perchloric acid
aqueous solution (5 to 20 mL per 1.0 g live weight of sample)
were added to the samples, which were thoroughly milled and
extracted at ambient temperature in an omnimixer. The milled
solution was centrifuged at 35,000 x g at 4°C for 20 minutes, a
supernatant solution was collected, and the solution was made a
free polyamine solution. The free polyamine solution (400 piL) ,
200 (IL of saturated aqueous solution of sodium carbonate and 200
[iL of dansyl chloride/acetone solution (10 mg/mL) were added into
a microtube with a screw cap, and gently mixed. The cap of the
tube was tightly sealed followed by being covered with aluminium
foil, and the mixture was heated in a water bath at 60°C for one
hour to perform dansylation. After cooling the tube, 200 piL of an
aqueous solution of proline (100 mg/mL) was added and mixed. The
tube was covered with aluminium foil and heated again in the
water bath for 30 minutes. After cooling, acetone was removed by
blowing nitrogen gas, then 600 of toluene was added and mixed
vigorously. The tube was left stand to separate two phases, and
subsequently, 300 to 400 L of an upper layer toluene layer was
dispensed in a microtube. Toluene was completely removed by
blowing the nitrogen gas to the dispensed toluene. The dansylated
free polyamine was dissolved by adding 200 of methanol to the
tube. Putrescine, sperrnidine and free polyamine of spermine were
quantified by an inner standard method using high performance
liquid chromatography connecting a fluorescence detector
(excitation wavelength: 365 run, luminescence wavelength: 510 run).
The HPLC column was a fiBondapak CIS (027324 by Waters, 3.9 x 300
mm, 10 pirn particle diameter). The polyamine content of the
(Table Removed)

samples was calculated by determining the peak area of the
internal standard and each polyamine based on the HPLC chart of
the standard solutions and samples. The results are given in
As is shown in Table 2, it has been demonstrated that the
amounts of putrescine, spermidine and spermine contained in the
cell lines in which FSPD1, FSAM24, FADC76, FSPM5 or OSPD2 which
was the spermidine synthase gene had been introduced in the sense
direction were signi ficantly increased compared with those in the
wild type (WT) and that the amounts of total polyamine contained
were also significantly increased compared with that in the wild
type (WT) . In particular, the amounts of contained spermidine and
spermine were remarkably increased. It has been shown that the
amounts of contained spermidine and spermine were increased in
the range of 1.1 to 3.0 times compared with those in the wild
type (non-transformant) by introducing FSPD1, FSAM24, FADC76,
FSPM5 or OSPD2 into the plant. Adverse effects such as growth
inhibition and fertility reduction were not observed in the
transformants (cell lines) in which the amounts of contained
spermidine and spermine had been increased in the range of 1.1 to
3.0 times compared with those in the wild type (non-transformant).
Example 3: Microarray analysis of transgenic Arabidopsis
3 hornozygous cell lines in which the amounts of contained
spermidine and spermine had been increased in the range of 1.1 to
3.0 times compared with those in the wild type were selected in
the T3 transformants. The seeds from the wild type (WT) and the
T3 homozygous cell lines (TSP-16, OSP-2) were seeded in plastic
pots containing the potting compost (Metromix 250 supplied from
Hyponex Japan). Sufficient water was given to the soil, and the
pots were covered with Saran wrap, to which the cold temperature
treatment (synchronization) was given for 2 days. After the cold
temperature treatment, the pots were transferred to the
cultivation room, and the acclimation for about one week was
performed under the long day condition (22°C, lighten for 16 hours,
50 molsec"'1 PPFD) . After one week, the Saran wrap was
removed, and the cultivation was started under the above long day
condition. On the 50th day (just before internode elongation)
after the start of the cultivation, an overground part and a root
part were separately sampled. Their fresh weights were measured,
and immediately they were frozen in liquid nitrogen and stored at
-80°C. Total RNA was extracted using TRIZOL reagent (supplied
from G1BCO-BRL) in accordance with its protocol. Furthermore, the
total RNA was purified using RNeasy column (supplied from Qiagen)
in accordance with its protocol. Probes were prepared from 40 fig
of 3 kinds of total RNA (WT, TSP-16, OSP-2). The probes were
prepared using LabelStar Array labeling kit (supplied from
Qiagen) by Cyanine 3-dUTP and Cyanine 5-dUTP in accordance with
its protocol. cDNA array chips (donated by Professor Takayuki
Kawauchi of Nara Institute of Science and Technology Graduate
University) and/or DNA array chips (Arabidopsis supplied from
Agilent Technologies) were used for the array analysis. The array
chips were prehybridized in prehybridization buffer (4*SSC, 1%
BSA, 0.1% SOS) at 37°C for one hour. The array chips were washed
with highly purified Mill! Q water. This manipulation was
repeated twice. Contained water was removed using the plate
centrifuge (1500 rpm, 5 minutes). The array chips were dried in
the incubator set at 65°C for one or more hours. Hybridization
was performed using the probe produced by the LabelStar Array
labeling kit. The array chips were hybridized with hybridization
buffer (4 x SSC, 10 x Denhart solution, 1% BSA, 0.2% SDS, 1 ng/fiL
of poly A, 0.03 .g/(xL of yeast tRNA) containing 35 of the
probe at 60 to 65°C for 17 hours. After the hybridization, the
array chips were washed with washing solutions starting from 1 x
SSC and 0.2% SDS at 65°C to finally 0.2 x SSC at room temperature.
Scanning and data analysis were performed using Scan Array5000
and QuantArray software (supplied from GSI Lumonics) or
ScanArray4000XL (supplied from Packard Biochip Technologies). The
fluorescence value of the negative control was used for the
background, and the background was subtracted from the
fluorescence value of each spot. Either the median normalization
method or the global normalization method was used for the
normalization. In order to increase reliability of the microarray
analysis, the analysis was repeated several times for each array
chip. The stress defense genes whose expression amounts had been
increased (Ratio of expression amount is in the range of 1.5 to
5.0 times) in the transformants (TSP-16, OSP-2) compared with
those in the wild type (WT) were shown in Table 3.
(Table Removed)

From the results in Table 3, it has been shown that the
expression levels in the stress defense gene group (Gene Numbers
1 to 20) are increased 1.5 to 5.0 times compared with those in
the wild type (WT) which is the non-transformant by transforming
the plant with the spermidine synthase gene (FSPD1) derived from
Cucurbit a fid folia Bouch or the spermidine synthase gene (OSPD2)
derived from the rice plant.
It has been reported that a CBF1/DREB1B transcription
factor of Gene Number 1 is one of the transcription factors
induced by stress and that tolerance against various
environmental stress such as drought, salt, freeze and cold
temperature is enhanced by introducing it into the plants (The
Plant; Cell, 10, 1391-1406, 1998, Nature Biotechnology, 17, 287-
291, 1999, Plant Physiology, 124, 1854-1865, 2000, Plant
Physiology, 130, 639-648, 2002, Plant Physiology, 130, 618-
2002) .
It has been known that an old regulated protein/LEA protein
of Gene Number 2 is a late embryogenesis abundant (LEA) protein
and induced by stress, and it has been reported that the
tolerance against drought stress and salt stress is enhanced by
introducing HVI which is the LEA protein gene into the rice plant
(Plant Physiology, 110, 249-257, 1996).
It has been reported that a cold regulated protein/cor!5 of
Gene Number 3 is the gene induced by cold temperature stress and
that it is deeply involved in the freeze stress tolerance (Pro.
Natl. Acad. Sci. USA, 93, 13404-13409, 1996, Pro. Natl. Acad. Sci.
USA, 95, 14570-14575, 1998).
It. has been reported that a pathogen related PR-1 protein
of Gene Number 4 is the protein induced by pathogen infection and
that the tolerance against heavy metal stress and pathogen
infection stress is enhanced by introducing the CABPR1 gene which
is one of PR-1(pathogenesis-related protein 1) into tobacco
(Plant Cell Rep., Feb 18, 2005).
It has been reported that an early response dehydration
protein/ERD15 of Gene Number 5 is the gene induced by drought
stress and is deeply involved in the drought stress tolerance
(Plant Physiology, 106, 1707, 1994).
It: has been reported that a salt stress induced tonoplast
intrinsic protein/aquaporin of Gene Number 6 and a water channel
protein/aquaporin are the water channel proteins induced by
stress and are deeply involved in the tolerance against osmotic
stress and low temperature stress (Mol. Cells., 9(1), 84-90, 1999,
Foods Food Ingredients J. Jpn., 176, 40-45).
It: has been reported that a dehydration induced
protein/RD22, rd22 of Gene Number 8 is the protein induced by
drought stress and is deeply involved in the drought stress
tolerance (Plant Cell., 15(1), 63-78, 2003).
The proteins of Gene Numbers 9, 10, 11, 12 and 13 are the
proteins induced by stress, arid it has been suggested that they
are involved in stress tolerance, but their functions are not
elucidated sufficiently.
The proteins of Gene Numbers 14, 15, 16, 17 and 18 are the
proteins induced by disease stress, and it has been suggested
that they are involved in the disease stress tolerance, but their
functions are not elucidated sufficiently.
It has been known that peroxidase of Gene Number 19 is one
(EC l.U.1.7) of cell wall enzymes and induced by disease stress,
and it has been reported that tolerance against oxidative
stressoxidative stress and pest stress is enhanced by introducing
it into the plant (Plant Physiology, 132, 1177-1185, 2003, J.
Econ. Entomol., 95(1), 81-88, 2002).
It has been reported that a senescence associated protein
senl of Gene Number 20 is the protein induced by aging stress,
salt stress, osmotic stress and low temperature stress and is
deeply involved in tolerance against aging stress, salt stress,
osmotic stress and low temperature stress (Plant Physiology, 130,
2129-2141, 2002).
From the above results, it has been demonstrated that the
expression amount of the stress defense gene involved in the
stress tolerance is increased (induction or increase) compared
with that in the wild type by transforming the plant with the
polyamine synthase gene, particularly the spermidine synthase
(SPDS) gene.
Next, the expression amounts of the stress defense genes
under the stress condition were compared between the
transformants and the wild type. T3 homozygous cell lines in
which the amounts of contained spermidine and spermine had been
increased in the range of 1.1 to 3.0 times compared with that in
the wild type were selected in the T3 transformants. The seeds
from the wild type (WT) and the T3 homozygous cell lines (TSP-16,
OSP-2) were seeded in plastic pots containing the potting compost
(Metromix 250 supplied from Hyponex Japan). Sufficient water was
given to the soil, and the pots were covered with Saran wrap, to
which the cold temperature treatment (synchronization) was given
for 2 days. After the cold temperature treatment, the pots were
transferred to the cultivation room, and the acclimation for
about one week was performed under the long day condition (22°C,
lighten for 16 hours, 50 mol m2 sec"1 PPFD) . After one week, the
Saran wrap was removed, and the cultivation was started under the
above long day condition. On the 48th day (just before internode
elongation) after the start of the cultivation, the pots were
transferred under the low temperature stress condition (5/5°C:
day/night, lighten for 16 hours, 240 mol nf2 sec'1 PPFD) to
perform the stress treatment for 2 days. After the treatment, an
overground part and a root part were separately sampled. Their
fresh weights were measured, and immediately they were frozen in
liquid nitrogen and stored at -80°C. Total RNA was extracted
using TRIZOL reagent (supplied from GIBCO-BRL) in accordance with
its protocol. Furthermore, the total RNA was purified using
RNeasy column (supplied from Qiagen) in accordance with its
protocol. Probes were prepared from 40 (g of 3 kinds of total RNA
(WT, TSP-16, OSP-2). The probes were prepared using LabelStar
Array labeling kit (supplied from Qiagen) by Cyanine 3-dUTP and
Cyanine 5-dUTP in accordance with its protocol. cDNA array chips
(donated by Professor Takayuki Kawauchi of Nara Institute of
Science and Technology Graduate University) and/or DNA array
chips (Arabidopsis supplied from Agilent Technologies) were used
for the array analysis. The array chips were prehybridized in
prehybridization buffer (4xSSC, 1% BSA, 0.1% SDS) at 37°C for one
hour. The array chips were washed with highly purified Milli Q
water. This manipulation was repeated twice. Contained water was
removed using the plate centrifuge (1500 rpm, 5 minutes) . The
array chips were dried in the incubator set at 65°C for one or
more hours. Hybridization was performed using the probe produced
by the LabelStar Array labeling kit. The array chips were
hybridized with hybridization buffer (4 x SSC, 10 x Denhart
solution, 1% BSA, 0.2% SDS, 1 g/L of poly A, 0.03 (g/fiL of
yeast tRNA) containing 35 L of the probe at 60 to 65°C for 17
hours. After the hybridization, the array chips were washed with
o,. washing solutions starting from 1 x SSC and 0.2% SDS at 65°C to
finally 0.2 x SSC at room temperature. Scanning and data analysis
were performed using Scan Array5OOO and QuantArray software
(supplied from GSI Lumonics) or ScanArray4000XL (supplied from
Packard Biochip Technologies). The fluorescence value of the
negative control was used for the background, and the background
was subtracted from the fluorescence value of each spot. Either
the median normalization method or the global normalization
method was used for the normalization. In order to increase
reliability of the microarray analysis, the analysis was repeated
several times for each array chip. The stress defense genes whose
expression amounts had been increased (Ratio of expression amount
is in the range of 2.0 to 18.0 times) in the transformants (TSP-
16, OSP-2) compared with those in the wild type (WT) were shown
(Table Removed)

From the results in Table 4, it has been shown that the
expression levels in the stress defense gene group (gene Numbers
1 to 20) are increased 2.0 to 18.0 times compared with those in
the wild type (WT) even under the stress condition by
transforming the plant with the polyamine synthase gene,
Cucurbita ficifolia Bouch or the spermidine synthase gene (OSPD2)
derived from the rice plant.
It has been reported that the senescence associated
proteins of Gene Numbers 21 and 46 are induced by aging stress,
salt stress, osmotic stress and low temperature stress and are
deeply involved in the tolerance against aging stress, salt
stress, osmotic stress and low temperature stress (Plant
Physiology, 130, 2129-2141, 2002).
It has been reported that the nematode resistance
protein/Hslpro-1 of Gene Number 22 is deeply involved in nematode
stress tolerance (Science, 275, 832-834, 1997) .
It has been reported that a WRKY transcription factor of
Gene Number 23 is one of transcription factors and is deeply
involved in pathogen stress tolerance (EMBO J., 15, 5690-5700,
1996, Plant Mol., Biol., 29, 691-702, 1995, Mol. Plant-Microbe
Interact., 16, 295-305, 2003), drought stress tolerance and high
temperature stress tolerance (Plant Physiol., 130, 1143-1151,
2002) .
It has been reported that the zinc finger protein of gene
Number 24, 26, 31, 39, 40 or 48 is one of transcription factors
induced by salt stress, osmotic stress and low temperature stress
and is deeply involved in the tolerance against salt stress,
osmotic stress and low temperature stress (Plant Physiology, 130,
2129-2141, 2002).
It has been reported that a transcriptional activator CBF1,
a transcription activator CBF1 and transcription factor DREB of
Gene Numbers 25, 42, 27, 28 and 32 are the transcription factors
induced by various stresses such as salt stress, osmotic stress
and low temperature stress (Plant Physiology, 130, 2129-2141,
2002) and that the tolerance against various environmental
stresses such as drought, salt, freeze and low temperature is
enhanced by introducing them into the plants (The Plant Cell, 10,
1391-1406, 1998, Nature Biotechnology, 17, 287-291, 1999, Plant
Physiology, 124, 1854-1865, 2000, Plant Physiology, 130, 639-648,
2002, Plant Physiology, 130, 618-626, 2002) .
The stress induced protein stil and the stress responsive
protein of Gene Numbers 29 and 47 are the proteins induced by the
stress and it has been suggested that they are involved in the
stress tolerance, but their functions are not elucidated
sufficiently.
It: has been reported that the early response dehydration
protein/ERD15 of Gene Numbers 30 and 37 is induced by drought
stress and is deeply involved in drought stress tolerance (Plant
Physiology, 106, 1707, 1994).
It has been reported that the heat shock protein DnaJ
homolog of Gene Number 33 is the gene induced by high temperature
stress, drought stress and low temperature stress and is deeply
involved in the tolerance against high temperature stress,
drought stress and low temperature stress (The Plant Cell, 13,
61-72, 2001).
It: has been reported that the pathogenesis related protein
of Gene Number 34 is induced by pathogen infection and that the
tolerance against heavy metal stress and pathogen infection
stress is enhanced by introducing CABPR1 gene which is one of PR-
1(pathogenesis-related protein) into tobacco (Plant Cell Rep.,
Feb 18, 2005).
It. has been reported that the myb protein of gene Number 35
is one of transcription factors, is the protein induced by ABA
(abscisic acid), drought stress and low temperature stress, and
is deeply involved in the tolerance against drought stress and
low temperature stress (The Plant Cell, 5, 1529-1539, 1993, The
Plant Cell, 9, 1859-1868, 1997, Plant Physiology, 130, 2129-2141,
2002).
It has been reported that the jasmonic acid regulatory
protein of Gene Number 36 is induced by jasmonic acid and is
deeply involved in the tolerance against pathogen stress, pest
stress and disease stress because jasmonic acid is involved in
pathogen stress, pest stress and disease stress (Trends Plant
Sci., 2, 302-307, 1997).
11: has been reported that the low temperature induced
protein 78/LTI78, rd29A, COR78 of Gene Number 38 is induced by
low temperature stress, drought, stress and salt stress and is
deeply involved in the tolerance against low temperature stress,
drought stress and salt stress (Plant Cell, 6,251-264, 1994).
Cytochrome P450 of Gene Number 42 is the protein induced by
various stresses, and is suggested to be involved in various
stresses.
It has been reported that the AP2 domain transcription
factor and the AP2 domain protein of Gene Numbers 43 and 45 are
the transcription factors induced by low temperature stress and
are deeply involved in the tolerance against low temperature
stress (Plant Physiology, 130, 2129-2141, 2002, The Plant Journal,
38, 9820993, 2004).
It has been known that peroxidase of Gene Number 44 is one
(EC 1.11.1.7) of cell wall enzymes and induced by the disease
stress, and it has been reported that the tolerance against
oxidative stressoxidative stress and pest stress is enhanced by
introducing it into the plant (Plant Physiology, 132, 1177-1185,
2003, J. Econ. Entomol., 95(1), 81-88, 2002).
Gene Number 49 is the protein induced by the disease stress
and has been suggested to be involved in the disease stress
tolerance, but its functions are not elucidated sufficiently.
Example 4: Evaluation of stress defense capacity
(1) Evaluation of Osmotic stress Tolerance
The surfaces of seeds of the transformants (TSP-15, 16, 17)
obtained in Example 2 and the wild type (WT: Columbia strain)
were sterilized in the same manner as in section (6) of Example 2.
Germination growth media containing 100 mM and 200 mM sorbitol (1
x MS salt, 10 g/L sucrose, 0.1 g/L myo-inositol, 5% MES, 8 g/L
Phytagar, pH 5.7) was inoculated with the sterilized seeds one at
a time. Inoculation was followed by about 2 days of low
temperature treatment at 4°C, and then by the start of cultivation
at 22°C under conditions involving long days (16 hour long days/8
hour nights). The state of growth after inoculation was monitored,
particularly the state of the growth of plants on germination
growth media during weeks 6 and 10. The results are given in
Figure 2.
Several days following inoculation, TSP-15, 16, and 17
showed improved germination than the wild type (WT) on growth
medium containing 100 mM and 200 mM sorbitol, revealing improved
growth. In week 6 after inoculation, TSP-15, 16, and 17 plants on
medium containing 100 mM and 200 mM sorbitol were larger than the
WT, with significantly less impaired growth. The results for TSP-
17 in particular are given in Figure 2. After week 7 following
inoculation, the plants on TSP-15, 16, and 17 containing 200 mM
sorbitol in particular exhibited far improved growth,
particularly the roots, compared to WT. In week 10 following
inoculation, there were significant differences in both the parts
above ground and the roots. The results for TSP-16 in particular
are given in Figure 2. Some of the WT were found to have yellowed
and died due to impaired growth.
From the above results, it has been demonstrated that the
osmotic stress defense effect can be imparted to the plant by
introducing the spermidine synthase gene (FSPD1, OSPD2).
(2) Evaluation of drought stress tolerance
The transformants (T3 homozygous cell lines) selected from
the transformants (TSP-15, TSP-16) were used. The seeds from the
obtained transformants and the wild type (WT: Columbia strain)
were seeded in plastic pots containing the potting compost,
Metromix (supplied from Hyponex Japan). Inoculation was followed
by about 2 days of low temperature treatment at 4°C, and the pots
were then placed in plastic vats to start cultivation at 23°C
under conditions involving long days (16 hour long days/8 hour
nights). Rosette leaves had fully developed by about week 3 after
inoculation. Individuals characterized by uniform growth at the
time the rosette leaves had fully developed were selected, water
was then fed into the vats to ensure uniform soil moisture, and
water was filled to the middle of the plastic pots. After 5 days,
a constant soil moisture was confirmed, and drought stress
treatment was started (termination of water feed). The state of
growth was monitored immediately after water termination.
Withering from drought stress damage was noted in the wild
type (WT) on day 13 after the start of drought treatment. 50% of
the WT plants had died by Day 14 of drought treatment. Meanwhile,
in the transformants, 20% of the plants died of drought treatment
and higher survival rate than in the WT was shown. By Day 15,
100% of WT withered whereas 30 to 40% of the transformants
survived. The results are given in Figure 3. From the results in
Fig. 3, it has been evidently confirmed that the wild type: WT
(left) withered whereas the transformants: T3 homozygous cell
lines (middle and right) survived.
Two transformants (T3 homozygous cell lines) selected from
the transformant (OSP-2) were used. The seeds from the obtained
two transformants and the wild type (WT: Columbia strain) were
seeded in plastic pots containing the potting compost, Metromix
(supplied from Hyponex Japan). Inoculation was followed by about
2 days of low temperature treatment at 4°C, and the pots were then
placed in plastic vats to start cultivation at 23°C under
conditions involving long days (16 hour long days/8 hour nights).
Rosette leaves had fully developed by about week 4 after
inoculation. Individuals characterized by uniform growth at the
time the rosette leaves had fully developed were selected, water
was then fed into the vats to ensure uniform soil moisture, and
water was filled to the middle of the plastic pots. After 5 days,
a constant soil moisture was confirmed, and drought stress
treatment was started (termination of water feed). The state of
growth was monitored immediately after water termination.
Withering from drought stress damage was noted in the wild
type (WT) on day 14 after the start of drought treatment. 50% of
the WT plants had died by Day 15 of drought treatment. Meanwhile,
in two transformants, 20% of the plants died of drought treatment,
and the higher survival rate than in WT was shown. By Day 18
after the start of the treatment, 100% of WT withered whereas 50%
of the transformants survived. The results are given in Figure 4.
From the results in Fig. 4, it has been evidently confirmed that
the wild type: WT (left) withered whereas the transformants: T3
homozyqous cell lines (middle and right) survived.
From the above results, it has been demonstrated that the
drought: stress defense effect can be imparted to the plant by
introducing the spermidine synthase gene (FSPD1, OSPD2).
(3) Evaluation of Cold stress Tolerance (Freeze Stress Tolerance)
The transformants (T3 homozygous cell lines) selected from
the transformants (TSP-15, TSP-16) were used. The seeds from the
obtained transformants and the wild type (WT: Columbia strain)
were seeded in plastic pots containing the potting compost,
Metromix (supplied from Hyponex Japan). For about 2 days after
seeding, the cold temperature treatment at 4°C was performed,
subsequently the pots was placed in a plastic tray, and the
cultivation was started at 23°C under the long day condition
(lighten for 16 hours and darken for 8 hours). Until about 4
weeks after seeding and until rosette leaves were completely
developed, the plants were grown. At a time point when the
rosette leaves were completely developed, individuals at the same
growth stage were selected, and then transferred to a growth
chamber at -5°C to start the freeze stress treatment. The freeze
stress treatment was performed in a dark phase for 40 hours.
After the treatment, the plants were returned to the room at
ambient temperature at 23°C under the long day condition, and the
state of growth was observed.
From immediately after returning to the ambient temperature,
in the wild type (WT), a submerged state and wilting which were
freeze stress disorders were observed. By Day 5 after returning
to the ambient temperature, all plants withered in WT. Meanwhile,
30 to 40% of the plants survived in the transformants. The
results were given in Fig. 5. From the results in Fig. 5, it has
been evidently confirmed that the wild type: WT (left) withered
whereas the transformants: T3 homozygous cell lines (middle and
right) survived. Similar results were obtained in the cell line
(OSP-2) in which the spermidine synthase gene (OSPD2) derived
from the rice plant had been introduced.
From the above results, it has been demonstrated that the
low temperature stress (freeze stress) defense effect can be
imparted to the plant by introducing the spermidine synthase gene
(SPDS) into the plant.
(4) Evaluation of Salt Stress Tolerance
The surfaces of seeds of the transformants (TSP-16)
obtained in Example 2 and the wild type (WT: Columbia strain)
were sterilized in the same manner as in section (6) of Example 2.
Germination growth medium containing 75 mM NaCl (75 mM NaCl, 1 x
MS salt, 10 g/L sucrose, 0.1 g/L myo-inositol, 5% MES, 5 g/L
Gellan gum, pH 5.7) was inoculated with the sterilized seeds one
at a time. Inoculation was followed by about 2 days of low
temperature treatment at 4°C, and then by the start of cultivation
at 22°C under conditions involving long days (16 hour long days/8
hour nights). In week 6 after inoculation, the extent of plant
growth on the germination growth medium was observed. The results
are given in Figure 6.
From the results in Fig. 6, the remarkable growth
inhibition was observed in WT which was control on the medium
containing 75 mM NaCl, and the entire plant was whitened or
yellowed to stop the growth and wither. Meanwhile, in the
transformants in which the spermidine synthase gene had been
introduced, the growth inhibition was given, but true leaves were
developed and the growth continued as they were although it was
late. Similar results were obtained in the cell line (OSP-2) in
which the spermidine synthase gene (OSPD2) derived from the rice
plant had been introduced.
From the above results, it has been demonstrated that the
salt stress defense effect can be imparted to the plant by
introducing the spermidine synthase gene (SPDS) into the plant.
(5) Evaluation of Herbicide stress Tolerance
The; surfaces of seeds (cell lines: pBI121 (35S-GUS) , TSP-15,
TSP-16, TSA-1, TAD-3) and the wild type (WT: Columbia strain)
were sterilized in the same manner as in section (6) of Example 2.
Germination growth medium containing 2 \iM paraquat (PQ) (2 [M
paraquat, 1 x MS salt, 1C) g/L sucrose, 0.1 g/L myo-inositol, 5%
MES, 5 g/L Gellan gum, pH 5.7) was inoculated with the sterilized
seeds one at a time. Inoculation was followed by about 2 days of
low temperature treatment at 4°C, and then by the start of
cultivation at 22°C under conditions involving long days (16 hour
long days/8 hour nights). The number of germinating individuals
(germination rate) was observed on day 10 after inoculation, and
the number of individuals surviving (survival rate) was observed
on day 20. The results are given in Table 5.
(Table Removed)

The results of Table 5 show that the wild type and vector
control line (pBI121) had extremely low germination and survival
rates as a result of toxicity caused by paraquat, whereas cell
lines TSP-15, TSP-16, TSA-1, TAD-3 which contained polyamine
synthase genes retained high germination and survival rates.
From the above results, it has been demonstrated that the
herbicide stress defense effect can be imparted to the plant by
introducing the polyamine synthases (SPDS, SAMDC, ADC) genes into
the plant.
Exarnp_le_5: Production of transgenic sweet potato
Sweet potato Kokei 14 (donated by Professor Takiko Shimada
at Ishikawa Agricultural College, Agricultural Resource Institute,
hereinafter referred to as "Kokei 14" or "wild type") was grown
and cultivated in a container under usual cultivation management
to collect tens of cane tops (about 5 cm in length) containing
shoot apex. The cane tops were immersed for two minutes in a 300-
ml beaker to which 150ml of ethanol had been added, followed by
dipped for 2 minutes in a beaker to which 150ml of disinfecting
solution (5% sodium hypochlorite, 0.02% Triton X-100) had been
added. Sterilized cane tops were washed with an aqueous
sterilized solution placed in a sterilization beaker. After
washing, about 0.5 mm of tissue containing meristematic tissue
was aseptically removed under stereoscopic microscope. The tissue
was then plated in embryogenic callus induction medium [4F1
plate:LS medium (1.9g/l KN03, 1.65g/l NH4N03, 0.32g/l MgS04-7H20,
0.44g/l CaCl2-2H20, 0.17g/l KH2P04, 22.3mg/l MnS04-4H20, 8.6mg/l
ZnS04-7H20, 0.025mg/l CuS04-5H20, 0.025mg/l CoCl2-6H20, 0.83mg KI,
6.2mg H3F303, 27.8mg FeS04-7H20, 37.3mg/l Na2-EDTA, 100mg/l myoinositol,
0.4mg/l thiamine hydrochloride), Img/L 4-
fluorophenoxyacetic acid(4FA), 30g/L sucrose, 3.2g/l gellan gum,
pH5.8] and then cultured in plant incubators (MLR-350HT, by
Sanyo) under dark condition at 26°C. After one month culture,
embryogenic calli capable of regeneration to plant body were
selected from proliferated tissue. The selected embryogenic calli
were continued to proliferate with transferred to a new 4F1 plate
every month. Agrobacterium was infected as follows. Transformed
Agrobacterium strains, EHA101/pBI35S-FSPDl+/-, EHAl01/pBIC2-
FSPD1+/- (CaMV35S promoter had been replaced with the peroxidase
promoter derived from the horseradish), EHA101/pBl35S-FSAM24+/-,
EHA101/pBIC2-FSAM24+/- (CaMV35S promoter had been replaced with
the peroxidase promoter derived from the horseradish),
EHA101/pBI35S-FADC76+/-, EHA101/pBIC2-FADC76+/- (CaMV35S promoter
had been replaced with the peroxidase promoter derived from the
horseradish) were cultured in LB medium containing 50 mg/L of
kanamycin and 50 mg/L of hygromycin at 27°C for two nights.
Subsequently, about two rice grains of microbial cells were
picked up, suspended in 50 mL of an infection medium (LS medium,
20 mg/L of acetosyringone, 1 mg/L of 4FA, 30 g/L of sucrose, pH
8), and shaken at 26°C at 100 rpm under complete darkness for one
hour. The suspension was transferred to 300-ml sterilized beaker
in which stainless steel basket was placed. The embryogenic calli
which were cultured two to three weeks were placed on the basket
of the beaker for dipping for two minutes. The calli together
with the basket were placed on doubled sterilized filter paper to
remove the excess moisture. The calli were transferred on coculture
medium (4F1A20 plate: LS medium, lmg/1 4FA, 20mg/l 3,5'-
dimethcxy-4x-hydroxy-acetophenone, 30g/l sucrose, 3.2g/l gellari
gum, pH5.8) and co-cultured for three days at 22 °C under dark
condition. The embryogenic calli which were co-cultured for three
days were transferred on the basket of the 300-ml beaker in which
the sterilized stainless basket was placed. A 50 ml of
disinfection solution containing carbenicillin in a final
concentration of 500mg/l in sterilized water was added to the
beaker. The calli was fully washed for several minutes by
anchoring the basket with tweezers. The embryogenic calli
together with the basket were placed in the 300-ml beaker to
which the disinfection solution was added for further washing.
After repeating the same washing procedure, the excess moisture
of the calli was removed on a sterilized filter paper to arrange
and culture the calli in selection (4FlHmCar plate: LS medium,
lmg/1 4FA, 25mg/l hygromycin, 500mg/l carbenicillin, 30g/l
sucrose, 3.2g/l gellan gum, pH5.8) at 26 °C under dark condition.
For selection of the transformed callus, the embryonic callus
cultured for 2 weeks was cultured by transferring to a new
4FlHmCar plate every two weeks. Non-transformed calli turned
brown, but part of transformants were embryogenic calli with pale
yellow.After 60 days of culture on the selection medium, the
transformed embryogenic calli were transferred to somatic cell
embryogenic medium (A4GlHmCar plate: LS medium, 4mg/l ABA, lmg/1
GA3, 25mg/l hygromycin, 500mg/l carbenicillin, 30g/l sucrose,
3.2g/l gellan gum, pH5.8) to culture for two weeks at 26 °C under
weak light, and all long-day condition (30~40/lmol/m'Ys) and then
transferred to plant body forming medium (AO.O5HmCar plate: LS
medium, 0.05mg/l ABA, 25mg/l hygromycin, 500mg/l carbenicillin,
30g/l sucrose, 3.2g/l gellan gum, pH5.8) to sulture in the same
condition. The transformants were transferred to a new A0.05HmCar
plate every two weeks. Since the transformed cells turned green
to form somatic cell embryo derived from embryogenic calli,
somatic cell embryo was transferred to plant grouth medium (OG
plate: LS medium, 30g/l sucrose, 3.2g/l gellan gum, pH5.8) to
form shoot. For the constructs in which FSAM24 and FADC76 had
been controlled by the 35S promoter, the number of obtained
transformants was obviously lower compared with the constructs
controlled by the C2 promoter. Since the SAMDC gene and the ADC
gene which act upstream of the polyamine metabolism largely
affect the polyamine metabolism, when FSAM24 and FADC76 were
controlled the constitutive promoter such as 35S promoter, it was
likely that the excessive or rapid change of the polyamine amount
adversely affected the growth. For the constructs in which FSAM24
and FADC76 had been controlled by the 35S promoter, many
transformants as possible were assured, and among them, the
transformants where the increase of the polyamine amount had been
in the range of 1.1 to 4.0 times which had little or no effect on
the growth and development were selected. The introduced gene was
confirmed and the expression was analyzed for the obtained
transformants. Specifically, to confirm the introduced gene,
genomic DNA was prepared and then analyzed by PCR method arid
Southern hybridization. For the expression of the introduced gene,
RNA was prepared, and then analyzed by Northern hybridization. As
a result, the transformed sweet potatoes (transformants) in which
the objective gene had been introduced and expressed could be
obtained. Furthermore, the expression level and the translation
level were examined in detail by Northern blotting and Western
blotting, and cell lines in which the polyamine synthase gene had
been introduced and the gene was stably expressed or translated
were selected. Cell lines TSP-SS-1, TSP-SS-2, TSP-SS-3, TSP-SS-4,
TSP-SS-5, TSP-SS-6. TSP-SS-7, TSP-CS-1, TSP-CS-3, TSP-CS-4 and
TSP-CS-5 in which FSPD1 was introduced in a sense direction (+)
were selected. TSP-SS-1, TSP-SS-2, TSP-SS-3, TSP-SS-4, TSP-SS-5,
TSP-SS-6 and TSP-SS-7 were cell lines in which CaMV 35S promoter
was introduced. TSP-CS-1, TSP-CS-3, TSP-CS-4 and TSP-CS-5 were
cell lines in which a peroxidase promoter (C2 promoter) derived
from horseradish was introduced. The cell lines, TSA-SS-1, TSASS-
2, TSA-SS-5, TSA-SS-6, TSA-CS-1, TSA-CS-7 and TSA-CS-9 in
which E'SAM24 had been introduced in the sense direction were
selected, TSA-SS-1, TSA-SS-2, TSA-SS-5 and TSA-SS-6 were the cell
lines in which the CaMV35S promoter had been introduced as the
promoter, and TSA-CS-1, TSA-CS-7 and TSA-CS-9 were the cell lines
in which the peroxidase promoter (C2 promoter) derived from the
horseradish had been introduced. The cell lines, TAD-SS-1, TADSS-
2, TAD-SS-4, TAD-CS-1, TAD-CS-8 and TAD-CS-10 in which FADC76
had been introduced in the sense direction were selected, TAD-SS-
1, TAD-SS-2 and TAD-SS-4 were the cell lines in which the CaMV35S
promoter had been introduced as the promoter, and TAD-CS-1, TADCS-
8 and TAD-CS-10 were the cell lines in which the peroxidase
promoter (C2 promoter) derived from the horseradish had been
introduced.
(2) Analysis of polyamine
For the transformed sweet potatoes produced in (1), the
cell lines controlled by the constitutive 35S promoter or the
inducible C2 promoter were selected, and polyamine was analyzed
under non-stress condition. Approximately 0.3 to 0.9 g of young
leaves from the transgenic plants (TSP, TSA, TAD) and the wild
type plants (WT), which were cultivated at the same time, were
sampled, frozen and stored. Dilution internal standard solution
(1, 6-hexanediamine, internal standard content = 7.5 or 12 nmol)
and 5% perchloric acid aqueous solution (5 to 10 mL per 1.0 g
specimen live body weight) were added to the sampled specimen,
and was thoroughly ground down and extracted using an omnimixer
at room temperature. The ground solution was centrifuged at 4°C,
35,000 x g for 20 minutes, and the supernatant was collected and
was taken as the free polyamine solution. Four hundred
microliters of free polyamine solution, 200 of saturated
sodium carbonate aqueous solution, and 200 of dansyl
chloride/acetone solution (10 mg/mL) were added into a microtube
with a screw cap, and lightly mixed. After firmly closing with a
tube stopper and covering with aluminum foil, dansylation was
conducted by heating for 1 hour in a 60°C water bath. After
allowing the tube to cool, 200 [XL of proline aqueous solution
(100 mg/mL) was added and mixed. The tube was covered with
aluminum foil and heated again for 30 minutes in a water bath.
After standing to cool, the acetone was removed by spraying
nitrogen gas, and then 600 (XL of toluene was added and vigorously
mixed. After allowing the tube to stand quietly and separate into
2 phases, 300 [XL of toluene in the upper layer was separated into
a microtube. The toluene was completely removed by spraying
nitrogen gas. 200 (XL of methanol was added to the tube and the
dansylated free polyamine was dissolved. The free polyamine
content of putrescine, spermidine and spermine was assayed by the
internal standard method using high performance liquid
chromatography connected to a fluorescence detector (excitation
wavelength: 365nm • emission wavelength: 510nm) . A (XBondapak
C18 (manufactured by Waters, Co.: 027324, 3.9x300 mm, particle
size 10 urn) was used for the HPLC column. The polyamine content
in the specimens was calculated by deriving the peak areas of the
polyamine and internal standard from the HPLC charts of the
standard solution and specimens. A part of the results is shown
in Table 6.
As is evident in Table 6, it has been demonstrated that the
amounts of contained putrescine, spermidine and spermine were
(Table Removed)

significantly increased and the total amounts of contained
polyamine were also significantly increased in the cell lines
(TSP-SS, TSA-SS and TSD-SS) in which the polyamine synthase gene
(FSPD1, FSAM24 and FADC76) had been introduced in the sense
direction under the control of the 35S promoter and the cell
lines (TSP-CS) in which the gene had been introduced in the sense
direction under the control of the C2 peroxidase promoter
compared with the wild type (WT). Although, in TSA (FSAM24), the
S-adenosylmethionine decarboxylase (SAMDC) gene largely affects
the polyamine metabolism, the translation level was suppressed
and no excessive increase of contained polyamine was observed
because the SAMDC gene containing all 5'-non-translated region
(uORF) had been introduced into the plant. In TAD (FADC76), the
arginine decarboxylase (ADC) gene also largely affects the
polyamine metabolism as with the SAMDC gene, but the translation
level was suppressed and no excessive increase of contained
polyamine was observed because the ADC gene containing all 5'-
non-translated region (uORF) had been introduced into the plant.
Example 6: Evaluation of stress tolerance of transgenic sweet
potato
(1) Evaluation of salt stress tolerance
The cell lines (TSA-CS-1, TAD-CS-1) were selected in the
transformed cell lines. Stem cuttings from two transformed cell
lines and the wild type (WT) were planted to the growth medium
(MS medium, 20 g/L of sucrose, 3.2 g/L of gellan gum, pH 5.8) in
the presence or absence of high concentration of 150 mM NaCl, and
transferred to the growth chamber (temperature at 25°C, lighten
for 16 hours, 50 punol m"2 s"1 PPFD) to start the salt stress study.
One month after the start of the study, salt stress diseases were
examined. As a result, in the leaves in the wild type (WT), a
yellowing phenomenon which was one of the salt stress diseases
was noticeably observed. Meanwhile, in the leaves in the two
transformed cell lines (TSA-CS-1, TAD-CS-1), the yellowing
phenomenon was not observed at all. A leaf sight of TSA-CS-1 was
shown in Fig. 7. From the above results, it has been shown that
growth disorder due to the salt stress is milder and the
tolerance against the salt stress is remarkably excellent in the
transformants in which the SAMDC gene or the ADC gene was
excessively expressed under the control of the stress/ diseaseinducible
promoter compared with wild type when encountered to
the salt stress for a short period with higher salt concentration
(150 mM) than the usual salt stress condition.
(2) Evaluation of tolerance against moderate environment (low
temperature, weak light) stress
The cell Lines (TSP-SS-1, TSP-SS-2, TSP-SS-4) were selected
in the transformed cell lines. Stem cuttings (15 to 20 per line)
from the three transformed lines and the wild type (WT) were
planted in plastic pots filled with the commercially available
potting compost (Sansan bed soil) to radicate. After radicating,
the plants were cultivated in a closed system glass green house
(temperature at 22 to 25°C, humidity at 55%, natural day length)
for about; one month until the fifth leaf was completely developed.
Thereafter, the plants were transferred to the moderate
environment of 21 to 22°C and light intensity of 40 rno! m2 s~l
PPFD (16 hours day length) , and after 3 months, root tuber
formation was surveyed. A formation rate of the root tubers and a
root sight: upon survey were shown in Figs. 8 and 9, respectively
(fine roots were removed to show the root tubers easily in the
transformants) . In the wild type, the root tuber was not formed
at all. Meanwhile, the root tubers were formed in the
transformants, and the formation rates of the root tubers in
three transformants were 33, 80 and 89%, respectively. In a
reproducibility study in which the plants were cultivated under
the moderate environment of the same condition, the similar
results were obtained. In the wild type, the root tuber was not
formed at all, but the root tubers were formed in the
transformants, and the formation rates of the root tubers were 50
to 83% depending on the cell lines.
(3) Evaluation of tolerance against salt/drought/water stresses
Two cell lines (TSP-SS-1, TSP-SS-4) were selected from the
transformed cell lines. The two transformed cell lines and the
wild type (WT: Kokei 14) were used to perform the cultivation
experiment in the closed system glass green house. Stem cuttings
with one sprout were prepared, and planted in the commercially
available bed soil (Sansan bed soil) to radicate followed by
being grown in the closed system glass green house (temperature
at 23°C/21°C, humidity at 55%, natural day length) until the fifth
leaf was completely developed. Seedlings at the same growth stage
were selected in these seedlings after 3 weeks. Four stems were
planted in each 6 per line of 30 L planters filled with 20 L
potting compost (Sansan bed soil) (2 planters per treatment
group), and cultivated in the closed system glass green house
(set temperature at 23 to 24°C/21 to 22°C, humidity at 55%,
natural day length). As fertilizers, 3.6 g of potassium sulfate
and 13.4 g of Ecolong (14-12-14, 100 day type) per planter were
(DM-8M supplied from Sansyo) in all planters. Groups were divided
into a non-stress group (control group), a salt stress group and
a drought: stress group, and two planters were given to each group.
In the salt stress group, 80 g of NaCl per 100 L of the potting
compost was mixed with soil all layers when planted, and further
1.5 months after planting, 40 g of NaCl per 100 L of the potting
compost was additionally given. The NaCl concentration was
totally about 21 mmol/L per planter. The salt stress was started
when fix planted. For affusion in the salt stress group and the
control group, pF 2.3 of a soil water suction pressure was an
affusion point, the amount of tap water (1.5 to 6 L/time per
planter) where pF was lowered to a field capacity (pF 1.5) was
given. The drought stress was given by limiting the affusion, and
0.75 to 3 L of water was given to the planter when pF became 2.9.
The drought stress treatment was started one week after fix
planting in consideration of taking roots after fix planting.
About 4 months (harvesting stage) after fix plating, wet weight
of the root tuber and the number of root tubers were surveyed.
For poiyamine analysis, the amounts of free polyamine contained
in the Leaves and the root tubers at the harvest were examined.
The growth (wet; weight) of the underground part (root tubers) by
about 4 months after fix planting and a root sight were shown in
Figs. 10 and 11, respectively. The wet weight of the root tubers
in the transformants was significantly heavier than in the wild
type for the control group, and the root tuber weight per plant
was about 40 g heavier in the transformants than in the wild type.
The wet weight of the root tubers in the transformants was
significantly heavier than in the wild type for the salt stress
group, and the root tuber weight per plant was about 60 g heavier
in the transformants than in the wild type. The wet weight of the
root tubers in the transformants was significantly heavier than
in the wild type for the drought group, and the root tuber weight
pe.r plant was about 30 g heavier in the transformants than in the
wild type. Further, the numbers of the root tubers were shown in
Table b. 'In the transformants, the number of root tubers per
plant was 2 more regardless of the presence and absence of the
stress treatment. It was confirmed that the yield of the root
tubers and the number of root tubers were obviously increased in
the transformants compared with the wild type. Free polyamine in
the leaf and the root tuber was analyzed 2 months after fix
planting and at the harvest. The results at the harvest were
shown in Fig. 12. Particularly, the amounts of spermidine (spd)
contained in the leaf and the root tuber in the transformants
were significantly increased about 2 times compared with the wild
type regardless of the presence or absence of the stress
treatment. Depending on the treatment group, the amounts of
contained putrescine (Put) and spermine (Spm) were also increased.
From the above results, under both non-stress condition and
stress condition, it was shown that the formation of the root
tubers (roots) was improved to increase the root tuber yield and
the number of root tubers by increasing the polyamine level in
the sweet potato. Next, the amount of contained starch which was
a major component of the root tuber was examined. The root tuber
with similar size was selected from each plant, and about 100 to
200 g of a root tuber piece was sampled from a vicinity of a
center of the root tuber. The root tuber piece was cut finely,
and 500 ml, of distilled water was added to pulverize by a blender
mixer for 1.5 minutes. A pulverization solution was filtrated
with a sieve of 75 [m, and a filtrated solution was collected.
The sieve was washed with 500 mL of distilled water and the
filtrate was collected. The filtrated solution was left stand for
several hours to precipitate starch, and a supernatant was
discarded. Subsequently, 500 mL of distilled water was added,
stirred to wash starch, and left stand to precipitate starch
followed by discarding the supernatant. This manipulation was
repeated three times or more to thoroughly wash starch. The
precipitated starch after washing was dried in air at room
temperature for 2 days or more. The dried starch was collected
and weighed to calculate the amount of contained starch per wet
weight and a starch yield (g/plant). As a result, in all
treatment groups, the amount of contained starch per root tuber
wet weight was higher in the transformants than in the wild type,
and particularly, in the drought stress group, the amount of
contained starch in the wild type was 12% whereas that in the
both transformants (TSP-SS-1, TSP-SS-4) was 17% which was high.
The starch yields were 39.6 g/plant in the wild type, 48.3
g/plant in TSP-SS-1 and 49.8 g/plant in TSP-SS-4 in the salt
stress group, as well as 13.8 g/plant in the wild type, 21.1
g/plant in TSP-SS-1 and 21.8 g/plant in TSP-SS-4 in the drought
stress group. In both treatment groups, the starch yield was
higher in the transformants than in the wild type. From the above,
it has been demonstrated that the amount of contained starch
which is the major component of the root tuber and the starch
yield are increased by increasing the polyamine level in the
plant.
According to the present invention, by being capable of
imparting the stress defense effects of the plant, it is possible
to avoid the diseases due to various stresses which the plant
encounters in its growth and development process and reduce the
growth inhibition, it is also possible to anticipate the
stabilization of cultivation, the enhancement of productivity,
the enlargement of cultivation regions and the enlargement of
cultivation periods, and it is anticipated to largely contribute
to the industry. It becomes possible to cultivate the plants in
barren lands and salt accumulated soils, and it can be
anticipated to contribute to the global warming and the food
problem.





Claims
1. A method of inducing an expression of at least two stress
defense genes in a plant, characterized in that an expression
amount of at least one stress defense gene is increased compared
with a non-transformant by transforming the plant with an
exogenous spermidine synthase (SPDS) gene, an exogenous S-
adenosylmethionine decarboxylase (SAMDC) gene, an exogenous
arginine decarboxylase (ADC) gene, an ornithine decarboxylase
(ODC) gene and/or a spermine synthase (SPMS) gene under the
control of a promoter capable of functioning in the plant.
2. The method according to claim 1 characterized by further
including a step of selecting a transformed plant in which
expression levels of at least two stress defense genes have been
increased compared with the non-transformant.
3. The method according to claim 1 characterized in that the
stress defense gene is selected from the group consisting of 49
genes with specific Accession Number selected from the group
consisting of the followings and genes having 60% or more
homology to these genes.
(Table Removed)
4. The method according to claim 1 characterized in that the
stress defense gene is selected from the group consisting of the
followings:
I. CBF1, DREB1B
II. CBF3, DREB1A
III. DREB2B
IV. LTI78, COR78, rd29A
V. RD22, rd22
VI. Corlb
VII. ERD15
VIII. LEA protein
IX. PR-1
X. Peroxidase
XI. Hslpro-1
5. The; method according to claim 1 wherein the expression
amount of the stress defense gene is augmented 1.3 to 10 times
compared with a plant before being transformed.
6. The method according to claim 1 wherein the expression
amount of the stress defense gene is augmented 1.4 to 8 times
compared with the non-transformant.
7. The method according to claim 1 wherein the expression
amount; of the stress defense gene is augmented 1.5 to 6 times
compared with the non-transformant.
8. The method according to claim 1 characterized in that a
gene introduced into the plant is the exogenous spermidine
synthase (SPDS) gene derived from a plant.
9. The method according to claim 1 characterized in that the
exogenous spermidine synthase (SPDS) gene is a spermidine
synthase gene having a base sequence of the following (a) or (b)
or (c):
(a) a base sequence represented by base numbers 77 to 1060 in a
base sequence represented by SEQ ID N0:l (SPDS, 1328);
(b) a base sequence which hybridizes with the above base sequence
(a) or a complementary chain thereto under a stringent condition
and encodes a protein having a spermidine synthase activity; and
(c) a base sequence which is composed of a sequence having one or
more base deletions, substitutions, insertions or additions in
the base sequence (a) or (b) and encodes the protein having the
spermidine synthase activity.
10. The method according to claim 1 characterized in that the
exogenous spermidine synthase (SPDS) gene is a spermidine
synthase gene having a base sequence of the following (a) or (b)
or (c):
(a) a base sequence represented by base numbers 118 to 1281 in a
base sequence represented by SEQ ID NO:3 (SPDS, 1560);
(b) a base sequence which hybridizes with the above base sequence
(a) or a complementary chain thereto under a stringent condition
and encodes a protein having a spermidine synthase activity; and
(c) a base sequence which is composed of a sequence having one or
more base deletions, substitutions, insertions or additions in
the base sequence (a) or (b) and encodes the protein having the
spermidine synthase activity.
11. The method according to claim 1 characterized in that the
exogenous S-adenosylmethionine decarboxylase (SAMDC) gene is an
S-adenosylmethionine decarboxylase gene having a base sequence of
the following (a) or (b) or (c):
(a) a base sequence represented by base numbers 456 to 1547 in a
base sequence represented by SEQ ID N0:5 (SAMDC, 1814);
(b) a base sequence which hybridizes with the above base sequence
(a) or a complementary chain thereto under a stringent condition
and encodes a protein having a S-adenosylmethionine decarboxylase
activity; and
(c) a base sequence which is composed of a sequence having one or
more base deletions, substitutions, insertions or additions in the base sequence (a) or (b) and encodes the protein having an S-adenosylmethionine decarboxylase activity.
12. The method according to claim 1 characterized in that the
exogenous arginine decarboxylase (ADC) gene is an arginine
decarboxylase gene having a base sequence of the following (a) or
(b) or (c) :
(a) a base sequence represented by base numbers 541 to 2661 in a
base sequence represented by SEQ ID N0:7 (ADC, 3037);
(b) a base sequence which hybridizes with the above base sequence
(a) or a complementary chain thereto under a stringent condition
and encodes a protein having a arginine decarboxylase activity;
and
(c) a base sequence which is composed of a sequence having one or
more base deletions, substitutions, insertions or additions in
the base sequence (a) or (b) and encodes the protein having an
arginine decarboxylase activity.
13. The method according to claim 1 characterized in that the
exogenous spermine synthase (SPMS) gene is a spermine synthase
gene having a base sequence of the following (a) or (b) or (c):
(a) a base sequence represented by base numbers 1 to 1020 in a
base sequence represented by SEQ ID NO:9 (SPMS, 1020);
(b) a base sequence which hybridizes with the above base sequence
(a) or a complementary chain thereto under a stringent condition
and encodes a protein having a spermine synthase activity; and
(c) a base sequence which is composed of a sequence having one or
more base deletions, substitutions, insertions or additions in
the base sequence (a) or (b) and encodes the protein having a
spermine synthase activity.
14. The method according to claim 1 wherein an introduced
polyamine synthase gene is a gene encoding an arginine
decarboxyjase (ADC) and/or a gene encoding an S
adenosylmethionine decarboxylase (SAMDC) comprising uORF upstream of the gene.
15. The method according to claim 1 wherein one or more stress
defense effects selected from the group consisting of the
following (i) to (xiii) can be imparted:
(i) low temperature stress
(ii) high temperature stress
(iii) salt stress
(iv) osmotic stress
(v) oxidative stress
(vi) herbicide stress
(vii) freeze stress
(viii) drought stress
(ix) pathogen infection stress
(x) pest: stress
(xi) disease stress
(xii) aging stress and
(xiii) heavy metal stress.
16. A method of imparting stress defense effects to a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) gene, an exogenous arginine (ADC) decarboxylase gene, an
ornithine decarboxylase (ODC) gene and/or a spermine synthase
(SPMS) gene under the control of a promoter capable of
functioning in the plant.
17. A method of imparting stress defense effects to a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant; with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) gene, an exogenous arginine (ADC) decarboxylase gene, an ornithine decarboxylase (ODC) gene and/or a spermine synthase
(SPMS) gene under the control of a promoter capable of functioning in the plant, and a transformed plant in which expression levels of the stress defense genes have been increased compared with a non-transformed plant (wild type) is selected
18. A method of enhancing productivity of a plant characterized
in that expression amounts of at least two stress defense genes
are increased compared with a non-transformant by transforming
the plant with an exogenous spermidine synthase (SPDS) gene, an
exogenous S-adenosylmethionine decarboxylase (SAMDC) gene, an
exogenous arginine (ADC) decarboxylase gene, an ornithine
decarboxylase (ODC) gene and/or a spermine synthase (SPMS) gene
under the control of a promoter capable of functioning in the
plant.
19. A method of enhancing stress tolerance in a plant
characterized in that expression amounts of at least two stress
defense genes are increased compared with a non-transformant by
transforming the plant with an exogenous spermidine synthase
(SPDS) gene, an exogenous S-adenosylmethionine decarboxylase
(SAMDC) gene, an exogenous arginine (ADC) decarboxylase gene, an
ornithine decarboxylase (ODC) gene and/or a spermine synthase (SPMS) gene under the control of a promoter capable of functioning in the plant.

Documents:

632-del-2006-Abstract-(14-02-2013).pdf

632-del-2006-abstract.pdf

632-del-2006-assignments.pdf

632-del-2006-Claims-(14-02-2013).pdf

632-del-2006-Claims-(26-08-2013).pdf

632-del-2006-claims.pdf

632-del-2006-Correspondence-Others-(14-02-2013).pdf

632-del-2006-Correspondence-Others-(26-08-2013).pdf

632-del-2006-correspondence-others.pdf

632-del-2006-description (complete).pdf

632-del-2006-drawings.pdf

632-del-2006-form-1.pdf

632-del-2006-Form-2-(14-02-2013).pdf

632-del-2006-form-2.pdf

632-del-2006-form-3.pdf

632-del-2006-form-5.pdf

632-del-2006-GPA-(14-02-2013).pdf

632-del-2006-gpa.pdf

632-del-2006-Petition-137-(14-02-2013).pdf


Patent Number 257095
Indian Patent Application Number 632/DEL/2006
PG Journal Number 36/2013
Publication Date 06-Sep-2013
Grant Date 02-Sep-2013
Date of Filing 10-Mar-2006
Name of Patentee TOYO BOSEKI KABUSHIKI KAISHA.
Applicant Address 2-8, DOJIMA HAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA-FU, JAPAN.
Inventors:
# Inventor's Name Inventor's Address
1 IZUMI IHARA C/O TOYO BOSEKI KABUSHIKI KAISHA 1-1, KATATA 2-CHOME, OTSU-SHI, SHIGA-KEN, JAPAN.
2 SHOJI TACHIBANA 745, HAGINO, GEINO-CHO, TSU-SHI, MIE-KEN, JAPAN.
3 ATSUSHI SOGABE C/O TOYO BOSEKI KABUSHIKI KAISHA 1-1, KATATA 2-CHOME, OTSU-SHI, SHIGA-KEN, JAPAN.
4 YOSHIHISA KASUKABE C/O TOYO BOSEKI KABUSHIKI KAISHA 1-1, KATATA 2-CHOME, OTSU-SHI, SHIGA-KEN, JAPAN.
PCT International Classification Number C12N 15/82
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/685,490 2005-05-31 U.S.A.
2 60/666,177 2005-03-30 U.S.A.
3 2005-180644 2005-06-21 U.S.A.