Note: Descriptions are shown in the official language in which they were submitted.
CA 02323756 2007-06-27
GENETIC ENGINEERING SALT TOLERANCE IN CROP PLANTS
BACKGROUND OF THE INVENTION
Environmental stress due to salinity is one of the most serious factors
limiting the
productivity of agricultural crops, which are predominantly sensitive to the
presence of
high concentrations of salts in the soil. Large terrestrial areas of the world
are affected
by levels of salt inimical to plant growth. It is estimated that 35-45% of the
279 million
hectares of land under irrigation is presently affected by salinity. This is
exclusive of the
regions classified as and and desert lands, (which comprises 25% of the total
land of
our planet). Salinity has been an important factor in human history and in the
life spans
of agricultural systems. Salt impinging on agricultural soils has created
instability and
has frequently destroyed ancient and recent agrarian societies. The Sumerian
culture
faded as a power in the ancient world due to salt accumulation in the valleys
of the
Euphrates and Tigris rivers. Large areas of the Indian subcontinent have been
rendered unproductive through salt accumulation and poor irrigation practices.
In this
century, other areas, including vast regions of Australia, Europe, southwest
USA, the
Canadian prairies and others have seen considerable declines in crop
productivity.
Although there is engineering technology available to combat this problem,
though drainage and supply of high quality water, these measures are extremely
costly.
In most of the cases, due to the increased need for extensive agriculture,
neither
improved irrigation efficiency nor the installation of drainage systems is
applicable.
Moreover, in the and and semi-arid regions of the world water evaporation
exceeds
precipitation. These soils are inherently high in salt and require vast
amounts of
irrigation to become productive. Since irrigation water contains dissolved
salts and
minerals, an application of water is also an application of salt that
compounds the
salinity problem.
Increasing emphasis is being given to modify plants to fit the restrictive
growing
conditions imposed by salinity. If economically important crops could be
manipulated
and made salt resistant, this land could be farmed resulting in greater sales
of seed and
greater yield of useful crops. Conventional breeding for salt tolerance has
been
attempted for a long time. These breeding practices have been based mainly on
the
following strategies: a) the use of wide crosses between crop plants and their
more salt-
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tolerant wild relatives [1 ], b) screening and selecting for variation within
a particular
phenotype [2], c) designing new phenotypes through recurrent selection [3].
The lack of
success in generating tolerant varieties (given the low number of varieties
released and
their limited salt tolerance) [4] would suggest that conventional breeding
practices are
not enough and that in order to succeed a breeding program should include the
engineering of transgenic crops [5].
Several biochemical pathways associated with stress tolerance have been
characterized in different plants and a few of the genes involved in these
processes
have been identified and in some cases the possible role of proteins has been
investigated in transgenic/overexpression experiments. Several compatible
solutes
have been proposed to play a role in osmoregulation under stress. Such
compatible
solutes, including carbohydrates [6], amino acids [7] and quaternary N-
compounds [8)
have been shown to increase osmoregulation. under stress. Also, proteins that
are
normally expressed during seed maturation (LEAs, Late Embriogenesis Abundant
proteins) have been suggested to play a role in water retention and in the
protection of
other proteins during stress. The overexpression of LEA in rice provided a
moderate
benefit to the plants during water stress [9,10]. A single gene (sod2) coding
for a Na+/H+
antiport has been shown to confer sodium tolerance in fission yeast [11,12],
although
the role of this plasma membrane-bound protein appears to be only limited to
yeast.
One of the main disadvantages of using this gene for transformation of plants
is
associated with the typical problems encountered in heterologous gene
expression, i.e.
incorrect folding of the gene product, targeting of the protein to the target
membrane
and regulation of the protein function.
Plants that tolerate and grow in saline environments have high intracellular
salt
levels. A major component of the osmotic adjustment in these cells is
accomplished by
ion uptake. The utilization of inorganic ions for osmotic adjustment suggests
that salt-
tolerant plants must be able to tolerate high levels of salts within their
cells. However,
enzymes extracted from these plants show high sensitivity to salt. The
sensitivity of the
cytosolic enzymes to salt would suggest that the maintenance of low cytosolic
sodium
concentration, either by compartmentation in cell organelles or by exclusion
through the
plasma membrane, must be necessary if the enzymes in the cell are to be
protected
from the inimical effects of salt.
Plant cells are structurally well suited to the compartmentation of ions.
Large
membrane-bound vacuoles are the site for a considerable amount of
sequestration of
ions and other osmotically active substances. A comparison of ion distribution
in cells
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R' -V^%' = coe _4UENCHEN_04 _ 25- EC. ..v 4'16 941 9443 CA 00990021
25-05-2000 and tissues of various plant species indicates that a primary
characteristic of salt
tolerant plants is their ability to exclude sodium out of the cell and to take
up sodium
and to sequester it in the cell vacuoles. Transport mechanisms could actively
move
ions into the vacuole, removing the potentially harmful ions from the cytosol.
These
ions, in turn, could act as an osmoticum within the vacuole, which would then
be
responsible for maintaining water flow into the cell. Thus, at the cellular
level both
specific transport systems for sodium accumulation in the vacuole and sodium
extrusion out of the cell are correlated with salt tolerance.
Transporter polypeptides and sequences having some similarity to transporter
sequences are known in the art (e.g. Ohki et al., AC D49589, EMBL Database, 22
March 1995; Borgese et al., "Cloning and expression of a cAMP-activated Na+/H+
exchanger: evidence that the cytoplasmic domain mediates hormonal regulation",
(1992) PNAS, 89:6765-6769).
A review of the efforts to engineer salt tolerance is found in Bohnert and
Jensen ("Strategies for engineering water-stress tolerance in plants" (1996)
Trends in
Biotechnology, 3:1489-97) and in Rausch at al ("Salt stress responses of
higher
plants: The role of proton pumps and Na'/H4- antiporters," (1996) J. Plant
Physiol
148:425-433).
Barkla et al ( "The plant vacuolar Na`/H' antiport," (1954) Symp. Soc. Exp.
Biol. 48:141-53) describes Na'!H' antiport activity in sugar beet cell
suspensions. It
also discloses an antiport associated polypeptide. -However, the disclosed
polypeptide is not an antiport. It is not suitable for preparation of salt
tolerant
transgenic plants or cells. In another paper Barkla. et al physiologically
characterized
NA'!H' antiporter activity in M. crystallinum. No gene or polypeptide was
isolated.
WO 91/06651 (Young at al.) discloses a yeast sod2 gene that is stated to be
useful for making transgenic yeast and plants. Schachtman et al. discloses a
low
affinity ion transporter. However, these genes have not been useful for making
salt
tolerant plants.
Dante (AC 004655 EMBL Datbase 1 July 1997) purports to show a
polypeptide sequence stated to be "similar to sodium hydrogen exchanger.' This
sequence was generated by sequencing software and It is incorrect. The
sequence
does not exist in nature.
Newman (AC T75860 EMBL Database, 25 March 1995) encodes a fragment
of a transporter gene. The fragment is too short to be useful in preparing
salt tolerant
plants.
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AMENDED SHEET
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CA 00990021 c
25-05-2000
SUMMARY OF THE INVENTION
We have isolated the first such system of Intracellular sat management. We
identified the presence of a functional "vacuolar Na*/W antiport in the
vacuolar
membrane of higher plants [13,14,15,16,17,18].
We have demonstrated the Na'1H+ antiPort function in isolated tonoplast
membranes and in intact vesicles and we showed that the activity of antiport
molecules was salt dependent. Neither a protein sequence nor a gene encoding
the
antiport were identified in previously published work. We have now identified
nucleic
acid molecules coding for plant Na''M" antiports, the nucleic acid molecules
and
polypeptides produced by the nucleic acid molecules being the subject of the
present
invention. These polypeplides are useful for the extrusion of sodium ions from
the
cytosol, either through the accumulation of sodium ions into the vacuoles or
into the
extracellular space, thus providing the most important trait for salt
tolerance in plants.
These nucleic acid molecules, preferably genes= are useful for the engineering
of salt
tolerant plants by transformation of salt-sensitive crops overexpressing one
or more
of these nucleic acid molecules under the control of constitutively active
promoters or
under the control of conditionally-induced promoters. Agrobacterium
tumefaciens-
mediated transformation or particle-bombardment-mediated transformation are
useful
for depending upon the plant species.
The invention includes an isolated nucleic acid molecule encoding a PNHX
transporter polypeptide, or a fragment of a polypeptide having Na'/H'
transporter
activity and capable of increasing salt tolerance in a cell.
The invention also relates to an isolated nucleic acid molecule encoding a
THX transporter polypeptide, PNHX transporter polypeptide, or a fragment of a
polypeptide having Na`/H` transporter activity and capable of increasing salt
tolerance in a cell, comprising a nucleic acid molecule selected from the
group
consisting of.
35 3a
AMENDED SHEET
CA 02323756 2000-09-18
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(a) a nucleic acid molecule that hybridizes to all or part of a nucleic
molecule
in [SEQ ID NO:1], [SEQ ID NO:31, [SEQ ID NO:17], [SEQ ID NO:19], or a
complement thereof under moderate or high stringency hybridization
conditions, wherein the nucleic acid molecule encodes a TNHX
transporter polypeptide, a PNHX transporter polypeptide or a polypeptide
having Na+/H+ transporter activity and capable of increasing salt
tolerance in a cell;
(b) a nucleic acid molecule degenerate with respect to (a), wherein the
nucleic molecule encodes a TNHX transporter polypeptide, a PNHX
transporter polypeptide or a polypeptide having Na+/H+ transporter
activity and capable of increasing salt tolerance in a cell.
The hybridization conditions preferably comprise moderate (also called
intermediate) or high stringency conditions selected from the conditions in
Table 4.
The invention also includes an isolated nucleic acid molecule encoding a THX
transporter polypeptide or a PNHX transporter polypeptide, or a fragment of a
polypeptide having Na+/H+ transporter activity and capable of increasing salt
tolerances
in a cell, comprising a nucleic acid molecule selected from the group
consisting of:
(a) the nucleic acid molecule of the coding strand shown in [SEQ ID NO:1
[SEQ ID NO:3], [SEQ ID NO:17], [SEQ ID NO:19] or a complement
thereof;
(b) a nucleic acid molecule encoding the same amino acid sequence as a
nucleotide sequence of (a); and
(c) a nucleic acid molecule having at least 17% identity with the nucleotide
sequence of (a) and which encodes a THX transporter polypeptide or the
PNHX transporter polypeptide or a polypeptide having Na+/H+ transporter
activity.
The THX transporter polypeptide or the PNHX transporter polypeptide preferably
comprises an AtNHX transporter polypeptide having Na+/H+ transporter activity
and
capable of increasing salt tolerance in a cell. The nucleic acid molecule may
comprise
all or part of a nucleotide sequence in [SEQ ID NO:1], [SEQ ID NO:3], [SEQ ID
NO:17]
or [SEQ ID NO: 19] (or the coding region therof).
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The invention also includes an AtNHX nucleic acid molecule isolated from
Arabidopsis thaliana, or a fragment thereof encoding a transporter polypeptide
having
Na'/H` transporter activity and capable of increasing salt tolerance in a
cell.
Another aspect of the invention relates to a recombinant nucleic acid molecule
comprising a nucleic acid molecule and a constitutive promoter sequence or an
inducible promoter sequence, operatively linked so that the promoter enhances
transcription of the nucleic acid molecule in a host cell.
The nucleic acid molecule preferably comprises genomic DNA, cDNA or RNA.
In another aspect, the nucleic acid molecule is chemically synthesized. The
nucleic acid
molecule is preferably isolated from Arabidopsis thaliana.
The nucleic acid molecule preferably encodes a TNHX transporter polypeptide or
PNHX transporter polypeptide that is capable of extruding monovalent cations
out of the
cytosol of a cell to provide the cell with increased salt tolerance, wherein
the monovalent
cations are selected from at least one of the group consisting of sodium,
lithium and
potassium. The cell preferably comprises a plant cell. The monovalent cations
are
preferably extruded into a vacuole or into the extracellular space.
The invention also includes an isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of 8 to 10
nucleotides of the
nucleic acid molecules described above, 11 to 25 nucleotides of the nucleic
acid
molecules described above, and 26 to 50 nucleotides of the nucleic acid
molecules
described above.
The invention also includes an isolated oligonucleotide comprising at least
about
10 nucleotides from a sequence selected from the group consisting of 5'-
GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ ID NO:11], 5'-
CCGAATTCTCAAAGCTTTTCTTCCACG-3' [SEQ ID NO:12], 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ ID NO:13], 5'-
GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ ID NO:14], 5'-
CCGAATTCTCAAAGCTTTI'CTTCCACG-3' [SEQ ID NO:15], 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ ID NO: 161 or another
oligonucleotide described in this application.
Another aspect of the invention relates to a vector comprising a nucleic acid
molecule of the invention. The vector preferably comprises a promoter selected
from
the group consisting of a super promoter, a 35S promoter of cauliflower mosaic
virus, a
drought-inducible promoter, an ABA-inducible promoter, a heat shock-inducible
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promoter, a salt-inducible promoter, a copper-inducible promoter, a steroid-
inducible
promoter and a tissue-specific promoter.
The invention also includes a host cell comprising a recombinant nucleic acid
molecule of the invention, or progeny of the host cell.
The host cell is preferably selected from the group consisting of a fungal
cell, a
yeast cell, a bacterial cell, a microorganism cell and a plant cell. The
plant, a plant part,
a seed, a plant cell or progeny thereof preferably comprises the recombinant
nucleic
acid molecule of the invention. The plant part preferably comprises all or
part of a leaf,
a flower, a stem, a root or a tuber. The plant, plant part, seed or plant cell
is preferably
of a species selected from the group consisting of potato, tomato, brassica,
cotton,
sunflower, strawberries, spinach, lettuce, rice, soybean, corn, wheat, rye,
barley,
atriplex, sorgum, alfalfa, salicornia and the plant species or types in Table
5.
The plant, plant part, seed or plant cell preferably comprises a dicot plant
or a
monocot plant.
The invention also relates to a method for producing a recombinant host cell
capable of expressing the nucleic acid molecule of the invention, the method
comprising
introducing into the host cell a vector of the invention. The invention also
includes a
method of producing a genetically transformed plant which expresses TNHX or
PNHX
transporter polypeptide, comprising regenerating a genetically transformed
plant from a
plant cell, seed or plant part of the invention. In one method, the genome of
the host
cell also includes a functional TNHX or PNHX gene. In another method, the
genome of
the host cell does not include a functional TNHX or PNHX gene. The invention
also
includes a transgenic plant produced according to a method of the invention.
Another aspect of the invention relates to a method for expressing a TNHX or
PNHX transporter polypeptide in the host cell of the invention, a the plant,
plant part,
seed or plant cell of the invention, the method comprising culturing the host
cell under
conditions suitable for gene expression. A method for producing a transgenic
plant that
expresses elevated levels of PNHX transporter polypeptide relative to a non-
transgenic
plant, comprising transforming a plant with the vector of the invention. The
invention
also relates to an isolated polypeptide encoded by and/or produced from a
nucleic acid
molecule of the invention, or the vector of the invention.
The invention also relates to an isolated PNHX transporter polypeptide or a
fragment thereof having Na'/H+ transporter activity and capable of increasing
salt
tolerance in a cell. The polypeptide of the invention preferably comprises an
AtNHX
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WO 99/47679 PCT/CA99/00219
transporter polypeptide. The polypeptide of the invention preferably comprises
all or
part of an amino acid sequence in [SEQ ID NOS: 2, 4, 6, 8, 18, or 20] (figure
1). The
invention also includes a polypeptide fragment of the AtNHX transporter
polypeptide of
the invention, or a peptide mimetic of the AtNHX transporter polypeptide,
having Na+/H+
transporter activity and capable of increasing salt tolerance in a cell. The
polypeptide
fragment of the invention, preferably consists of at least 20 amino acids,
which fragment
has Na+/H+ transporter activity and is capable of increasing salt tolerance in
a cell. The
fragment or peptide mimetic'of the invention is preferably capable of being
bound by an
antibody to a polypeptide of the invention. In one embodiment, the polypeptide
of the
invention is recombinantly produced.
The invention also includes an isolated and purified transporter polypeptide
comprising the amino acid sequence of a TNHX transporter polypeptide or a PNHX
transporter polypeptide, wherein the transporter polypeptide is encoded by a
nucleic
acid molecule that hybridizes under moderate or stringent conditions to a
nucleic acid
molecule in [SEQ ID NOS: 1, 3, 5, 7, 17, or 19] (figure 1), a degenerate form
thereof or a
complement. The invention also includes a polypeptide comprising a sequence
having
greater than 28% sequence identity to a polypeptide of the invention
(preferably a
polypeptide in figure 1, such as [SEQ ID NOS: 2, 4, 6, 8, 18, or 20]).
The polypeptide of the invention, preferably comprises a Na+/H+ transporter
polypeptide. The polypeptide is preferably isolated from Arabidopsis thaliana.
The invention also includes an isolated nucleic acid molecule encoding
polypeptide of the invention (preferably a polypeptide in figure 1: [SEQ ID
NOS: 2, 4, 6,
8, 18, or 20]).
Another aspect of the invention relates to an antibody directed against a
polypeptide of the invention. The antibody of the invention, preferably
comprises a
monoclonal antibody or a polyclonal antibody.
The invention also relates to an isolated nucleic acid molecule encoding a
TNHX
transporter polypeptide or a PNHX transporter polypeptide, or a fragment of a
polypeptide having Na+/H+ transporter activity and capable of increasing salt
tolerance in
a cell, comprising a nucleic acid molecule selected from the group consisting
of:
(a) a nucleic acid molecule that hybridizes to all or part of a nucleic
molecule
in [SEQ ID NO:5], [SEQ ID NO:7], [SEQ ID NO:9] or to a nucleic acid
molecule comprising about nucleotides 1-1487 of [SEQ ID NO:9], or a
complement thereof under moderate or high stringency hybridization
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conditions, wherein the nucleic acid molecule encodes a TNHX
transporter polypeptide, a PNHX transporter polypeptide or a polypeptide
having Na+/H+ transporter activity and capable of increasing salt
tolerance in a cell;
(b) a nucleic acid molecule degenerate with respect to (a), wherein the
nucleic molecule encodes a TNHX polypeptide, a PNHX polypeptide or a
polypeptide having Na+/H+ transporter activity and capable of increasing
salt tolerance in a cell.
(c) the nucleic acid molecule of the coding strand shown in [SEQ ID NO:5],
[SEQ ID NO:7], [SEQ ID NO:9], nucleotides 1-1487 of [SEQ ID NO:9], or
an isolated nucleic acid molecule including about 1614 nucleic acids
including [SEQ ID NO:5], [SEQ ID NO:7], nucleotides 1 to 1487 of the
nucleic acid molecule in [SEQ ID NO:91 or the complement thereof;
(d) a nucleic acid molecule encoding the same amino acid sequence as a
nucleotide sequence of (c); and
(e) a nucleic acid molecule having at least 17% sequence identity with the
nucleotide sequence of (c) and which encodes a TNHX transporter
polypeptide, a PNHX transporter polypeptide or a polypeptide having
Na+/H+ transporter activity and capable of increasing salt tolerance in a
cell.
The invention also includes a polypeptide produced from a nucleic acid
molecule
of the invention. The invention includes a polypeptide comprising (a) the
amino acid
sequence in [SEQ ID NO:6], [SEQ ID NO:8], [SEQ ID NO:10]; (b) amino acids 1 to
496
of [SEQ ID NO:10]; and (c) a sequence having greater than 28% homology to the
polypeptide in (a) or (b). The invention includes a polypeptide comprising a
Na+/H+
transporter polypeptide capable of increasing salt tolerance in a cell. The
invention also
includes a DNA molecule encoding the polypeptides of the invention.
The invention relates to a method of producing a genetically transformed plant
which expresses or overexpresses a TNHX transporter polypeptide, a PNHX
transporter
polypeptide or a polypeptide having Na+/H+ transporter activity and capable of
increasing salt tolerance in a cell and wherein the plant has increased salt
tolerance,
comprising:
a) cloning or synthesizing a TNHX nucleic acid molecule, a PNHX nucleic
acid molecule or a nucleic acid molecule which codes for a Na+/H+
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WO 99/47679 PCT/CA99/00219
transporter polypeptide, wherein the polypeptide is capable of providing
salt tolerance to a plant;
b) inserting the nucleic acid molecule in a vector so that the nucleic acid
molecule is operably linked to a promoter;
c) inserting the vector into a plant cell or plant seed;
d) regenerating the plant from the plant cell or plant seed, wherein salt
tolerance in the plant is increased compared to a wild type plant.
The invention includes a transgenic plant produced according to a method of
the
invention.
The nucleic acid molecules have several uses which will be discussed in more
detail below. The nucleic acid molecules and the polypeptides are used in a
method for
protecting a plant from the adverse affects of a saline environment by
incorporating a
nucleic acid molecule for salt tolerance and/or the polypeptide of the
invention into a
plant. The nucleic acid molecules of the invention are also useful for the
identification of
homologous nucleic acid molecules from plant species, preferably salt tolerant
species
and genetically engineering salt tolerant plants of agricultural and
commercial interest.
The invention relates to isolated nucleic acid molecules encoding a
polypeptide
for extrusion of sodium ions from the cytosol of a cell to provide the cell
with salt
tolerance. The nucleic acid molecules preferably comprise the nucleotide
sequence in
figure 1(a) or (b). The nucleic acid molecules may be DNA or RNA. The nucleic
acid
molecules may be used to transform a cell selected from the group consisting
of a plant
cell, a yeast cell and a bacterial cell. The sodium ions are extruded into a
vacuole or out
of the cell. The nucleic acid molecules encode a Na`/H+ exchanger polypeptide.
In a preferred embodiment, the nucleic acid molecules are isolated from
Arabidopsis thaliana.
The invention includes an isolated nucleic acid molecule, comprising the DNA
sequence in figure 1(a), (b), (c)(i), (c)(ii), (d) or (e). The invention also
relates to an
isolated nucleic acid molecule, comprising a sequence having greater than 17%
homology to the sequences of the invention described in the preceding
paragraphs.
In an alternate embodiment, the nucleic acid molecule consists of a sequence
selected from the group consisting of 8 to 10 nucleotides of the nucleic acid
molecules
of the invention, 11 to 25 nucleotides of the nucleic acid molecule and 26 to
50
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nucleotides of the nucleic acid molecules. These nucleic acid molecules
hybridize to
nucleic acid molecules described in the preceding paragraphs.
The nucleic acid molecule of the invention may have a sense or an antisense
sequence.
In another embodiment, the invention is an isolated oligonucleotide consisting
of
a sequence selected from the group consisting of 5'-
GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ ID NO:11], 5'-
CCGAATTCTCAAAGCTiTTCTTCCACG-3' [SEQ ID NO:121, 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ ID NO:13], an oligonucleotide
with an antisense sequence of 5'-GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ ID
NO:14], an oligonucleotide with an antisense sequence of 5'-
CCGAATTCTCAAAGCTTTTCTTCCACG-3' [SEQ ID NO:15] and an oligonucleotide
with an antisense sequence of 5'-CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ
ID NO:16]. The invention includes an isolated oligonucleotide consisting of 5
to 15
nucleotides of these oligonucleotides. The invention includes an isolated
oligonucleotide consisting of a sequence homologous to the oligonucleotide of
claim 15
or claim 16.
In an alternate embodiment, the invention is an expression vector comprising a
nucleic acid molecule of the invention. The expression vector preferably
consists of a
promoter selected from the group consisting of a super promoter, a 35S
promoter of
cauliflower mosaic virus, a drought-inducible promoter, an ABA-inducible
promoter, a
heat shock-inducible promoter, a salt-inducible promoter, a copper-inducible
promoter, a
steroid-inducible promoter and a tissue-specific promoter.
The invention is a polypeptide produced from the nucleic acid molecules of the
invention. The invention is also a polypeptide produced from the expression
vector.
The polypeptide is used for extrusion of sodium ions from the cytosol of a
cell to provide
the cell with salt tolerance.
In a preferred embodiment, the polypeptide has the amino acid sequence in
figure 1(a)-(e). The polypeptides may be homologous to the polypeptide in
figure 1(a)-
(e). In an alternate embodiment, the polypeptides comprise a sequence having
greater
than 28% homology to the polypeptide in figure 1 (a)-(e). The polypeptides are
Na+/H+
exchanger polypeptides.
CA 02323756 2000-09-18
WO 99/47679 PCT/CA99/00219
The polypeptides are preferably isolated from Arabidopsis thaliana.
The invention includes peptides consisting of at least 5 amino acids of the
polypeptides described in the preceding paragraphs. In another embodiment, the
peptides consist of 41 to 75 amino acids of the polypeptides described in the
preceding
paragraphs.
The invention also includes isolated nucleic acid molecules encoding the
polypeptides of the invention. The isolated nucleic acid molecule preferably
encodes
the polypeptide of figure 1 (a)-(e).
The polypeptides of the invention that extrude sodium ions from the cytosol of
a
cell to provide the cell with salt tolerance, preferably consist of an
amiloride binding
domain. The amiloride binding domain is between amino acids 82 to 90 in both
AtNHX1
and AtNHX2. in figure 1(a)-(e) and between amino acids 58 to 66 in both AtNHX3
and
AtNHX4 in figures (d) and (e).
The invention also includes a monoclonal antibody or polyclonal antibody
directed against a polypeptide of the invention.
Another embodiment of the invention includes a transformed microorganism
comprising an isolated nucleic acid molecule of the invention. The invention
also
includes a transformed microorganism including an expression vector.
The invention includes a plant cell transformed with a nucleic acid molecule
of
the invention. The invention also includes a yeast cell transformed with the
nucleic acid
molecule of the invention. In another embodiment, the invention is a plant,
plant part or
seed, generated from a plant cell transformed with a nucleic acid molecule of
the
invention. The invention also relates to a plant, plant part, seed or plant
cell transfected
with a nucleic acid molecule of the invention. The plant, plant part, seed or
plant cell is
preferably selected from a species selected from the group consisting of
potato, tomato,
brassica, cotton, sunflower, strawberries, spinach, lettuce, rice, soybean,
corn, wheat,
rye, barley, atriplex, sorgum, alfalfa and salicornia and other plants in
Table 5.
The invention also includes a method for producing a polypeptide of the
invention by culturing a plant, plant part, seed or plant cell of the
invention and
recovering the expressed polypeptide from the culture.
The invention includes an isolated nucleic acid molecule encoding a
polypeptide
capable of extruding monovalent cations from the cytosol of a cell to provide
the cell
with increased salt tolerance. The nucleic acid molecule preferably includes
the
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nucleotide sequence in figure 1(a)-(e). The nucleic acid molecule is
preferably DNA or
RNA. The cell is preferably a plant cell, a yeast cell or a bacterial cell.
The monovalent
cations are preferably sodium, lithium or potassium. The monovalent cations
are
preferably extruded into a vacuole or out of the cell. The nucleic acid
molecules
preferably encode a Na`/H+ exchanger polypeptide. The nucleic acid molecule is
preferably isolated from Arabidopsis thaliana.
The invention also includes an isolated nucleic acid molecule, including a
sequence having greater than 17% homology to a sequence referred to in the
preceding
paragraph.
The invention also includes a nucleic acid molecule of 8 to 10 nucleotides, 11
to
25 or 26 to 50 nucleotides of a nucleic acid molecule of the invention.
The invention also includes a nucleic acid molecule which nucleic acid
molecule
hybridizes a nucleic acid molecule of the invention. The nucleic acid molecule
comprises a sense or an antisense sequence.
The invention also includes an isolated oligonucleotide including a sequence
selected from the group consisting of 5'-GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ
ID NO:11], 5'- CCGAATTCTCAAAGCTTTTCTTCCACG-3' [SEQ ID NO:12], 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ ID NO:13], 5'-
GCCATGTTGGATTCTCTAGTGTCG-3 [SEQ ID NO:14], 5'-
CCGAATTCTCAAAGCTTTTCTTCCACG-3' [SEQ ID NO: 15] and 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3' [SEQ ID NO:16] or 5 to 15 nucleotides
of one of these oligonucleotides. The invention also includes an isolated
oligonucleotide
having a sequence homologous to one of these oligonucleotides.
The invention also includes an expression vector including a nucleic acid
molecule of the invention. The expression vector preferably comprises a
promoter
selected from the group consisting of a super promoter, a 35S promoter of
cauliflower
mosaic virus, a drought-inducible promoter, an ABA-inducible promoter, a heat
shock-
inducible promoter, a salt-inducible promoter, a copper-inducible promoter, a
steroid-
inducible promoter and a tissue-specific promoter.
The invention also includes a polypeptide produced from a nucleic acid
molecule
or expression vector of the invention. The invention also includes a
polypeptide for
extrusion of monovalent cations ions from the cytosol of a cell to provide the
cell with
salt tolerance. The invention also includes a polypeptide including the amino
acid
sequence in figure 1 (a)-(e) or a polypeptide homologous to one of these
sequences.
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The invention also includes a polypeptide including a sequence having greater
than
28% homology to one of these polypeptides. The polypeptide is preferably a
Na`/H+
exchanger polypeptide isolated from Arabidopsis thaliana. The invention also
includes
a peptide including at least 5 amino acids or 41 to 75 amino acids of the
polypeptide of
the invention. The invention also includes nucleic acid molecules these
polypeptides.
The invention includes a polypeptide for extrusion of monovalent cations ions
from the cytosol of a cell to provide the cell with salt tolerance, including,
but not
necessarily having, an amiloride binding domain.
Another aspect of the invention relates to a monoclonal or polyclonal antibody
directed against a polypeptide of the invention.
Another variation includes a transformed microorganism including an isolated
nucleic acid molecule of the invention. The transformed microorganism
preferably
includes an expression vector of the invention:
A plant cell, yeast cell transformed or transfected with a nucleic acid
molecule or
a plant, plant part or seed, generated from the plant cell. The plant, plant
part, seed or
plant cell is preferably from a species selected from the group consisting of
potato,
tomato, brassica, cotton, sunflower, strawberries, spinach, lettuce, rice,
soybean, corn,
wheat, rye, barley, atriplex, sorgum, alfalfa, salicomia and other plants in
Table 5. The
invention also includes a method for producing a peptide, by culturing the
plant, plant
part, seed or plant cell and recovering the expressed peptide from the
culture.
The invention includes a nucleic acid molecule that encodes all or part of a
polypeptide capable of extruding monovalent cations from the cytosol of a cell
to provide
the cell with salt tolerance, wherein the sequence hybridizes to the nucleic
acid
molecule of all or part of [SEQ ID NO:1] or [SEQ ID NO:3], [SEQ ID NO:17],
[SEQ ID
NO:19] , figure 5(b) or a nucleic acid molecule including nucleotides 1-1487
of figure
5(b) under low, medium and high stringency conditions. The high stringency
conditions
preferably comprise a wash stringency of selected from the group of
hybridization and
wash stringencies in Table 4.
The invention includes an isolated nucleic acid molecule encoding a
polypeptide
capable of extruding monovalent cations from the cytosol of a cell to provide
the cell
with salt tolerance, including the nucleic acid molecule in figure 5(b). The
invention also
includes an isolated nucleic acid molecule encoding a polypeptide capable of
extruding
monovalent cations from the cytosol of a cell to provide the cell with salt
tolerance,
including nucleotides 1 to 1487 of the nucleic acid molecule in figure 5(b).
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Another aspect of the invention relates to an isolated nucleic acid molecule
including about 1640 (or preferably about 1600 or 1700) nucleic acids encoding
a
polypeptide capable of extruding monovalent cations from the cytosol of a cell
to provide
the cell with salt tolerance, the nucleic acid molecule including nucleotides
1 to 1487 (or
preferably about nucleotides 1 to 1470, 1480, 1490 or 1500) of the nucleic
acid
molecule in figure 5(b). The molecule is preferably DNA or RNA. The cell is
preferably
selected from the group consisting of a plant cell, a yeast cell and a
bacterial cell. The
molecule preferably encodes a Na+/H+ exchanger polypeptide. The nucleic acid
molecule is preferably isolated from Arabidopsis thaliana.
The invention also includes the nucleic acid molecule in figure 5(b) or a
nucleic
acid molecule having greater than 17% homology to the sequence in 5(b). The
invention includes polypeptides produced from this one of these nucleic acid
molecules.
The invention also relates to a polypeptide including the amino acid sequence
in figure
5(b) or amino acids I to 496 of figure 5(b). (note: polypeptide including 1 to
496 is
preferably about 530, 540 or 550 amino acids, most preferably about 538 amino
acids)
or a homologous polypeptide, preferably having greater than 28% homology. The
polypeptide is preferably a Na+/H+ exchanger polypeptide, isolated from
Arabidopsis
thaliana. The invention also includes a DNA molecule encoding one of these
polypeptides.
The invention includes an isolated nucleic acid molecule encoding a
polypeptide
capable of extruding monovalent cations from the cytosol of a cell to provide
the cell
with salt tolerance, including at least one of the nucleic acid molecules in
figure 1(c).
The molecule is preferably DNA or RNA. The cell is preferably selected from
the group
consisting of a plant cell, a yeast cell and a bacterial cell and encodes a
Na+/H+
exchanger polypeptide isolated from Arabidopsis thaliana.
The invention includes an isolated nucleic acid molecule, including the
nucleic
acid molecule in figure 1 (c)(i) or I (c)(ii) or a polypeptide produced from a
nucleic acid
molecule of the invention. The invention also includes a polypeptide including
the
amino acid sequence in figure 1(c)(i) or 1(c)(ii) or homologous to this
polypeptide,
preferably having greater than 28% homology. The polypeptide is preferably a
Na+/H+
exchanger polypeptide, isolated from Arabidopsis thaliana. The invention
includes a
DNA molecule encoding one of these polypeptides.
It will be clear to one skilled in the art that the sequences in figures 1(c)
and 5
are useful in isolating other salt tolerant nucleic acid molecules (for
example probes may
be made from the sequences in figures 1(c) and 5), preparing transgenic plants
and
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performing many of the other methods of the invention that are described with
respect to
sequences in figures 1(a), (b), (d) and (e). Variants and modifications of
figure 1(c) and figure 5
sequences are also included within the invention as are methods using varied
or modified
sequences (the same preferred percentages of identity and sequence described
with respect to
figures 1(a), (b), (d) and (e) also apply to figures 1(c) and 5). Nucleic acid
molecules including a
portion of the nucleic acid molecule in figure 5 preferably include about
nucleotides 1-1487 (or a
partial sequence thereof, preferably starting from the coding region, which
will be apparent to a
skilled person, at about nucleotide 286). The nucleotide sequence including
all or part of
sequence in figure 1(c) or figure (5) will be preferably about 1683 or about
1614 nucleotides in
length, respectively, (or 1683 or 1614 nucleotides minus part or all of the 5'
or 3' untranslated
region nucleotides). The nucleic acid molecules are most preferably 1600 to
1620 or 1670 to
1690 nucleotides in length. Polypeptides including a portion of the nucleic
acid molecule in
figure 5 preferably include about amino acids I to 496 (or a partial sequence
thereof) in figure 5.
The sequences encoding all or part of the polypeptide in figure 5 or encoding
a polypeptide
corresponding to either of the nucleic acid molecule sequences in figure 1(c)
are preferably
about 538 amino acids (preferably about 60 kda) and about 318 amino acids in
length,
respectively. Preferred polypeptides are about 530-550 amino acids in length
or 310-330 amino
acids. Polypeptides including a portion of the nucleic acid molecule in figure
1(c) preferably
include part or all of either of the sequences in 1(c) (nucleic acid molecules
preferably include all
or part of the corresponding nucleic acid molecule sequences).
BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described in relation to the
drawings in which:
Figure 1. (a) Shows the nucleic acid molecule that is [SEQ ID NO:1] and the
polypeptide that is [SEQ ID NO:2].
In a preferred embodiment, the figure shows isolated AtNHX1 cDNA encoding a
Na+/H`
exchanger from Arabidopsis thaliana showing cDNA sequence and the
corresponding amino
acid sequence for AtNHX1. Twelve transmembrane domains are present, a
conserved
amiloride-binding domain is present, and a relatively hydrophilic C-terminal
region is also
present. The predicted open reading frame begins at nucleotide 286. The amino
acids are
centred below the corresponding codon and are numbered on the left;
(b) Shows the nucleic acid molecule that is [SEQ ID NO:3] and the polypeptide
that
is [SEQ ID NO:4].
In a preferred embodiment, the figure shows isolated AtNHX2 cDNA encoding a
Na'/H' exchanger from Arabidopsis thaliana showing cDNA sequence and the
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corresponding predicted amino acid sequence for AtNHX2. The predicted open
reading
frame begins at nucleotide 61. The amino acids are centred below the
corresponding
codon and are numbered on the left;
(c) (i) Shows the nucleic acid molecule that is [SEQ ID NO:5] and the
polypeptide
that is [SEQ ID NO:6]. (ii) Shows the nucleic acid molecule that is [SEQ ID
NO:7] and
the polypeptide that is [SEQ ID NO:8].
In a preferred embodiment, the figure shows AtNHX3 partial cDNA sequences. The
amino acids are centred below the corresponding codon and are numbered on the
left
(i) 5' sequence of the partial AtNHX3 cDNA and amino acid sequence; (ii) In a
preferred
embodiment, the figure shows 3' sequence of the partial AtNHX3 cDNA and amino
sequence;
(d) Shows the nucleic acid molecule that is [SEQ ID NO:17] and the polypeptide
that
is [SEQ ID NO:18].
In a preferred embodiment, the figure shows isolated AtNHX3 cDNA encoding a
Na+/H+ exchanger from Arabidopsis thaliana showing cDNA sequence and the
corresponding predicted amino acid sequence for AtNHX3. The predicted open
reading
frame begins at nucleotide 67. The amino acids are centred below the
corresponding
codon and are numbered on the left. (e) Isolated AtNHX4 cDNA encoding a Na+/H+
exchanger from Arabidopsis thaliana showing cDNA sequence [SEQ ID NO:19] and
the
corresponding predicted amino acid sequence [SEQ ID NO:20] for AtNHX4. The
predicted open reading frame begins at nucleotide 55. The amino acids are
centred
below the corresponding codon and are numbered on the left.
(e). Shows the nucleic acid molecule that is [SEQ ID NO: 191 and the
polypeptide that
is [SEQ ID NO:20].
In a preferred embodiment, the figure shows isolated AtNHX4 cDNA encoding a
Na+/H+ exchanger from Arabidopsis thaliana.
Figure 2. (a) Alignment of the predicted amino acid sequences of Arabidopsis
AtNHX1,
from Arabidopsis thaliana with other Na+/H+ exchangers from other organisms.
Sequences were aligned using the Clustal W program [19] using default
parameters
(fixed gap penalty=10, floating gap penalty=l0, protein weight matrix
BLOSUM62).
Sequences and GenBank accession numbers are: ScNHX1, late endosomal Na+/H+
exchanger S. cerevisiae (GenBank accession #927695); CeNHEI, C. elegans
(GenBank accession # 3877723; HsNHE6, Homo sapiens mitochondrial Na+/H+
exchanger (GenBank accession # 2944237); (b) Alignment of the predicted amino
acid
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sequences of Arabidopsis AtNHX1, AtNHX2 and AtNXH3 cDNAs from Arabidopsis
thaliana. Sequences were aligned using the Clustal W program [19] using
default
parameters (fixed gap penalty=10, floating gap penalty=10, protein weight
matrix
BLOSUM62). (c) Alignment of the predicted amino acid sequences of Arabidopsis
AtNHX3 and AtNXH4 cDNAs from Arabidopsis thaliana. Sequences were aligned
using
the Clustal W program (19] using default parameters (fixed gap penalty= 10,
floating gap
penalty=10, protein weight matrix BLOSUM62); (b) Alignment of the predicted
amino
acid sequences of AtNHX1, AtNHX2 and AtNHX3 cDNAs from Arabidopsis thaliana.
Sequences were aligned using the Clustal W prgoram using default parameters
(fixed
gap penalty=10, floating gap penalty=l0, protein weight matrix BLOSUM62); (c)
Alignment of the predicted amino acid sequences of AtNHX3 and AtNHX4 cDNAs
from
Arabidopsis thaliana. Sequences were aligned using the Clustal W program using
default parameters (fixed gap penalty=l0, floating gap penalty=10, protein
weight matrix
BLOSUM62).
Figure 3. A Southern blot of Arabidopsis genomic DNA. Genomic DNA (10 g per
lane) was digested with various restriction enzymes, separated on a 1.0%
agarose gel,
transferred onto a GeneScreen Plus membrane (Amersham), and hybridized to a
radiolabelled AtNHX1 cDNA as described in Materials and Methods. Restriction
enzymes used were; C, Clal; E, ECoRI; X, Xbal; H, Hindlll.
Figure 4. RNA blot of AtNHX1 expression in different tissues. Total RNA (40
g) was
separated on a 1.0% agarose gel, transferred to a GeneScreen Plus membrane
(Amersham) and hybridized to a radiolabelled AtNHX1
cDNA probe as described in Materials and Methods. Tissues in each lane were as
follows: 1, mature leaf; 2, flower (including sepals); 3, infloresence stem;
4, seedling shoot; 5, seedling root.
Figure 5. (a) and (b) show the nucleic acid molecule that is [SEQ ID NO:9] and
the
polypeptide that is [SEQ ID NO:10].
In a preferred embodiment, (a) and (b) show modified arabidopsis
sodium/proton antiporter cDNA and polypeptide sequence.
Figure 6. RNA blot comparing transcript levels in Arabidopsis thaliana leaf
tissue from
wild type and different transgenic lines overexpressing AtNHX1. RNA was
extracted
from 4 week-old plants. Total RNA (30 p.g per lane) was separated on a 1.0 %
agarose
gel, transferred to a GeneScreen Plus membrane (Amersham) and hybridized to a
radiolabelled AtNHX1 cDNA probe as described in Materials and Methods. An
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endogenous 2.1 kb transcript was detected in the transgenic lines as well as
in wild
type. An overexpressedl.8 kb transcript was only seen in the transgenic lines.
The 1.8
kb transcript corresponds to the open reading frame coding for AtNHX1, lacking
the 5'-
and 3'-untranslated regions present in the original cDNA (2.1 kb). Ribosomal
RNA
(rRNA) was used to confirm equal loading of the gels, as seen by methylene-
blue
staining of the blot.
wt: wild-type; X1-21, X1-3' and X1-4': independent transgenic lines.
Figure 7. Twenty 3-week old kanamycin-resistant Arabidopsis thaliana plants
for each
of the 3 independent transgenic lines (X1.2', X1.3' and X1.4') transformed
with AtNHXI,
as well as 20 wild-type plants of the same age were used for assessment of
salt
tolerance. Plants were watered with 25 ml of 1/8 strength MS salts (control
solution)
supplemented with different concentrations of NaCl. The following schedule was
used
for a total of 16 days, at which point pictures of representative plants were
taken: a)
wild-type: A=OmM NaCl, B=50mM NaCl, C=1OOmM NaCl, D=150mM NaCl, E=200mM
NaCl; b) X1.2' transgenic line: A=OmM NaCl, B=50mM NaCl, C=1 OOmM NaCl,
D=150mM NaCl, E=200mM NaCl; c) X1.3' transgenic line; d) X1.4' transgenic
line:
A=OmM NaCl, B=50mM NaCl, C=1OOmM NaCl, D=150mM NaCl, E=200mM NaCl; e)
wild type: A=OmM NaCl, E=200mM NaCI vs. transgenic strain 2': A=OmM NaCl,
E=200mM NaCl; f) wild type: A=OmM NaCl, E= 200mM NaCl vs. transgenic strain
4':
A=OmM NaCl, E=200mM NaCl; g) wild type: A=OmM NaCl, E=200mM NaCI vs.
transgenic strain 2': A=OmM NaCl, E=200mM NaCl and transgenic strain 4': A=OmM
NaCl, E=200mM NaCl.
Treatments:
A) watered with a control solution (1/8 MS strength solution, 0mM NaCl) eight
times
(once every two days)
B) watered with a control solution supplemented with 50mM NaCl eight times
(once
every two days)
C) watered twice (once every two days) with a control solution supplemented
with
50mM NaCl, then with a control solution supplemented with 100mM NaCl six
times (once every two days).
D) watered twice (once every two days) with a control solution supplemented
with
50mM NaCl, then with a control solution supplemented with 100mM NaCl twice
(once every two days) followed by a control solution supplemented with 150mM
NaCl four times (once every two days).
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E) watered twice (once every two days) with a control solution supplemented
with
50mM NaCl, then with a control solution supplemented with 100mM NaCl twice
(once every two days) followed by a control solution supplemented with 150mM
NaCl twice (once every two days) and a control solution supplemented with 200
mM NaCl twice (once every two days).
Figure 8. (a) shows [SEQ ID NO:21] (b) shows [SEQ ID NO:22] (c) shows [SEQ ID
NO:231 (d) shows [SEQ ID NO:24] (e) shows [SEQ ID NO:25] (f) shows [SEQ ID
NO:26]
(g) shows [SEQ ID NO:27] (h) shows [SEQ ID NO:28].
In preferred embodiments, (a)-(h) show sequences from Table 2: (a) GenBank
Accession No. 3850064 569 a.a.; (b) GenBank Accession No. 927695 633 a.a.; (c)
GenBank Accession No. C91832 378 bp mRNA EST; (d) GenBank Accession No.
C91861 268 bp mRNA EST; (e) GenBank Accession No. AU032544 380 bp mRNA
EST; (f) GenBank Accession No. AA660573 596 bp mRNA EST; (g) GenBank
Accession No. L44032 522 bp mRNA STS; (h) GenBank Accession No. T75860 (EST)
330 bp mRNA EST.
DETAILED DESCRIPTION OF THE INVENTION
Salt Tolerance Nucleic Acid Molecules and Polypeptides
The invention relates to nucleic acid molecules and polypeptides which
increase
salt tolerance in cells and plants. PNHX polypeptides are plant Na'/H+
transporter
polypeptides that are capable of increasing and enhancing salt tolerance in a
cell,
preferably a plant cell. These transporters (also referred to as exchangers,
antiports or
antiporters) extrude monovalent cations (preferably potassium ions or lithium
ions, most
preferably sodium ions) out of the cytosol. The cations are preferably
extruded into the
vacuoles or extracellular space. The affinity for particular ions varies
between
transporters. The listed preferences refer to the cations that are most likely
to be
abundant in the cytosol and therefore most likely to be extruded. It is not
necessarily a
reflection of transporter affinity for particular cations. The PNHX nucleic
acid molecules
which encode PNHX polypeptides are particularly useful in producing transgenic
plants
which have increased salt tolerance compared to a wild type plant.
It will also be apparent that there are polypeptide and nucleic acid molecules
from other organisms, such as yeast, microorganisms, fish, birds or mammals,
that are
similar to PNHX polypeptides and nucleic acid molecules. The entire group of
Na'/H+
transporter polypeptides and nucleic acid molecules that are capable of
increasing salt
tolerance in a cell (including PNHX and AtNHX polypeptides and nucleic acid
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molecules) are collectively referred to as ("TNHX polypeptides" and "TNHX
nucleic acid
molecules"). TNHX polypeptides are Na+/H+ transporters that are capable of
increasing
salt tolerance in a cell, preferably a plant cell, because they extrude
monovalent cations
(preferably potassium ions or lithium ions, most preferably sodium ions) out
of the
cytosol.
The role of TNHX and PNHX nucleic acid molecules and polypeptides in
maintaining salt tolerance was not shown before this invention. The ability of
these
compounds to increase salt tolerance of transgenic host cells (particularly
plant cells)
and transgenic plants compared to wild type cells and plants was unknown.
PNHX and TNHX polypeptides need not necessarily have the primary function of
providing salt tolerance. All nucleotides and polypeptides which are suitable
for use in
the methods of the invention, such as the preparation of transgenic host cells
or
transgenic plants, are included within the scope of the invention. Genomic
clones or
cDNA clones are preferred for preparation of transgenic cells and plants.
In a preferred embodiment, the invention relates to cDNAs encoding Na+/H+
exchangers from Arabidopsis thaliana. The cDNA sequences and the corresponding
amino acid sequences for AtNHX1, AtNHX2, AtNHX3 and AtNHX4 are presented in
Figure 1. AtNHX1 and AtNHX2 are homologs that are physically located at
different
places in the genome. The invention also includes splice variants of the
nucleic acid
molecules as well as polypeptides produced from the molecules. For example,
AtNHX3
and AtNHX4 are homologs of AtNHX1 and AtNHX2. AtNHX3 and AtNHX4 are identical
for a long sequence beginning at the N-terminus. This indicates that the
difference in
sequence at the C-terminus is due to alternative splicing of a nucleic acid
molecule (also
known as splicing variants). This allows a single nucleic acid molecule to
produce
varying polypeptides.
Characterization of Salt Tolerance Nucleic Acid Molecules and Polypeptides
The longest open reading frame of 1614 base pairs in AtNHX1 encodes a
poiypeptide of 538 amino acids with a predicted molecular weight ("MW') of
about 60
Kda. A comparison of this full length cDNA with the Arabidopsis genome
sequence (A-
TM021804.4) revealed the presence of 13 introns and 14 exons. This polypeptide
encoded by the open reading frame was about 19% larger than the sequence
predicted
by the Arabidopsis genomic sequence (A_TM021 B04.4). This sequence encodes the
full length exchanger given that the cDNA region immediately upstream of the
start
codon contains predicted stop codons in all three reading frames. In addition,
a
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transcript of approximately 2 kb, which corresponds roughly in size to the
predicted
mRNA for AtNHX1, was observed on RNA blots. Based on the amino acid sequence
of
AtNHX1, 12 transmembrane domains are predicted, a conserved amiloride-binding
domain is present, and a relatively hydrophilic C-terminal region is also
predicted.
AtNHX1 shows some similarity at the amino acid level to Na+/H'' exchangers
isolated
from a variety of organisms ranging from yeast (about 27% identity) to humans
(about
20%). A second salt tolerance cDNA and polypeptide, AtNHX2, was obtained from
Arabidopsis thaliana (Figure 1(b)). We characterized a third salt tolerance
nucleic acid
molecule, AtNHX3, by obtaining 5' and 3' cDNA and N-terminal and C-terminal
sequences from Arabidopsis thaliana (Figure 1(c)). In one variation, the
invention
includes DNA sequences (and the corresponding polypeptide) including at least
one of
the sequences shown in figure 1(c) in a nucleic acid molecule of preferably
about: less
than 1000 base pairs, less than 1250 base pairs, less than 1500 base pairs,
less than
1750 base pairs, less than 2000 base pairs, less than 2250 base pairs, less
than 2500
base pairs, less than 2750 base pairs or less than 3000 base pairs. We also
identified
the full AtNHX3 sequence (Figure 1(d)). A fourth sequence, AtNHX4, was also
identified (figure .1(e)).
The coding regions of the nucleic acid molecules are as follows:
Table I
Nucleic Acid Molecule Start Nucleotide End Nucleotide
AtNHX1 286 1902
AtNHX2 61 1707
AtNHX3 67 1024
AtNHX4 55 813
It will be apparent that these may be varied, for example, by shortening the
5'
untranslated region or shortening the nucleic acid molecule so that the end
nucleotide is
in a different position.
The discussion of the nucleic acid molecules, sequence identity, hybridization
and other aspects of nucleic acid molecules included within the scope of the
invention is
intended to be applicable to either the entire nucleic acid molecules in
figures 1(a), (b),
(d) and (e) and the coding regions of these molecules, shown in Table 1. One
may use
the entire molecule in figure 1 or only the coding region. Other possible
modifications to
the sequence will also be apparent.
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Southern Blot Analysis (figure 3) suggests that AtNHX1 is likely present as a
single copy gene in Arabidopsis. A Northern blot (figure 4) showed that AtNHX
polypeptide (particularly AtNHX1) was expressed in all tissues examined (root,
shoot
(shoot includes leaves and stems), flower, inflorescence stem).
Function of Salt Tolerance Nucleic Acid Molecules
The polypeptides of the invention allow the extrusion of monovalent cations
(preferably potassium ions or lithium ions, most preferably sodium ions) from
the
cytosol, which in this application preferably refers to the transport and
accumulation of
sodium ions into the vacuoles or into the extracellular space (outside of the
cell), thus
providing the most important trait for salt tolerance in plants. Antiport
polypeptides from
organisms other than plants have shown different specificity for monovalent
ions (e.g.
D.G. Warnock, A.S. Pollock, "Sodium Proton Exchange in Epithelial Cells",
pages 77-90,
in S. Grinstein ed. Sodium Proton Exchange, (1987, CRC Press, USA).) TNHX and
PNHX transporters will also show different specificity between transporters.
The nucleic
acid molecules of the invention allow the engineering of salt tolerant plants
by
transformation of crops with this nucleic acid molecule under the control of
constitutively
active promoters or under the control of conditionally-inducible promoters.
The resulting
expression or overexpression of these nucleic acid molecules confers increased
salt
tolerance in plants grown in soil, solid, semi-solid medium or hydroponically.
The PNHX Nucleic Acid Molecule and Polypeptide is Conserved in Plants
Sequence Identity
This is the first isolation of a nucleic acid molecule encoding a Na+/Hf
exchanger
from plant species. It is widely known amongst those skilled in the art that
Arabidopsis
thaliana is a model plant for many plant species. Nucleic acid sequences
having
sequence identity to the AtNHX sequences are found in other plants, in
particular
halophytes such as Beta Vulgaris and Atriplex (see Examples 2 and 7).
Sequences
from Arabidopsis thaliana and other plants are collectively referred to as
"PNHX" nucleic
acid sequences and polypeptides. We isolate PNHX nucleic acid molecules from
plants
having nucleic acid molecules that are similar to those in Arabidopsis
thaliana, such as
beet, tomato, rice, cucumber, radish and other plants as in Table 5 and using
techniques described in this application. The invention includes methods of
isolating
these nucleic acid molecules and polypeptides as well as methods of using
these
nucleic acid molecules and polypeptides according to the methods described in
this
application, for example those used with respect to AtNHX.
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Table 2 below shows several sequences with sequence identity and sequence
similarity to the AtNHX polypeptides. Where polypeptides are shown, a suitable
corresponding DNA encoding the polypeptide will be apparent. These sequences
code
for polypeptides similar to portions of AtNHX polypeptides. The sequences in
Table 2
are useful to make probes to identity full length sequences or fragments (from
the listed
species or other species). One skilled in the art would be able to design a
probe based
on a polypeptide or peptide fragment. The invention includes nucleic acid
molecules of
about: 10 to 50 nucleotides, 50 to 200 nucleotides, 200 to 500 nucleotides,
500 to 1000
nucleotides, 1000 to 1500 nucleotides, 1500 to 1700 nucleotides, 1700 to 2000
nucleotides, 2000 to 2500 nucleotides or at least 2500 nucleotides and which
include all
or part of the sequences (or corresponding nucleic acid molecule) in Table 2.
The
invention also includes peptides and polypeptides of about: 10 to 50 amino
acids, 50 to
200 amino acids, 200 to 500 amino acids, 500 to 750 amino acids or at least
750 amino
acids which encode all or part of the polypeptides in Table 2 (wherein the
polypeptide is
produced according to a reading frame aligned with an AtNHX polypeptide).
Possible
modifications to these sequences will also be apparent. The polypeptide and
nucleic
acid molecules are also useful in research experiments or in bioinformatics to
locate
other sequences. The nucleic acid molecules and polypeptides preferably
provide
Na+/H+ transporter activity and are capable of moving monovalent cations from
the
cytosol of the cell into vacuoles or the extracellular space (in this
application,
extracellular space refers to the space outside a cell in an organism or the
space
outside a cultured cell).
Table 2
Organism GenBank Accession No.
Yeast (S. pombe) (Fig. 8(a)) 3850064
Yeast (Saccharomyces cervisae) (Fig. 8(b)) 927695
Rice EST (Fig. 8(c)) C 91832
Rice EST (Fig. 8(d)) C 91861
Rice EST (Fig. 8(e)) AV032544
Medicago Trunculata EST (Fig. 8(f)) AA660573
Hordeum Vulgare STS (Fig. 8g)) L 44032
As shown in Table 3 below, many nucleic acid molecules identified in
Arabidopsis thaliana have striking DNA sequence similarity to nucleic acid
molecules
encoding the homologous polypeptide in other plant species. Using the
techniques
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WO 99/47679 PCT/CA99/00219
described in this application and others known in the art, it will be apparent
that the
nucleic acid molecule encoding the homologous Na+/H+ exchanger in other plant
species including, but not limited to plants of agricultural and commercial
interest, will
have DNA sequence identity (homology) at least about > 17%, >20%, >25%, >35%
to a
DNA sequence shown in figure 1 or 5 (or a partial sequence thereof). Some
plants
species may have DNA with a sequence identity (homology) at least about: >50%,
>60%, >70%, >80% or >90% more preferably at least about >95%, >99% or >99.5%,
to
a DNA sequence in figure 1 or 5 (or a partial sequence thereof). The invention
also
includes modified nucleic acid molecules from plants other than Arabidopsis
thaliana
which have sequence identity at least about: > 17%, >20%, >25%, >35%, >50%,
>60%,
>70%, >80% or >90% more preferably at least about >95%, >99% or >99.5%, to an
AtNHX sequence in figure 1 or 5 (or a partial sequence thereof). Modified
nucleic acid
molecules are discussed below. Preferably about 1, 2, 3, 4, 5, 6 to 10, 10 to
25, 26 to
50 or 51 to 100, or 101 to 250 nucleotides or amino acids are modified.
Sequence
identity is most preferably calculated as the number of identical amino acid
residues
expressed as a percentage of the length of the shorter of the two sequences in
a
pairwise alignment. The pairwise alignment is constructed preferably using the
Clustal
W program preferably using the following parameter settings: fixed gap
penalty=1 0,
floating gap penalty=l 0, protein weight matrix=BLOSUM62. For example, if a
nucleotide sequence (called "Sequence A") has 90% identity to a portion of the
nucleotide sequence in Figure 1(a), then Sequence A will be identical to the
referenced
portion of the nucleotide sequence in Figure 8, except that Sequence A may
include up
to 10 point mutations, such as substitutions with other nucleotides, per each
100
nucleotides of the referenced portion of the nucleotide sequence in Figure 8.
Polypeptides having sequence identity may be similarly identified.
The invention also includes nucleic acid molecules encoding polypeptides
having
sequence similarity taking into account conservative amino acid substitutions.
Sequence similarity (and preferred percentages) are discussed below.
It will be apparent that nucleic acid molecule encoding the homologous Na+/H+
exchanger in other species (preferably plants) including, but not limited to
plants of
agricultural and commercial interest, will hybridize to all or part of a
sequence in figure 1
or 5 (or a partial sequence thereof) under low, moderate (also called
intermediate
conditions) or high stringency conditions. Preferred hybridization conditions
are
described below.
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WO 99/47679 PCT/CA99/00219
The invention includes the nucleic acid molecules from other plants as well as
methods of obtaining the nucleic acid molecules by, for example, screening a
cDNA
library or other DNA collection with a probe of the invention (such as a probe
comprising
at least about: 10 or preferably at least 15 or 30 nucleotides of AtNHX1,
AtNHX2,
AtNHX3 or AtNHX4 or a sequence in figure 5) and detecting the presence of a
TNHX or
PNHX nucleic acid molecule. Another method involves comparing the AtNHX
sequences (eg in figure 1 or 5) to other sequences, for example using
bioinformatics
techniques such as database searches or alignment strategies, and detecting
the
presence of a TNHX or PNHX nucleic acid molecule or polypeptide. The invention
includes the nucleic acid molecule and/or polypeptide obtained according to
the
methods of the invention. The invention also includes methods of using the
nucleic acid
molecules, for example to make probes, in research experiments or to transform
host
cells or make transgenic plants. These methods are as described below.
The polypeptides encoded by the homologous TNHX or PNHX nucleic acid
molecules in other species will have amino acid sequence identity. The
preferred
percentage of sequence identity for sequences of the invention includes
sequences
having identity of at least about: 30% to AtNHX1, 31% to AtNHX2, 36% to
AtNHX3, and
36% to AtNHX4. Sequence identity may be at least about: >20%, >25%, >28%,
>30%,
>35%, >40%, >50% to an amino acid sequence shown in figure 1 or 5 (or a
partial
sequence thereof). Some polypeptides may have a sequence identity of at least
about:
>60%, >70%, >80% or >90%, more preferably at least about: >95%, >99% or >99.5%
to
an amino acid sequence in figure 1 or 5 (or a partial sequence thereof).
Identity is
calculated according to methods known in the art. Sequence identity is most
preferably
assessed by the Clustal W program. The invention also includes modified
polypeptides
from plants which have sequence identity at least about: >20%, >25%, >28%,
>30%,
>35%, >40%, >50%, >60%, >70%, >80% or >90% more preferably at least about
>95%, >99% or >99.5%, to an AtNHX sequence in figure 1 or 5 (or a partial
sequence
thereof). Modified polypeptides molecules are discussed below. Preferably
about: 1, 2,
3, 4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides
or amino acids
are modified.
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Table 3
Plant Vacuolar H`-PPiase (vacuolar pvrophosphatase)
Polypeptide DNA
Arabidopsis (Accession # 282878) 100% 100%
Beet (Accession # 485742) 88.7% 72.8%
Tobacco (Accession # 1076627) 89.9% 68.4%
Rice (Accession # 1747296) 85% 70.4%
Tonoplast Intrinsic Polypeptide (water channel)
Polypeptide DNA
Arabidopsis (Accession # X63551) 100% 100%
Curcubita (Cucumber) (Accession # D45078) 66.5% 39.1%
Raphanus (radish) (Accession # D84669) 56.7% 37.4%
Helianthus (Accession # X95951) 50.4% 35.2%
High Affinity Ammonium Transporter
Polypeptide DNA
Arabidopsis (Accession # X75879) 100% 100%
Tomato (Accession # X95098) 73.5% 62.9%
Rice (Accession # AF001505) 66.6% 58.1%
Nucleic Acid Molecules and Polypeptides Similar to AtNHX
Those skilled in the art will recognize that the nucleic acid molecule
sequences
in figure 1(a), (b), (d) and (e) are not the only sequences which may be used
to provide
increased salt tolerance in plants. The genetic code is degenerate so other
nucleic acid
molecules which encode a polypeptide identical to an amino acid sequence in
figure
1(a), (b), (d) or (e) may also be used. The sequence of the other nucleic acid
molecules
of this invention may also be varied without changing the polypeptide encoded
by the
sequence. Consequently, the nucleic acid molecule constructs described below
and in
the accompanying examples for the preferred nucleic acid molecules, vectors,
and
transformants of the invention are merely illustrative and are not intended to
limit the
scope of the invention.
The sequences of the invention can be prepared according to numerous
techniques. The invention is not limited to any particular preparation means.
For
example, the nucleic acid molecules of the invention can be produced by cDNA
cloning,
genomic cloning, DNA synthesis, polymerase chain reaction (PCR) technology, or
a
combination of these approaches ([31] or Current Protocols in Molecular
Biology (F. M.
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WO 99/47679 PCT/CA99/00219
Ausbel et al., 1989).). Sequences may be synthesized using well known methods
and
equipment, such as automated synthesizers. Nucleic acid molecules may be
amplified
by the polymerase chain reaction. Polypeptides may,. for example, be
synthesized or
produced recombinantly.
Sequence Identity
The invention includes modified nucleic acid molecules with a sequence
identity
at least about: >17%, >20%, >30%, >40%, >50%, >60%, >70%, >80% or >90% more
preferably at least about >95%, >99% or >99.5%, to a DNA sequence in figure 1
or 5
(or a partial sequence thereof). Preferably about 1, 2, 3, 4, 5, 6 to 10, 10
to 25, 26 to 50
or 51 to 100, or 101 to 250 nucleotides or amino acids are modified. Identity
is
calculated according to methods known in the art. Sequence identity is most
preferably
assessed by the Clustal W program. For example, if a nucleotide sequence
(called
"Sequence A") has 90% identity to a portion of the nucleotide sequence in
Figure 1(a),
then Sequence A will be identical to the referenced portion of the nucleotide
sequence
in Figure 1, except that Sequence A may include up to 10 point mutations, such
as
deletions or substitutions with other nucleotides, per each 100 nucleotide of
the
referenced portion of the nucleotide sequence in Figure 1. Nucleotide
sequences
functionally equivalent to the PNHX or AtNHX sequences can occur in a variety
of forms
as described below. Polypeptides having sequence identity may be similarly
identified.
The polypeptides encoded by the homologous NHX, PNHX Na+/H+ exchange
nucleic acid molecule in other species will have amino acid sequence identity
(also
known as homology) at least about: >20%, >25%, >28%, >30%, >40% or >50% to an
amino acid sequence shown in figure 1 or 5 (or a partial sequence thereof).
Some
plants species may have polypeptides with a sequence identity (homology) of at
least
about: >60%, >70%, >80% or >90%, more preferably at least about: >95%, >99% or
>99.5% to all or part of an amino acid sequence in figure 1 or 5 (or a partial
sequence
thereof). Identity is calculated according to methods known in the art.
Sequence identity
is most preferably assessed by the Clustal W program. Preferably about: 1, 2,
3, 4, 5, 6
to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides or amino
acids are
modified.
The invention includes nucleic acid molecules with mutations that cause an
amino acid change in a portion of the polypeptide not involved in providing
salt tolerance
and ion transport or an amino acid change in a portion of the polypeptide
involved in
providing salt tolerance so that the mutation increases or decreases the
activity of the
polypeptide.
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Hybridization
Other functional equivalent forms of the AtNHX nucleic acid molecules encoding
nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA
hybridization
techniques. These nucleic acid molecules and the AtNHX sequences can be
modified
without significantly affecting their activity.
The present invention also includes nucleic acid molecules that hybridize to
one
or more of the sequences in figure 1 or 5 (or a partial sequence thereof) or
their
complementary sequences, and that encode expression for peptides or
polypeptides
exhibiting substantially equivalent activity as that of an AtNHX polypeptide
produced by
the DNA in figure 1 or their variants. Such nucleic acid molecules preferably
hybridize
to the sequences under low, moderate (intermediate), or high stringency
conditions.
(see Sambrook et al. (Most recent edition) Molecular Cloning: A Laboratory
Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Preferable
hybridization conditions are about those in Table 4.
The present invention also includes nucleic acid molecules from any source,
whether modified or not, that hybridize to genomic DNA, cDNA, or synthetic DNA
molecules that encode the amino acid sequence of an AtNHX polypeptide, or
genetically degenerate forms, under salt and temperature conditions equivalent
to those
described in this application, and that code for a peptide, polypeptide or
polypeptide that
has Na+/H+ transporter activity. Preferably the polypeptide has the same or
similar
activity as that of an AtNHX polypeptide. The nucleic acid molecules may
encode
TNHX or PNHX polypeptides. A nucleic acid molecule described above is
considered to
be functionally equivalent to an AtNHX nucleic acid molecule (and thereby
having
Na+/H+ transporter activity) of the present invention if the polypeptide
produced by the
nucleic acid molecule displays the following characteristics: the polypeptide
mediates
the proton-dependent sodium transport and sodium-dependent proton transport in
intact
cells, isolated organelles and purified membrane vesicles. These sodium/proton
movements should be higher (preferably at least about 50% higher and most
preferably
at least about 100% higher) than the proton movements observed in the presence
of a
background of potassium ions and/or other monovalent cations (i.e. rubidium,
cesium,
etc., but most preferably not lithium) (13,14).
The invention also includes nucleic acid molecules and polypeptides having
sequence similarity taking into account conservative amino acid substitutions.
Sequence similarity (and preferred percentages) are discussed below.
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Modifications to Nucleic Acid Molecule or Polynertide Sequence
Changes in the nucleotide sequence which result in production of a chemically
equivalent or chemically similar amino acid sequences are included within the
scope of
the invention. Variants of the polypeptides of the invention may occur
naturally, for
example, by mutation, or may be made, for example, with polypeptide
engineering
techniques such as site directed mutagenesis, which are well known in the art
for
substitution of amino acids. For example, a hydrophobic residue, such as
glycine can
be substituted for another hydrophobic residue such as alanine. An alanine
residue
may be substituted with a more hydrophobic residue such as leucine, valine or
isoleucine. A negatively charged amino acid such as aspartic acid may be
substituted
for glutamic acid. A positively charged amino acid such as lysine may be
substituted for
another positively charged amino acid such as arginine.
Therefore, the invention includes polypeptides having conservative changes or
substitutions in amino acid sequences. Conservative substitutions insert one
or more
amino acids which have similar chemical properties as the replaced amino
acids. The
invention includes sequences where conservative substitutions are made that do
not
destroy Na+/H+ transporter activity of the transporter polypeptide. The
preferred
percentage of sequence similarity for sequences of the invention includes
sequences
having at least about: 48% similarity to AtNHX1, 48% similarity to AtNHX2, 56%
similarity to AtNHX3, and 56% similarity to AtNHX4. The similarity may also be
at least
about: 60% similarity, 75% similarity, 80% similarity, 90% similarity, 95%
similarity, 97%
similarity, 98% similarity, 99% similarity, or more preferably at least about
99.5%
similarity, wherein the polypeptide Na+/H+ has transporter activity. The
invention also
includes nucleic acid molecules encoding polypeptides, with the polypeptides
having at
least about: at least about: 48% similarity to AtNHX1, 48% similarity to
AtNHX2, 56%
similarity to AtNHX3, and 56% similarity to AtNHX4. The similarity may also be
at least
about: 60% similarity, 75% similarity, 80% similarity, 90% similarity, 95%
similarity, 97%
similarity, 98% similarity, 99% similarity, or more preferably at least about
99.5%
similarity, wherein the polypeptide Na+/H+ has transporter activity, to an
amino acid
sequence in figure 1 or 5 (or a partial sequence thereof) considering
conservative amino
acid changes, wherein the polypeptide has Na+/H+ transporter activity.
Sequence
similarity is preferably calculated as the number of similar amino acids in a
pairwise
alignment expressed as a percentage of the shorter of the two sequences in the
alignment. The pairwise alignment is preferably constructed using the Clustal
W
program, using the following parameter settings: fixed gap penalty= 10,
floating gap
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WO 99/47679 PCT/CA99/00219
penalty=l0, protein weight matrix=BLOSUM62. Similar amino acids in a pairwise
alignment are those pairs of amino acids which have positive alignment scores
defined
in the preferred protein weight matrix (BLOSUM62). The protein weight matrix
BLOSUM62 is considered appropriate for the comparisons described here by those
skilled in the art of bioinformatics. (The reference for the clustal w program
(algorithm) is
Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting,
positions-specific gap penalties and weight matrix choice. Nucleic Acids
Research,
22:4673-4680; and the reference for BLOSUM62 scoring matrix is Henikoff, S.
and
Henikoff, J.G. (1993) Performance evaluation of amino acid substitution
matrices.
Proteins, 7:49-61.)
Polypeptides comprising one or more d-amino acids are contemplated within the
invention. Also contemplated are polypeptides where one or more amino acids
are
acetylated at the N-terminus. Those of skill in the art recognize that a
variety of
techniques are available for constructing polypeptide mimetics with the same
or similar
desired biological activity (Na'/H+ transporter activity) as the corresponding
polypeptide
compound of the invention but with more favorable activity than the
polypeptide with
respect to solubility, stability, and/or susceptibility to hydrolysis and
proteolysis. See, for
example, Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989). Examples
of
polypeptide mimetics are described in U.S. Patent Nos. 5,643,873. Other
patents
describing how to make and use mimetics include, for example in, 5,786,322,
5,767,075, 5,763,571, 5,753,226, 5,683,983, 5,677,280, 5,672,584, 5,668,110,
5,654,276, 5,643,873. Mimetics of the polypeptides of the invention may also
be made
according to other techniques known in the art. For example, by treating a
polypeptide
of the invention with an agent that chemically alters a side group by
converting a
hydrogen group to another group such as a hydroxy or amino group. Mimetics
preferably include sequences that are either entirely made of amino acids or
sequences
that are hybrids including amino acids and modified amino acids or other
organic
molecules.
The invention also includes hybrid nucleic acid molecules and polypeptides,
for
example where a nucleotide sequence from one species of plant is combined with
a
nucleotide sequence from another sequence of plant, mammal or yeast to produce
a
fusion polypeptide. The invention includes a fusion protein having at least
two
components, wherein a first component of the fusion protein comprises a
polypeptide of
the invention, preferably a full length AtNHX polypeptide. The second
component of the
CA 02323756 2000-09-18
WO 99/47679 PCT/CA99/00219
fusion protein preferably comprises a tag, for example GST, an epitope tag or
an
enzyme. The fusion protein may comprise lacZ.
The invention also includes polypeptide fragments of the polypeptides of the
invention which may be used to confer salt tolerance if the fragments retain
Na'/H'
transporter activity. The invention also includes polypeptides fragments of
the
polypeptides of the invention which may be used as a research tool to
characterize the
polypeptide or its activity. Such polypeptides preferably consist of at least
5 amino
acids. In preferred embodiments, they may consist of 6 to 10, 11 to 15, 16 to
25, 26 to
50, 51 to 75,76 to 100 or 101 to 250 amino acids of the polypeptides of the
invention (or
longer amino acid sequences). The fragments preferably have sodium/proton
transporter activity. Fragments may include sequences with one or more amino
acids
removed, for example, C-terminus amino acids in an AtNHX sequence.
The invention also includes a composition comprising all or part of an
isolated
TNHX or PNHX (preferably AtNHX) nucleic acid molecule of the invention and a
carrier,
preferably in a composition for plant transformation. The invention also
includes a
composition comprising an isolated TNHX or PNHX polypeptide (preferably AtNHX)
and
a carrier, preferably for studying polypeptide activity.
Recombinant Nucleic Acid Molecules
The invention also includes recombinant nucleic acid molecules comprising a
nucleic acid molecule of the invention and a promoter sequence, operatively
linked so
that the promoter enhances transcription of the nucleic acid molecule in a
host cell (the
nucleic acid molecules of the invention may be used in an isolated native gene
or a
chimeric gene (for example, where a nucleic acid molecule coding region is
connected
to one or more heterologous sequences to form a gene). The promoter sequence
is
preferably a constitutive promoter sequence or an inducible promoter sequence,
operatively linked so that the promoter enhances transcription of the DNA
molecule in a
host cell. The promoter may be of a type not naturally associated with the
cell.
Transcription is enhanced with promoters known in the art such as the "Super-
promoter"
[20] or the 35S promoter of cauliflower mosaic virus [21].
Inducible promoters are also used. These include:
a) drought- and ABA-inducible promoters which may include ABA-
responsive elements [22,23]
b) heat shock-inducible promoters which may contain HSEs (heat shock
elements) as well as CCAAT box sequences [24]
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c) salt-inducible promoters which may include AT and PR elements [25]
d) Copper-inducible promoter that includes ACE1 binding sites [26]
e) steroid-inducible promoter that includes the glucocorticoid response
element along with an expression vector coding for a mammalian steroid
receptor [27].
In addition, tissue specific expression is achieved with the use of tissue-
specific
promoters such as, the Fd (Ferredoxin) promoter that mediates high levels of
expression in green leaves [28] and peroxidase promoter for root-specific
expression
[29]. These promoters vary in their transcription initiation rate and/or
efficiency.
A recombinant nucleic acid molecule for conferring salt tolerance may also
contain suitable transcriptional or translational regulatory elements.
Suitable regulatory
elements may be derived from a variety of sources, and they may be readily
selected by
one with ordinary skill in the art. Examples of regulatory elements include: a
transcriptional promoter and enhancer or RNA polymerase binding sequence, a
ribosomal
binding sequence, including a translation initiation signal. Additionally,
depending on the
vector employed, other genetic elements, such as selectable markers, may be
incorporated into the recombinant molecule. Markers facilitate the selection
of a
transformed host cell. Such markers include genes associated with temperature
sensitivity, drug resistance, or enzymes associated with phenotypic
characteristics of
the host organisms.
Nucleic acid molecule expression levels are controlled with a transcription
initiation region that regulates transcription of the nucleic acid molecule or
nucleic acid
molecule fragment of interest in a plant, bacterial or yeast cell. The
transcription
initiation region may be part of the construct or the expression vector. The
transcription
initiation domain or promoter includes an RNA polymerase binding site and an
mRNA
initiation site. Other regulatory regions that may be used include an enhancer
domain
and a termination region. The regulatory elements described above may be from
animal, plant, yeast, bacterial, fungal, viral or other sources, including
synthetically
produced elements and mutated elements.
Methods of modifying DNA and polypeptides, preparing recombinant nucleic acid
molecules and vectors, transformation of cells, expression of polypeptides are
known in
the art. For guidance, one may consult the following US patent nos. 5,840,537,
5,850,025, 5,858,719, 5,710,018, 5,792,851, 5,851,788, 5,759,788, 5,840,530,
5,789,202, 5,871,983, 5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693,
5,847,258, 5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211,
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5,767,375, 5,750,848, 5,859,337, 5,563,246, 5,346,815, and W09713843. Many of
these patents also provide guidance with respect to experimental assays,
probes and
antibodies, methods, transformation of host cells and regeneration of plants,
which are
described below.
Host Cells Including a Salt Tolerance Nucleic Acid Molecule
In a preferred embodiment of the invention, a plant or yeast cell is
transformed
with a nucleic acid molecule of the invention or a fragment of a nucleic acid
molecule
and inserted in a vector.
Another embodiment of the invention relates to a method of transforming a host
cell with a nucleic acid molecule of the invention or a fragment of a nucleic
acid
molecule, inserted in a vector. The invention also includes a vector
comprising a
nucleic acid molecule of the invention. The TNHX, PNHX and AtNHX nucleic acid
molecules can be cloned into a variety of vectors by means that are well known
in the
art. The recombinant nucleic acid molecule may be inserted at a site in the
vector
created by restriction enzymes. A number of suitable vectors may be used,
including
cosmids, plasmids, bacteriophage, baculoviruses and viruses. Suitable vectors
are
capable of reproducing themselves and transforming a host cell. The invention
also
relates to a method of expressing polypeptides in the host cells. A nucleic
acid
molecule of the invention may be used to transform virtually any type of
plant, including
both monocots and dicots. The expression host may be any cell capable of
expressing
TNHX, PNHX, such as a cell selected from the group consisting of a seed (where
appropriate), plant cell, bacterium, yeast, fungus, protozoa, algae, animal
and animal
cell.
Levels of nucleic acid molecule expression may be controlled with nucleic acid
molecules or nucleic acid molecule fragments that code for anti-sense RNA
inserted in
the vectors described above.
Agrobacterium fumefaciens-mediated transformation, particle-bombardment-
mediated transformation, direct uptake, microinjection, coprecipitation and
electroporation-mediated nucleic acid molecule transfer are useful to transfer
a Na+/H+
transporter nucleic acid molecule into seeds (where appropriate) or host
cells, preferably
plant cells, depending upon the plant species. The invention also includes a
method for
constructing a host cell capable of expressing a nucleic acid molecule of the
invention,
the method comprising introducing into said host cell a vector of the
invention. The
genome of the host cell may or may not also include a functional TNHX or PNHX
gene.
The invention also includes a method for expressing a TNHX or PNHX transporter
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polypeptide in the host cell or a plant, plant part, seed or plant cell of the
invention, the
method comprising culturing the host cell under conditions suitable for gene
expression.
The method preferably also includes recovering the expressed polypeptide from
the
culture.
The invention includes the host cell comprising the recombinant nucleic acid
molecule and vector as well as progeny of the cell. Preferred host cells are
fungal cells,
yeast cells, bacterial cells, mammalian cells, bird cells, reptile cells,
amphibious cells,
microorganism cells and plant cells. Host cells may be cultured in
conventional nutrient
media. The media may be modified as appropriate for inducing promoters,
amplifying
genes or selecting transformants. The culture conditions, such as temperature,
composition and pH will be apparent. After transformation, transformants may
be
identified on the basis of a selectable phenotype. A selectable phenotype can
be
conferred by a selectable marker in the vector.
Transgenic Plants and Seeds
Plant cells are useful to produce tissue cultures, seeds or whole plants. The
invention includes a plant, plant part, seed, or progeny thereof including a
host cell
transformed with a PNHX nucleic acid molecule. The plant part is preferably a
leaf, a
stem, a flower, a root, a seed or a tuber.
The invention includes a transformed (transgenic) plant having increased salt
tolerance, the transformed plant containing a nucleic acid molecule sequence
encoding
for Na+/H+ transporter polypeptide activity and the nucleic acid molecule
sequence
having been introduced into the plant by transformation under conditions
whereby the
transformed plant expresses a Na+/H+ transporter in active form.
The methods and reagents for producing mature plants from cells are known in
the art. The invention includes a method of producing a genetically
transformed plant
which expresses PNHX or TNHX polypeptide by regenerating a genetically
transformed
plant from the plant cell, seed or plant part of the invention. The invention
also includes
the transgenic plant produced according to the method. Alternatively, a plant
may be
transformed with a vector of the invention.
The invention also includes a method of preparing a plant with increased salt
tolerance, the method comprising transforming the plant with a nucleic acid
molecule
which encodes a TNHX transporter polypeptide, a PNHX transporter polypeptide
or a
polypeptide encoding a Na+/H+ transporter polypeptide capable of increasing
salt
tolerance in a cell, and recovering the transformed plant with increased salt
tolerance.
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WO 99/47679 PCT/CA99/00219
The invention also includes a method of preparing a plant with increased salt
tolerance,
the method comprising transforming a plant cell with a nucleic acid molecule
which
encodes a TNHX transporter polypeptide, a PNHX transporter polypeptide or a
polypeptide encoding a Na+/H+ transporter polypeptide capable of increasing
salt
tolerance in a cell., and producing the transformed plant with increased salt
tolerance.
Overexpression of Na+/H+ exchangers leads to an improved ability of the
transgenic plants to uptake more monovalent cations from the growth media
(soil)
leading to an increased or enhanced tissue expansion. Figure 7 shows that
transformed
plants have grown larger even where no NaCl is added to soil. Therefore, the
invention
also relates to methods of producing or growing plants with increased tissue
expansion
(this could be manifested as enhanced size, growth or growth potential and may
appear
as increased or enhanced root, crown, shoot, stem, leaf, flower size or
abundance in
comparison to a wild type plant). The methods of preparing plants that have
increased
tissue expansion are the same as the methods for preparing a plant with
increased salt
tolerance described in this application (or the methods are easily adapted, to
the extent
that there is a difference in the methods).
The plants whose cells may be transformed with a nucleic acid molecule of this
invention and used to produce transgenic plants include, but are not limited
to the
following:
Target plants:
Group I (transformable preferably via Agrobacterium tumefaciens)
Arabidopsis
Potato
Tomato
Brassica
Cotton
Sunflower
Strawberries
Spinach
Lettuce
Rice
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Group II (transformable preferably via biolistic particle delivery systems
(particle
bombardment)
Soybean
Rice
Corn
Wheat
Rye
Barley
Atriplex
Salicornia
The nucleic acid molecule may also be used with other plants such as oat,
barley, hops, sorgum, alfalfa, sunflower, alfalfa, beet, pepper, tobacco,
melon, squash,
pea, cacao, hemp, coffee plants and grape vines. Trees may also be transformed
with
the nucleic acid molecule. Such trees include, but are not limited to maple,
birch, pine,
oak and poplar. Decorative flowering plants such as carnations and roses may
also be
transformed with the nucleic acid molecule of the invention. Plants bearing
nuts such as
peanuts may also be transformed with the salt tolerance nucleic acid molecule.
A list of
preferable plants is in Table 5.
In a preferred embodiment of the invention, plant tissue cells or cultures
which
demonstrate salt tolerance are selected and plants which are salt tolerant are
regenerated from these cultures. Methods of regeneration will be apparent to
those
skilled in the art (see Examples below, also). These plants may be reproduced,
for
example by cross pollination with a plant that is salt tolerant or a plant
that is not salt
tolerant. If the plants are self-pollinated, homozygous salt tolerant progeny
may be
identified from the seeds of these plants, for example by growing the seeds in
a saline
environment, using genetic markers or using an assay for salt tolerance. Seeds
obtained from the mature plants resulting from these crossings may be planted,
grown
to sexual maturity and cross-pollinated or self-pollinated.
The nucleic acid molecule is also incorporated in some plant species by
breeding methods such as back crossing to create plants homozygous for the
salt
resistance nucleic acid molecule.
A plant line homozygous for the salt tolerance nucleic acid molecule may be
used as either a male or female parent in a cross with a plant line lacking
the salt
tolerance nucleic acid molecule to produce a hybrid plant line which is
uniformly
heterozygous for the nucleic acid molecule. Crosses between plant lines
homozygous
36
Rev \J(MI:PPA-MUEVC4F4 04 :25- 5CA 02323756 2000-09-18416 941 9443-. +49 89 25-
05-2000 - - - - - CA 009900219
for the salt resistance nucleic add molecule are fused to generate hybrid seed
homozygous for the resistance nucleic acid molecule.
The nucleic acid molecule of the invention may also be used as a marker in
transformation experiments with plants. A salt sensitive plant may be
transformed with
a salt tolerance nucleic acid molecule and a nucleic acid molecule of interest
which are
linked. Plants transformed with the nucleic acid; molecule of interest will
display
improved growth in a saline environment relative to the non-transformed
plants.
FragmentslProbes
Preferable fragments include
10 to 50, 50 to 100, 100 to 250, 250 to 500, "500 to 1000, 1000 to 1500, or
1500 or
more nucleotides of a nucleic acid molecule of the invention. A fragment may
be
generated by removing a single nucleotide from:a sequence in figure 1 or 5 (or
a partial
sequence thereof). Fragments may or may not have Na'/H+ transporter activity.
The nucleic acid molecules of the invention (including a fragment of a
sequence
in figure 1 or 5 (or a partial sequence thereof) (such as [SEQ ID NO: I], [SEQ
ID NO;31,
[SEQ ID NO:5] or [SEQ ID NO:7j) can be used as probes to detect nucleic acid
molecules according to techniques known in the art (for example, see US patent
nos.
5,792,851 and 5,851,788). The probes may be used to detect nucleic acid
molecules
that encode polypeptides similar to the polypeptides of the invention. For
example, a
probe having at least about 10 bases will hybridize to similar sequences under
stringent
hybridization conditions (Sambrook at at. 1989, Molecular Cloning, A
Laboratory
Manual, Cold Spring Harbor)-
The invention includes oligonucleotide probes made from the AtNHX sequences
described in this application or other nucleotide sequences of the invention.
The probes
may be about 10 to 30 or 15 to 30 nucleotides in length and are preferably at
least 30 or
more nucleotides. A preferred probe is 5'-TTCTTCATATATCTTTTGCCACCC-3'
(coding for the amiloride binding domain) or at least about 10 or 15
nucleotides of this
sequence. The invention also includes an oligonudeotide including at least 30
consecutive nucleotides of an AtNHX molecule in Figure 1 or 5 (or a partial
sequence
thereof). The probes are useful to identify nucleic acids encoding AtNHX,
polypeptides
and proteins other than those described in the application, as well as
peptides,
polypeptides, and proteins have Na'/H+ transporter activity and preferably
functionally
equivalent to AtNHX. The oligonucleotide probes are capable of hybridizing to
one or
37
AMENDED SHEET i
CA 02323756 2000-09-18
WO 99/47679 PCT/CA99/00219
more of the sequences shown in Figure 1 or 5 (or a partial sequence thereof)
or the
other sequences of the invention under low, moderate or high stringency
hybridization
conditions. A nucleotide sequence encoding a polypeptide of the invention may
be
isolated from other organisms by screening a library under low, moderate or
high
stringency hybridization conditions with a detectable probe (e.g. a labeled
probe). The
activity of the polypeptide encoded by the nucleotide sequence may be assessed
by
cloning and expression of the DNA. After the expression product is isolated,
the
polypeptide is assayed for Na+/H+ transporter activity as described in this
application.
Functionally equivalent AtNHX, TNHX or PNHX nucleic acid molecules from
other cells, or equivalent AtNHX, TNHX or PNHX -encoding cDNAs or synthetic
DNAs,
can also be isolated by amplification using Polymerase Chain Reaction (PCR)
methods.
Oligonucleotide primers, including degenerate primers, based on the amino acid
sequence of the sequences in Figures 1 or 5 (or a partial sequence therof) can
be
prepared and used in conjunction with PCR technology employing reverse
transcriptase
to amplify functionally equivalent DNAs from genomic or cDNA libraries of
other
organisms. Alternatively, the oligonucleotides, including degenerate
nucleotides, can be
used as probes to screen cDNA libraries.
Thus, the invention includes an oligonucleotide probe comprising all or part
of a
nucleic acid in figure 1 or 5 (or a partial sequence thereof), or a
complementary strand
thereof. The probe is preferably labeled with a detectable marker. The
invention also
includes an oligonucleotide comprising at least 10, 15 or 30 nucleotides
capable of
specifically hybridizing with a sequence of nucleic acids of the nucleotide
sequence set
forth in figure 1 or 5 (or a partial sequence thereof). The invention also
includes a single
strand DNA primer for amplification of PNHX nucleic acid, wherein the primer
is selected
from a nucleic acid sequence derived from a nucleic acid sequence in figure 1
or 5 (or a
partial sequence thereof).
The invention also includes a method for identifying nucleic acid molecules
encoding a TNHX, PNHX or AtNHX polypeptide. Techniques for performing the
methods are described in, for example, US Patent Nos. 5,851,788 and 5,858,719.
A
preferred method includes contacting a sample containing nucleic acids with an
oligonucleotide, wherein said contacting is effected under low, moderate or
high
stringency hybridization conditions, and identifying nucleic acids which
hybridize thereto.
Hybridization forms a hybridization complex. The presence of a complex
correlates with
the presence of a nucleic acid molecule encoding TNHX, plant PNHX polypeptide
or
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WO 99/47679 PCT/CA99/00219
AtNHX in the sample. In a preferred method, the nucleic acid molecules are
amplified by
the polymerase chain reaction prior to hybridization.
KITS
The invention also includes a kit for conferring increased salt tolerance to a
plant
or a host cell including a nucleic acid molecule of the invention (preferably
in a
composition fo the invention) and preferably reagents for transforming the
plant or host
cell.
The invention also includes a kit for detecting the presence of a TNHX or a
PNHX nucleic acid molecule, comprising at least one oligonucleotide of the
invention.
Kits may be prepared according to known techniques, for example, see patent
nos.
5,851,788 and 5,750,653.
Antibodies
The invention includes an isolated antibody immunoreactive with a polypeptide
of the invention (see Example 1). The antibody may be labeled with a
detectable
marker or unlabeled. The antibody is preferably a monoclonal antibody or a
polyclonal
antibody. TNHX, PNHX or AtNHX antibodies can be employed to screen organisms
containing TNHX, PNHX or AtNHX polypeptides. The antibodies are also valuable
for
immuno-purification of polypeptides from crude extracts.
The isolated antibody is preferably specifically reactive with a TNHX or PNHX
transporter, preferably an AtNHX transporter. The transporter is preferably
encoded by
a nucleic acid molecule in figure 1 (or molecules that hybridize to a molecule
in figure 1
under low, moderate or high stringency hybridization conditions or molecules
having at
least about: 17%, at least 20%, at least 25%, or at least 35% sequence
identity (or the
other preferred percentages of identity or sequence similarity described
above) to a
molecule in figure 1 or 5 (or a partial sequence thereof). The transporter is
preferably a
polypeptide in figure 1 (or polypeptides having at least about: 28%, 35%
sequence
identity (or the other preferred percentages of identity or sequence
similarity described
above) to a polypeptide in figure 1 or 5 (or a partial sequence thereof). The
antibody
preferably does not cross-react with other transporter polypeptides. The
antibody is
preferably specifically reactive with a polypeptide having an amino acid
sequence
encoded by a nucleic acid molecule set forth in figure 1 or 5 (or a partial
sequence
thereof).
Examples of the preparation and use of antibodies are provided in US Patent
Nos. 5,792,851 and 5,759,788. For other examples of methods of the preparation
and
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WO 99/47679 PCT/CA99/00219
uses of monoclonal antibodies, see US Patent Nos. 5,688,681, 5,688,657,
5,683,693,
5,667,781, 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345
and
5,496,705. Examples of the preparation and uses of polyclonal antibodies are
disclosed
in US Patent Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147.
The invention also includes methods of using the antibodies. For example, the
invention includes a method for detecting the presence of TNHX, PNHX or AtNHX
transporter polypeptide, by: a) contacting a sample containing one or more
polypeptides
with an antibody of the invention under conditions suitable for the binding of
the
antibody to polypeptides with which it is specifically reactive; b) separating
unbound
polypeptides from the antibody; and c) detecting antibody which remains bound
to one
or more of the polypeptides in the sample.
Research Tool
Cell cultures, seeds, plants and plant parts transformed with a nucleic acid
molecule of the invention are useful as research tools. For example, one may
obtain a
plant cell (or a cell line, such as an immortalized cell culture or a primary
cell culture)
that does not express AtNHX1, insert an AtNHX1 nucleic acid molecule in the
cell, and
assess the level of AtNHX1 expression and activity. Alternatively, PNHX
nucleic acid
molecules may be overexpressed in a plant that expresses a PNHX nucleic acid
molecule. In another example, experimental groups of plants may be transformed
with
vectors containing different types of PNHX nucleic acid molecules (or PNHX
nucleic
acid molecules similar to PNHX or fragments of PNHX nucleic acid molecules) to
assess the levels of protein produced, its functionality and the phenotype of
the plants
(for example, phenotype in saline soil). The polypeptides are also useful for
in vitro
analysis of TNHX, PNHX or AtNHX activity or structure. For example, the
polypeptides
produced can be used for microscopy or X-ray crystallography studies.
The TNHX, PNHX or AtNHX nucleic acid molecules and polypeptides are also
useful in assays. Assays are useful for identification and development of
compounds to
inhibit and/or enhance polypeptide function directly. For example, they are
useful in an
assay for evaluating whether test compounds are capable of acting as
antagonists for
PNHX polypeptides by: (a) culturing cells containing: a nucleic acid molecule
which
expresses PNHX polypeptides (or polypeptides having PNHX or Na+/H+ activity)
wherein the culturing is carried out in the presence of: increasing
concentrations of at
least one test compound whose ability to inhibit transport activity of PNHX
polypeptide is
sought to be determined, and a fixed concentration of salt; and (b) monitoring
in the
cells the level of salt transported out of the cytosol as a function of the
concentration of
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WO 99/47679 PCT/CA99/00219
the test compound, thereby indicating the ability of the test compound to
inhibit PNHX
transporter activity. Alternatively, the concentration of the test compound
may be fixed
and the concentration of salt may be increased.
Another experiment is an assay for evaluating whether test compounds are
capable of acting as agonists for PNHX polypeptide characterized by being able
to
transport salt across a membrane, (or polypeptides having PNHX or Na+/H+
transporter
activity) by (a) culturing cells containing: a nucleic acid molecule which
expresses PNHX
polypeptide or (or polypeptides having PNHX activity) thereof, wherein said
culturing is
carried out in the presence of: fixed concentrations of at least one test
compound whose
ability to increase or enhance salt transport activity of PNHX polypeptide is
sought to be
determined, and an increasing concentration of salt; and (b) monitoring in the
cells the
level of salt transported out of the cytosol as a function of the
concentration of the test
compound, thereby indicating the ability of the test compound compound to
increase or
enhance PNHX polypeptide activity. Alternatively, the concentration of the
test
compound may be fixed and the concentration of salt may be increased. Suitable
assays may be adapted from, for example, US patent no. 5,851,788. It is
apparent that
TNHX and AtNHX may also be used in assays.
Bioremediation
Soils containing excessive salt may be unable to grow plants in a manner
suitable for agriculture. The invention includes a method for removing salt
from a
growth medium, comprising growing a plant transformed with a nucleic acid
molecule of
the invention and expressing a salt tolerance Na+/H` transporter polypeptide
in the
growth medium for a time period sufficient for the plant root to uptake and
accumulate
salt in the root or shoot biomass. The growth medium may be a solid medium,
semi-
solid medium, liquid medium or a combination thereof. It may include soil,
sand, sludge,
compost, or artificial soil mix. The shoot (leaf or stem) or and root biomass
may be
harvested. Preferably, a sufficient portion of the shoot biomass is not
harvested and is left
in the growth media to permit continued plant growth.
Using Exogenous Agents in Combination with a Vector
The nucleic acid molecules of the invention may be used with other nucleic
acid
molecules that relate to salt tolerance, for example, osmoregulant genes. Host
cells or
plants may be transformed with these nucleic acid molecules. Osmoregulants are
disclosed, for example, in US Patent Nos. 5,563,324 and 5,639, 950.
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It will be clear to those skilled in the art that sequences in figure 1(c) and
5(a)
and (b) are also useful, for example in preparation of probes or as
experimental tools or
as antigens to which antibodies may be directed. The following Examples are
intended
to illustrate and assist in the further understanding of the invention.
Particular materials
employed, species, conditions and the like are not intended to limit the
reasonable
scope of the invention.
Example 1
Preparation of golyclonal and monoclonal antibodies.
Hydropathy profiles of the Arabidopsis Na+/H+ antiport revealed a relatively
hydrophilic domain (at the C-terminus) with possible regulatory functions. The
C-
terminus was sub-cloned into the pGEX - 2TK vector (Pharmacia) to allow the
overexpression of the C-terminus polypeptide as a GST-fusion polypeptide in E.
coli.
The fusion polypeptide was purified by glutathione-affinity chromatography and
used as
an antigen in rabbits to obtain polyclonal antibodies [30].
Monoclonal antibodies are prepared in mice hybridomas according to
established techniques [30] using the C-terminus polypeptide as described
above.
Polyclonal and monoclonal antibodies raised against other regulatory regions
of the
Arabidopsis Na+/H+ antiport are also obtained as described above. The
invention
includes the antibodies and the hybridoma which secretes the monoclonal
antibodies.
Example 2
Identification of homologous nucleic acid molecules from other plant species,
preferably
salt tolerant species.
Several experimental approaches are used to identify homologous nucleic acid
molecules from salt tolerant species. a) We screen cDNA and genomic libraires
from
sugar beets (a moderate salt-tolerant crop, also known as red beet) under low-
stringency conditions with an Arabidopsis Na+/H+ antiport cDNA as a probe [31
]; b) We
apply PCR techniques using degenerate oligonucleotide primers designed
according to
the conserved regions of the Arabidopsis Na+/H+ antiport [32]; c) We screen
cDNA
expression libraries from different plants (salt-tolerant and salt-sensitive)
using
antibodies raised against an Arabidopsis Na+/H+ antiport [31]. We also use
bioinformatics techniques to identify nucleic acid molecules. The invention
includes
methods of using such a nucleic acid molecule, for example to express a
recombinant
polypeptide in a transformed cell.
42
CA 02323756 2007-09-10
The techniques described above for isolating nucleic acid molecules from
Arabidopsis and sugar beet are used to isolate a salt tolerance nucleic acid
molecule from
Atriplex and other plants.
Example 3
Overexpression of the PNHX transporter, preferably Arabidopsis transporter
(AtNHX).
The Na+/H+ antiport is expressed in Arabidopsis plants, although the wild type
plants show impaired growth at NaCl concentrations higher than 75 mM. The
Na+/H+
antiport is overexpressed in these plants in order to improve their tolerance
to high salt
concentrations. A full length cDNA (preferably coding for the AtNHX1
polypeptide
(AtNHX2, AtNHX3 or AtNHX4) cloned from an Arabidopsis thaliana (Columbia)
seedling
cDNA library is ligated into a pBINS1 vector [33]. This vector contains a
constitutively
strong promotor ("super-promotor [20]). Also, T-DNA vectors (pBECKS) are used
[34].
Constructs containing the AtNHX1 cDNA with the full Na+/H+ antiport open
reading frame
in a sense orientation were selected by colony hybridization using the AtNHX1
as a probe
and by restriction-digest analysis using Bglll restriction endonuclease. These
constructs
are used to transform Agrobacterium tumefaciens, and these transformed
Agrobacterium
tumefaciens are used for transformation of Arabidopsis plants. The
Agrobacterium for
inoculation is grown at 28 C in a medium containing 5g/l BactoTM Beef Extract,
5g/I Bacto-
PeptoneTM, 1g/l BactoTM Yeast Extract, 240 mg MgSO4 and 5g/l sucrose. The pH
will be
adjusted to 7.2 with NaOH.
Arabidopsis seeds are washed and surface-sterilized in 5% (w/v) sodium
hypochlorite containing 0.15% (v/v) Tween-20TH. The seeds are rinsed
thoroughly with
sterile distilled water. Seed aliquots are dispensed in flasks containing 45
ml of
cocultivation medium (MS salts, 100 mM sucrose, 10 mg/I thiamine, 0.5 mg/I
pyridoxine,
0.5 mg/I nicotinic acid, 100 mg/I inositol and the pH adjusted to 6.0 with
KOH. The
flasks are incubated at 22 C under constant rotation (190 rpm) and constant
light. After
10-18 h (time needed to break clumps of seeds) 5 ml of log phase of
Agrobacterium
(OD600=0.75) carrying the AtNHX1 construct are added. Twenty-four hours
following the
inoculation, the seeds are dried by filtration and sown into pre-soaked
vermiculite. The
flats containing the seeds are irrigated as required with a half-Hoagland
solution. The
flats are covered with plastic to prevent desiccation and maintained at low
artificial
illumination. After 3 days the flats are transferred to the greenhouse (the
plastic cover
removed) under a 16/8 day/night cycle. Supplementary light is provided by high
pressure sodium vapor lights. Seven weeks after sowing, the plants are dried
thoroughly and the seeds (T2) harvested. Transformation efficiency is
estimated by
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plating 100,000 seeds (approximately 2.5 g of seeds) on agar plates containing
50 mg/I
kanamycin in a medium containing 1% (w/v) sucrose, 0.8 (w/v) agar, MS salts
and a pH
6.0 adjusted with KOH. The plates are transferred to a growth room at 25 C
under
continuous light. After 10 days the kanamycin-resistant seedlings are
transferred to new
growth medium for 2 weeks and then transferred to small pots containing
vermiculite. At
senescence (8 weeks) the seeds are collected from single plants (T3). These
seeds are
germinated and used to assess salt tolerance of the transgenic plants.
Example 4
Overexpression of TNHX or PNHX in other plants.
In a preferred method, overexpression of PNHX, preferably AtNHX1, AtNHX2,
AtNHX3 or AtNHX4, in a number of plants (potato, tomato, brassica, cotton,
sunflower,
strawberries, spinach, lettuce, rice, soybean, corn, wheat, rye, barley,
atriplex,
salicornia, and others) is achieved by Agrobacterium tumefaciens-based
transformation
and/or particle bombardment (AtNHX2, AtNHX3, AtNHX4 are also useful in this
example). The full length cDNA (coding for the AtNHX1) is ligated into the
pBINS1
vector or pBECKS (as described above) and these constructs are used to
transform
Agrobacterium tumefaciens strain LBA4404. Agrobacterium used for inoculation
is
grown as described above. Cultured cells (callus), leaf explants, shoot and
root cultures
are used as targets for transformation. The targeted tissues are co-cultivated
with the
bacteria for 1 - 2 days. Afterwards, the tissue is transferred to a growth
media containing
kanamycin. After one week the tissue is transferred to a regeneration medium
containing MS salts, 1 % sucrose, 2.5 mg/I 3-benzyladenine, 1 mg/I zeatin,
0.75% agar
and kanamycin. Weekly transfers to fresh regeneration media are performed.
In another preferred embodiment, overexpression constructs carrying the
AtNHX1 cDNA are introduced into an electro-competent Agrobacterium tumefaciens
(LBA4404) by electroporation. The Agrobacteria are plated on LB plates
containing 50
mg/L kanamycin and grown for -2 days at 30 C to select for bacteria carrying
the
overexpression constructs. One liter liquid LB+kanamycin (50mg/L) is
inoculated with a
single Agrobacterium colony selected from the LB (kanamycin 50mg/L) plates.
The
culture is grown to a minimum of OD=1 (600nm) for 2-3 days. The Agrobacteria
are then
pelleted and resuspended in 1L infiltration medium (IM - 0.5XMS salts; 0.5 g/L
MES; 5%
sucrose; 0.03% Silwet L-77). Flowering Arabidopsis plants with primary bolts
reaching
-15cm are used for the transformation procedure (T1). Pots of Arabidopsis
plants are
dunked into the IM solution containing the Agrobacteria and left submerged for
2-6
minutes. The same procedure can be repeated after 8-12 days on the same
plants.
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Plants are allowed to senesce, the plants are dried thoroughly and the seeds
harvested.
Seeds are plated on agar plates containing 25 mg/L kanamycin in a medium
containing
MS salts, 0.8% (w/v) agar adjusted to pH 6.0 with KOH. The plates are
transferred to a
growth room at 25 C under continuous light. After 10 days the kanamycin-
resistant
seedlings (T2) are transferred to small pots containing vermiculite. At
senescence (-8
weeks) the seeds are collected from single plants and plated on agar plates
containing
MS salts and 25mg/L kanamycin. After 10 days the kanamycin-resistant seedlings
(T3)
are transferred to small pots containing vermiculite. Seeds produced by these
plants
are germinated and used to assess salt tolerance of the transgenic plants.A
biolistic
particle delivery system (particle bombardment) is also used for the
overexpression of
NHX (AtNHXI, AtNHX2, AtNHX3 or AtNHX4 are useful for this example). Constructs
made using a plasmid vector preferably carrying a constitutive promoter, the
AtNHX1
open reading frame in a sense orientation and a NOS termination site are used.
Plasmid DNA is precipitated into 1.25 mg of 1-2 pm gold particles using 25 l
of 2.5 M
CaCl2 and 10 l of 0.1 M thiamine (free base). DNA-coated particles are washed
with
125 i of 100% ethanol and then resuspended in 30 pl ethanol. The samples are
sonicated to obtain an efficient dispersion, and the samples are aliquoted to
obtain
delivery disks containing 3 pg of DNA each. Particle bombardment is optimized
according to the specific tissue to be transformed. Tissue samples are placed
in Petri
dishes containing 4.5 g/l basal MS salts, 1 mg/l thiamine, 10 mg/l
myoinositol, 30 g/i
sucrose, 2.5 mg/I amphotericin and 10 mM K2HP04 at pH 5.7. After bombardment
the
petri dishes are incubated for 18 - 24 hours. Tissue is regenerated in plates
with growth
media containing the selective marker. Rooting is initiated and transformed
plants are
grown under optimal growth conditions in growth chambers. After 2 - 4 weeks
the
seedlings are transferred to new growth medium for 2 weeks and then
transferred to
small pots containing vermiculite. At senescence the seeds are collected from
single
plants. These seeds are germinated and used to assess salt tolerance of the
transgenic
plants.
Example 5
Overexpression of AtNHX1-homologs in other plants.
Overexpression of AtNHX1-homologs from other plant species, preferably salt
tolerant species (i.e., sugar beet) in other plants (potato, tomato, brassica,
cotton,
sunflower, strawberries, spinach, lettuce, rice, soybean, corn, wheat, rye,
barley,
atriplex, salicornia, and others) is achieved by Agrobacterium tumefaciens-
based
transformation and/or particle bombardment as described above (in Examples 3
and 4).
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Regeneration of the transformed plants is performed as described in Examples 3
and 4
(AtNHX2, AtNHX3 or AtNHX4).
Example 6
Expression of PNHX, AtNHX1, AtNHX1 homologs and AtNHX1 derivatives in
Saccharomyces cerevisiae.
Expression of TNHX or PNHX, preferably AtNHX1, AtNHX1 homologs (such as
AtNHX2, AtNHX3, AtNHX4), and AtNHX1 derivatives in yeast is useful to assess
and
characterize the biochemical properties of the recombinant and native
polypeptides.
Expression in yeast also facilitates the study of interactions between AtNHX1,
its
homologs and derivatives with regulatory polypeptides. We have made
conditional
expression constructs by ligating the coding region of the AtNHX1 cDNA into
two
vectors, pYES2 (Invitrogen) and pYEP434 [35]. Both constructs provide
galactose-
inducible expression, but pYES2 has a URA3 selectable marker while pYEP434 has
LEU2 as a selectable marker. Transformation by lithium acetate [36], 1994), is
followed
by selection on solid media containing amino acids appropriate for the
selection of cells
containing the transformation vector. For integrative transformation, the
YXplac series
of vectors for integrative transformation are used [37].
Example 7
Molecular characterization and functional analysis of Na/H+ exchangers from
Arabidopsis and other plants, preferably salt-tolerant (halophytes) plants.
We do molecular and biochemical characterization of the different Na+/H+
exchangers from Arabidopsis and other plants, preferably salt tolerant plants
(halophytes). We determine the expression patterns of the different
Arabidopsis
putative exchangers. Using Northern blot analysis with isoform-specific cDNA
probes
under high stringency conditions and standard molecular biology protocols, we
determine the tissue-specificity, developmental and salt-inducibility gene
expression
profiles of each isoform.
We employ common molecular biology procedures to isolate Na'/H' exchangers
from other plants (Table 5), in particular halophytes (such as Beta vulgaris,
Atriplex,
Messembryanthemum chrystalinum, etc.). We designed degenerate oligonucleotide
PCR primers, based upon highly conserved regions within Na`/H+ exchangers (one
within the amiloride-binding domain, and another within a region about 200
amino acid
residues further downstream) from Arabidopsis, yeast, mammals, and C. elegans,
to
generate a 600 - 1,000 bp DNA fragments by PCR. Sequencing of these products
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WO 99/47679 PCT/CA99/00219
revealed significant homology to AtNHXI and they are therefore being used as a
probe
to screen the different halophyte cDNA libraries to isolate the full-length
cDNAs by
standard methods. We use the nucleic acid molecules obtained in this procedure
in
methods of producing transgenic host cells and plants as described above.
We have subcloned unique regions from AtNHX1, AtNHX2 and AtNHX3
isoforms into a prokaryotic expression vector (pGEX2TK, Pharmacia) for the
production
of recombinant GST-fusion proteins that are being used for the generation of
isoform-
specific polyclonal antibodies in rabbits. Briefly, sequence-specific
oligonucleotides,
with 5' BamHl (sense strand) and 3' EcoRl (antisense strand) flanking
restriction sites,
were used for PCR-mediated amplification of the unique (partial) coding
regions from
each isoform, and the digested PCR products were ligated into EcoRl/BamHl-
digested
pGEX2TK vector. pGEX2TK plasmids containing the inserts corresponding to each
AtNHX isoform were sequenced on both strands to verify the fidelity of the PCR
reaction
and were used for expression and purification of the recombinant GST-fusion
proteins in
E.coli (BL21 pLysS) as per manufacturers instructions (Pharmacia). We follow
an
identical procedure to that described above to produce recombinant halophyte-
PNHX
GST-fusion protein in E. coll. Antibodies against the fusion proteins are
produced in
rabbits by standard procedures and their isoform-specificity are confirmed by
western
blotting using the different GST-fusion proteins. The antibodies are used in
conjunction
with subcellular membrane fractions (prepared from sucrose density gradients)
[15] from
various Arabidopsis and other plant tissues, preferably halophyte tissues and
western
blots to determine the subcellular localization of each Na'/H+ exchanger
isoform. These
localization studies assign functions to the various isoforms.
Example 8
Biochemical characterization and functional analysis of Na;/H; exchangers from
Arabidopsis and other plants, preferably salt-tolerant (halophytes) plants.
Biochemical characterization of the Na'/H+ exchanger isoforms is performed in
(1) heterologous eukaryotic expression systems (baculovirus expression system
in Sf9
insect cells, transgenic yeast); and in (ii) transgenic plants.
The use of heterologous expression systems allows the fast characterization of
the kinetic properties of each exchanger isoform (Km, V1i181%t ion
specificity). Baculovirus-
infected Sf9 cells have proven to be a useful and adaptable system for high-
level
expression of correctly folded eukaryotic membrane proteins, thus they are an
ideal tool
for the study of membrane-bound proteins. The large size of the cells,
combined with
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the relatively short time needed for the expression of the foreign plasma
membrane-
bound proteins (3-4 days) provides an excellent experimental system for the
application
of isotope exchange techniques. For expression in Sf9 insect cells, the
Invitrogen
baculovirus Sf9 insect cell system is used. Expression vector constructs
(pBluBac4.5,
Invitrogen) encoding full-length AtNHX exchanger proteins are prepared for
each AtNHX
and other PNHX isoforms using a PCR-based subcloning approach similar to that
described above for the generation of GST-fusion proteins. Initially, the
suitability of the
insect cell expression system for uptake analysis is performed using a single
AtNHX
isoform. The other PNHX isoforms are studied in a similar manner. Cultures of
Sf9
insect cells are infected with baculovirus containing expression vector
constructs
encoding the different PNHX isoforms. Infection and selection of transformants
are
performed as per manufacturer's instructions (Invitrogen). The isoform-
specific
antibodies described above aid in the assessment of recombinant protein
expression
and localization within the insect cells.
Equally important is the use of transgenic yeast as a tool for the expression
of
recombinant eukaryotic proteins, particularly because of post-translational
modifications
and targeting to endomembranes. In addition, functional complementation of
yeast
mutant strains with plant proteins is often possible. We have subcloned the
AtNHX1
cDNA into a yeast expression vector (pYES2) using a PCR-based approach as
described above. Yeast (strain w303a) have been transformed with this
construct and
expression of the recombinant plant protein is confirmed once the antiserum is
available. In addition, salt-tolerance of transformed yeast isassessed for
each AtNHX
isoform by comparing growth rates at different NaCl concentrations. Methods
for the
isolation of transport-competent plasma membranes and tonoplast and the
isolation of
intact vacuoles are performed. The kinetics of H+/Na+ exchange is measured in
intact
insect cells and yeast, intact yeast vacuoles, and isolated plasma membranes
and
tonoplast vesicles according to known methods. Na+ influx in intact cells is
monitored by
isotopic exchange using [22Na+]CI and fast-filtration techniques [17,i,ii].
Kinetics of H+-
dependent Na+ fluxes in vesicles is monitored by following the pH-dependent
fluorescent quench of acridine dyes [13,17].
The results of these kinetic characterization studies provides information
about
the ion specificity, affinity, and optimal activity conditions for each AtNHX
isoform. We
assign the activity of each isoform to the corresponding target membrane,
andwe also
determine which of the isoforms have a higher affinity for sodium. We
characterize the
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mechanisms of salt tolerance in general and tissue-specificity and
developmental
expression in particular.
In transgenic plants, expression of the different Na+/H+ antiports is verified
with
western blots using the isoform-specific antibodies described above. The
kinetics of
H'/Na+ exchange is measured in intact vacuoles, isolated plasma membranes and
tonoplast vesicles (from roots and leaves) as described above.
Example 9
Identification of positive and negative regulators of Na+/H+ antiport
activity.
Heterologous expression of plant transport molecules in Saccharomyces
cerevisiae has been used successfully in recent years in numerous studies. The
availability of yeast mutants with salt-sensitive phenotypes (generated
by'knock-outs' of
sodium transport molecules such as denal-4 - the plasma membrane Na+- ATPase
pumps) makes it an especially suitable system for the study of sodium
transport
molecules. This heterologous expression facilitates kinetic studies of the
antiport
activity in yeast cells using radiolabelled 22Na+.
Successful suppression of yeast mutants, incapable of sodium detoxification
allows for the genetic identification of positive and negative regulators of
these Na+/H+
antiports. Mutant yeast cells having a suppressed phenotype as a result of the
expression of a plant Na+/H+ antiport are transformed with an Arabidopsis cDNA
library
for the purpose of identifying particular regulators of these antiport
molecules. A
phenotype of increased sodium tolerance in yeast identifies particular
positive regulators
of the antiport activity while negative regulators are identified by a
phenotype of
decreased sodium tolerance. These phenotypes depend on the co-expression of
the
particular cDNAs identified along with that of the Na+/H+ antiport under
investigation.
Identification of essential amino acid residues regulating the activity of
Na+/H+
exchanger molecules is investigated by random mutagenesis of the antiport
molecule
which is achieved by PCR using a commercially available low fidelity Taq
enzyme. The
constructs generated are used in transforming sodium-related yeast mutants to
identify
particular Na+/H+ antiport residues that affect suppression of the mutant
yeast
phenotype. Both gain-of-function and loss-of-function mutations are examined
and
mapped to the particular mutant residue by sequencing. Gain-of-function
mutations are
of particular interest since they represent constitutive activation of the
antiport activity
allowing for increased sodium detoxification.
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Example 10
Transformation of Arabidopsis thaliana using overexpression of different
putative isoforms
and antiports from other plants, preferably salt tolerant plants and
evaluation of salt-
tolerance.
Arabidopsis represents a readily transformable model organism with the
particular advantage of having a short generation time. Agrobacterium
tumefaciens-
mediated genetic transformation is utilized for Arabidopsis (ecotype
Columbia). Studies
include the overexpression of PNHX transgenes in a wild-type background,
combined
overexpression of more than one PNHX transgene, and suppression of endogenous
PNHX expression using antisense PNHX expression. Stable transformation of
progeny
is confirmed by Southern blotting. Overexpression of transgenes, or
suppression of
expression using antisense constructs, is confirmed by Northern and western
blotting.
In all cases, salt-tolerance of transgenic plants is compared to wild-type
plants, and
control plants transformed with empty transformation vectors. Separate
transformations
are performed on Arabidopsis plants using expression vector constructs for
each of the
different AtNHX isoforms. In addition, Arabidopsis plants are transformed with
PNHX
genes from other plants, preferably salt tolerant plants in order to assess
the effect on
salt tolerance of the expression of a Na`/H' exchanger in a glycophytic plant.
For overexpression studies, full-length AtNHX1, AtNHX2, AtNHX3 and AtNHX4
cDNAs are subcloned in a sense orientation into the expression vector
containing a
"superpromoter" [20]. A PCR based subcloning strategy is used for each AtNHX
cDNA
as described above for the production of NHXGST-fusion constructs. For the
production
of vector constructs containing PNHX cDNAs in an antisense orientation,
oligonucleotides with Sall and Sacl restriction sites flanking the C-terminal
and N-
terminal PNHX regions respectively, are used for PCR amplification. All
plasmid
constructs are sequenced on both strands to confirm the fidelity of the PCR
amplification
before transformation of Agrobacterium tumefaciens (strain LBA4404). For each
PNHX-
pBISN1 construct, approximately 1L of Agrobacterium culture, grown under
antibiotic
selection at 28 C, is used for the transformation of Arabidopsis. Plants are
ready for
transformation when primary bolts are approximately 15cm. About 2 flats of
plants (~ 80
plants per flat) are used per transformation. A highly efficient, vacuum-less
infiltration
transformation method [iii] is used. Harvested Agrobacterium cultures are
resuspended
in an infiltration media containing a mild surfactant (Silwet L-77, Lehle
Seeds), and each
pot of Arabidopsis is simply submerged in the Agrobacterium for 2-6 minutes.
Plants
are thereafter drained, and returned to the growth chamber until the seeds are
ready for
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harvesting (about 4 weeks). Seeds (T1 generation) are collected and after
surface
sterilization, are plated on sterile, selective media containing kanamycin,
vernalized, and
then grown under optimal conditions. Healthy seedlings showing kanamycin
resistence
after about 7 days are transplanted to soil and the presence of the transgene
confirmed
by Southern blotting. Seeds from T1 transformants (ie T2 generation) are
harvested,
sown, and T2 plants used for Northern and western blotting to determine the
expression
patterns of the transgenes and PNHX proteins. Representative transgenic lines
(e.g.
showing low, medium, or high transgene expression) is used for studies of salt-
tolerance. A similar approach is used for transformation of Arabidopsis with
the PNHXs
from other plants.
Salt tolerance is assessed by measuring the growth rate of the plants at
increasing salt concentrations. Plant biomass, root/shoot ratios, tissue ion
content is
measured. Root and hypocotyl growth rates is measured and correlated with
tissue
water content of plants growing at different NaCl concentrations.
Example 11
Transformation of crop plants with A. thaliana and/or other exchangers under
constitutive
and inducible promoters and evaluation of salt-tolerance.
a) Agrobacterium tumefaciens-mediated transformation of crop plants
We assess whether or not homologues of the AtNHX genes exist in the plant of
choice. We use degenerate oligonucleotide PCR-primers (as described for other
plants)
and a cDNA library to isolate the full-length cDNA. The high efficiency
Agrobacterium-
mediated transformation method developed specifically for Brassica by Moloney
et al [iv]
is used to introduce and overexpress foreign nucleic acid molecules and/or
overexpress
the endogenous PNHX nucleic acid molecule in the crop plant(s). This method
takes
advantage of the fact that cut cotyledonary petioles from, which are capable
of
undergoing organogenesis (ie generating explants), are very susceptible to
Agrobacterium infection. Shortly after germination (-' 5 days) cotlyedons are
excised
and imbedded into Murashige-Skoog medium (Gibco) enriched with benyzladenine.
Expression vector constructs are prepared using a PCR-based subcloning
approach as
described above using the pCGN5059 binary plasmid (which employs the CaMV 35S
promoter to drive constitutively high expression) engineered for gentamycin
resistance
[iv] and cDNAs of the various AtNHX clones and/or the halophyte PNHX clones,
and the
choosen plant PNHX clones. Excised cotyledons are infected with Agrobecterium
cultures (strain EHA101), containing the vector construct of interest, by
brief dipping and
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then co-cultivated with the Agrobacterium for a 72h. Subsequently, cotlyedons
are
transferred to regeneration medium containing gentamycin as the selective
agent. After
explant regeneration, and subculturing, on selective media (- 4 weeks)
explants are
transferred to rooting medium and then into soil once a root mass has
developed.
Tissue samples are examined from growing plants to confirm transgene presence
by
Southern blotting as described above for the transformation of Arabidopsis.
Transformed plants (T1 generation) are allowed to flower and set seed and
these seeds
are germinated (T2) under selective conditions and transformants used for
expression
analysis of the transgenes and evaluation of salt-tolerance as described
above. Also,
biochemical analysis of the plants is performed. These include, Na`/K` ratios,
sugar,
amino acid and quaternary N-compounds. Salt-tolerance is also evaluated in
fields
trials.
b) Microprojectile bombardment-mediated transformation of crop plants.
A microprojectile bombardment-mediated transformation of crop plants is used
when Agrobacterium tumefaciens-mediated transformation is not successful. We
assess whether or not homologues of the AtNHX genes exist in the plant of
choice. We
use degenerate oligonucleotide PCR-primers (as described above) and a cDNA
library
to isolate the full-length cDNA. Expression vector constructs, using the pBAR
vector for
high level expression of AtNHX or the halophyte PNHX or the endogenous PNHX
from
the plant of choice, are used in conjunction with the microprojectile
bombardment
system as described by Tomes et al. [v]. Bombardment procedures is carried out
in
callus tissue. Plant calli are initiated by culturing immature embryos on
Callus medium
[vi]. After about 2 weeks, friable calli that are growing rapidly are
subcultured and grown
for an additional 2 weeks and then used for transformation. Calli for
transformation are
transferred to fresh medium, incubated for 24 h and bombarded with tungsten
microprojectiles carrying the pBARNHX vector construct. Bombardment conditions
is
performed according to manufacturer's instructions. Calli that show visible
growth 10
days after bombardment are transferred to selective media (containing either
Bialaphos
or Ignite) in order to identify putative transformants. The growth of
transformed plant
calli on this selective media is continued for 3-4 months. Each putative
stable transgenic
event becomes apparent as a mass of friable embyogenic callus growing in the
presence of the selection agent. Stable transformation is verified by Southern
blots.
Selected calli are transferred onto a regeneration medium [v], kept in the
dark at 28 C
for 7 days and then transferred to growth chambers under a 16-h photoperiod
until
green shoots appear. Plantlets (1-2 cm long) are transferred to individual
tubes
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containing germination medium to allow continued development. At the three to
four
leaf stage, plants are transferred to soil and into the greenhouse. At the
eight-leaf
stage, these plants are sprayed with 1 % (w/v) Ignite herbicide to detect the
presence of
the BAR gene. This herbicide kills those plants not carrying the BAR gene.
Confirmed
transgenic plants (T1) are allowed to mature, flower, set seed, and seeds used
for the
production of T2 plants. Transgenic T2 plants are used for the evaluation of
salt-
tolerance as described above. Transgenic T2 and T3 plants are used in field
trials for
the evaluation of salt tolerance.
METHODS
Cloning of the Arabidopsis Na`/H+ antiport cDNA (AtNHX1)
The full-length cDNA of AtNHX1 was cloned by us from an Arabidopisis thaliana
(Columbia) seedling cDNA library [38]. The library was initially screened with
an EST
(GenBank # T75860; Figure 8(h)) obtained from the Arabidopsis Biological
Resource
Center (ABRC) that showed homology to Arabidopsis genomic sequence (A-
TM021 B04.4). The invention includes nucleic acid molecules of between about:
500-
1000, 1000-1500 1500-1600, 1600-1700, 1700-2000 or 2000-2500 or greater than
2500
nucleotides including the EST sequence (or a sequence having at least about:
35, 35,
55, 65, 75, 85, 90, 95, 99, 99.5 sequence identity to the EST sequence or the
polypeptide encoded by the EST sequence) and which encodes a polypeptide that
extrudes monovalent (preferably potassium ions or lithium ions, most
preferably sodium
ions) out of the cytosol for preparation of transgenic plants and host cells,
and in the
other methods of the invention described below. These sequences are useful in
the
methods of the invention described above (for example as a probe, research
uses,
hybridization). The Arabidopsis genomic sequence predicted a polypeptide of
457
amino acids. Plaques that hybridized with the labeled EST probe were subjected
to a
secondary screen using the PCR product from the nested amplification of a
region
coding for the N-terminal portion of the predicted polypeptide. The forward
primer,
based on the predicted start codon of the polypeptide (Primer-NT), 5-
GCCATGTTGGATTCTCTAGTGTCG-3 and the reverse primer, based on the stop
codon predicted from the EST (Primer-CT), 5'-
CCGAATTCTCAAAGCTTTTCTTCCACG-3', were used to amplify a 1.7 kb product from
the seedling library. This product was purified by agarose gel electrophoresis
and used
as the template for a second amplification using primer-NT and a reverse
primer
(primer-C) based on the genomic sequence, 5'-
CGGAATTCACAGAAAAACACAGTGAGGAT-3'. The resulting 900 bp fragment served
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WO 99/47679 PCT/CA99/00219
as the template for the probe used in the secondary screen. The pure plaques
obtained
in the secondary screen were tested by PCR using the primer-NT, primer-CT
combination. Three of the plaques, from which a 1.7 kb product was amplified,
were
selected for excision of the phagemid. Single colonies containing the excised
phagemid
were grown in liquid culture. Aliquots of each of these cultures were used as
templates
for the PCR amplification of the region bound by the library plasmid to the 5'
side of the
clone (T3 promoter) and the reverse primer C. In one clone, a 1.2 kb fragment
was
amplified, which implied that the clone had an upstream untranslated region of
approximately 300 bp. This clone was selected for complete sequencing.
Cloning of the Arabidopsis AtNHX2 Na`/H' antiport cDNA
The full-length AtNHX2 cDNA was cloned from an Arabidopsis thaliana
(Columbia) seedling cDNA library. PCR primers were designed for the
amplification of
the AtNHX2 sequence based on a BAC DNA sequence (MTE17) with a predicted amino
acid sequence showing homology to AtNHX1. The forward primer (X6F), 5'-
CCTCAGGTGATACCAATCTCA-3 and the reverse primer (X6REV), 5 -
GATCCAATGTAACACCGGAG-3 were used to amplify a 1.2 kb product from the
seedling library by PCR. This product was purified by agarose gel
electrophoresis and
used as a probe in hybridization screening of the seedling cDNA library.
Plaques that
hybridized with the iabeled probe were subjected to a secondary screen using
the 1.2
kb PCR product as a probe. Pure plaques obtained in the secondary screen were
tested
by PCR using primer - X6F, primer - X6REV combination. Only one of the plaques
had
the 1.2 kb product amplified from it. This plaque was used for excision of the
phagemid.
This clone was used for complete sequencing.
Cloning of the Arabidopsis AtNHX3 and AtNHX4 Na*/H+ antiport cDNAs
Full length AtNHX3 and AtNHX4 cDNAs were cloned by us from an Arabidopsis
thaliana (Columbia) seedling cDNA libraries (CD4-15 and CD4-1 6; Arabidopsis
Stock
Center, Columbus, Ohio). PCR primers were designed for the amplification of a
genomic sequence based on a BAC DNA sequence (F20D21) with a predicted amino
acid sequence showing homology to both AtNHX1 and AtNHX2. The forward primer
(NHX7F), 5-TTCGTTCTCGGCCATGTCC-3 and the reverse primer (NHX7REV), 5'-
CGGAGAGACCAACACCTTCTGC-3 were used to amplify a 2.2 kb product using
Arabidopsis thaliana (Columbia) genomic DNA as a template. This product was
purified
by agarose gel electrophoresis and used as a probe in hybridization screening
of the
seedling cDNA libraries. Plaques that hybridized with the labeled probe were
subjected
to a secondary screen using the 2.2 kb PCR product as a probe. Pure plaques
were
54
CA 02323756 2007-06-27
used as templates for the PCR amplification of the region bound by the library
plasmid
using the T3 and T7 promoter sequences as primers. Two independent clones
(insert
sizes of 1.7kb and 2.1 kb) were selected for phagemid excision and complete
sequencing.
Southern Blot Analysis
Genomic DNA was isolated from mature leaf tissue of Arabidopsis thaliana
(Columbia). 10 ug of this genomic DNA was digested with Clal, EcoRl, Xbal, or
Hindi 11,
fractionated on 0.7% agarose gel, and transferred to HybondTM N+ membrane
(Amersham) according to manufacturers instructions. Overnight hybridization
was
performed at 65 C in Amersham hybridization buffer with AtNHX1 cDNA fragments
labeled with 32P by the random priming method. The final wash was in 0.1X
SSPE,
0.1% SDS at 65 C. Hybridization signals were detected by autoradiography on
BioMaxTM hyperfilm (Kodak).
Northern Blot Analysis
Arabidopsis thaliana ecotype Columbia was grown either on vertical plates on
medium containing 0.5X MS salts and 1% agar at 20-25 C under continuous
fluorescent
light for 1.5 weeks or in soil at 20-25 C under fluorescent light and
incandescent light
with a 14 hour photo period for 3-4 weeks. Total RNA was isolated from flower,
leaf,
and inflorescence stems of the mature plants and from root and shoot tissues
of the
vertically grown seedlings using TRIZOLTM reagent (GibcoBRL). 40 ug of RNA was
electrophoresed and transferred to Hybond' N+ membrane (Amersham) according to
manufacturers instructions. Methylene blue was used to visualize the 26S and
18S
ribosomal RNA for quantitation. The blotted RNA was hybridized and washed as
described for the southern blot analysis.
The present invention has been described in terms of particular embodiments
found or proposed by the present inventor to comprise preferred modes for the
practice
of the invention. It will be appreciated by those of skill in the art that, in
light of the
present disclosure, numerous modifications and changes can be made in the
particular
embodiments exemplified without departing from the intended scope of the
invention. All
such modifications are intended to be included within the scope of the
appended claims.
Generation of Transgenic Arabidopsis Plants Overexpressing AtNHX1
An AtNHX1 PCR product was amplified using VentTM DNA polymerase (New
England Biolabs) with the following primers (SE-ATX1-Sall 5'-
CGCGTCGACATGTTGGATTCTCTAGTGTCG-3' and ATXCT2 5'-
CA 02323756 2007-06-27
CCGAATTCTCAAAGCTTTTCTTCCACG-3'). This product was digested with Sall gel
purified and used in a ligation reaction along with pBISNI prevously digested
with Sall
and Smal and gel purified. The resulting vector pBISNI-AtNHX1 contained the
AtNHX1
open reading frame in a sense orientation under the control of the super
promoter.
Overexpression constructs carrying the AtNHX1 cDNA are introduced into an
electro-competent Agrobacterium tumefaciens (LBA4404) by electroporation. The
Agrobacteria are plated on LB plates containing 50 mg/L kanamycin and grown
for -2
days at 30 C to select for bacteria carrying the overexpression constructs.
One liter
liquid LB+kanamycin (50mg/L) is inoculated with a single Agrobacterium colony
selected
from the LB (kanamycin 50mg/L) plates. The culture is grown to a minimum of
OD=1
(600nm) for 2-3 days. The Agrobacteria are then pelleted and resuspended in 1
L
infiltration medium (IM - 0.5XMS salts; 0.5 g/L MES; 5% sucrose; 0.03% Silwet
L-77).
Flowering Arabidopsis plants with primary bolts reaching -15cm are used for
the
transformation procedure (T1). Pots of Arabidopsis plants are dunked into the
IM
solution containing the Agrobacteria and left submerged for 2-6 minutes. The
same
procedure can be repeated after 8-12 days on the same plants. Plants are
allowed to
senesce, the plants are dried thoroughly and the seeds harvested. Seeds are
plated on
agar plates containing 25 mg/L kanamycin in a medium containing MS salts, 0.8%
(w/v)
agar adjusted to pH 6.0 with KOH. The plates are transferred to a growth room
at 25 C
under continuous light. After 10 days the kanamycin-resistant seedlings (T2)
are
transferred to small pots containing vermiculite. At senescence (-8 weeks) the
seeds
are collected from single plants and plated on agar plates containing MS salts
and
25mg/L kanamycin. After 10 days the kanamycin-resistant seedlings (T3) are
transferred to small pots containing vermiculite. Seeds produced by these
plants are
germinated and used to assess salt tolerance of the transgenic plants.
Assessment of Salt Tolerance in Transpenic Plants
This procedure is described in the legend for Figure 7.
The present invention has been described in detail and with particular
reference
to the preferred embodiments; however, it will be understood by one having
ordinary
skill in the art that changes can be made thereto without departing from the
spirit and
scope of the invention.
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CA 02323756 2007-06-27
Table 4
High stringency ( very similar sequences)
Hybridization Wash
55-65 C 60-65'C
5xSSC 0.1xSSC
2% SDS 0.1% SDS
100 glml SSDNA
Intermediate stringency (similar sequences)
(Moo Att S~f;Y~~GV-Ly~
Hybridization I Wash
40-50 C 50-50 C
5xSSC 0.1xSSC
2% SDS 01% SDS
100 g/ml SSDNA
Low stringency ( low similarity among sequences, i.e.
many sequences similar)
Hybridization Wash
30-40 C 40-50 C
5xSSC 2xSSC
2% SDS 02% SDS
100 gfml SSDNA
Abbreviations:
SSC = sodium chloride-sodium citrate buffer
SSDNA = single stranded DNA
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Table 5 - List of Plants
Alfalfa Melon
Almond Mustard
Apple Oak
Apricot Oat
Arabidopsis Olive
Artichoke Onion
Atriplex Orange
Avocado Pea
Barley Peach
Beet Pear
Birch Pepper
Brassica Pine
Cabbage Plum
Cacao Poplar
Cantaloup/cantalope Potato
Carnations Prune
Castorbean Radish
Caulifower Rape
Celery Rice
Clover Roses
Coffee Rye
Corn Sorghum
Cotton Soybean
Cucumber Spinach
Garlic Squash
Grape Strawberries
Grapefruit Sunflower
Hemp Sweet corn
Hops Tobacco
Lettuce Tomato
Maple Wheat
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[20] Ni et al., (1995) Plant Journal 7:661-676
[21] Shah et at., (1986) Science 233:478-481.
[22] Ono et at., (1996) Plant Physiol 112:483-491
[23] Abe et al., (1997) Plant Cell 9:1859-1868.
[24] Rieping M and Schoffl F (1992) Mol Gen Genet 231:226-232].
[25] Raghothama et at., (1997) Plant Mol Biol 34:393-402].
[26] Mett et al., (1996) Transgenic Res 5:105-113
[27] Schena et at., (1991) PNAS 88:10421-10425.
[28] Vorst et al. (1990) Plant Mol Biol 14:491-499.
[29] Wanapu & Shinmyo (1996) Ann. NY Acad. Sci. 782:107-114.
[30] Harlow E & Lane D (1988). Antibodies: a laboratory manual. Cold Spring
Harbor
Laboratory Press. New York.
[31] Sambrook, J, Fritsch, E.E. & Maniatis, T. (1989). Molecular Cloning: A
laboratory manual. Cold Spring Harbor Laboratory Press. New York.
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CA 02323756 2000-09-18
WO 99/47679 PCT/CA99/00219
[32] Lee, C.C. & Caskey, T. in: PCR Protocols: A guide to Methods and
Applications.
Academic Press, Inc. San Diego. pp.46-53
[33] Narasimhulu, SB, et al., Plant Cell 8:873-886 [1996])
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[35] Ma H, et al., (1987) Gene 58:202-226
[36] Gietz RD & Woods, RA (1994). High efficiency transformation with lithium
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New York: IRL Press. pp. 121-134.
[37] Gietz, RD & Sugino, A (1988), Gene 74: 527-534.
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[i] Blumwald, E. (1987). Physiol. Plant 69, 731-734.
[ii] Blumwald, E. & Poole, R.J. (1986). Plant Physiol. 80,727-731.
(iii] Clough, M. & Bent, A. (1997). Arabidopsis Meeting, Madison, WI,
[iv] Moloney, M.M., Walker, J.M., & Sharma, K.K. (1989). Plant Cell Rep. 8,
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[v] Tomes, D.T., Ross, M.C., & Songstad, D.D. (1995). ln:Plant Cell, Tissue
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Culture (O.L. Gamborg & G.C. Phillips, eds). Springer, New York. pp 197-213.
[vi] Amstrong, C.L., & Green, C.E. (1985). Planta 164,207-214.
CA 02323756 2000-09-18
SEQUENCE LISTING
<110> BLUMWALD, Eduardo
APSE, Maris
SNEDDEN, Wayne
AHARON, Gilad
<120> GENETIC ENGINEERING SALT TOLERANCE IN CROP PLANTS
<130> 1110/0058
<150> PCT/CA99/00219
<151> 1999-03-18
<150> US 60/078,474
<151> 1998-04-01
<150> US 60/116,111
<151> 1999-01-15
<160> 37
<170> Patentln Ver. 2.1, Word 97
<210> 1
<211> 2178
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (286)..(1899)
<223> Figure 1(a)
<400> 1
cctctctgtt tcgttcctcg tagacgaaga agaagaagaa tctcaggttt tagctttcga 60
agcttccaaa attttgaatt ttgatcttct gggctctttt gtaaatcaga ctgaagatat 120
ttagattacc cagaagttgt tcaaggaatg gtttcagtgg acagcacgga aagataaaag 180
agactttttt ttccagattt tgctgatcca aaatctgaat agttgttcat gttcttggat 240
caaatctgga aagaggaagt ttgttggatc tagaagaaga taaca atg ttg gat tct 297
Met Leu Asp Ser
1
cta gtg tcg aaa ctg cct tcg tta tcg aca tct gat cac get tct gtg 345
Leu Val Ser Lys Leu Pro Ser Leu Ser Thr Ser Asp His Ala Ser Val
10 15 20
gtt gcg ttg aat ctc ttt gtt gca ctt ctt tgt get tgt att gtt ctt 393
Val Ala Leu Asn Leu Phe Val Ala Leu Leu Cys Ala Cys Ile Val Leu
25 30 35
61
CA 02323756 2000-09-18
ggt cat ctt ttg gaa gag aat aga tgg atg aac gaa tcc atc acc gcc 441
Gly His Leu Leu Glu Glu Asn Arg Trp Met Asn Glu Ser Ile Thr Ala
40 45 50
ttg ttg att ggg cta ggc act ggt gtt acc att ttg ttg att agt aaa 489
Leu Leu Ile Gly Leu Gly Thr Gly Val Thr Ile Leu Leu Ile Ser Lys
55 60 65
gga aaa agc tcg cat ctt ctc gtc ttt agt gaa gat ctt ttc ttc ata 537
Gly Lys Ser Ser His Leu Leu Val Phe Ser Glu Asp Leu Phe Phe Ile
70 75 80
tat ctt ttg cca ccc att ata ttc aat. gca ggg ttt caa gta aaa aag 585
Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly Phe Gln Val Lys Lys
85 90 95 100
aag cag ttt ttc cgc aat ttc gtg act. att atg ctt ttt ggt get gtt 633
Lys Gln Phe Phe Arg Asn Phe Val Thr Ile Met Leu Phe Gly Ala Val
105 110 115
ggg act att att tct tgc aca atc ata tct cta ggt gta aca cag ttc 681
Gly Thr Ile Ile Ser Cys Thr Ile Ile Ser Leu Gly Val Thr Gln Phe
120 125 130
ttt aag aag ttg gac att gga acc ttt: gac ttg ggt gat tat ctt get 729
Phe Lys Lys Leu Asp Ile Gly Thr Phe Asp Leu Gly Asp Tyr Leu Ala
135 140 145
att ggt gcc ata ttt get gca aca gat: tca gta tgt aca ctg cag gtt 777
Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser Val Cys Thr Leu Gln Val
150 155 160
ctg aat caa gac gag aca cct ttg ctt: tac agt ctt gta ttc gga gag 825
Leu Asn Gln Asp Glu Thr Pro Leu Leu Tyr Ser Leu Val Phe Gly Glu
165 170 175 180
ggt gtt gtg aat gat gca acg tca gtt: gtg gtc ttc aac gcg att cag 873
Gly Val Val Asn Asp Ala Thr Ser Val Val Val Phe Asn Ala Ile Gln
185 190 195
agc ttt gat ctc act cac cta aac cac: gaa get get ttt cat ctt ctt 921
Ser Phe Asp Leu Thr His Leu Asn His Glu Ala Ala Phe His Leu Leu
200 205 210
gga aac ttc ttg tat ttg ttt ctc cta agt acc ttg ctt ggt get gca 969
Gly Asn Phe Leu Tyr Leu Phe Leu Leu Ser Thr Leu Leu Gly Ala Ala
215 220 225
acc ggt ctg ata agt gcg tat gtt atc aag aag cta tac ttt gga agg 1017
Thr Gly Leu Ile Ser Ala Tyr Val Ile Lys Lys Leu Tyr Phe Gly Arg
230 235 240
cac tca act gac cga gag gtt gcc ctt atg atg ctt atg gcg tat ctt 1065
His Ser Thr Asp Arg Glu Val Ala Leu Met Met Leu Met Ala Tyr Leu
245 250 255 260
tct tat atg ctt get gag ctt ttc gac ttg agc ggt atc ctc act gtg 1113
62
CA 02323756 2000-09-18
Ser Tyr Met Leu Ala Glu Leu Phe Asp Leu Ser Gly Ile Leu Thr Val
265 270 275
ttt ttc tgt ggt att gtg atg tcc cat tac aca tgg cac aat gta acg 1161
Phe Phe Cys Gly Ile Val Met Ser His Tyr Thr Trp His Asn Val Thr
280 285 290
gag agc tca aga ata aca aca aag cat acc ttt gca act ttg tca ttt 1209
Glu Ser Ser Arg Ile Thr Thr Lys His Thr Phe Ala Thr Leu Ser Phe
295 300 305
ctt gcg gag aca ttt att ttc ttg tat gtt gga atg gat gcc ttg gac 1257
Leu Ala Glu Thr Phe Ile Phe Leu Tyr Val Gly Met Asp Ala Leu Asp
310 315 320
att gac aag tgg aga tcc gtg agt gac aca ccg gga aca tcg atc gca 1305
Ile Asp Lys Trp Arg Ser Val Ser Asp Thr Pro Gly Thr Ser Ile Ala
325 330 335 340
gtg agc tca atc cta atg ggt ctg gtc atg gtt gga aga gca gcg ttc 1353
Val Ser Ser Ile Leu Met Gly Leu Val Met Val Gly Arg Ala Ala Phe
345 350 355
gtc ttt ccg tta tcg ttt cta tct aac tta gcc aag aag aat caa agc 1401
Val Phe Pro Leu Ser Phe Leu Her Asn Leu Ala Lys Lys Asn Gln Ser
360 365 370
gag aaa atc aac ttt aac atg cag gtt gtg att tgg tgg tct ggt ctc 1449
Glu Lys Ile Asn Phe Asn Met Gln Val Val Ile Trp Trp Ser Gly Leu
375 380 385
atg aga ggt get gta tct atg get ctt gca tac aac aag ttt aca agg 1497
Met Arg Gly Ala Val Ser Met Ala Leu Ala Tyr Asn Lys Phe Thr Arg
390 395 400
gcc ggg cac aca gat gta cgc ggg aat gca atc atg atc acg agt acg 1545
Ala Gly His Thr Asp Val Arg Gly Asn Ala Ile Met Ile Thr Ser Thr
405 410 415 420
ata act gtc tgt ctt ttt agc aca gtg gtg ttt ggt atg ctg acc aaa 1593
Ile Thr Val Cys Leu Phe Ser Thr Val Val Phe Gly Met Leu Thr Lys
425 430 435
cca ctc ata agc tac cta tta ccg cac cag aac gcc acc acg agc atg 1641
Pro Leu Ile Ser Tyr Leu Leu Pro His Gln Asn Ala Thr Thr Ser Met
440 445 450
tta tct gat gac aac acc cca aaa tcc ata cat atc cct ttg ttg gac 1689
Leu Her Asp Asp Asn Thr Pro Lys Her Ile His Ile Pro Leu Leu Asp
455 460 465
caa gac tcg ttc att gag cct tca ggg aac cac aat gtg cct cgg cct 1737
Gin Asp Ser Phe Ile Glu Pro Ser Gly Asn His Asn Val Pro Arg Pro
470 475 480
gac agt ata cgt ggc ttc ttg aca cgg ccc act cga acc gtg cat tac 1785
Asp Ser Ile Arg Gly Phe Leu Thr Arg Pro Thr Arg Thr Val His Tyr
63
CA 02323756 2000-09-18
485 490 495 500
tac tgg aga caa ttt gat gac tcc ttc atg cga ccc gtc ttt gga ggt 1833
Tyr Trp Arg Gln Phe Asp Asp Ser Phe Met Arg Pro Val Phe Gly Gly
505 510 515
cgt ggc ttt gta ccc ttt gtt cca ggt. tct cca act gag aga aac cct 1881
Arg Gly Phe Val Pro Phe Val Pro Gly Ser Pro Thr Glu Arg Asn Pro
520 525 530
cct gat ctt agt aag get tgagggtaac gtggaagaaa agctttgatt 1929
Pro Asp Leu Ser Lys Ala
535
ttttttggta gaaaagggtg attcaaatta tgcttttgtg taaattatcc atttgtaata 1989
ttgtttgtga ggacagaaat ctgtcctaac gttttgagag cagaaagcaa aacatggcaa 2049
ctttgaagtg tttgattgat gtatgtaatt atattcatat ttgttttgtt gtaacacaaa 2109
ctacacattt gtttatgttt tgaatttggt ttttgcttcg aaaaaaaaaa aaaaaaaaaa 2169
aaaaaaaaa 2178
<210> 2
<211> 538
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(a)
<400> 2
Met Leu Asp Ser Leu Val Ser Lys Leu Pro Ser Leu Ser Thr Ser Asp
1 5 10 15
His Ala Ser Val Val Ala Leu Asn Leu Phe Val Ala Leu Leu Cys Ala
20 25 30
Cys Ile Val Leu Gly His Leu Leu Glu Glu Asn Arg Trp Met Asn Glu
35 40 45
Ser Ile Thr Ala Leu Leu Ile Gly Leu Gly Thr Gly Val Thr Ile Leu
50 55 60
Leu Ile Ser Lys Gly Lys Ser Ser His Leu Leu Val Phe Ser Glu Asp
65 70 75 80
Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly Phe
85 90 95
Gln Val Lys Lys Lys Gin Phe Phe Arg Asn Phe Val Thr Ile Met Leu
100 105 110
Phe Gly Ala Val Gly Thr Ile Ile Ser Cys Thr Ile Ile Ser Leu Gly
64
CA 02323756 2000-09-18
115 120 125
Val Thr Gln Phe Phe Lys Lys Leu Asp Ile Gly Thr Phe Asp Leu Gly
130 135 140
Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser Val Cys
145 150 155 160
Thr Leu Gln Val Leu Asn Gin Asp Glu Thr Pro Leu Leu Tyr Ser Leu
165 170 175
Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Val Phe
180 185 190
Asn Ala Ile Gln Ser Phe Asp Leu Thr His Leu Asn His Glu Ala Ala
195 200 205
Phe His Leu Leu Gly Asn Phe Leu Tyr Leu Phe Leu Leu Ser Thr Leu
210 215 220
Leu Gly Ala Ala Thr Gly Leu Ile Ser. Ala Tyr Val Ile Lys Lys Leu
225 230 235 240
Tyr Phe Gly Arg His Ser Thr Asp Arq Glu Val Ala Leu Met Met Leu
245 250 255
Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Asp Leu Ser Gly
260 265 270
Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser His Tyr Thr Trp
275 280 285
His Asn Val Thr Glu Ser Ser Arg Ile Thr Thr Lys His Thr Phe Ala
290 295 300
Thr Leu Ser Phe Leu Ala Glu Thr Phe Ile Phe Leu Tyr Val Gly Met
305 310 315 320
Asp Ala Leu Asp Ile Asp Lys Trp Arg Ser Val Ser Asp Thr Pro Gly
325 330 335
Thr Ser Ile Ala Val Ser Ser Ile Leu Met Gly Leu Val Met Val Gly
340 345 350
Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Ala Lys
355 360 365
Lys Asn Gln Ser Glu Lys Ile Asn Phe Asn Met Gln Val Val Ile Trp
370 375 380
Trp Ser Gly Leu Met Arg Gly Ala Val Ser Met Ala Leu Ala Tyr Asn
385 390 395 400
Lys Phe Thr Arg Ala Gly His Thr Asp Val Arg Gly Asn Ala Ile Met
405 410 415
Ile Thr Ser Thr Ile Thr Val Cys Leu Phe Ser Thr Val Val Phe Gly
CA 02323756 2000-09-18
420 425 430
Met Leu Thr Lys Pro Leu Ile Ser Tyr Leu Leu Pro His Gln Asn Ala
435 440 445
Thr Thr Ser Met Leu Ser Asp Asp Asn Thr Pro Lys Ser Ile His Ile
450 455 460
Pro Leu Leu Asp Gln Asp Ser Phe Ile Glu Pro Ser Gly Asn His Asn
465 470 475 480
Val Pro Arg Pro Asp Ser Ile Arg Gly Phe Leu Thr Arg Pro Thr Arg
485 490 495
Thr Val His Tyr Tyr Trp Arg Gln Phe Asp Asp Ser Phe Met Arg Pro
500 505 510
Val Phe Gly Gly Arg Gly Phe Val Pro Phe Val Pro Gly Ser Pro Thr
515 520 525
Glu Arg Asn Pro Pro Asp Leu Ser Lys Ala
530 535
<210> 3
<211> 1788
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (61)..(1647)
<223> Figure 1(b)
<400> 3
tcttcgtttg cgattggtgt tttcaaaatc gacgaaatcg aaaacattat cgagtgaaaa 60
atg agt atc gga tta aca gag ttt gtg acg aat aaa cta gca get gag 108
Met Ser Ile Gly Leu Thr Glu Phe Val Thr Asn Lys Leu Ala Ala Glu
1 5 10 15
cat cct cag gtg ata cca atc tca gtg ttc atc gcc att ctc tgt cta 156
His Pro Gln Val Ile Pro Ile Ser Val Phe Ile Ala Ile Leu Cys Leu
20 25 30
tgt tta gtt atc ggc cac ttg ctt gaa gag aat cga tgg gtt aat gaa 204
Cys Leu Val Ile Gly His Leu Leu Glu Glu Asn Arg Trp Val Asn Glu
35 40 45
tct att acc gcc att tta gta gga gca gca tca gga aca gtg atc tta 252
Ser Ile Thr Ala Ile Leu Val Gly Ala Ala Ser Gly Thr Val Ile Leu
50 55 60
ctt att agt aaa gga aaa agt tca cat att ttg gtg ttt gat gaa gaa 300
Leu Ile Ser Lys Gly Lys Ser Ser His Ile Leu Val Phe Asp Glu Glu
65 70 75 80
66
CA 02323756 2000-09-18
ctc ttc ttc att tac ctt ctt cct cca ata atc ttc aat get ggg ttc 348
Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly Phe
85 90 95
caa gtt aag aaa aag aag ttt ttt cac aac ttt tta acc atc atg tcc 396
Gln Val Lys Lys Lys Lys Phe Phe His Asn Phe Leu Thr Ile Met Ser
100 105 110
ttt ggt gtg att gga gtt ttc atc tcc act gtc att atc tcg ttt ggg 444
Phe Gly Val Ile Gly Val Phe Ile Ser Thr Val Ile Ile Ser Phe Gly
115 120 125
act tgg tgg ctg ttt ccc aag ttg gga ttt aag ggg ttg agt get aga 492
Thr Trp Trp Leu Phe Pro Lys Leu Gly Phe Lys Gly Leu Ser Ala Arg
130 135 140
gac tat ctt gcc ata gga acg att ttc:: tca tca act gat act gtt tgc 540
Asp Tyr Leu Ala Ile Gly Thr Ile Phe Ser Ser Thr Asp Thr Val Cys
145 150 155 160
act cta cag att ctc cat caa gat gaa aca cca ttg cta tac agc tta 588
Thr Leu Gln Ile Leu His Gln Asp Glu Thr Pro Leu Leu Tyr Ser Leu
165 170 175
gtc ttt gga gaa gga gtg gtg aat gat gca acc tca gtt gta ctg ttc 636
Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Leu Phe
180 185 190
aac gcc gtg caa aag att caa ttt gaa agc cta acc ggt tgg acg gcg 684
Asn Ala Val Gln Lys Ile Gln Phe Glu Ser Leu Thr Gly Trp Thr Ala
195 200 205
ctg caa gta ttt ggg aac ttt ttg tac ctc ttc tca aca agc aca ctt 732
Leu Gln Val Phe Gly Asn Phe Leu Tyr Leu Phe Ser Thr Ser Thr Leu
210 215 220
ctc gga att ggt gtg ggg cta ata aca tct ttt gtt ctt aaa acc ttg 780
Leu Gly Ile Gly Val Gly Leu Ile Thr Ser Phe Val Leu Lys Thr Leu
225 230 235 240
tat ttt gga aga cat tct act aca cgc gaa ctc gcc atc atg gtt cta 828
Tyr Phe Gly Arg His Ser Thr Thr Arg Glu Leu Ala Ile Met Val Leu
245 250 255
atg get tac ctt tca tat atg ttg get gag ctc ttc tca tta agt gga 876
Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Ser Leu Ser Gly
260 265 270
att ctt act gtt ttc ttc tgt ggt gtt tta atg tcg cat tat gca tca 924
Ile Leu Thr Val Phe Phe Cys Gly Val Leu Met Ser His Tyr Ala Ser
275 280 285
tat aac gtg aca gag agc tca aga atc act tcc agg cat gta ttt gca 972
Tyr Asn Val Thr Glu Ser Ser Arg Ile Thr Ser Arg His Val Phe Ala
290 295 300
67
CA 02323756 2000-09-18
atg ttg tcc ttt att gcg gag aca ttc ata ttt ctg tat gtt gga aca 1020
Met Leu Ser Phe Ile Ala Glu Thr Phe Ile Phe Leu Tyr Val Gly Thr
305 310 315 320
gat get ctt gat ttt aca aag tgg aag aca agc agc tta agc ttt ggg 1068
Asp Ala Leu Asp Phe Thr Lys Trp Lys Thr Ser Ser Leu Ser Phe Gly
325 330 335
ggt act ctg ggt gtc tcc ggt gtc ata acc gca tta gta ttg ctt gga 1116
Gly Thr Leu Gly Val Ser Gly Val Ile Thr Ala Leu Val Leu Leu Gly
340 345 350
cga gca gca ttt gtc ttt cca ctc tcg gtc tta aca aat ttc atg aac 1164
Arg Ala Ala Phe Val Phe Pro Leu Ser. Val Leu Thr Asn Phe Met Asn
355 360 365
agg cac act gaa aga aac gag tct atc: aca ttt aag cat cag gtg atc 1212
Arg His Thr Glu Arg Asn Glu Ser Ile Thr Phe Lys His Gln Val Ile
370 375 380
att tgg tgg gca ggt cta atg cga ggt get gtc tca att get ctg get 1260
Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Ile Ala Leu Ala
385 390 395 400
ttc aag cag ttc aca tac tcc ggt gtt aca ttg gat cct gtg aat get 1308
Phe Lys Gln Phe Thr Tyr Ser Gly Val Thr Leu Asp Pro Val Asn Ala
405 410 415
gcc atg gtc acc aac acc act atc gtt gtt ctc ttt act aca ctg gtc 1356
Ala Met Val Thr Asn Thr Thr Ile Val Val Leu Phe Thr Thr Leu Val
420 425 430
ttt ggt ttc ctc aca aaa cca ctt gtg aat tat ctc ctt cct caa gat 1404
Phe Gly Phe Leu Thr Lys Pro Leu Val Asn Tyr Leu Leu Pro Gln Asp
435 440 445
gca agt cac aac acc gga aat aga ggt aaa cgc act gag cca ggt tct 1452
Ala Ser His Asn Thr Gly Asn Arg Gly Lys Arg Thr Glu Pro Gly Ser
450 455 460
ccg aaa gaa gat gcg aca ctt cct ctt ctt tcc ttt gac gag tct get 1500
Pro Lys Glu Asp Ala Thr Leu Pro Leu Leu Ser Phe Asp Glu Ser Ala
465 470 475 480
tcc acc aac ttc aat aga get aga gat agt att tcc ctt ctg atg gaa 1548
Ser Thr Asn Phe Asn Arg Ala Arg Asp Ser Ile Ser Leu Leu Met Glu
485 490 495
caa cct gtg tac acc atc cac cgc tac tgg aga aag ttt gac gac aca 1596
Gln Pro Val Tyr Thr Ile His Arg Tyr Trp Arg Lys Phe Asp Asp Thr
500 505 510
tac atg agg cct atc ttc ggt gga cct cgt cga gaa aac caa cca gaa 1644
Tyr Met Arg Pro Ile Phe Gly Gly Pro Arg Arg Glu Asn Gln Pro Glu
515 520 525
tgc tagaattgat ccgggttctc cgcggggaaa tcatgatgag ttagtttttt 1697
68
CA 02323756 2000-09-18
Cys
ttatagtcaa gaaagtagga tagttggttt agctaaaaca gtttcttaaa gtttttgtta 1757
aatgtataca acaaggttct tctatatacg c 1788
<210> 4
<211> 529
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(b)
<400> 4
Met Ser Ile Gly Leu Thr Glu Phe Val Thr Asn Lys Leu Ala Ala Glu
1 5 10 15
His Pro Gin Val Ile Pro Ile Ser Val Phe Ile Ala Ile Leu Cys Leu
20 25 30
Cys Leu Val Ile Gly His Leu Leu Glu Glu Asn Arg Trp Val Asn Glu
35 40 45
Ser Ile Thr Ala Ile Leu Val Gly Ala Ala Ser Gly Thr Val Ile Leu
50 55 60
Leu Ile Ser Lys Gly Lys Ser Ser His Ile Leu Val Phe Asp Glu Glu
65 70 75 80
Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly Phe
85 90 95
Gin Val Lys Lys Lys Lys Phe Phe His Asn Phe Leu Thr Ile Met Ser
100 105 110
Phe Gly Val Ile Gly Val Phe Ile Ser Thr Val Ile Ile Ser Phe Gly
115 120 125
Thr Trp Trp Leu Phe Pro Lys Leu Gly Phe Lys Gly Leu Ser Ala Arg
130 135 140
Asp Tyr Leu Ala Ile Gly Thr Ile Phe Ser Ser Thr Asp Thr Val Cys
145 150 155 160
Thr Leu Gin Ile Leu His Gin Asp Glu Thr Pro Leu Leu Tyr Ser Leu
165 170 175
Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser Val Val Leu Phe
180 185 190
Asn Ala Val Gin Lys Ile Gin Phe Glu Ser Leu Thr Gly Trp Thr Ala
195 200 205
Leu Gin Val Phe Gly Asn Phe Leu Tyr Leu Phe Ser Thr Ser Thr Leu
69
CA 02323756 2000-09-18
210 215 220
Leu Gly Ile Gly Val Gly Leu Ile Thr Ser Phe Val Leu Lys Thr Leu
225 230 235 240
Tyr Phe Gly Arg His Ser Thr Thr Arg Glu Leu Ala Ile Met Val Leu
245 250 255
Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Ser Leu Ser Gly
260 265 270
Ile Leu Thr Val Phe Phe Cys Gly Val Leu Met Ser His Tyr Ala Ser
275 280 285
Tyr Asn Val Thr Glu Ser Ser Arg Ile Thr Ser Arg His Val Phe Ala
290 295 300
Met Leu Ser Phe Ile Ala Glu Thr Phe Ile Phe Leu Tyr Val Gly Thr
305 310 315 320
Asp Ala Leu Asp Phe Thr Lys Trp Lys Thr Ser Ser Leu Ser Phe Gly
325 330 335
Gly Thr Leu Gly Val Ser Gly Val Ile Thr Ala Leu Val Leu Leu Gly
340 345 350
Arg Ala Ala Phe Val Phe Pro Leu Ser Val Leu Thr Asn Phe Met Asn
355 360 365
Arg His Thr Glu Arg Asn Glu Ser Ile Thr Phe Lys His Gln Val Ile
370 375 380
Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Ile Ala Leu Ala
385 390 395 400
Phe Lys Gln Phe Thr Tyr Ser Gly Val Thr Leu Asp Pro Val Asn Ala
405 410 415
Ala Met Val Thr Asn Thr Thr Ile Val Val Leu Phe Thr Thr Leu Val
420 425 430
Phe Gly Phe Leu Thr Lys Pro Leu Val Asn Tyr Leu Leu Pro Gln Asp
435 440 445
Ala Ser His Asn Thr Gly Asn Arg Gly Lys Arg Thr Glu Pro Gly Ser
450 455 460
Pro Lys Glu Asp Ala Thr Leu Pro Leu Leu Ser Phe Asp Glu Ser Ala
465 470 475 480
Ser Thr Asn Phe Asn Arg Ala Arg Asp Ser Ile Ser Leu Leu Met Glu
485 490 495
Gin Pro Val Tyr Thr Ile His Arg Tyr Trp Arg Lys Phe Asp Asp Thr
500 505 510
Tyr Met Arg Pro Ile Phe Gly Gly Pro Arg Arg Glu Asn Gln Pro Glu
CA 02323756 2000-09-18
515 520 525
Cys
<210> 5
<211> 714
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (55)..(612)
<223> Figure 1(c)(i)
<400> 5
acaaaagctg gagctccacc gcggtggcgg ccgctctaga actagtggat cccc cgg 57
Arg
1
get gca gga att cgc ggc cgc ctc ggc cat gtc ctc cgc cgt cat cga 105
Ala Ala Gly Ile Arg Gly Arg Leu Gly His Val Leu Arg Arg His Arg
10 15
ttc cac tat ctt cct gaa gcc agc ggt tcg ctt ctc att ggt tta atc 153
Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile Gly Leu Ile
20 25 30
gtc ggt ata ctt get aat atc tcc gat act gag act agc att agg acg 201
Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser Ile Arg Thr
35 40 45
tgg ttt aat ttc cac gaa gag ttc ttc ttc ttg ttt ttg ttg cct ccc 249
Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu Leu Pro Pro
50 55 60 65
atc ata ttc cag tca ggt ttc agt ctt caa cct aaa cca ttc ttt tct 297
Ile Ile Phe Gln Ser Gly Phe Ser Leu Gln Pro Lys Pro Phe Phe Ser
70 75 80
aac ttt gga gcc att gtt acc ttt get atc atc gga act ttt gtc get 345
Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr Phe Val Ala
85 90 95
tca gtt gtt act ggt ggt ctg gtt tat ctt ggc ggc tct atg tat ctc 393
Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser Met Tyr Leu
100 105 110
atg tat aaa ctt ccc ttt gtt gag tgt ctt atg ttt ggt gca ctt ata 441
Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly Ala Leu Ile
115 120 125
tca get acg gac cct gtc act gta ctc tct ata ttc cag gat gtg ggc 489
Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gln Asp Val Gly
130 135 140 145
71
CA 02323756 2000-09-18
acc gat gtt aac ctg tat get ttg gtc ttt gga gaa tca gtt ctg aat 537
Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser Val Leu Asn
150 155 160
gat get atg gca ata tca ttg tac aga aca atg tcc tta gta aac cgc 585
Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu Val Asn Arg
165 170 175
cag tcc tcg tct ggg gaa cat ttt tca tggtggtgat caggtttttt 632
Gln Ser Ser Ser Gly Glu His Phe Ser
180 185
gagactttgc tggctcaatg tcgcaggggt tggggttgga ttcacttcag cttaatatcc 692
tcctcgatcc tcctatttcc to 714
<210> 6
<211> 186
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(c)(i)
<400> 6
Arg Ala Ala Gly Ile Arg Gly Arg Leu Gly His Val Leu Arg Arg His
1 5 10 15
Arg Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile Gly Leu
20 25 30
Ile Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser Ile Arg
35 40 45
Thr Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu Leu Pro
50 55 60
Pro Ile Ile Phe Gin Ser Gly Phe Ser Leu Gln Pro Lys Pro Phe Phe
65 70 75 80
Ser Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr Phe Val
85 90 95
Ala Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser Met Tyr
100 105 110
Leu Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly Ala Leu
115 120 125
Ile Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gln Asp Val
130 135 140
Gly Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser Val Leu
145 150 155 160
72
CA 02323756 2000-09-18
Asn Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu Val Asn
165 170 175
Arg Gln Ser Ser Ser Gly Glu His Phe Ser
180 185
<210> 7
<211> 420
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (52)..(393)
<223> Figure 1(c)(ii)
<400> 7
ggacttcgag ggccatggca tttgcacttg cacttcaata cttcatgatc t acc aga 57
Thr Arg
1
ggt cac ggc cca atc atc ttt tac tgc acc aca act att gtt gtt gtc 105
Gly His Gly Pro Ile Ile Phe Tyr Cys Thr Thr Thr Ile Val Val Val
10 15
acg gtt tta cta ata gga ggt tcg aca ggt aaa atg ttg gaa get ttg 153
Thr Val Leu Leu Ile Gly Gly Ser Thr Gly Lys Met Leu Glu Ala Leu
20 25 30
gaa gtt gta ggt gac gat ctt gat gac tcc atg tct gaa ggc ttt gaa 201
Glu Val Val Gly Asp Asp Leu Asp Asp Ser Met Ser Glu Gly Phe Glu
35 40 45 50
gag agc gat cat cag tat gtc cct cc: cct ttt agc att gga get tca 249
Glu Ser Asp His Gln Tyr Val Pro Pro Pro Phe Ser Ile Gly Ala Ser
55 60 65
tct gac gag gat aca tca tca tca gga agc agg ttc aag atg aag ctg 297
Ser Asp Glu Asp Thr Ser Ser Ser Gly Ser Arg Phe Lys Met Lys Leu
70 75 80
aag gag ttt cac aaa acc act aca tca ttc acc gcg ttg gac aaa aac 345
Lys Glu Phe His Lys Thr Thr Thr Ser Phe Thr Ala Leu Asp Lys Asn
85 90 95
ttt ctg act ccg ttc ttc aca act aat agt gga gat gga gat gga gat 393
Phe Leu Thr Pro Phe Phe Thr Thr Asn Ser Gly Asp Gly Asp Gly Asp
100 105 110
ggggagtagc atggaaaaga tgtgtat 420
<210> 8
<211> 114
73
CA 02323756 2000-09-18
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(c)(ii)
<400> 8
Thr Arg Gly His Gly Pro Ile Ile Phe Tyr Cys Thr Thr Thr Ile Val
1 5 10 15
Val Val Thr Val Leu Leu Ile Gly Gly Ser Thr Gly Lys Met Leu Glu
20 25 30
Ala Leu Glu Val Val Gly Asp Asp Leu Asp Asp Ser Met Ser Glu Gly
35 40 45
Phe Glu Glu Ser Asp His Gin Tyr Val Pro Pro Pro Phe Ser Ile Gly
50 55 60
Ala Ser Ser Asp Glu Asp Thr Ser Sear Ser Gly Ser Arg Phe Lys Met
65 70 75 80
Lys Leu Lys Glu Phe His Lys Thr Thar Thr Ser Phe Thr Ala Leu Asp
85 90 95
Lys Asn Phe Leu Thr Pro Phe Phe Thr Thr Asn Ser Gly Asp Gly Asp
100 105 110
Gly Asp
<210> 9
<211> 2284
<212> DNA
<213> Arabidopsis thaliana
<220>
<223> Figure 5(a) and (b)
<400> 9
ttcgcggccg cgtctctctc tatttccagt aaaaaatcga aatttcgtat aatttcctca 60
gtcccgtaat tttctccttt tttttcttcc ccaattcctt caattttcga attcgcctct 120
ctgtttcgtt cctcgtagac gaagaagaag aagaatctca ggttttagct ttcgaagctt 180
ccaaaatttt gaattttgat cttctgggct cttttgtaaa tcagactgaa gatatttaga 240
ttacccagaa gttgttcaag gaatggtttc agtggacagc acggaaagat aaaagagact 300
tttttttcca gattttgctg atccaaaatc tgaatagttg ttcatgttct tggatcaaat 360
ctggaaagag gaagtttgtt ggatctagaa gaagataaca atgttggatt ctctagtgtc 420
74
CA 02323756 2000-09-18
gaaactgcct tcgttatcga catctgatca cgcttctgtg gttgcgttga atctctttgt 480
tgcacttctt tgtgcttgta ttgttcttgg tcatcttttg gaagagaata gatggatgaa 540
cgaatccatc accgccttgt tgattgggct aggcactggt gttaccattt tgttgattag 600
taaaggaaaa agctcgcatc ttctcgtctt tagtgaagat cttttcttca tatatctttt 660
gccacccatt atattcaatg cagggtttca agtaaaaaag aagcagtttt tccgcaattt 720
cgtgactatt atgctttttg gtgctgttgg gactattatt tcttgcacaa tcatatctct 780
aggtgtaaca cagttcttta agaagttgga cattggaacc tttgacttgg gtgattatct 840
tgctattggt gccatatttg ctgcaacaga ttcagtatgt acactgcagg ttctgaatca 900
agacgagaca cctttgcttt acagtcttgt attcggagag ggtgttgtga atgatgcaac 960
gtcagttgtg gtcttcaacg cgattcagag ctttgatctc actcacctaa accacgaagc 1020
tgcttttcat cttcttggaa acttcttgta tttgtttctc ctaagtacct tgcttggtgc 1080
tgcaaccggt ctgataagtg cgtatgttat caagaagcta tactttggaa ggcactcaac 1140
tgaccgagag gttgccctta tgatgcttat ggcgtatctt tcttatatgc ttgctgagct 1200
tttcgacttg agcggtatcc tcactgtgtt tttctgtggt attgtgatgt cccattacac 1260
atggcacaat gtaacggaga gctcaagaat aacaacaaag catacctttg caactttgtc 1320
atttcttgcg gagacattta ttttcttgta tgttggaatg gatgccttgg acattgacaa 1380
gtggagatcc gtgagtgaca caccgggaac atcgatcgca gtgagctcaa tcctaatggg 1440
tctggtcatg gttggaagag cagcgttcgt ctttccgtta tcgtttctat ctaacttagc 1500
caagaagaat caaagcgaga aaatcaactt taacatgcag gttgtgattt ggtggtctgg 1560
tctcatgaga ggtgctgtat ctatggctct tgcatacaac aagtttacaa gggccgggca 1620
cacagatgta cgcgggaatg caatcatgat cacgagtacg ataactgtct gtctttttag 1680
cacagtggtg tttggtatgc tgaccaaacc actcataagc tacctattac cgcaccagaa 1740
cgccaccacg agcatgttat ctgatgacaa caccccaaaa tccatacata tccctttgtt 1800
ggaccaagac tcgttcattg agccttcagg gaaccacaat gtgcctcggc ctgacagtat 1860
acgtggcttc ttgacacggc ccactcggaa ccgtgcatta ctaactggag acaatttgat 1920
gactctttca tgcgacccgt ctttggaggt cgtggctttg taccctttgt tccaggttct 1980
ccaactgaga gaaaccctcc tgatcttagt aaggcttgag ggtaacgtgg aagaaaagct 2040
ttgatttttt ttggtagaaa agggtgattc aaattatgct tttgtgtaaa ttatccattt 2100
gtaatattgt ttgtgaggac agaaatctgt cctaacgttt tgagagcaga aagcaaaaca 2160
CA 02323756 2000-09-18
tggcaacttt gaagtgtttg attgatgtat gtaattatat tcatatttgt tttgttgtaa 2220
cacaaactac acatttgttt atgttttgaa tttggttttt gcttcgaaaa aaaaaaaaaa 2280
aaaa 2284
<210> 10
<211> 547
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 5(a)and (b)
<400> 10
Met Leu Asp Ser Leu Val Ser Lys Leu Pro Ser Leu Ser Thr Ser Asp
1 5 10 15
His Ala Ser Val Val Ala Leu Asn Leu Phe Val Ala Leu Leu Cys Ala
20 25 30
Cys Ile Val Leu Gly His Leu Leu Glu Glu Asn Arg Trp Met Asn Glu
35 40 45
Ser Ile Thr Ala Leu Leu Ile Gly Leu Gly Thr Gly Val Thr Ile Leu
50 55 60
Leu Ile Ser Lys Gly Lys Ser Ser His Leu Leu Val Phe Ser Glu Asp
65 70 75 80
Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe Asn Ala Gly Phe
85 90 95
Gin Val Lys Lys Lys Gin Phe Phe Arg Asn Phe Val Thr Ile Met Leu
100 105 110
Phe Gly Ala Val Gly Thr Ile Ile Ser Cys Thr Ile Ile Ser Leu Gly
115 120 125
Val Thr Gin Phe Phe Lys Lys Leu Asp Ile Gly Thr Phe Asp Leu Gly
130 135 140
Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ala Ala Thr Asp Ser Val Cys
145 150 155 160
Thr Leu Gin Val Leu Asn Gin Asp Glu Thr Pro Leu Leu Tyr Ser Leu
165 170 175
Val Phe Gly Glu Giy Val Val Asn Asp Ala Thr Ser Val Val Val Phe
180 185 190
Asn Ala Ile Gin Ser Phe Asp Leu Thr His Leu Asn His Glu Ala Ala
195 200 205
76
CA 02323756 2000-09-18
Phe His Leu Leu Gly Asn Phe Leu Tyr Leu Phe Leu Leu Ser Thr Leu
210 215 220
Leu Gly Ala Ala Thr Gly Leu Ile Ser Ala Tyr Val Ile Lys Lys Leu
225 230 235 240
Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala Leu Met Met Leu
245 250 255
Met Ala Tyr Leu Ser Tyr Met Leu Ala Glu Leu Phe Asp Leu Ser Gly
260 26.5 270
Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser His Tyr Thr Trp
275 280 285
His Asn Val Thr Glu Ser Ser Arg Ile Thr Thr Lys His Thr Phe Ala
290 295 300
Thr Leu Ser Phe Leu Ala Glu Thr Phe Ile Phe Leu Tyr Val Gly Met
305 310 315 320
Asp Ala Leu Asp Ile Asp Lys Trp Arg Ser Val Ser Asp Thr Pro Gly
325 330 335
Thr Ser Ile Ala Val Ser Ser Ile Leu Met Gly Leu Val Met Val Gly
340 345 350
Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser Asn Leu Ala Lys
355 360 365
Lys Asn Gin Ser Glu Lys Ile Asn Phe Asn Met Gin Val Val Ile Trp
370 375 380
Trp Ser Gly Leu Met Arg Gly Ala Val Ser Met Ala Leu Ala Tyr Asn
385 390 395 400
Lys Phe Thr Arg Ala Gly His Thr Asp Val Arg Gly Asn Ala Ile Met
405 410 415
Ile Thr Ser Thr Ile Thr Val Cys Leu Phe Ser Thr Val Val Phe Gly
420 425 430
Met Leu Thr Lys Pro Leu Ile Ser Tyr Leu Leu Pro His Gin Asn Ala
435 440 445
Thr Thr Ser Met Leu Ser Asp Asp Asn Thr Pro Lys Ser Ile His Ile
450 455 460
Pro Leu Leu Asp Gin Asp Ser Phe Ile Glu Pro Ser Gly Asn His Asn
465 470 475 480
Val Pro Arg Pro Asp Ser Ile Arg Gly Phe Leu Thr Arg Pro Thr Arg
485 490 495
Asn Arg Ala Leu Leu Thr Gly Asp Asn Leu Met Thr Leu Ser Cys Asp
500 505 510
77
CA 02323756 2000-09-18
Pro Ser Leu Glu Val Val Ala Leu Tyr. Pro Leu Phe Gln Val Leu Gln
515 520 525
Leu Arg Glu Thr Leu Leu Ile Leu Val Arg Leu Glu Gly Asn Val Glu
530 535 540
Glu Lys Leu
545
<210> 11
<211> 24
<212> DNA
<213> Synthetic
<220>
<223> Page 5 and 53 - Forward primer - Isolated
oligonucleotide sequence
<400> 11
gccatgttgg attctctagt gtcg 24
<210> 12
<211> 27
<212> DNA
<213> Synthetic
<220>
<223> Pages 5 and 53 -Reverse primer - isolated
oligonucleotide
<400> 12
ccgaattctc aaagcttttc ttccacg 27
<210> 13
<211> 29
<212> DNA
<213> Synthetic
<220>
<223> Pages 5 and 53 - Isolated oligonucleotide
<400> 13
cggaattcac agaaaaacac agtgaggat 29
<210> 14
<211> 24
<212> DNA
<213> Synthetic
<220>
78
CA 02323756 2000-09-18
<223> Page 5 - Isolated oligonucleotide
<400> 14
gccatgttgg attctctagt gtcg 24
<210> 15
<211> 27
<212> DNA
<213> Synthetic
<220>
<223> Page 5 - Isolated oligonucleotide
<400> 15
ccgaattctc aaagcttttc ttccacg 27
<210> 16
<211> 29
<212> DNA
<213> Synthetic
<220>
<223> Page 5 - Isolated oligonucleotide
<400> 16
cggaattcac agaaaaacac agtgaggat 29
<210> 17
<211> 1683
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (67)..(1041)
<223> Figure 1(d)
<400> 17
cgccacgacc ctcagggcca ggttaagcag cagcaagcgg ccggcgttgg tatactgctt 60
cagatt atg atg ctc gtg ctt tcc ttc gtt ctc ggc cat gtc ctc cgc 108
Met Met Leu Val Leu Ser Phe Val Leu Gly His Val Leu Arg
1 5 10
cgt cat cga ttc cac tat ctt cct gaa gcc agc ggt tcg ctt ctc att 156
Arg His Arg Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile
15 20 25 30
ggt tta atc gtc ggt ata ctt get aat atc tcc gat act gag act agc 204
Gly Leu Ile Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser
79
CA 02323756 2000-09-18
35 40 45
att agg acg tgg ttt aat ttc cac gaa gag ttc ttc ttc ttg ttt ttg 252
Ile Arg Thr Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu
50 55 60
ttg cct ccc atc ata ttc cag tca ggt ttc agt ctt caa cct aaa cca 300
Leu Pro Pro Ile Ile Phe Gln Ser Gly Phe Ser Leu Gln Pro Lys Pro
65 70 75
ttc ttt tct aac ttt gga gcc att gtt acc ttt get atc atc gga act 348
Phe Phe Ser Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr
80 85 90
ttt gtc get tca gtt gtt act ggt ggt ctg gtt tat ctt ggc ggc tct 396
Phe Val Ala Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser
95 100 105 110
atg tat ctc atg tat aaa ctt ccc ttt gtt gag tgt ctt atg ttt ggt 444
Met Tyr Leu Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly
115 120 125
gca ctt ata tca get acg gac cct gtc act gta ctc tct ata ttc cag 492
Ala Leu Ile Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gln
130 135 140
gat gtg ggc acc gat gtt aac ctg tat get ttg gtc ttt gga gaa tca 540
Asp Val Gly Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser
145 150 155
gtt ctg aat gat get atg gca ata tca ttg tac aga aca atg tcc tta 588
Val Leu Asn Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu
160 165 170
gta aac cgc cag tcc tcg tct ggg gaa cat ttt ttc atg gtg gtg atc 636
Val Asn Arg Gln Ser Ser Ser Gly Glu His Phe Phe Met Val Val Ile
175 180 185 190
agg ttt ttt gag act ttt get ggc tca atg tct gca ggg gtt ggg gtt 684
Arg Phe Phe Glu Thr Phe Ala Gly Ser Met Ser Ala Gly Val Gly Val
195 200 205
gga ttc act tca get tta ctc ttt aag tat gca gga ttg gac acc gag 732
Gly Phe Thr Ser Ala Leu Leu Phe Lys Tyr Ala Gly Leu Asp Thr Glu
210 215 220
aat ctt cag aac ttg gag tgt tgt ctc ttt gta ctt ttc ccg tat ttt 780
Asn Leu Gln Asn Leu Glu Cys Cys Leu Phe Val Leu Phe Pro Tyr Phe
225 230 235
tca tac atg ctt gca gaa ggt gtt ggt ctc tcc ggc att gtt tct ata 828
Ser Tyr Met Leu Ala Glu Gly Val Gly Leu Ser Gly Ile Val Ser Ile
240 245 250
ctc ttc aca gga att gtt atg aag cgoo tac act ttc tca aat ctc tca 876
Leu Phe Thr Gly Ile Val Met Lys Arg Tyr Thr Phe Ser Asn Leu Ser
255 260 265 270
CA 02323756 2000-09-18
gaa get tca cag agt ttc gta tct tct ttt ttt cac ttg ata tct tcg 924
Glu Ala Ser Gln Ser Phe Val Ser Ser Phe Phe His Leu Ile Ser Ser
275 280 285
cta gca gaa act ttc acg ttc att tac atg gga ttt gat att gcc atg 972
Leu Ala Glu Thr Phe Thr Phe Ile Tyr Met Gly Phe Asp Ile Ala Met
290 295 300
gag cag cat agc tgg tcc cat gtt ggg ttt atc ctt ttc tct att gta 1020
Glu Gln His Ser Trp Ser His Val Gly Phe Ile Leu Phe Ser Ile Val
305 310 315
tcc tca ttt act gat cgt cag tgattgzatg cagtggctgt caatgtattt 1071
Ser Ser Phe Thr Asp Arg Gln
320 325
gggtgtgcat atttggtcaa cctatttaga caggagaacc agaagatacc tatgaagcac 1131
caaaaagccc tttggtatag tggacttcga ggggcaatgg catttgcact tgcacttcaa 1191
tcacttcatg atctaccaga gggtcacggc caaatcatct ttactgcaaa ccacaactat 1251
tgttgttgtc acggttttac taataggagg ttcgacaggt aaaatgttgg aagctttgga 1311
agttgtaggt gacgatcttg atgactccat gtctaaaggc tttgaagaga gcgatcatca 1371
gtatgtccct cctcctttta gcattggagc ttcatctgac gaggatacat catcatcagg 1431
aagcaggttc aagatgaagc tgaaggagtt tcacaaaacc actacatcat tcaccgcgtt 1491
ggacaaaaac tttctgactc cgttcttcac aactaatagt ggagatggag atggagatgg 1551
ggagtagcat ggaaaagatg tgtatttgtg gtccaggcca agctataatt agagtacaca 1611
tatgtctatg taagattaac actggttgat tttacctctc gcaaaatgcc cactataaag 1671
ttgacgattt cc 1683
<210> 18
<211> 325
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(d)
<400> 18
Met Met Leu Val Leu Ser Phe Val Leu Gly His Val Leu Arg Arg His
1 5 10 15
Arg Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile Gly Leu
20 25 30
Ile Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser Ile Arg
81
CA 02323756 2000-09-18
35 40 45
Thr Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu Leu Pro
50 55 60
Pro Ile Ile Phe Gin Ser Gly Phe Ser Leu Gin Pro Lys Pro Phe Phe
65 70 75 80
Ser Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr Phe Val
85 90 95
Ala Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser Met Tyr
100 105 110
Leu Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly Ala Leu
115 120 125
Ile Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gin Asp Val
130 135 140
Gly Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser Val Leu
145 150 155 160
Asn Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu Val Asn
165 170 175
Arg Gin Ser Ser Ser Gly Glu His Phe Phe Met Val Val Ile Arg Phe
180 185 190
Phe Glu Thr Phe Ala Gly Ser Met Ser Ala Gly Val Gly Val Gly Phe
195 200 205
Thr Ser Ala Leu Leu Phe Lys Tyr Ala Gly Leu Asp Thr Glu Asn Leu
210 215 220
Gin Asn Leu Glu Cys Cys Leu Phe Val Leu Phe Pro Tyr Phe Ser Tyr
225 230 235 240
Met Leu Ala Glu Gly Val Gly Leu Ser Gly Ile Val Ser Ile Leu Phe
245 250 255
Thr Gly Ile Val Met Lys Arg Tyr Thr Phe Ser Asn Leu Ser Glu Ala
260 265 270
Ser Gin Ser Phe Val Ser Ser Phe Phe His Leu Ile Ser Ser Leu Ala
275 280 285
Glu Thr Phe Thr Phe Ile Tyr Met Gly Phe Asp Ile Ala Met Glu Gin
290 295 300
His Ser Trp Ser His Val Gly Phe Ile Leu Phe Ser Ile Val Ser Ser
305 310 315 320
Phe Thr Asp Arg Gin
325
82
CA 02323756 2000-09-18
<210> 19
<211> 2122
<212> DNA
<213> Arabidopsis thaliana
<220>
<221> CDS
<222> (55)..(750)
<223> Figure 1(e) AtNHX4 CDNA sequence
<400> 19
cagggccagg ttaagcagca gcaagcggcc ggcgttggta tactgcttca gatt atg 57
Met
1
atg ctc gtg ctt tcc ttc gtt ctc ggc cat gtc ctc cgc cgt cat cga 105
Met Leu Val Leu Ser Phe Val Leu Gly His Val Leu Arg Arg His Arg
10 15
ttc cac tat ctt cct gaa gcc agc ggt= tcg ctt ctc att ggt tta atc 153
Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile Gly Leu Ile
20 25 30
gtc ggt ata ctt get aat atc tcc gat act gag act agc att agg acg 201
Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser Ile Arg Thr
35 40 45
tgg ttt aat ttc cac gaa gag ttc ttc ttc ttg ttt ttg ttg cct ccc 249
Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu Leu Pro Pro
50 55 60 65
atc ata ttc cag tca ggt ttc agt ctt caa cct aaa cca ttc ttt tct 297
Ile Ile Phe Cln Ser Gly Phe Ser Leu Gln Pro Lys Pro Phe Phe Ser
70 75 80
aac ttt gga gcc att gtt acc ttt get atc atc gga act ttt gtc get 345
Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr Phe Val Ala
85 90 95
tca gtt gtt act ggt ggt ctg gtt tat ctt ggc ggc tct atg tat ctc 393
Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser Met Tyr Leu
100 105 110
atg tat aaa ctt ccc ttt gtt gag tgt ctt atg ttt ggt gca ctt ata 441
Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly Ala Leu Ile
115 120 125
tca get acg gac cct gtc act gta ctc tct ata ttc cag gat gtg ggc 489
Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gln Asp Val Gly
130 135 140 145
acc gat gtt aac ctg tat get ttg gtc ttt gga gaa tca gtt ctg aat 537
Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser Val Leu Asn
150 155 160
gat get atg gca ata tca ttg tac aga aca atg tcc tta gta aac cgc 585
83
CA 02323756 2000-09-18
Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu Val Asn Arg
165 170 175
cag tcc tcg tct ggg gaa cat ttt ttc: atg gtg gtg atc agg ttt ttt 633
Gln Ser Ser Ser Gly Glu His Phe Phe Met Val Val Ile Arg Phe Phe
180 185 190
gag act ttt get ggc tca atg tct gca ggg gtt ggg gtt gga ttc act 681
Glu Thr Phe Ala Gly Ser Met Ser Ala Gly Val Gly Val Gly Phe Thr
195 200 205
tca get tta ata tcc ttc ctc gaa tcc tct att ttt ctt att aga tgt 729
Ser Ala Leu Ile Ser Phe Leu Glu Sei. Ser Ile Phe Leu Ile Arg Cys
210 215 220 225
cac atg gcc aaa aat gta ttg taaaatctta actcagaaca cctctttaag 780
His Met Ala Lys Asn Val Leu
230
tatgcaggat tggacaccga gaatcttcag aacttggagt gttgtctctt tgtacttttc 840
ccgtattttt cgtaagtaga caaaacaact ctcctcctgt ctcttcgtat ttatgacaac 900
acttcttccc cctaatgtat tctggttatt ctgtaagata catgcttgca gaaggtgttg 960
gtctctccgg cattgtttct atactcttca caggaattgt aatcgccgag tcattgtagc 1020
ttttacatct tagttgatgt taatatcttg gaaagacata tttaggctgc ctaatatagt 1080
gctactgtag gttatgaagc gctacacttt ctcaaatctc tcagaagctt cacagagttt 1140
cgtatcttct ttttttcact tgatatcttc gctagcagaa actttcacgt tcatttacat 1200
gggatttgat attgccatgg agcagcatag ctggtcccat gttgggttta tccttttctc 1260
tattgtatcc tcatttactg atcgtcagtg attgtatgca gtgttagtca gtgttgtaaa 1320
tccttgactt taccttttgc ttctgcgttt catgactgac atcagttgtt tattggcgtg 1380
gctaggtgac taaatgcttt tttatcctgg ctgatcgctt cattatcacc atggttttcg 1440
attcggattt acctatatgt tctgcaatgc ttttctcacg cagggctgtc aatgtatttg 1500
ggtgtgcata tttggtcaac ctatttagac aggagaacca gaagatacct atgaagcacc 1560
aaaaagccct ttggtatagt ggacttcgag gggcaatggc atttgcactt gcacttcaat 1620
cacttcatga tctaccagag ggtcacggcc aaatcatctt tactgcaacc acaactattg 1680
ttgttgtcac ggttttacta ataggaggtt cgacaggtaa aatgttggaa gctttggaag 1740
ttgtaggtga cgatcttgat gactccatgt ctgaaggctt tgaagagagc gatcatcagt 1800
atgtccctcc tccttttagc attggagctt catctgacga ggatacatca tcatcaggaa 1860
gcaggttcaa gatgaagctg aaggagtttc acaaaaccac tacatcattc accgcgttgg 1920
84
CA 02323756 2000-09-18
acaaaaactt tctgactccg ttcttcacaa ctaatagtgg agatggagat ggagatgggg 1980
agtagcatgg aaaagatgtg tatttgtggt ccaggccaag ctataattag agtacacata 2040
tgtctatgta agattaacac tggttgattt tacctctcgc aaaatgccca ctataaagtt 2100
gacgatttcc aagacatttc ga 2122
<210> 20
<211> 232
<212> PRT
<213> Arabidopsis thaliana
<220>
<223> Figure 1(e)
<400> 20
Met Met Leu Val Leu Ser Phe Val Leu Gly His Val Leu Arg Arg His
1 5 10 15
Arg Phe His Tyr Leu Pro Glu Ala Ser Gly Ser Leu Leu Ile Gly Leu
20 25 30
Ile Val Gly Ile Leu Ala Asn Ile Ser Asp Thr Glu Thr Ser Ile Arg
35 40 45
Thr Trp Phe Asn Phe His Glu Glu Phe Phe Phe Leu Phe Leu Leu Pro
50 55 60
Pro Ile Ile Phe Gln Ser Gly Phe Ser Leu Gln Pro Lys Pro Phe Phe
65 70 75 80
Ser Asn Phe Gly Ala Ile Val Thr Phe Ala Ile Ile Gly Thr Phe Val
85 90 95
Ala Ser Val Val Thr Gly Gly Leu Val Tyr Leu Gly Gly Ser Met Tyr
100 105 110
Leu Met Tyr Lys Leu Pro Phe Val Glu Cys Leu Met Phe Gly Ala Leu
115 120 125
Ile Ser Ala Thr Asp Pro Val Thr Val Leu Ser Ile Phe Gln Asp Val
130 135 140
Gly Thr Asp Val Asn Leu Tyr Ala Leu Val Phe Gly Glu Ser Val Leu
145 150 155 160
Asn Asp Ala Met Ala Ile Ser Leu Tyr Arg Thr Met Ser Leu Val Asn
165 170 175
Arg Gln Ser Ser Ser Gly Glu His Phe Phe Met Val Val Ile Arg Phe
180 185 190
Phe Glu Thr Phe Ala Gly Ser Met Ser Ala Gly Val Gly Val Gly Phe
195 200 205
CA 02323756 2000-09-18
Thr Ser Ala Leu Ile Ser Phe Leu Glu Ser Ser Ile Phe Leu Ile Arg
210 215 220
Cys His Met Ala Lys Asn Val Leu
225 230
<210> 21
<211> 569
<212> PRT
<213> Schizosaccharomyces pombe
<220>
<223> Figure 8(a)
<400> 21
Met Pro Asp Ser Lys His Trp Val Ile Leu Leu Phe Arg Arg Asp Gly
1 5 10 15
Asp Asp Asp Asp Asp Asp Gly Gln Asp Pro Ala Leu Gln Glu Leu Tyr
20 25 30
Ser Ser Trp Ala Leu Phe Ile Leu Leu Val Leu Leu Ile Gly Ala Leu
35 40 45
Leu Thr Ser Tyr Tyr Val Gln Ser Lys Lys Ile Arg Ala Ile His Glu
50 55 60
Thr Val Ile Ser Val Phe Val Gly Met Val Val Gly Leu Ile Ile Arg
65 70 75 80
Val Ser Pro Gly Leu Ile Ile Gln Asn Met Val Ser Phe His Ser Thr
85 90 95
Tyr Phe Phe Asn Val Leu Leu Pro Pro Ile Ile Leu Asn Ser Gly Tyr
100 105 110
Glu Leu His Gln Ser Asn Phe Phe Arg Asn Ile Gly Thr Ile Leu Thr
115 120 125
Phe Ala Phe Ala Gly Thr Phe Ile Ser Ala Val Thr Leu Gly Val Leu
130 135 140
Val Tyr Ile Phe Ser Phe Leu Asn Phe Glu Asn Leu Ser Met Thr Phe
145 150 155 160
Val Glu Ala Leu Ser Met Gly Ala Thr Leu Ser Ala Thr Asp Pro Val
165 170 175
Thr Val Leu Ala Ile Phe Asn Ser Tyr Lys Val Asp Gln Lys Leu Tyr
180 185 190
Thr Ile Ile Phe Gly Glu Ser Ile Leu Asn Asp Ala Val Ala Ile Val
195 200 205
86
CA 02323756 2000-09-18
Met Phe Glu Thr Leu Gin Gin Phe Gin Gly Lys Thr Leu His Phe Phe
210 215 220
Thr Leu Phe Ser Gly Ile Gly Ile Phe Ile Ile Thr Phe Phe Ile Ser
225 230 235 240
Leu Leu Ile Gly Val Ser Ile Gly Leu Ile Thr Ala Leu Leu Leu Lys
245 250 255
Tyr Ser Tyr Leu Arg Arg Tyr Pro Ser Ile Glu Ser Cys Ile Ile Leu
260 265 270
Leu Met Ala Tyr Thr Ser Tyr Phe Phe Ser Asn Gly Cys His Met Ser
275 280 285
Gly Val Val Ser Leu Leu She Cys Gly Ile Thr Leu Lys His Tyr Ala
290 295 300
She She Asn Met Ser Tyr Lys Ala Lys Leu Ser Thr Lys Tyr Val She
305 310 315 320
Arg Val Leu Ala Gin Leu Ser Glu Asn Phe Ile She Ile Tyr Leu Gly
325 330 335
Met Ser Leu Phe Thr Gin Val Asp Leu Val Tyr Lys Pro Ile Phe Ile
340 345 350
Leu Ile Thr Thr Val Ala Val Thr Ala Ser Arg Tyr Met Asn Val She
355 360 365
Pro Leu Ser Asn Leu Leu Asn Lys She His Arg Gin Arg Asn Gly Asn
370 375 380
Leu Ile Asp His Ile Pro Tyr Ser Tyr Gin Met Met Leu Phe Trp Ala
385 390 395 400
Gly Leu Arg Gly Ala Val Gly Val Ala Leu Ala Ala Gly Phe Glu Gly
405 410 415
Glu Asn Ala Gin Thr Leu Arg Ala Thr Thr Leu Val Val Val Val Leu
420 425 430
Thr Leu Ile Ile She Gly Gly Thr Thr Ala Arg Met Leu Glu Ile Leu
435 440 445
His Ile Glu Thr Gly Val Ala Ala Asp Val Asp Ser Asp Thr Glu Ile
450 455 460
Gly Met Leu Pro Trp Gin Gin Ser Pro Glu Phe Asp Leu Glu Asn Ser
465 470 475 480
Ala Met Glu Leu Ser Asp Ala Ser Ala Glu Pro Val Val Val Asp Gin
485 490 495
Gin Phe Thr Thr Glu His Phe Asp Glu Gly Asn Ile Ala Pro Thr Leu
500 505 510
87
CA 02323756 2000-09-18
Ser Lys Lys Val Ser Ser Thr Phe Glu Gin Tyr Gin Arg Ala Ala Gly
515 520 525
Ala Phe Asn Gin Phe Phe His Ser Ser Arg Asp Asp Gin Ala Gin Trp
530 535 540
Leu Thr Arg Phe Asp Glu Glu Val Ile Lys Pro Val Leu Leu Glu Arg
545 550 555 560
Asp Asn Leu Lys Asn Gly Thr Lys Lys
565
<210> 22
<211> 633
<212> PRT
<213> Saccharomyces cerevisiae
<220>
<223> Figure 8(b)
<400> 22
Met Leu Ser Lys Val Leu Leu Asn Ile Ala Phe Lys Val Leu Leu Thr
1 5 10 15
Thr Ala Lys Arg Ala Val Asp Pro Asp Asp Asp Asp Glu Leu Leu Pro
20 25 30
Ser Pro Asp Leu Pro Gly Ser Asp Asp Pro Ile Ala Gly Asp Pro Asp
35 40 45
Val Asp Leu Asn Pro Val Thr Glu Glu Met Phe Ser Ser Trp Ala Leu
50 55 60
Phe Ile Met Leu Leu Leu Leu Ile Ser Ala Leu Trp Ser Ser Tyr Tyr
65 70 75 80
Leu Thr Gin Lys Arg Ile Arg Ala Val His Glu Thr Val Leu Ser Ile
85 90 95
Phe Tyr Gly Met Val Ile Gly Leu Ile Ile Arg Met Ser Pro Gly His
100 105 110
Tyr Ile Gin Asp Thr Val Thr Phe Asn Ser Ser Tyr Phe Phe Asn Val
115 120 125
Leu Leu Pro Pro Ile Ile Leu Asn Sear Gly Tyr Glu Leu Asn Gin Val
130 135 140
Asn Phe Phe Asn Asn Met Leu Ser Ile Leu Ile Phe Ala Ile Pro Gly
145 150 155 160
Thr Phe Ile Ser Ala Val Val Ile Gly Ile Ile Leu Tyr Ile Trp Thr
165 170 175
Phe Leu Giy Leu Glu Ser Ile Asp Ile Ser Phe Ala Asp Ala Met Ser
88
CA 02323756 2000-09-18
180 185 190
Val Gly Ala Thr Leu Ser Ala Thr Asp Pro Val Thr Ile Leu Ser Ile
195 200 205
Phe Asn Ala Tyr Lys Val Asp Pro Lys Leu Tyr Thr Ile Ile Phe Gly
210 215 220
Glu Ser Leu Leu Asn Asp Ala Ile Ser Ile Val Met Phe Glu Thr Cys
225 230 235 240
Gin Lys Phe His Gly Gin Pro Ala Thr Phe Ser Ser Val Phe Glu Gly
245 250 255
Ala Gly Leu Phe Leu Met Thr Phe Ser Val Ser Leu Leu Ile Gly Val
260 265 270
Leu Ile Gly Ile Leu Val Ala Leu Leu Leu Lys His Thr His Ile Arg
275 280 285
Arg Tyr Pro Gin Ile Glu Ser Cys Leu Ile Leu Leu Ile Ala Tyr Glu
290 295 300
Ser Tyr Phe Phe Ser Asn Gly Cys His Met Ser Gly Ile Val Ser Leu
305 310 315 320
Leu Phe Cys Gly Ile Thr Leu Lys His Tyr Ala Tyr Tyr Asn Met Ser
325 330 335
Arg Arg Ser Gin Ile Thr Ile Lys Tyr Ile Phe Gin Leu Leu Ala Arg
340 345 350
Leu Ser Glu Asn Phe Ile Phe Ile Tyr Leu Gly Leu Glu Leu Phe Thr
355 360 365
Glu Val Glu Leu Val Tyr Lys Pro Leu Leu Ile Ile Val Ala Ala Ile
370 375 380
Ser Ile Cys Val Ala Arg Trp Cys Ala Val Phe Pro Leu Ser Gin Phe
385 390 395 400
Val Asn Trp Ile Tyr Arg Val Lys Thr Ile Arg Ser Met Ser Gly Ile
405 410 415
Thr Gly Glu Asn Ile Ser Val Pro Asp Glu Ile Pro Tyr Asn Tyr Gin
420 425 430
Met Met Thr Phe Trp Ala Gly Leu Arq_ Gly Ala Val Gly Val Ala Leu
435 440 445
Ala Leu Giy Ile Gin Gly Glu Tyr Lys Phe Thr Leu Leu Ala Thr Val
450 455 460
Leu Val Val Val Val Leu Thr Val Ile Ile Phe Gly Gly Thr Thr Ala
465 470 475 480
Gly Met Leu Glu Val Leu Asn Ile Lys Thr Gly Cys Ile Ser Glu Glu
89
CA 02323756 2000-09-18
485 490 495
Asp Thr Ser Asp Asp Glu Phe Asp Ile Glu Ala Pro Arg Ala Ile Asn
500 50`) 510
Leu Leu Asn Gly Ser Ser Ile Gln Thr Asp Leu Gly Pro Tyr Ser Asp
515 520 525
Asn Asn Ser Pro Asp Ile Ser Ile Asp Gln Phe Ala Val Ser Ser Asn
530 535 540
Lys Asn Leu Pro Asn Asn Ile Ser Thr Thr Gly Gly Asn Thr Phe Gly
545 550 555 560
Gly Leu Asn Glu Thr Glu Asn Thr Ser Pro Asn Pro Ala Arg Ser Ser
565 570 575
Met Asp Lys Arg Asn Leu Arg Asp Lys Leu Gly Thr Ile Phe Asn Ser
580 585 590
Asp Ser Gln Trp Phe Gln Asn Phe Asp Glu Gln Val Leu Lys Pro Val
595 600 605
Phe Leu Asp Asn Val Ser Pro Ser Leu Gln Asp Ser Ala Thr Gln Ser
610 615 620
Pro Ala Asp Phe Ser Ser Gln Asn His
625 630
<210> 23
<211> 378
<212> DNA
<213> Oryza sativa
<220>
<223> Figure 8(c)
<400> 23
caagaagcta tacattggaa ggcattctac tgaccgtgag gttgccctta tgatgctcat 60
ggcttacctt tcatatatgc tggctgagtt gctagatttg agcggcattc tcaccgtatt 120
cttctgtggt attgtaatgt cacattacac ttggcataac gtcacagaga gttcaagagt 180
tacaacaaag cacgcatttg caactctgtc cttcattgct gagacttttc tcttcctgta 240
tgttgggatg gatgcattgg atattgaaaa atgggagntt nccagtgaca gacctggnaa 300
atccattngg gtaagctcaa ttttgctagg gattggttcc tgattggaag ngctgctttt 360
gnaattcccc tggtggtc 378
<210> 24
<211> 268
<212> DNA
<213> Oryza sativa
<220>
<223> Figure 8 (d)
CA 02323756 2000-09-18
<400> 24
gtttggtaat tggaggaggt ggagtaatgg agctcgggtt ggggatgggg atggggctgg 60
gcgacccgnc tgcggactac ggctcgatcg cggcggtggg gatgttcgtg gcgctcatct 120
gcgtctgcat cgtcgtcggc cacctcctcg aggagagccg atggatgaac gagtccatca 180
ccgcgctaat catcgggttg ggtacttgga ggagtgnttt tgnatggtgt cgagctggaa 240
gcactcggna tactggtgtt cagcgagg 268
<210> 25
<211> 380
<212> DNA
<213> Oryza sativa
<220>
<223> Figure 8(e)
<400> 25
acattccctg aaagnactgc tggacntttg agggctcgga tgcctgtaga tccaggactc 60
aaaggatgnt gagctagagg ttgttgggat ggtgaagttt gcttaccaag ggccatttac 120
attgtctggc atcaaactat gcccagccac tgatggcacg gctcagttta atgaggctgg 180
ccacaccttc tccagtggga gttatctgtg catctaattg gtaccttctt tgtattgtag 240
ttgttacttt acccttgatt tgttcggttt gcttctaaag caggttgtga aattcctatt 300
gtatgtngtg acgcttgttt gttttttgag gctggaaatt acatcatgtt tttgatttgt 360
ctattaaaaa aaaaaaaaaa 380
<210> 26
<211> 596
<212> DNA
<213> Medicago truncatula
<220>
<223> Figure 8(f)
<400> 26
gtcaaaactc atccctcctc ttccatttgc atattcttct ttatcatctt ttcttcccta 60
aattagagtc tatccttccg cccatagtct ttgacaccct tttcaaaatt ctagaacaag 120
aattttattc ttcatatata tatatatata tatccaatta accatctcaa tctcatattc 180
acatatacct cataaaccat ccataacatc cttaaaaacc ctctaagccc tttcaaactt 240
tgatttgtaa ttgtttctct tataagtctt aacctgcaca aatcaatttt aatttcttat 300
gttcatatag ttatgaatga ttgaaaaaaa cacaaatgac tccagttatc tgtgagatct 360
ctatgataaa ctctactctc cagacgcagg acacatttag ttcaatcttt ctctgttgtt 420
ttcctctact ggttctatat tttctcatga attattaatt aatcctatat tctttctttt 480
caatacaaat ttagtttcat taattctatc aacataatca attaaactac atagttagaa 540
aaatagtact attaccacga tcactcaaag ttttttagtt tttaacaaac antctg 596
<210> 27
<211> 522
<212> DNA
<213> Hordeum vulgare
<220>
<223> Figure 8(g)
91
CA 02323756 2000-09-18
<400> 27
atttacatgg ttataccagt tatcttgagc acttatgcat catccagtga tcagttttgc 60
ttccattcag actgatgggt ctggcagaag taatgtattc tggtggactt acatctatca 120
gcgatgatga aacttgatga tcagtttttt tagttgaaaa attctgcaag aacagctact 180
taatgctcta ttgtgtatcg caggcacaca tcagctgctg atgtctgcta tacttctgta 240
ctctcactat agctcatcta tgacgtctag acatgctagc gtatgtgtan nnnacatcgc 300
gctagtatgt atactctcac atcatatgct acctgttctat atagaactat gtgatagcta 360
ctgctatact gctgtcatac agagtcccgt taatatcaat gctattttgc tttcctcaaa 420
gaaaaaagga aatgactttc cttttgatta tatatttgat ccaggttttc ggcttgctga 480
ctaagcctct gattaatctc ctcgtcccac caagacctgg ca 522
<210> 28
<211> 330
<212> DNA
<213> Arabidopsis thaliana
<220>
<223> Figure 8(h)
<400> 28
tttccgttat cgtttctatc taacttagcc aagaagaatc aaagcgagaa aatcaacttt 60
aacatgcagg ttgtgatttg gtggtctggt ctcatgagag gtgctgtatc tatggctctt 120
gcatacaaca agtttacaag ggccgggcac acagatgtac gngggaatgc aatcatgatc 180
acgngtacgn taactgtctg tntttttagc acagtggtgt ttggtatgct gaccaaacca 240
ntcataagct acctatttac cgnaccanga accttcatca acgnggcatg tttatcttgn 300
attncaaata acccnaanaa tccnatacca 330
<210> 29
<211> 633
<212> PRT
<213> Saccharomyces cerevisiae
<220>
<223> Figure 2(a)
<400> 29
Met Leu Ser Lys Val Leu Leu Asn Ile Ala Phe Lys Val Leu Leu Thr
1 5 10 15
Thr Ala Lys Arg Ala Val Asp Pro Asp Asp Asp Asp Glu Leu Leu Pro
20 25 30
Ser Pro Asp Leu Pro Gly Ser Asp Asp Pro Ile Ala Gly Asp Pro Asp
35 40 45
Val Asp Leu Asn Pro Val Thr Glu Glu Met Phe Ser Ser Trp Ala Leu
50 55 60
Phe Ile Met Leu Leu Leu Leu Ile Ser Ala Leu Trp Ser Ser Tyr Tyr
65 70 75 80
92
CA 02323756 2000-09-18
Leu Thr Gin Lys Arg Ile Arg Ala Val His Glu Thr Val Leu Ser Ile
85 90 95
Phe Tyr Gly Met Val Ile Gly Leu Ile Ile Arg Met Ser Pro Gly His
100 105 110
Tyr Ile Gin Asp Thr Val Thr Phe Asn Ser Ser Tyr Phe Phe Asn Val
115 120 125
Leu Leu Pro Pro Ile Ile Leu Asn Ser Gly Tyr Glu Leu Asn Gin Val
130 135 140
Asn Phe Phe Asn Asn Met Leu Ser Ile Leu Ile Phe Ala Ile Pro Gly
145 150 155 160
Thr Phe Ile Ser Ala Val Val Ile Gly Ile Ile Leu Tyr Ile Trp Thr
165 170 175
Phe Leu Gly Leu Glu Ser Ile Asp Ile Ser Phe Ala Asp Ala Met Ser
180 18.5 190
Val Gly Ala Thr Leu Ser Ala Thr Asp Pro Val Thr Ile Leu Ser Ile
195 200 205
Phe Asn Ala Tyr Lys Val Asp Pro Lys Leu Tyr Thr Ile Ile Phe Gly
210 215 220
Glu Ser Leu Leu Asn Asp Ala Ile Ser Ile Val Met Phe Glu Thr Cys
225 230 235 240
Gin Lys Phe His Gly Gin Pro Ala Thr Phe Ser Ser Val Phe Glu Gly
245 250 255
Ala Gly Leu Phe Leu Met Thr Phe Ser Val Ser Leu Leu Ile Gly Val
260 265 270
Leu Ile Gly Ile Leu Val Ala Leu Leu Leu Lys His Thr His Ile Arg
275 280 285
Arg Tyr Pro Gin Ile Glu Ser Cys Leu Ile Leu Leu Ile Ala Tyr Glu
290 295 300
Ser Tyr Phe Phe Ser Asn Gly Cys His Met Ser Gly Ile Val Ser Leu
305 310 315 320
Leu Phe Cys Gly Ile Thr Leu Lys His Tyr Ala Tyr Tyr Asn Met Ser
325 330 335
Arg Arg Ser Gin Ile Thr Ile Lys Tyr Ile Phe Gin Leu Leu Ala Arg
340 345 350
Leu Ser Glu Asn Phe Ile Phe Ile Tyr Leu Gly Leu Glu Leu Phe Thr
355 360 365
Glu Val Glu Leu Val Tyr Lys Pro Leu Leu Ile Ile Val Ala Ala Ile
370 375 380
93
CA 02323756 2000-09-18
Ser Ile Cys Val Ala Arg Trp Cys Ala Val Phe Pro Leu Ser Gln Phe
385 390 395 400
Val Asn Trp Ile Tyr Arg Val Lys Thr Ile Arg Ser Met Ser Gly Ile
405 410 415
Thr Gly Glu Asn Ile Ser Val Pro Asp Glu Ile Pro Tyr Asn Tyr Gln
420 425 430
Met Met Thr Phe Trp Ala Gly Leu Arg Gly Ala Val Gly Val Ala Leu
435 440 445
Ala Leu Gly Ile Gln Gly Glu Tyr Lys Phe Thr Leu Leu Ala Thr Val
450 455 460
Leu Val Val Val Val Leu Thr Val Ile Ile Phe Gly Gly Thr Thr Ala
465 470 475 480
Gly Met Leu Glu Val Leu Asn Ile Lys Thr Gly Cys Ile Ser Glu Glu
485 490 495
Asp Thr Ser Asp Asp Glu Phe Asp Ile Glu Ala Pro Arg Ala Ile Asn
500 505 510
Leu Leu Asn Gly Ser Ser Ile Gln Thr Asp Leu Gly Pro Tyr Ser Asp
515 520 525
Asn Asn Ser Pro Asp Ile Ser Ile Asp Gln Phe Ala Val Ser Ser Asn
530 535 540
Lys Asn Leu Pro Asn Asn Ile Ser Thr Thr Gly Gly Asn Thr Phe Gly
545 550 555 560
Gly Leu Asn Glu Thr Glu Asn Thr Ser Pro Asn Pro Ala Arg Ser Ser
565 570 575
Met Asp Lys Arg Asn Leu Arg Asp Lys Leu Gly Thr Ile Phe Asn Ser
580 585 590
Asp Ser Gln Trp Phe Gln Asn Phe Asp Glu Gln Val Leu Lys Pro Val
595 600 605
Phe Leu Asp Asn Val Ser Pro Ser Leu Gln Asp Ser Ala Thr Gln Ser
610 615 620
Pro Ala Asp Phe Ser Ser Gln Asn His
625 630
<210> 30
<211> 669
<212> PRT
<213> Homo sapiens
<220>
<223> Figure 2(a) HsNHE6 Na+/H+ exchanger (GenBank
Accession No. 2944237)
94
CA 02323756 2000-09-18
<400> 30
Met Ala Arg Arg Gly Trp Arg Arg Ala Pro Leu Arg Arg Gly Val Gly
1 5 10 15
Ser Ser Pro Arg Ala Arg Arg Leu Met Arg Pro Leu Trp Leu Leu Leu
20 25 30
Ala Val Gly Val Phe Asp Trp Ala Gly Ala Ser Asp Gly Gly Gly Gly
35 40 45
Glu Ala Arg Ala Met Asp Glu Giu Ile Val Ser Glu Lys Gin Ala Glu
50 55 60
Glu Ser His Arg Gin Asp Ser Ala Asn Leu Leu Ile Phe Ile Leu Leu
65 70 75 80
Leu Thr Leu Thr Ile Leu Thr Ile Trp Leu Phe Lys His Arg Arg Ala
85 90 95
Arg Phe Leu His Glu Thr Gly Leu Ala Met Ile Tyr Gly Leu Leu Val
100 105 110
Gly Leu Val Leu Arg Tyr Gly Ile His Val Pro Ser Asp Val Asn Asn
115 120 125
Val Thr Leu Ser Cys Glu Val Gin Ser Ser Pro Thr Thr Leu Leu Val
130 135 140
Thr Phe Asp Pro Glu Val Phe Phe Asn Ile Leu Leu Pro Pro Ile Ile
145 150 155 160
Phe Tyr Ala Gly Tyr Ser Leu Lys Arg Arg His Phe Phe Arg Asn Leu
165 170 175
Gly Ser Ile Leu Ala Tyr Ala Phe Leu Gly Thr Ala Ile Ser Cys Phe
180 185 190
Val Ile Gly Ser Ile Met Tyr Gly Cys Val Thr Leu Met Lys Val Thr
195 200 205
Gly Gin Leu Ala Gly Asp Phe Tyr Phe Thr Asp Cys Leu Leu Phe Gly
210 215 220
Ala Ile Val Ser Ala Thr Asp Pro Val Thr Val Leu Ala Ile Phe His
225 230 235 240
Glu Leu Gin Val Asp Val Glu Leu Tyr Ala Leu Leu Phe Gly Glu Ser
245 250 255
Val Leu Asn Asp Ala Val Ala Ile Val Leu Ser Ser Ser Ile Val Ala
260 265 270
Tyr Gin Pro Ala Gly Asp Asn Ser His Thr Phe Asp Val Thr Ala Met
275 280 285
CA 02323756 2000-09-18
Phe Lys Ser Ile Gly Ile Phe Leu Gly Ile Phe Ser Gly Ser Phe Ala
290 295 300
Met Gly Ala Ala Thr Gly Val Val Thr Ala Leu Val Thr Lys Phe Thr
305 310 315 320
Lys Leu Arg Glu Phe Gln Leu Leu Glu Thr Gly Leu Phe Phe Leu Met
325 330 335
Ser Trp Ser Thr Phe Leu Leu Ala Glu Ala Trp Gly Phe Thr Gly Val
340 345 350
Val Ala Val Leu Phe Cys Gly Ile Thr Gln Ala His Tyr Thr Tyr Asn
355 360 365
Asn Leu Ser Thr Glu Ser Gln His Arq_ Thr Lys Gln Leu Phe Glu Leu
370 375 380
Leu Asn Phe Leu Ala Glu Asn Phe Ile Phe Ser Tyr Met Gly Leu Thr
385 390 395 400
Leu Phe Thr Phe Gln Asn His Val Phe Asn Pro Thr Phe Val Val Gly
405 410 415
Ala Phe Val Ala Ile Phe Leu Gly Arg Ala Ala Asn Ile Tyr Pro Leu
420 425 430
Ser Leu Leu Leu Asn Leu Gly Arg Arg Ser Lys Ile Gly Ser Asn Phe
435 440 445
Gln His Met Met Met Phe Ala Gly Leu Arg Gly Ala Met Ala Phe Ala
450 455 460
Leu Ala Ile Arg Asp Thr Ala Thr Tyr Ala Arg Gln Met Met Phe Ser
465 470 475 480
Thr Thr Leu Leu Ile Val Phe Phe Thr Val Trp Val Phe Gly Gly Gly
485 490 495
Thr Thr Ala Met Leu Ser Cys Leu His Ile Arg Val Gly Val Asp Ser
500 505 510
Asp Gln Glu His Leu Gly Val Pro Glu Asn Glu Arg Arg Thr Thr Lys
515 520 525
Ala Glu Ser Ala Trp Leu Phe Arg Met Trp Tyr Asn Phe Asp His Asn
530 535 540
Tyr Leu Lys Pro Leu Leu Thr His Ser Gly Pro Pro Leu Thr Thr Thr
545 550 555 560
Leu Pro Ala Cys Cys Gly Pro Ile Ala Arg Cys Leu Thr Ser Pro Gln
565 570 575
Ala Tyr Glu Asn Gln Glu Gln Leu Lys Asp Asp Asp Ser Asp Leu Ile
580 58.5 590
96
CA 02323756 2000-09-18
Leu Asn Asp Gly Asp Ile Ser Leu Thr Tyr Gly Asp Ser Thr Val Asn
595 600 605
Thr Glu Pro Ala Thr Ser Ser Ala Pro Arg Arg Phe Met Gly Asn Ser
610 615 620
Ser Glu Asp Ala Leu Asp Arg Glu Leu Ala Phe Gly Asp His Glu Leu
625 630 635 640
Val Ile Arg Gly Thr Arg Leu Val Leu Pro Met Asp Asp Ser Glu Pro
645 650 655
Pro Leu Asn Leu Leu Asp Asn Thr Arq His Gly Pro Ala
660 665
<210> 31
<211> 541
<212> PRT
<213> C. elegans
<220>
<223> Figure 2(a) CeNHEl (GenBank Accession No. 3877723)
<400> 31
Met Lys Val Glu Ser Leu Phe Phe Met Ser Gin Thr Phe Asp Val Ile
1 5 10 15
Thr Lys Asn Lys Thr Ile Val Lys Glu Pro Pro Asp Tyr Leu Met Leu
20 25 30
Glu Val Lys Pro Glu Gly Gly Ser Arg Val Ser Phe His Tyr Glu Leu
35 40 45
Ile Glu Gly Phe Phe Ala Asp Lys Arg Lys Lys Ile Glu Gin Gin Ile
50 55 60
Glu Gin Lys Ser Val Phe Ser Pro Glu Val Phe Phe Asn Met Leu Ile
65 70 75 80
Pro Pro Ile Ile Phe Asn Ala Gly Tyr Ser Leu Lys Lys Arg His Phe
85 90 95
Phe Arg Asn Ile Gly Ser Ile Leu Ala Ile Val Phe Ile Gly Thr Thr
100 105 110
Ile Ser Cys Phe Gly Thr Gly Cys Leu Met Phe Val Phe Thr Ser Ile
115 120 125
Phe Gin Met Gly Tyr Ser Phe Lys Giu Leu Leu Phe Phe Gly Ala Leu
130 135 140
Ile Ser Ala Thr Asp Pro Val Thr Ile Ile Ser Val Phe Asn Asp Met
145 150 155 160
Asn Val Glu Ala Asp Leu Phe Ala Leu Ile Phe Gly Glu Ser Ala Leu
97
CA 02323756 2000-09-18
165 170 175
Asn Asp Ala Val Ala Ile Val Leu Ser Glu Val Ile Glu Asn Phe Ser
180 185 190
Thr Ser Ser Glu Ala Ile Thr Leu Gin Asp Phe Gly Ser Ala Ile Ala
195 200 205
Gly Phe Ala Gly Val Phe Phe Gly Ser Leu Met Leu Gly Phe Met Ile
210 215 220
Gly Cys Met Asn Ala Phe Leu Thr Lys Met Thr Leu Ile Ser Glu His
225 230 235 240
Ala Leu Leu Glu Ser Ser Leu Phe Val Leu Ile Ser Tyr Ile Ser Phe
245 250 255
Leu Val Ala Glu Val Cys Gly Leu Thr Gly Ile Val Ser Val Leu Phe
260 265 270
Cys Gly Ile Ala Gin Ala His Tyr Thr Tyr Asn Asn Leu Ser Asp Glu
275 280 285
Ser Gin Ser Asn Thr Lys His Phe Phe His Met Val Ser Phe Ile Met
290 295 300
Glu Ser Phe Ile Phe Cys Tyr Ile Gly Val Ser Val Phe Val Thr Asn
305 310 315 320
Asn Gin Arg Trp Ser Phe Ser Phe Leu. Leu Phe Ser Leu Ile Ser Ile
325 330 335
Thr Ala Ser Arg Ala Leu Phe Val Tyr Pro Leu Ser Trp Leu Leu Asn
340 345 350
Ile Arg Arg Arg Pro Lys Ile Pro Lys Arg Tyr Gin His Met Ile Leu
355 360 365
Phe Ala Gly Leu Arg Gly Ala Met Ala Phe Ala Leu Ala Gly Arg Asn
370 375 380
Thr Ser Thr Glu Asn Arg Gin Met Ile Phe Ala Thr Thr Thr Ala Val
385 390 395 400
Val Ile Val Thr Val Leu Val Asn Gly Gly Leu Thr Ser Trp Met Ile
405 410 415
Asp Tyr Leu Gin Ile Lys His Gly Lys Asp Ala Ile Glu Glu Gly Gin
420 425 430
Arg Leu Glu Asn Ser Met Ser Ser Ser Pro Ala Asp Gin His Ser Asp
435 440 445
Leu Asp Glu Ser Val Pro Val Thr Met Ser Pro Gly Leu Asn Pro Trp
450 455 460
Asp Lys Ala Phe Leu Pro Arg Lys Trp Tyr His Phe Asp Ala Arg Trp
98
CA 02323756 2000-09-18
465 470 475 480
Gln Leu Leu Lys Leu Val Phe Gln Phe His Glu Thr Ser Thr Asp Pro
485 490 495
Cys Asp Ala Ile She Gly Thr Asn Thr Pro Thr Val Leu Ser Ser Ile
500 505 510
Asp She Leu Val Asp Phe Lys Pro Ser Thr Arg Val Arg Gin Cys Arg
515 520 525
Ala Leu Gln Tyr Asn Cys Thr Ile Arg Asp Ser Ile Asp
530 535 540
<210> 32
<211> 21
<212> DNA
<213> Synthetic
<220>
<223> Page 54 - PCR forward primer (X6F)
<400> 32
cctcaggtga taccaatctc a 21
<210> 33
<211> 20
<212> DNA
<213> Synthetic
<220>
<223> Page 54 - PCR reverse primer (X6REV)
<400> 33
gatccaatgt aacaccggag 20
<210> 34
<211> 19
<212> DNA
<213> Synthetic
<220>
<223> Page 54 - PCR forward primer (NHX7F)
<400> 34
ttcgttctcg gccatgtcc 19
<210> 35
<211> 22
<212> DNA
99
CA 02323756 2000-09-18
<213> Synthetic
<220>
<223> Page 54 - PCR reverse primer (NHX7REV)
<400> 35
cggagagacc aacaccttct gc 22
<210> 36
<211> 24
<212> DNA
<213> Synthetic
<220>
<223> Page 37 - preferred oligonucleotide probe
<400> 36
ttcttcatat atcttttgcc accc 24
<210> 37
<211> 30
<212> DNA
<213> Synthetic
<220>
<223> Page 55 - Primer
<400> 37
cgcgtcgaca tgttggattc tctagtgtcg 30
1
100
