Note: Descriptions are shown in the official language in which they were submitted.
CA 02350186 2001-05-08
VOSSI US & PARTN ER
Patentanwglte
SIEBERTSTRASSE 4 - 81675 MUNCHEN
TEL.: +49-89-41 30 40 - FAX: +49-89-47 30 41 11 - FAX (Marken-Trademarks) :
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PCT application
ETH Zurich
Our ref.: C 2645 PCT
Promoters for gene expression in roots of plants
Description
The present invention relates to promoters causing a root-specific expression
of
coding nucleotide sequences which are controlled by the promoters for tissue-
specific gene expression in plants, expression cassettes, recombinant vectors
and
microorganisms comprising such promoters, transgenic plants transformed with
them, a method for the production of transgenic plants and a method for the
isolation
of root-specific promoters.
In the following, documents from the state of the art are cited the disclosure
content
of which is incorporated in this application by reference.
The use of plants modified in their genotype by means of genetic engineering
has
proven to be advantageous in many areas of agriculture and often to be the
only way
to transmit certain properties to useful plants. The principal aims are plant
protection
as well as an increase of quality of the harvestable products.
Various methods for the genetic modification of dicotyledonous and
monocotyledonous plants are known (cf., amongst others, Gasser and Fraley,
Science 244 (1989), 1293-1299; Potrykus, Ann. Rev. Plant Mol. Biol. Plant
Physiol.
42 (1991 ), 205-225). All the methods are based on the transmission of gene
constructs which, in most cases, are new combinations of specific coding
regions of
structural genes with promoter regions of other structural genes as well as
transcription terminators.
The provision of promoters plays a great role for the production of transgenic
plants
since the specificity of a promoter is crucial for the point in time, the type
of tissue
and the intensity in which a structural gene transmitted by means of genetic
engineering is expressed.
A multitude of promoters controlling the expression of foreign genes in plants
is
known. The promoter most commonly used is the 35S CaMV promoter (Franck et
al.,
Cell 1 (1980), 285-294) leading to a constitutive expression of the gene
introduced.
Often, however, inducible promoters are used, for example for wound induction
(DE-
\\Ntvossiusl\Allgemein\Daten-1\ast\translations\Eng\C\C2645PCT.doc
CA 02350186 2001-05-08
1
V
A-3843628), chemical induction (Ward et al., Plant Molec. Biol. 22 (1993), 361-
366)
or light induction (Fluhr et al., Science 232 (1986), 1106-1112).
Also, the use of cell- and tissue-specific promoters was described: guard cell-
specific
gene expression (DE-A-4207358), seed-, tuber- and fruit-specific gene
expression
(summarized in Edwards and Coruzzi, Annu. Rev. Genet. 24 (1990), 275-303; DE-A-
3843627), phloem-specific gene expression (Schmulling et al., Plant Cell 1
(1989),
665-670), root nodule-specific gene expression (DE-A-3702497) or meristem-
specific
gene expression (Ito et al., Plant Mol. Biol. 24 (1994), 863-878).
Promoters mediating the gene expression in roots include, for example, the
class II
patatin promoter (Koster-Topfer et al., Mol. Gen. Genet. 219 (1989), 390-396)
exhibiting a high expression in potato tubers and in specific cell layers of
root tips
after fusion with the reporter gene of the f3-glucuronidase (GUS) gene. GUS
fusion
experiments with the agropine synthase promoter (ags) (Inoguchi et al., Plant
Phys.
149 (1996), 73-78) exhibited a high GUS activity primarily in roots.
Transgenic
Arabidopsis plants containing an AKT1 (= potassium channel)-GUS-construct
(Lagarde et al., Plant J. 9 (1996), 195-203) particularly led to an expression
in the
outer cell layers of mature root segments. A portion with a length of 636 by
of the 5'
region of the TobRB7 (= water channel) gene (Yamamoto et al., Plant Cell 3
(1991 ),
371-382) mediated a root-specific GUS expression in transgenic tobacco plants.
Furthermore, GUS fusion experiments with the promoter of an extensin gene were
described wherein GUS activity in soy bean seedlings could be determined
depending on the developmental stage in the hypocotyl and in the root and/or
specific zones of the root (Ahn et al., Plant Cell 8 (1996), 1477-1490).
The use of the promoters described is often problematic. Promoters causing a
constitutive expression of the genes which are controlled by said promoters
can be
used, for example, for the production of plants which are tolerant to
herbicides and
resistant to pathogens. The disadvantage of these promoters, however, is that
the
products of the genes which are controlled by them are present in all parts of
the
plant, including the harvested parts of the plants, which can be undesirable
in some
cases. Inducible promoters are not unproblematic neither since the induction
conditions are typically difficult to control in plants which are used
agriculturally
outdoors.
If the various approaches of genetic modification in plants are to be carried
out
successfully, it is furthermore necessary that the genes which are to be
regulated in
different ways are subjected to the control of different promoters. It is,
thus,
necessary to provide different promoter systems with different specificities.
Only a limited number of promoters regulating the gene expression in roots has
been
known so far. if specific approaches of genetic modification in plants are to
be carried
2
CA 02350186 2001-05-08
out successfully, it is necessary to provide further alternative promoter
systems for
the gene expression in roots, which, in comparison to known systems, are
regulated
differently.
Therefore, the technical problem underlying the present invention is to
provide
means for an organ-specific, preferably root-specific gene expression of
plants.
These means should be suitable, for example, for the expression of genes
influencing the uptake of nutrients from the soil and for the expression of
genes
modifying the growth of roots.
This technical problem has been solved by the provision of the embodiments
characterized in the claims.
Thus, the present invention relates to a promoter selected from the group
consisting
of
a) promoters comprising the nucleic acid sequence indicated under SEQ ID No.
1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6;
b) promoters comprising a functional part of the nucleic acid sequence
indicated
under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
or SEQ ID No. 6 and causing a root-specific expression of a coding
nucleotide sequence controlled by them in plants;
c) promoters having a sequence which hybridizes with one of the sequences
shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ
ID No. 5 or SEQ ID No. 6 and which causes a root-specific expression of a
coding nucleotide sequence controlled by them in plants; and
d) promoters of genes encoding proteins the amino acid sequence of which
exhibits a homology of at least 60% to the amino acid sequence indicated
under SEQ ID No. 8, wherein these promoters cause a root-specific
expression of a coding nucleotide sequence controlled by them in plants.
In a preferred embodiment, the promoters of the invention are promoters from
plant
genes or derived therefrom.
Within the context of the present invention, a "promoter" is a DNA sequence
comprising the regulatory portion of a gene, preferably of a structural gene.
The
"regulatory portion" of a gene is understood to be the part which determines
the
expression of the gene. A regulatory portion has a sequence motif where
transcription factors and RNA polymerise assemble and induce the transcription
of
the coding portion of the gene. Furthermore, the regulatory portion may
comprise one
3
CA 02350186 2001-05-08
t
v
or more positive regulatory elements, so-called enhancers. In addition or
instead, it
may also contain negative regulatory elements, so-called silencers. A
"structural
gene" is a genetic unit of regulatory and coding portion whose gene product is
a
protein. The information for the primary amino acid sequence of the protein is
contained in the coding portion of the structural gene, while the regulatory
part
determines when and in which tissues and in which amounts the transcript of
the
coding portion according to which the gene product is synthesized is formed.
The
promoters according to the invention may originate from, for example, plant
genes,
be modified by recombinant DNA techniques and/or be produced synthetically.
Within the meaning of the present invention, the term "root-specific" means
that a
foreign gene which is under the control of a promoter according to the
invention is
expressed in the roots. Within the meaning of the present invention, root
specificity is
particularly given when the promoter according to the invention favours the
expression of a foreign gene in the roots in comparison to mature leaves and
causes
a significantly, for example at least 2- to 5-fold, preferably 5- to 10-fold,
particularly
preferred 10- to 100-fold increased expression.
In the context of the present invention, root specificity can be analysed via
reporter
gene experiments. For testing an isolated promoter sequence for promoter
activity in
roots, the promoter can, for example, be operatively linked to a reporter
gene, such
as the f3-glucuronidase gene from E. coli, in an expression cassette or in a
vector for
plant transformation. This construct is used for the transformation of plants.
Subsequently, the expression of the f3-glucuronidase in roots is determined in
comparison to mature leaves, as has been described, for example, by Martin et
al.
(The GUS Reporter System as a Tool to Study Plant Gene Expression, In: GUS
protocols: Using the GUS Gene as a Reporter of Gene Expression, Academic Press
(1992), 23-43).
The term "root" is known to the person skilled in the art. In addition, it is
referred to
Strasburger (Lehrbuch der Botanik fur Hochschulen: Begr. by Eduard Strasburger
u.a., new edition by Peter Sitte, Hubert Ziegler u.a., 34. Aufl., 1998,
Fischer (Gustav)-
Verlag, Stuttgart).
Surprisingly, it was found that a promoter with the nucleotide sequence shown
under
SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6 causes a root-specific expression of a coding nucleotide sequence which
is
controlled by said promoters in plants.
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CA 02350186 2001-05-08
This observation is particularly surprising since a genomic fragment (SEQ ID
No. 13)
of the promoter region (cf. Example 2) which has an extension of approximately
1000
by in comparison to SEQ ID No. 1 cannot mediate a root-specific GUS
expression.
Only shortened promoter fragments (SEQ ID No. 1 to 6) - in comparison to the
large
fragment (SEQ ID No. 13) - which were produced as described in Example 3C lead
to a root-specific gene expression of a coding nucleotide sequence controlled
by the
promoter.
During the analysis of the cDNA sequence (SEQ ID No. 7), three further ATG
translation initiation codons (positions 42-44, 65-67 and 142-144 of SEQ ID
No. 7)
are found in the 5' region upstream of the actual ATG (pos. 256-258, SEQ ID
No. 7),
which lead to several upstream open reading frames (uORFs). The presence of
such
uORFs can have a negative influence on the translation rate of specific mRNAs.
The
various promoter fragments are preferably constructed in a way so that they do
not
comprise this 5' upstream region (pos. 1-255, SEQ ID No. 7).
The promoter according to the invention allows for a root-specific gene
expression of
a coding nucleotide sequence controlled by the promoter. It represents an
interesting
alternative to other root-specific promoters since it can also mediate the
gene
expression in root hairs. Due to the bigger surface of the root hairs in
comparison to
the root, there is the possibility that the expression of such genes can be
manipulated
more effectively by means of the promoter according to the invention whose
gene
products, for example, mediate the transport of nutrients and metabolites via
the root
hair cells.
Various possible applications for the promoter according to the invention are
at
disposal. One possibility, for example, is the production of transgenic plants
which,
due to a modified root-hair metabolism that has a positive influence on the
root
surroundings (rhizosphere), show an increased yield. Furthermore, the
promoters
according to the invention can be used for the root-specific expression of
genes
mediating, for example, the absorption of heavy metal ions from contaminated
soil.
By means of the promoters according to the invention it would, thus, be
possible to
produce plants which can be used in phytoremediation.
In another embodiment of the invention, the promoters according to the
invention
allow for the expression of a coding nucleotide sequence controlled by said
promoters to be caused in specific root cells as, for example, in root cells
of the
primary root, in root hairs of the primary root, in root cells of the root tip
or in root
hairs of the primary root below the hypocotyl.
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CA 02350186 2001-05-08
Apart from a promoter exhibiting the complete sequence shown under SEQ ID No.
1,
SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6, the
present invention also relates to promoters exhibiting a functional part of
this
sequence and causing a root-specific expression of a coding nucleotide
sequence
controlled by these promoters in plants.
In this context, a "functional part" is to be understood as sequences which,
despite
deviating nucleotide sequence, still have the desired functions, for example
promoter
activity and tissue or organ specificity. A way of measuring promoter activity
is, for
example, the expression rate determined for a specific marker gene which is
subjected under the regulatory control of the promoter according to the
invention.
Suitable marker genes are, for example, the 13-glucuronidase (GUS) gene from
E.
coli or the green fluorescence protein (GFP) gene (Baulcombe et al., Plant J.
7 (16)
(1993), 1045-1053). The organ and tissue specificity can easily be established
by
comparing the expression rates determined for the individual tissues or organs
of the
plant for the above marker genes. Within the meaning of the present invention,
functional parts of the promoter sequences comprise naturally-occurring
variants of
the sequences described herein as well as artificial nucleotide sequences, for
example, obtained by chemical synthesis.
In particular, a functional part is also the natural or artificial mutation of
an originally
isolated promoter sequence which further exhibits the desired functions.
Mutations
comprise substitutions, additions, deletions, exchanges and/or insertions of
one or
more nucleotide residues. Thus, the present invention also comprises
nucleotide
sequences which can be obtained by modifications of the nucleotide sequence
depicted under SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID
No. 5 or SEQ ID No. 6. The aim of such a modification can be, for example, to
further
delimit the promoter sequence contained therein or, for example, to introduce
further
restriction sites.
Functional parts of a promoter sequence also comprise promoter variants whose
promoter activity, compared to the wild type, is decreased or increased.
In principle, the activity of a eukaryotic RNA polymerase II promoter is
caused by the
synergetic interaction of various trans-active factors (DNA binding proteins)
which
bind to the various cis-regulatory DNA elements present in the promoter. These
factors interact directly or indirectly with single or several factors of the
basal
transcription machinery which finally leads to the formation of a pre-
initiation complex
near the transcription start site (Drapkin et al., Current Opinion in Cell
Biology 5
(1993), 469-476). It could be called a modular set-up of eukaryotic RNA
polymerase
II promoters, wherein various cis-elements (modules) as partial components
each
determine the total activity of the promoter (Tjian and Maniatis, Cell 77
(1994), 5-8).
6
CA 02350186 2001-05-08
This modular set-up was elucidated, for example, by promoter studies with the
cauliflower mosaic virus (CaMV) 35S promoter (Benfey and Chua, Science 250
(1990), 959-966; Benfey et al., EMBO J. 9 (1990), 1677-1684; Benfey et al.,
EMBO
J. 9 (1990), 1685-1696). Due to the different tissue specificities that
different
restriction sub-fragments of the -343 to +8 (relative to the transcription
start site)
promoter mediate in transgenic tobacco plants, the promoter was divided into 6
sub-
domains. The strong, constitutive expression which the complete promoter
mediates
can, thus, be sectioned into tissue-specific partial activities.
Single sub-domains of the promoter according to the invention which mediate
potential tissue specificity can be identified, for example, by fusion with a
minimal
promoter reporter gene cassette. A minimal promoter is a DNA sequence
comprising
a TATA box, which is located about 20 to 30 base pairs upstream of the
transcription
start site, or an initiator sequence (Smale and Baltimore, Cell 57 (1989), 103-
113;
Zawel and Reinberg, Proc. Natl. Acad. Sci. 44 (1993), 67-108; Conaway and
Conaway, Annu. Rev. Biochem. 62 (1993), 161-190). Minimal promoters are, for
example, the -63 to +8 035S promoter (Frohberg, Dissertation at the FU Berlin
FB
Biologie (1994)), the -332 to +14 minimal patatin class I promoter as well as
the -176
to +4 minimal PetE promoter (Pwee et al., Plant J. 3 (1993), 437-449).
Furthermore, such sub-domains or cis-elements of the promoter according to the
invention can also be identified via deletion analyses and/or mutageneses
(Kawagoe
et al., Plant J. 5(6) (1994), 885-890). The test for functionality of such a
sub-domain
or cis-element can take place in pianta by the detection of the reporter
activity in
transformed cells. In the context of the present invention, the functional
part of the
promoter sequence also includes sub-domains and/or cis-elements of the
nucleotide
sequences depicted under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.
4, SEQ ID No. 5 or SEQ ID No. 6 which mediate a root specificity.
Moreover, the efficiency of a sub-region or of a cis-element can be increased
significantly by means of multimerisation. In the case of the CaMV 35S
promoter, the
dimerisation of a fragment with a length of 250 by in tandem, for example, led
to a
10-fold increase of the promoter activity (Kay et al., Science 230 (1987),
1299-1302).
In the case of the sub-domain B5 of the CaMV 35S promoter, there was a clear
increase in the activity of the promoter construct when this domain was
present as
tetramer and not as monomer (Benfey et al., EMBO J. 9 (1990), 1685-1696).
In another embodiment, the present invention particularly relates to di- and
multimers
of sub-domains and/or cis-elements of the nucleotide sequences depicted under
SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6.
CA 02350186 2001-05-08
a
In a further embodiment of the invention, the increase in promoter activity,
in
comparison with the wild type, is achieved by the combination of the promoter
according to the invention with a so-called enhancer.
In the literature, various enhancers have been described which, as a rule,
cause a
tissue-specific increase of the expression, wherein the tissue specificity is
usually
determined by the relevant enhancer used (Benfey et al., Science 250 (1990),
959-
966; Benfey et al., EMBO J. 8 (1989), 2195-2202; Chen et al, EMBO J. 7,
(1988),
297-302; Simpson et al., Nature 323 (1986), 551-554).
Moreover, there are enhancers, such as the PetE enhancer (Sandhu et al., Plant
Mol. Biol. 37 (1998), 885-896) which do not have a tissue-specific effect and
can,
thus, be put before the promoter according to the invention as purely
quantitative
enhancer elements in order to increase the expression in roots without
changing the
quality of the tissue specificity of the promoter according to the invention.
A root-specific enhancer which is based on the multimerisation of a specific
box
(Box II) was described, for example, in "DNA-Protein Interactions in the Auxin
regulated Promoter of T-DNA gene 5" (Author: Sirpa Nuotio, Acta Univeritatis
Ouluensis, A Scientiae Rerum Naturalium 299 (1997), Oulu University Press, pp.
38).
Furthermore, synthetic enhancers may be used, too, which are, for example,
derived
from naturally-occurring enhancers and/or can be obtained by combination of
various
enhancers.
The present invention also relates to promoters having a nucleotide sequence
which
hybridizes with the nucleotide sequence shown under SEQ ID No. 1, SEQ ID No.
2,
SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 and which cause a
root-
specific expression of a coding nucleotide sequence controlled by said
promoters in
plants. Such sequences are preferred to hybridize the sequence shown under SEQ
ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No.
6
under stringent conditions.
The term "stringent conditions" preferably refers to hybridization conditions
as
described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory
Manual,
2"d edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY). In
particular, hybridization takes place under the following conditions:
hybridisation buffer: 2 x SSC; 10 x Denhardt's solution (Fikoll 400 + PEG +
BSA; ratio
1:1:1 ); 0.1 % SDS; 5 mM EDTA; 50 mM Na2HP04; 250 ~g/ml herring sperm DNA;
50,ug/ml tRNA; or 0.25 M sodium phosphate buffer, pH 7.2, 1 mM EDTA, 7% SDS
hybridization temperature T = 65 to 68°C;
washing buffer 0.2 x SSC; 0.1 % SDS;
washing temperature T = 65 to 68°C.
s
CA 02350186 2001-05-08
a
Preferably, such promoters have a sequence identity of at least 30%,
preferably of at
least 40%, preferably of at least 50%, particularly preferably at least 60%,
more
particularly preferably of at least 70% and even more preferably of at least
80%,
preferably at least 90% and most preferably at least 95% to the promoter
sequence
shown under SEQ ID No. 1 or parts thereof. The sequence identity of such
promoter
sequences is preferably determined by comparison with the nucleotide sequence
shown under SEQ ID No. 1. If two sequences that are to be compared have a
different length, the sequence identity preferably refers to the percentage
proportion
of the nucleotide residues of the shorter sequence, which are identical to the
nucleotide residues of the longer sequence. The sequence identity can be
determined conventionally by using computer programs such as the Bestfit
program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive Madison, WI 53711 ).
Bestfit
uses the local homology algorithm by Smith and Waterman, Advances in Applied
Mathematics 2 (1981 ), 482-489 to find the segment with the highest sequence
identity between two sequences. When Bestfit or another sequence alignment
program is used to determine whether a specific sequence is, for example, 95%
identical to a reference sequence of the present invention, the parameters are
preferably adjusted in such a way that the percentage proportion of the
identity is
calculated over the complete length of the reference sequence and that gaps of
homology of up to 5% of the total number of nucleotides in the reference
sequence
are permitted. When Bestfit is used, the so-called optional parameters
preferably
keep their default values. The deviations which occur during comparison of a
given
sequence with the above-described sequences according to the invention, can be
caused, for example, by addition, deletion, substitution, insertion or
recombination.
Promoter sequences hybridizing, as described above, to the sequence shown
under
SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID
No. 6 or exhibiting a sequence identity to SEQ ID No. 1 are preferred to
originate
from plant organisms, preferably from higher plants, particularly preferably
from
dicotyledonous plants, most preferably from plants of the genus Lycopersicon.
In a preferred embodiment of the invention, the promoter according to the
invention
has the complete sequence shown under SEQ ID No. 3 (about 1.4 kb fragment),
SEQ ID No. 4 (about 1.1 kb fragment), SEQ ID No. 5 (0.9 kb fragment) or SEQ ID
No. 6 (0.55 kb fragment).
Furthermore, the present invention also relates to promoters exhibiting a
functional
part of said sequences and causing a root-specific expression of a coding
nucleotide
sequence controlled by said promoters in plants.
9
CA 02350186 2001-05-08
a
In a particularly preferred embodiment of the invention, the promoter
according to the
invention has the complete or a functional part of the sequence shown under
SEQ ID
No. 4 (1.1 kb fragment).
Without considering to be bound to a certain theory, it is assumed that the
promoter
shown under SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No.
or SEQ ID No. 6 originates from a plant gene belonging to a group of extensin-
like
proteins.
Thus, the present invention also relates to promoters of genes encoding a
protein,
preferably from the group of extensin-like proteins and exhibiting a homology,
i.e.
identity, of at least 60%, preferably of at least 70%, more preferably of at
least 80%,
particularly preferably of at least 90% and most preferably of at least 95% to
the
complete amino acid sequence depicted under SEQ ID No. 8, wherein these
promoters cause a root-specific expression of a coding nuleotide sequence
controlled by said promoters in plants.
In a particularly preferred Pmbodiment, the present invention relates to
promoters of
genes encoding a protein with an amino acid sequence indicated under SEQ ID
No.
8, wherein said promoters cause a root-specific expression of a coding
nuleotide
sequence controlled by said promoters in plants.
The nucleotide sequence depicted under SEQ ID No. 7 encodes a polypeptide (SEQ
ID No. 8) from L. esculentum which can be assumed to belong to the group of
the
extensin-like proteins.
In the context of the present invention, an extensin-like protein is a plant
protein
whose amino acid sequence exhibits at least one of the following sequence
motifs:
SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY, PPPPPTY,
PPPPPAY, PPPPPEY, PPPPPXY, SO000, SPPPPKH, SPPPPKK,
SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH,
SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK,
SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP,
SOOOOVKP.
Within the meaning of the present invention, in particular, plant proteins,
exhibiting at
least one of the SPPPP- and/or (P)PPPPYY-motifs contained in the SEQ ID No. 8
are called extensin-like proteins.
DNA sequences exhibiting homology to the sequence shown under SEQ ID No. 7
can be identified, for example, by means of computer-based sequence
comparisons
with known sequences or also by means of screening of, for example, cDNA or
io
CA 02350186 2001-05-08
s
genomic libraries with the sequence depicted under SEQ ID No. 7 or parts
thereof.
Such techniques are known to the person skilled in the art (see Sambrook et
al., loc.
cit.). Computer-based sequence comparisons can also be carried out on the
amino
acid level with the amino acid sequence indicated in SEQ ID No. 8 or parts
thereof.
The cell wall influences both the form and the function of the cell. The
protein fraction
of the cell wall contains both enzymes and structural proteins. The structural
proteins
of the cell wall that are best characterized include the so-called extensins
(Cassab
and Varner, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39 (1988), 321-353;
Showalter, Plant Cell 5 (1993), 9-23). Extensins belong to the family of the
hydroxyproline-rich glycoproteins (HRGPs). Genes and cDNAs encoding extensins
could so far be characterized from carrot (Chen and Varner, EMBO J. 4 (1985),
2145-2151 ), bean (Corbin et al., Mol. Cell Biol. 7 (1987), 4337-4344), rape
(Evans et
al., Mol. Gen. Genet. 223 (1990), 273-287), tomato (Showalter et al., Plant
Mol. Biol.
16 (1991 ), 547-565) and tobacco (Memelink et al., EMBO J. 6 (1987), 3579-
3583).
The gene expression of the extensins is development-dependent and is more
likely
to be regulated tissue-specifically than constitutively (Sommer-Knudsen et
al.,
Phytochemistry 47 (1998), 483-497; Ye et al., Plant Cell 3 (1991 ), 23-37).
During
comparison of tissue specificity of different extensin genes of different
plants, it is
found that the expression pattern of extensin genes can clearly vary dependent
on
the species of the plant analyzed, on the cell type and on the tissue type
(Showalter
et al., Plant Mol. Biol. 19 (1992), 205-215).
In soy bean, HRGPs are expressed most intensively in meristematic cells. The
HRGPnt3-gene from tobacco is expressed in the pericycle and in the endodermis,
particularly in specific cells which participate in the formation of side
roots (Keller et
al., Proc. Nat. Acad. Sci. 86 (1989), 1529).
It has been described that the gene expression of extensins can be regulated
by
environmental factors such as light, pathogen infestation, wounding, heat
stress (cf.
for example Cassab and Varner, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39
(1988),
321-353; Cortin et al., Mol. Cell Biol. 7 (1987), 4337-4344; Niebel et al.,
Plant Cell 5
(1993), 1697-1710). This, however, does not hold true for all extensins
(Sommer-
Knudsen et al., Phytochemistry 47 (1998), 483-497).
The mature extensin protein is normally rich in hydroxyproline (Hyp), serine
and
specific combinations of the amino acids valine, tyrosine, lysine and
histidine.
The primary structure of extensins (for a survey see, for example, Sommer-
Knudsen,
Phytochemistry 47 (1998), 483-497) is usually characterized by at least one
Ser-Pro3_
6-peptide unit which can also occur repeatedly or in connection with similar
sequences such as the following sequence motifs SO000, SPPPPKH, SPPPPKK.
SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH,
m
CA 02350186 2001-05-08
s
SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK,
SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP,
SOOOOVKP (cf. also Sommer-Knudsen et al., ibid.).
Extensins are subject to an intense post-translational modification. With
carrot-
extensin, for example, most proline residues are hydroxylated by prolyl-
hydroxylases.
The hydroxyproline residues serve as target for glycosylations (Lamport et
al.,
Biochem. J 133 (1972), 125-132; van Holst, Plant Physiol. 74 (1984), 247-251).
The
carbohydrates, especially galactose and arabinose (Smith et al.,
Phytochemistry 25
(1986), pp. 1021; Holst et al., Plant Physiol. 74 (1984), 247; Smith et al.,
Phytochemistry 23 (1984), 1233) serve the stabilization of the proteins
(Showalter,
Plant Cell 5 (1993), 9-23). The arabinose mainly occurs in the form of Hyp-
Ara~_4, the
galactose in the form of Ser-Gal.
Furthermore, a linkage of various extensins via isodityrosine bonds has been
discussed which, for example, can lead to a further strengthening of the cell
wall as a
reaction to pathogen attack (Brisson, Plant Cell 6 (1994), 1703-1712; Epstein,
Phytochemistry 23 (1984), 1241-1246). Intramolecular isodityrosine bonds could
be
detected in extensins (Epstein et al., Phytochemistry, 23 (1984), pp. 1241 ).
Intermolecular isodityrosine bonds, however, could only be detected in vitro
(Everdeen et al., Plant Physiol. 87 (1988), pp. 616; Huystee et al., Plant
Phys.
Biochem. 33 (1995), pp. 55).
By means of the proline analogue 3,4-dehydro-L-proline (Dhp), the prolyl-
hydroxylase can be selectively inhibited so that the biosynthesis of the
hydroxyproline can be reduced by means of Dhp. After treatment of root slices
of
carrot (Daucus carota) with Dhp, it could be shown that they synthesize HRGPs
being structurally modified. The treatment of tobacco protoplasts with Dhp
also led to
the regeneration of cell walls with a modified structure (Cooper, Plant
Physiol. 104
(1994), 747-752).
The present invention further relates to expression cassettes containing a
promoter
according to the invention. The term "expression cassette" relates to the
combination
of a promoter according to the invention with a nucleic acid sequence to be
expressed. This nucleic acid sequence may, for example, be a sequence encoding
a
polypeptide, i.e. a structural gene. It can be linked to the promoter in sense-
or in
antisense-orientation. The nucleic acid sequence can also encode a non-
translatable
RNA, for example an antisense-RNA or a ribozyme. These nucleic acid sequences
can be used in connection with the promoter according to the invention to
produce
plants with a modified, preferably improved phenotype. Moreover, the
metabolism of
the plant in the root can be influenced by means of the promoter according to
the
invention. Some examples of the heterologous (over)expression and of antisense-
12
CA 02350186 2001-05-08
inhibition with the aim to manipulate metabolism flows in transgenic plants
have been
summarized in Herbers and Sonnewald (TIBTECH 14 (1996), 198-205). An example
of ribozymes was published by Feyter (Mol. Gen. Genet. 250 (1996), 329-228).
Various possible applications of transgenic plants which can be produced by
means
of the promoters and vectors according to the invention are also described in
TIPTEC
Plant Product & Crop Biotechnology 13 (1995), 312-397.
The expression cassettes according to the invention can further contain a
transcription termination sequence downstream of the 3' end of the nucleic
acid
sequence linked to the promoter. In this context, "transcription termination
sequence"
is a DNA sequence which is located at the 3' end of a coding gene region and
which
is able to cause the termination of transcription and optionally the synthesis
of a
polyA-tail. An example of such a termination sequence is the one of the
octopine
synthase gene.
According to the invention, there can be one or more restriction sites between
the
promoter and the nucleic acid sequence and/or the nucleic acid sequence and
the
terminator.
Moreover, the present invention relates to vectors containing at least one
promoter
according to the invention.
In a preferred embodiment, the promoter according to the invention in such a
vector
is linked to a polylinker which allows for the integration of any sequences
downstream of the promoter. In this context, "polylinker" is a DNA sequence
containing the recognition sequences of at least one restriction enzyme,
preferably of
two or more restriction enzymes.
In a particularly preferred embodiment, the vector according to the invention
further
contains a sequence for the termination of the transcription, for example the
one of
the octopine synthase gene, downstream of the promoter and/or the polylinker.
The present invention also relates to vectors containing the expression
cassettes
according to the invention. Advantageously, the vectors according to the
invention
contain selection markers which are suitable to identify and optionally to
select cells
containing the vectors according to the invention.
In a preferred embodiment, the vectors according to the invention are suitable
for the
transformation of plant cells and particularly preferably for the integration
of foreign
DNA into the plant genome. Such vectors include, for example, binary vectors
which
partly are available commercially.
13
CA 02350186 2001-05-08
The present invention further relates to host cells genetically engineered
with a
promoter according to the invention or with an expression cassette or a vector
according to the invention.
In this context, "genetically engineered" means that the host cells contain a
promoter
according to the invention or an expression cassette or a vector according to
the
invention, preferably stably integrated into the genome and the promoter or
the
expression cassette was introduced as foreign DNA into the host cell or in a
predecessor of this cell. This means that the cells according to the invention
themselves can be the direct product of a transformation event or cells
descending
therefrom which contain a promoter according to the invention or an expression
cassette according to the invention. Both prokaryotic, particularly bacterial,
as well as
eukaryotic cells are possible as host cells. Eukaryotic cells can be, for
example,
fungal cells, in particular from the genus Saccharomyces, preferably from the
species
Saccharomyces cerevisiae.
In another embodiment, the invention relates to the use of vectors according
to the
invention, expression cassettes according to the invention or host cells
according to
the invention for the transformation of plants, plant cells, plant tissues or
parts.
In a particularly preferred embodiment, the host cells according to the
invention are
plant cells which are called transgenic plant cells in the following.
Moreover, the present invention also relates to plants containing plant cells
according
to the invention. These can belong to any plant species, genus, family, order
or class.
They may be both monocotyledonous and dicotyledonous plants. The plants
according to the invention are preferred to be useful plants, i.e. plants
which are of
interest to humans as regards agriculture, forestry and/or horticulture. In
this context,
agricultural useful plants such as cereals (e.g. wheat, oat, barley, rye),
maize, rice,
potato, turnips, tobacco, sugar cane, sugar beet, sunflower, banana, rape or
forage
or pasture grasses (such as alfalfa, white clover, red clover), flax, cotton,
soy, millet,
bean, pea etc., vegetable plants (such as tomato, cucumber, courgette,
aubergine,
cabbage species, artichoke, chicory etc.), fruit trees, hop, wine and so on.
Also of
interest are herbs and medicinal plants such as Catharanthus roseus, Datura
stramonium, Taxus SSP.I, Dioscorea deltoidea, Papaver somniferum, Atropa
belladonna, Rauwolfia serpentina, Hyoscyamus niger, Digitalis lanata, Datura
metel,
Digitalis purpurea, Pilocarpus jaborandi, Cinchona ledgeriana, Aconitum
napellus.
In another embodiment, the present invention also relates to methods for the
production of transgenic plants characterized in that plant cells, tissues or
parts or
14
CA 02350186 2001-05-08
w
protoplasts are transformed with a vector according to the invention or with
an
expression cassette according to the invention or with a microorganism
according to
the invention, the transformed cells, tissues, parts of plants or protoplasts
are
cultivated in a growth medium and optionally plants are regenerated from the
culture.
In another embodiment, the invention relates to the use of vectors according
to the
invention, expression cassettes according to the invention or host cells
according to
the invention for the production of transgenic hairy roots by means of
Agrobacterium
rhizogenes.
The plants according to the invention can be produced according to methods
known
to the person skilled in the art, for example by transformation of plant cells
or tissue
and regeneration of whole plants from the transformed cells and/or the tissue.
For the introduction of DNA in a plant host cell, there is a plurality of
techniques at
disposal. These techniques comprise the transformation of plant cells with T-
DNA
using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation
agent, the fusion of protoplasts, the injection, the electroporation of DNA,
the
introduction of DNA by means of the biolistic approach as well as other
possibilities.
Methods for the transformation of plant cells and plants, preferably
dicotyledonous
plants, preferably using the Agrobacterium-mediated transformation, have been
analysed intensively and described sufficiently in EP 0 120 516; Hoekema (In:
The
Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam
(1985),
chapter V); Fraley et al. (Crit. Rev. Plant Sci. 4 (1993), 1-46) and An et al.
(EMBO J.
4 (1985), 277-287). For the transformation of potato see e.g. Rocha-Sosa et
al.
(EMBO J. 8 (1989), 29-33).
The transformation of monocotyledonous plants by means of Agrobacterium-based
vectors has also been described (Chan et al., Plant Mol. Biol. 22 (1993), 491-
506;
Hiei et al., Plant J. 6 (1994), 271-282; Deng et al., Science in China 33
(1990), 28-34;
Wilmink et al., Plant Cell Reports 11 (1992), 76-80; May et al.,
Bio/Technology 13
(1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555;
Ritchie
et al., Transgenic Res. 2 (1993), 252-265}. Alternative systems for the
transformation
of monocotyledonous plants are the transformation by means of the biolistic
approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al.,
Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24
(1994), 317-
325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631 ), the protoplast
transformation, the electroporation of partially permeabilized cells or the
introduction
of DNA by means of glass fibres. The transformation of maize, in particular,
has been
described in the literature various times (e.g. WO 95/06128, EP 0 513 849, EP
0 465
875, EP 0 292 435; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm
et
CA 02350186 2001-05-08
al., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-
200;
Moroc et al., Theor. Appl. Genet. 80 (1990), 721-726).
The successful transformation of other cereals has also been described, e.g.
for
barley (Wan and Lemaux, loc. cit.; Ritala et al., loc. cit.; Krens et al.,
Nature 296
(1982), 72-74) and for wheat (Nehra et al., Plant J. 5 (1994), 285-297).
Various transformation methods have been described for rice, e.g. the
Agrobacterium-mediated transformation (Hiei et al., Plant J. 6 (1994), 271-
282; Hiei
et al., Plant Mol. Biol. 35 (1997), 205-218; Park et al., J. Plant Biol. 38
(1995), 365-
371 ), the protoplast transformation (Datta, In: "Gene transfer to plants",
Potrykus,
Spangenberg (eds.), Springer-Verlag, Berlin, Heidelberg, 1995, 66-75; Datta et
al.,
Plant Mol. Biol. 20 (1992), 619-629; Sadasivam et al., Plant Cell Rep. 13
(1994), 394-
396), the biolistic approach for the plant transformation (Li et al., Plant
Cell Rep. 12
(1993), 250-255; Cao et al., Plant Cell Rep. 11 (1992), 586-591; Christou,
Plant Mol.
Biol. (1997), 197-203) as well as the electroporation (Xu et al., In: "Gene
transfer to
plants", Potrykus, Spangenberg (eds.), Springer-Verlag, Berlin, Heidelberg,
1995,
201-208).
Moreover, the present invention also relates to propagation and harvest
material of
plants according to the invention containing plant cells according to the
invention. In
this context, the term "propagation material" comprises those parts of the
plant which
are suitable for the production of descendants via the vegetative or
generative way.
Suitable for the vegetative propagation are, for example, cuttings, callus
cultures,
rhizomes, root-stock or tubers. Further propagation material comprises, for
example,
fruits, seeds, seedlings, protoplasts, cell cultures etc. The propagation
material is
preferred to be tubers and seeds.
The present invention further relates to a method for the identification and
isolation of
promoters causing a root-specific expression of a coding nucleic acid sequence
controlled by them in plants, comprising the following steps:
a) hybridizing of a plant genomic library with a cDNA coding for an extensin-
like
protein;
b) isolating of positive clones;
c) testing the isolated clones for promoter activity
The hybridization carried out in step a) preferably takes place under
stringent
conditions. For this purpose, known sequences which encode an extensin-like
protein or parts thereof are used for the hybridization with a corresponding
library,
preferably under stringent conditions. The sequence shown under SEQ ID No. 7
or
16
CA 02350186 2001-05-08
parts thereof is/are preferred to be used. The methods are known to the person
skilled in the art and are described in detail, e.g. in Sambrook et al. (loc.
cit.).
As described earlier, within the meaning of the present invention, an extensin-
like
protein is a protein whose amino acid sequence exhibits at least one of the
sequence
motifs SPPPPP, SPPPPYY, SPPPPY, SPPPPVY, PPPPPYY, PPPPPSY,
PPPPPTY, PPPPPAY, PPPPPEY, PPPPPXY, SO000, SPPPPKH, SPPPPKK,
SPPPPKKPYYPP, SPPPPSP, SPPPPSPKYVYK, SPPPPSPSPPPP, SPPPPYYYH,
SPPPPYYYK, SOOOOTOVYK, SPPPPTPVYK, SOOOOVYK, SPPPPVYK,
SPPPPVYSPPPP, SPPPPVHSPPPPVA, SPPPPVK, SPPPPVKSPPPP,
SOOOOVKP and which exhibits a homology of at least 60%, preferably of at least
70%, preferably of at least 80%, particularly preferably of at least 90% and
most
particularly of at least 95% to the coding region indicated.under SEQ ID No.
7.
The identification of positive clones and isolation of the promoter sequence
stated in
step b) is carried out according to methods known to the person skilled in the
art and
are described, for example, by Sambrook et al. (loc. cit.).
The expression properties of the isolated promoter can be analyzed by means of
reporter gene experiments. For the test of the isolated promoter sequence
stated in
step c) for promoter activity in root cells, the promoter can, for example in
an
expression cassette or a vector for plant transformation, be operatively
linked to a
reporter gene such as the f3-glucoronidase gene from E. coli. This construct
is used
for the transformation of plants. Subsequently, the organ-specific expression
of the C3-
glucuronidase is determined, as e.g. by Martin et al. (The GUS Reporter System
as a
Tool to Study Plant Gene Expression, In: GUS Protocols: Using the GUS Gene as
a
Reporter of Gene Expression, Academic Press (1992), 23-43). The promoters
which
exhibit a root specificity are selected and then isolated.
In this context, an operative link is the sequential arrangement of promoter,
coding
sequence, terminator and optionally further regulatory elements with each of
the
elements mentioned being able to fulfil its function during gene expression in
accordance with the requirements.
The present invention further relates to the use of the promoters according to
the
invention or of the promoters identified by means of the method according to
the
invention for the root-specific expression of transgenes in plants.
In this context, the term "transgene" means a DNA sequence artificially
introduced
into a plant.
m
CA 02350186 2001-05-08
These and other embodiments are disclosed and obvious to the person skilled in
the
art and embraced by the description and the Examples of the present invention.
Additional literature regarding one of the methods, means and uses stated
above
which can be used within the meaning of the present invention can be obtained
from
the state of the art, e.g. from public libraries, e.g. using electronic aids.
For this
purpose, public data bases, such as the "Medfine", which are accessible via
the
Internet, e.g. under the address
http://www.ncbi.nlm.nih.gov/PubMed/medline.html,
can be used. Further data bases and addresses are known to the person skilled
in
the art and can be obtained from the Internet, e.g. under the address
http://www.lycos.com. A survey of sources and information with regard to
patents
and/or patent applications in biotechnology is given in Berks, TIBTECH 12
(1994),
352-364.
The Figures show:
Figure 1: Northern blot analysis of RNA in roots
,ug total RNA of stripped-off roots and root hairs and 10 ,ug total RNA of the
tomato
organs stated were applied per lane for the Northern blot analysis.
Hybridization of
the RNA with radioactively labelled LeExtl cDNA or 25S rDNA cDNA. The
transcript
sizes are given on the right-hand side. 25S rDNA served as control for an even
loading of the lanes and transfer onto the nylon membrane. Stripped-off roots
are
roots after root hair isolation with a clearly reduced number of root hairs.
The
isolation of plant RNA was carried out according to the hot phenol extraction
method
by Verwoerd et al. (Nucleic Acids Research 17 (1989), 2362). The RNA was
separated electrophoretically, transferred onto a nylon membrane and fixed
thereon
by UV radiation. After hybridizing and washing, the radioactive membrane was
exposed on X-ray film. This film was developed after an overnight exposition
at -
80°C. The intensity of the signals correlates with the concentration of
the specific
mRNA on the membrane.
Figure 2: Northern blot analysis of the expression of LeExt1
l0,ug total RNA (5,ug in the case of lanes 3 and 4: 18 days and soil) of 7, 14
and 18
days old seedlings (7 days, 14 days, 18 days), roots grown on soil (soil),
seedling
roots which were incubated in the dark (-light) or in light (+light), and
controls
(control), as well as wounded leaves (wounding) were used for the RNA Northern
blot analysis. Radioactively labelled LeExtl cDNA was used for the
hybridization.
The isolation of plant RNA was carried out according to Verwoerd et al. (1989)
(see
above). The RNA was separated electrophoretically, transferred onto a nylon
membrane and fixed thereon by UV radiation. After hybridizing and washing, the
is
CA 02350186 2001-05-08
radioactive membrane was exposed on X-ray film. This film was developed after
an
overnight exposition at -80°C. The intensity of the signals correlates
with the
concentration of the specific mRNA on the membrane.
Figure 3: In-situ detection of the LeExt1 mRNA
Localisation of the LeExt1 transcript in tomato roots. Light-microscopic
pictures of
young tomato roots under sterile conditions. A, B cuts imbedded in paraffin
were
hybridized to non-radioactively labelled LeExt1 anti-sense RNA, according to
the in
situ hybridizatiori method as described in Daram et al. (Planta 206 (1998),
225-223).
A Young seedling roots with growing root hairs in the differentiation zone of
the root.
B root hair zone of a young seedling root. The root tip is located on the
left. The size
of the bar is 200 ,um.
The following Examples illustrate the invention.
Example 1: Isolation of a root-specific gene
Plant material and extraction of root hairs
Various organs of tomato plants Lycopersicon esculentum Mill. cv. Moneymaker
grown on earth in the green house served as plant material. For the extraction
of
roots and root hairs, 8000 surface-sterilized seeds plated on a metal grid on
paper
(No. 0858, Schleicher & Schuell, Germany) in petri dishes under sterile
conditions.
Prior to that, the paper was moistened in 0.5 x Hoagland's solution.
Germination took
place at 22°C at a day/night cycle of 16h/8h. On day 3 after
germination, the grid was
lifted approximately 4 mm above the paper by pushing sterile glass beads
between
the grid and the paper. This allowed for vertical growth of the seedling roots
and
optimum root hair formation. On day 5, the seedlings were immersed with the
grid in
liquid nitrogen and the roots were stripped off the grid with a spatula. The
root hairs
were then brushed off the roots in liquid nitrogen and purified through
filters over a
250,um sieve for analysis, as described in Rohm and Werner (Physiologia
Plantarum
69 (1987), 129-136).
For the Northern blot analysis of seedling roots of different age, the
seedlings were
incubated over 7, 14 and 18 days, as described above, and the roots were
isolated
accordingly. For the isolation of hairy roots of plants grown on earth, plant
pots
containing densely grown seedlings from the green house were removed from the
earth. The roots growing on the surface of the earth on the side were
harvested with
tweezers and immediately frozen in liquid nitrogen. For light/dark tests,
seedlings
were incubated in a day/night cycle of 16h/8h exposure, as described above. 7-
day-
19
CA 02350186 2001-05-08
old roots were harvested after 4-hour exposure after beginning of the day
cycle. In
parallel, seedlings were incubated during 7 days in the permanent dark and,
subsequently, their roots were also harvested. Tomato leaves were wounded as
described in Peria-Cortes et al. (Planta 186 (1992), 495-502) and harvested
after 24
h. Non-wounded leaves were used as control.
RNA extraction and construction of a cDNA library
Total RNA from the organs mentioned was extracted according to the method by
Verwoerd et al. (Nucleic Acid Research 17 (1989), 2362). 7-60 ,ug total RNA
from
root hairs was further used for the isolation of the poly(A)+ RNA by using
magnetic
oligodT beads (Dynabeads, Dynal, Germany). 700 ng poly(A)+ RNA was used for
the
production of double-stranded cDNA (Pharmacia, Germany). After attaching an
EcoRllNotl linker, the cDNA was cloned into the expression vector AZAPII
(Stratagene, Germany). Packaging into the A phages took place using the
Gigapack
II Gold Packaging Extract Kit from Stratagene (Germany).
Differential screenina of the root hair-specific cDNA library
500,000 cDNAs packaged in phages were plated on YT agar in agar dishes and
screened, according to a standard protocol (Sambrook et al. (1989), Molecular
Cloning: A Laboratory Manual, 2"d edition, Cold Spring Harbour Press., Cold
Spring
Harbour, NY), with 680 ng radioactively labelled reverse-transcribed mRNA from
either root hairs or brushed-off roots. 100 putative positive plaques were
subjected to
a second screening to avoid artefacts. Finally, 76 positive plaques were used
for in
vivo excision. The bacteria cultures resulting therefrom were shaken in a 96-
well
microtitre plate in 2 x YT medium (Sambrook et al., loc. cit.) at 28°C
overnight.
Plasmid DNA from these cultures was used for a second screening by using a
kind of
reverse Southern method, as described in Bucher et al. (Plant Molecular
Biology 35
(1997), 497-508). After EcoRl digest of promising plasmids, the corresponding
inserts were isolated and further used for the Northern blot analysis.
Northern blot analysis and DNA seauencina
5-10 Ng total RNA from the organs mentioned were loaded onto a 1.2% agarose
glyoxal gel after glyoxylation (Hull (1985), Purification, biophysical and
biochemical
characterisation of viruses with special reference to plant viruses. In: Mahy,
(ed.),
Virology. A Practical Approach. IRL Press, Oxford, UK, pp. 1-24). Northern
blot
analysis and hybridization conditions were taken from standard protocols
(Sambrook
et al., loc. cit.). The blots were hybridized at 68°C. Detection of
LeExt1 mRNA was
achieved by using the LeExt1 cDNA as radioactive probe. Washing the blot for
the
last time took place using 0.1 x SSC at 68°C. After washing, the blots
were exposed
CA 02350186 2001-05-08
on X-ray film (Kodak, Germany) at -80°C. The transcript sizes were
determined by
comparison with glyoxylated marker DNA (BRL, Germany). The results are shown
in
Figures 1 and 2.
Sequencing of the LeExtt cDNA (=SEQ ID No. 7) took place by using dideoxy
sequencing with T7 DNA polymerase (Amersham, Germany). Sequence analysis
was carried out by means of the University of Wisconsin GCG Packet (Devereux
et
al., A comprehensive set of sequence analysis programs for the VAX. Nucleic
Acids
Research 12 (1984), 387-395).
Example 2: Isolation of a genomic fragment comprising the promoter region of
the root-specific gene
A genomic DNA library from tomato (Clontech Laboratories, USA) was screened
with
radioactively labelled LeExtl cDNA (cf. Example 1 ). 500,000 plaque forming
units
(PFU) of the DNA libraries were plated onto YT agar in agar dishes, incubated
for 6
to 8 hours at 37°C and transferred onto nylon membranes (Hybond N,
Amersham,
Germany) (Sambrook et al., see above). Hybridization conditions were taken
from
standard protocols (The specific activity Sambrook et al., see above). 30
plaques
from the first screening were selected for a second round. The phage
suspension of
two plaques selected after the second screening were plated again to produce
phage
lysates. From these A DNA was prepared according to Lockett (Analytical
Biochemistry 185 (1990), 230-234). 600 ng A DNA each was then digested with
various restriction enzymes and the fragments resulting therefrom were made
visible
on an agarose gel using ethidium bromide. After Southern blot hybridization
(Sambrook et al., see above) with radioactively labelled LeExt1 cDNA, at last,
a
genomic fragment with a length of approximately 3.4 kb was isolated which
hybridized to LeExt1 cDNA and which was cloned into pBluescript II SK~ plasmid
also
digested with Asp718. First, the fragment was partially sequenced via dideoxy
sequencing with T7 DNA polymerase (Amersham, Germany). It was found that the
genomic fragment overlapped with the LeExt'1 cDNA sequence over 204 base pairs
and further extended 5' upstream into the non-coding region of the LeExt1
gene.
Subsequently, both strands of the genomic fragment (approximately 3.4 kb) were
sequenced (SEQ ID No. 13).
21
CA 02350186 2001-05-08
Example 3: Production of GUS expression cassettes for the analysis of the
functionality of the promoter
In total, three different GUS expression cassettes were produced.
A. Translational fusion of the 3.4 kb genomic fragment with a GUS cassette,
wherein the
fragment contains approximately 200 base pairs of the coding sequence of the
LeExtl
cDNA. The genomic fragment was excised by a Kpnl digest from the pBluescript
II SK-
plasmid, blunted by a filling reaction using the T7 DNA polymerase (Ausubel et
al.,
Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York,
1998) and
cloned into the binary vector pB1101.3 (Clontech, USA) which was digested with
Smal
and dephosphorylated. pB1101.3 is a plasmid containing a GUS cassette without
a
promoter in the binary vector pBINl9. This construct was designated rohl .
B. Transcriptional fusion of a modified genomic fragment with a GUS cassette.
A
genomic fragment with a length of 3200 by was synthesized which lacks the ORF
(open
reading frame) which overlaps with the LeExtl cDNA using the oligonucleotides
42tnc3
(5'-GAGAGTCGACGATATCGGGCGAATTGGGTACC-3', SEQ ID No. 9) containing the
Sall restriction site, and 42trx4
(5'-GAGATCTAGAGGTACCGGACTTTATATAACATAAC-3', SEQ ID No. 10) containing
the Xbal restriction site by means of PCR (30 sec at 94°C melting of
the DNA, 1 min at
60°C annealing of the primers, 1 min at 72°C with 3 sec
extension per cycle DNA
synthesis by means of Taq DNA polymerase [Fermentas, Lithuania]. The fragment
was
digested with SaA and Xbal and cloned into the accordingly digested and
dephosphorylated pBluescript II SK- plasmid. After isolation of the plasmid
from an E. coli
DHSa culture (Sambrook et al., see above), the fragment was isolated by
SalllXbal digest
and blunted by a filling reaction using T7 DNA polymerase. The end product was
then
cloned into the binary vector pBl 101.3 which was digested with Smal and
deposphorylated. This construct was designated rohtnc.
C. A PCR product of approximately 3270 by was produced by means of the
following
primers:
42Xba (5'-GAGATCTAGACCATGGAGAAGAATTGG-3', SEQ ID No. 11) and 42Sa1 (5'-
GAGAGTCGACGGGCGAAlTGGGTACCG-3', SEQ ID No. 12). The PCR conditions
were: 20 sec at 94°C melting of the DNA, 45 sec at 50°C
annealing of primers, 2 min at
72°C DNA synthesis by means of Pfu DNA polymerase (Stratagene,
Germany). The
PCR product was directly cloned into the plasmid pCR-Script according to the
manufacturer's instructions (Stratagene, Germany). After SaA/Noi1 digest, the
ends of the
PCR product were blunted with the Klenow enzyme [Sambrook, 1989 #68] and
cloned in
pBluescript II SK- plasmid which was digested with Smal and dephosphorylated.
After
22
CA 02350186 2001-05-08
plasmid preparation (Sambrook et al., see above), the plasmid was digisted
with BsflCl
and linearized. By means of serial shortening of the PCR product via
exonuclease III and
S1 nuclease from the 5' end according to the manufacturer's protocol
(Fermentas,
Lithuania), shortened genomic fragments of approximately the following lenghts
were
produced: 2.2 kb, 1.7 kb, 1.4 kb, 1.1 kb, 0.9 kb and 0.55 kb. The AKT1-GUS-
3'NOS
cassette from the plasmid 5'-AKT1-320.X (Lagarde et al., Plant J. 9 (1996),
195-203) was
isolated using Sad, the ends were blunted by means of mung bean nuclease
treatment
(New Englands BioLabs Inc., Bioconcept, Switzerland) and afterwards digested
with
Ncol. The GUS-3'NOS cassette obtained in that way was cloned into NcollEcoRV
digested and dephosphorylated pBluescript II SK- plasmids containing the
shortened
genomic fragments. The promoter-GUS-3'NOS cassettes produced in that way were
isolated by digest with Sad and Sall from each plasmid and cloned into the
binary vector
Bini 9 (Bevan et al., Nucleic Acids Research 12 (1984), 8711 ) that was
Sad/SaA digested
and dephosphorylated. The constructs obtained were designated BIN-Ogenx-GUS
with x
indicating the corresponding length of the fragment 2.2 kb (SEQ ID No. 1), 1.7
kb (SEQ
ID No. 2), 1.4 kb (SEQ ID No. 3), 1.1 kb (SEQ ID No. 4), 0.9 kb (SEQ ID No. 5)
or 0.55 kb
(SEQ ID No. 6).
Example 4: Plant transformation
The constructs described which contain the promoter fragments of various sizes
were introduced, by means of electroporation, into the Agrobacterium strain
C58C1
containing the plasmid pGV2260 (Deblaere et al., Nucleic Acids Research 13
(1985),
4777-4788). The Agrobacteria were cultured on YEB medium (Vervliet et al.,
Journal
of General Virology 26 (1975), 33-48). The transformation of tobacco and
tomato
plants was carried out in accordance with the method of the Agrobacterium
tumefaciens-mediated gene transfer as described by Rosahl et al. (EMBO J. 6
(1987), 1155-1159) for tobacco and by Lillatti et al. (Biotechnology 5 (1987),
726-
730) for tomato.
Furthermore, the transformation of potato was carried out as described in
Rocha-
Sosa et al. (EMBO J. 8 (1989), 23-29).
Maize was transformed as described by Omirulleh et al. (in "Gene Transfer to
Plants", Potrykus, Spangenberg (eds.), Springer Verlag, Berlin, Heidelberg,
1995,
pp. 99).
Moreover, the constructs described above were introduced into the A.
rhizogenes
strain 15834 by electroporation (Jung et al., Biotechnol. Lett. 14 (1992), 695-
700) and
subsequently used for transforming Catharanthus roseus by means of
Agrobacterium
rhizogenes following the instructions in Toivonen et al. (Plant Cell Tissue
Org. Cult.
18 (1988), pp. 79).
23
CA 02350186 2001-05-08
A strong expression of GUS could be detected in the hairy roots. Rice was
transformed by means of particle bombardment as described, for instance, in
Christou (Plant Mol. Biol. 35 (1997), 197-203).
Example 5: Histochemical localisation of the LeExt1 promoter activity
The plant material was vacuum-infiltrated with an 0.1 % X-gluc solution (pre-
solving in
0.1 g X-gluc in 1 ml dimethylformamide, adding 1 ml 10% Triton and 5 ml 1 M
sodium
phosphate buffer, pH 7.2 and filling up with distilled water to obtain 100 ml)
and
incubated overnight at 37°C. After dyeing, the plants were fixed in
ethanol : acetic
acid (3:1 ) and decolorized in 100% ethanol. As a result, the green parts of
the plants
became colourless, whereas the blue colouring remained stable; cf. Fig. 3.
Example 6: Analysis of the specificity and the activity of the promoter
For analyzing the specificity of the promoter, the GUS activity in the leaves
of potato
and tomato plants was determined in comparison with the activity in roots
according
to the method by Jefferson et al. (EMBO J. 6 (1987), 3901-3907).
In tomato, the GUS activity in the roots is between 20 and 300 times higher
than the
activity in mature leaves. Most of the GUS-positive potato plants that were
examined
had a high GUS activity in the roots but no GUS activity in mature leaves.
For determining the activity of the promoter, the GUS activities were compared
to the
activities of the 35S promoter. The lines with the highest activity of the
promoter
exhibited an activity that was approximately half of the activity of the CaMV
35S
promoter.
24
CA 02350186 2001-05-08
1/9
SEQUENCE LISTING
<110> ETH Zurich
RIESMEIER, Jorg
WILLMITZER, Lothar
<120> Promoters for gene expression in roots of plants
<130> C 2645 PCT
<140>
<141>
<160> 13
<170> PatentIn Ver. 2.1
<210> 1
<211> 2205
<212> DNA
<213> Lycopersicon esculentum
<400> 1
aaagttatta acacaacttc catcacatta aagacatcat ttgataacat tcatgtcagc 60
atacttatac taaagttgac gggcttcaat ataaaattaa taaggataaa ttttaagaat 120
cacatagttt ttaactatga aattacaatc tattcgtctt tagtttgtaa ttacattaat 180
accttaaaaa gcaataaaaa tcgattatat taatatgcag cttgaataca ttaaagagta 240
tctcagatac attacaatat aaatcgggta aataatatgt agctcggata cattatatta 300
ataagtagct tatatacaat aaagaaataa gagattttag aggtttatag aaatagaagg 360
gaatattgaa aataaggaaa agaaaggtgt gtatttaagt attttttcca attaagaata 420
catttttcac gaaagaggtg ttcaagccaa atgaaagaag attgggccac ccaaaaccca 480
ataaaagagt ctaaaaagct gaacgcttta agtcaagagt tcctatttta aaagtttcct 540
agtttaaaaa gtttgttagt ttaaaaagtt tcctagttaa agtttttcag ctttcttagt 600
ttaatgtttc ctagtttaaa agttttcata aaagtaggat ttaaatcgag gaaaggctca 660
aaataatctc tcatctttag gttaagactc aaagtgatcc ttaagttttc acatggagca 720
ctagtaatct atcatgtttg caatattggt acacttttgg tcctaaaaaa actctaacat 780
acttcaaaaa taggtaacta agaataatac tattgatata ttacgcaaaa taatcgatac 840
aatttgtcta atctgtttga atttaaaaga agaaaaataa attgattgtt atagttataa 900
acatatgatt cctgagagat aaaagaaaca aaaggaatcg tagtgaatca ttttaatcaa 960
ataaaaagaa aatcgaagat aaatgttcca caaagataaa atatcacgat ttttttcata 1020
agcccttcaa tattaatcat ttagtgattg tttaggtgta tttgatttat attgcagaag 1080
ttttaagctc taagtatgaa tttttttaat atcggaactt ctaaaatatt ttcagaaaat 1140
tttcaaaata atttgatcta ctcaattttt ttccggtggg gttcgatatt cgggaatgaa 1200
gtaatatctc atacaacact tttttctcaa gactagatca tacgggacac aaatttgaat 1260
tattataatt aataaccaaa aatatgaaaa tatgacatta aaaacaatag cgtctaatta 1320
ttcccgttgg tatcattaac taatcaaacc caaaatattg tcattcttgg tgatgaaaag 1380
tatgtatggt actcgcaagg gtacgcaaaa taatcgatac aatttgtcta atctgtttga 1440
atttaaaaga agaaaaataa attgattgct attgttataa acatatgatt cctgagagat 1500
aaaagaaata aagcgaatcg tagtgaatca ttttaatcaa ataaaaacaa actcgtagat 1560
aaatgttcca caaagataaa atattacgat ttttttcata agcccttcaa tattcagtga 1620
atcgatttct tctatcaacg ttttttcagt tgatttgtca caacggagtt gctcaaagca 1680
gcttttatat gatttgcaag taaatgcaca tgagcaattt atcggcgggc acccgaagaa 1740
tagcttaccc atttattttt ttaaaaaaag attaagtaca ataccatgat gtggattgta 1800
agttgtgctc aacaagtaca aataattaat cgacaccaaa taatggacag tatttgttaa 1860
gcctacacat atctcaactt ttaaatatta attttatcaa tttttcacaa ccaaaagaaa 1920
tgaacaaaca acattcttgc aagtcaacaa ataatcgatt caaagtttag aaataggtga 1980
gtcaagcaaa tgtgtatgaa tagtttatga cttcccatta tctcaaaacc aaccttagtg 2040
gaacagcatt aaccaaaaaa cggctgatat gtttggatta tttaatttct aatttatcaa 2100
atcacatgtt agttatgtta tataaagtcc taattcctcc attctaaaac acacaagaaa 2160
aagaaaaaca aagatatatc tagccttaga atccaattct tctcc 2205
CA 02350186 2001-05-08
219
<210> 2
<211> 1727
<212> DNA
<213> Lycopersicon esculentum
<400> 2
caataaaaga gtctaaaaag ctgaacgctt taagtcaaga gttcctattt taaaagtttc 60
ctagtttaaa aagtttgtta gtttaaaaag tttcctagtt aaagtttttc agctttctta 120
gtttaatgtt tcctagttta aaagttttca taaaagtagg atttaaatcg aggaaaggct 180
caaaataatc tctcatcttt aggttaagac tcaaagtgat ccttaagttt tcacatggag 240
cactagtaat ctatcatgtt tgcaatattg gtacactttt ggtcctaaaa aaactctaac 300
atacttcaaa aataggtaac taagaataat actattgata tattacgcaa aataatcgat 360
acaatttgtc taatctgttt gaatttaaaa gaagaaaaat aaattgattg ttatagttat 420
aaacatatga ttcctgagag ataaaagaaa caaaaggaat cgtagtgaat cattttaatc 480
aaataaaaag aaaatcgaag ataaatgttc cacaaagata aaatatcacg atttttttca 540
taagcccttc aatattaatc atttagtgat tgtttaggtg tatttgattt atattgcaga 600
agttttaagc tctaagtatg aattttttta atatcggaac ttctaaaata ttttcagaaa 660
attttcaaaa taatttgatc tactcaattt ttttccggtg gggttcgata ttcgggaatg 720
aagtaatatc tcatacaaca cttttttctc aagactagat catacgggac acaaatttga 780
attattataa ttaataacca aaaatatgaa aatatgacat taaaaacaat agcgtctaat 840
tattcccgtt ggtatcatta actaatcaaa cccaaaatat tgtcattctt ggtgatgaaa 900
agtatgtatg gtactcgcaa gggtacgcaa aataatcgat acaatttgtc taatctgttt 960
gaatttaaaa gaagaaaaat aaattgattg ctattgttat aaacatatga ttcctgagag 1020
ataaaagaaa taaagcgaat cgtagtgaat cattttaatc aaataaaaac aaactcgtag 1080
ataaatgttc cacaaagata aaatattacg atttttttca taagcccttc aatattcagt 1140
gaatcgattt cttctatcaa cgttttttca gttgatttgt cacaacggag ttgctcaaag 1200
cagcttttat atgatttgca agtaaatgca catgagcaat ttatcggcgg gcacccgaag 1260
aatagcttac ccatttattt ttttaaaaaa agattaagta caataccatg atgtggattg 1320
taagttgtgc tcaacaagta caaataatta atcgacacca aataatggac agtatttgtt 1380
aagcctacac atatctcaac ttttaaatat taattttatc aatttttcac aaccaaaaga 1440
aatgaacaaa caacattctt gcaagtcaac aaataatcga ttcaaagttt agaaataggt 1500
gagtcaagca aatgtgtatg aatagtttat gacttcccat tatctcaaaa ccaaccttag 1560
tggaacagca ttaaccaaaa aacggctgat atgtttggat tatttaattt ctaatttatc 1620
aaatcacatg ttagttatgt tatataaagt cctaattcct ccattctaaa acacacaaga 1680
aaaagaaaaa caaagatata tctagcctta gaatccaatt cttctcc 1727
<210> 3
<211> 1357
<212> DNA
<213> Lycopersicon esculentum
<400> 3
taatctgttt gaatttaaaa gaagaaaaat aaattgattg ttatagttat aaacatatga 60
ttcctgagag ataaaagaaa caaaaggaat cgtagtgaat cattttaatc aaataaaaag 120
aaaatcgaag ataaatgttc cacaaagata aaatatcacg atttttttca taagcccttc 180
aatattaatc atttagtgat tgtttaggtg tatttgattt atattgcaga agttttaagc 240
tctaagtatg aattttttta atatcggaac ttctaaaata ttttcagaaa attttcaaaa 300
taatttgatc tactcaattt ttttccggtg gggttcgata ttcgggaatg aagtaatatc 360
tcatacaaca cttttttctc aagactagat catacgggac acaaatttga attattataa 420
ttaataacca aaaatatgaa aatatgacat taaaaacaat agcgtctaat tattcccgtt 480
ggtatcatta actaatcaaa cccaaaatat tgtcattctt ggtgatgaaa agtatgtatg 540
gtactcgcaa gggtacgcaa aataatcgat acaatttgtc taatctgttt gaatttaaaa 600
gaagaaaaat aaattgattg ctattgttat aaacatatga ttcctgagag ataaaagaaa 660
taaagcgaat cgtagtgaat cattttaatc aaataaaaac aaactcgtag ataaatgttc 720
cacaaagata aaatattacg atttttttca taagcccttc aatattcagt gaatcgattt 780
cttctatcaa cgttttttca gttgatttgt cacaacggag ttgctcaaag cagcttttat 840
atgatttgca agtaaatgca catgagcaat ttatcggcgg gcacccgaag aatagcttac 900
CA 02350186 2001-05-08
3/9
ccatttattt ttttaaaaaa agattaagta caataccatg atgtggattg taagttgtgc 960
tcaacaagta caaataatta atcgacacca aataatggac agtatttgtt aagcctacac 1020
atatctcaac ttttaaatat taattttatc aatttttcac aaccaaaaga aatgaacaaa 1080
caacattctt gcaagtcaac aaataatcga ttcaaagttt agaaataggt gagtcaagca 1140
aatgtgtatg aatagtttat gacttcccat tatctcaaaa ccaaccttag tggaacagca 1200
ttaaccaaaa aacggctgat atgtttggat tatttaattt ctaatttatc aaatcacatg 1260
ttagttatgt tatataaagt cctaattcct ccattctaaa acacacaaga aaaagaaaaa 1320
caaagatata tctagcctta gaatccaatt cttctcc 1357
<210> 4
<211> 1121
<212> DNA
<213> Lycopersicon esculentum
<400> 4
aagctctaag tatgaatttt tttaatatcg gaacttctaa aatattttca gaaaattttc 60
aaaataattt gatctactca atttttttcc ggtggggttc gatattcggg aatgaagtaa 120
tatctcatac aacacttttt tctcaagact agatcatacg ggacacaaat ttgaattatt 180
ataattaata accaaaaata tgaaaatatg acattaaaaa caatagcgtc taattattcc 240
cgttggtatc attaactaat caaacccaaa atattgtcat tcttggtgat gaaaagtatg 300
tatggtactc gcaagggtac gcaaaataat cgatacaatt tgtctaatct gtttgaattt 360
aaaagaagaa aaataaattg attgctattg ttataaacat atgattcctg agagataaaa 420
gaaataaagc gaatcgtagt gaatcatttt aatcaaataa aaacaaactc gtagataaat 480
gttccacaaa gataaaatat tacgattttt ttcataagcc cttcaatatt cagtgaatcg 540
atttcttcta tcaacgtttt ttcagttgat ttgtcacaac ggagttgctc aaagcagctt 600
ttatatgatt tgcaagtaaa tgcacatgag caatttatcg gcgggcaccc gaagaatagc 660
ttacccattt atttttttaa aaaaagatta agtacaatac catgatgtgg attgtaagtt 720
gtgctcaaca agtacaaata attaatcgac accaaataat ggacagtatt tgttaagcct 780
acacatatct caacttttaa atattaattt tatcaatttt tcacaaccaa aagaaatgaa 840
caaacaacat tcttgcaagt caacaaataa tcgattcaaa gtttagaaat aggtgagtca 900
agcaaatgtg tatgaatagt ttatgacttc ccattatctc aaaaccaacc ttagtggaac 960
agcattaacc aaaaaacggc tgatatgttt ggattattta atttctaatt tatcaaatca 1020
catgttagtt atgttatata aagtcctaat tcctccattc taaaacacac aagaaaaaga 1080
aaaacaaaga tatatctagc cttagaatcc aattcttctc c 1121
<210> 5
<211> 891
<212> DNA
<213> Lycopersicon esculentum
<400> 5
taattattcc cgttggtatc attaactaat caaacccaaa atattgtcat tcttggtgat 60
gaaaagtatg tatggtactc gcaagggtac gcaaaataat cgatacaatt tgtctaatct 120
gtttgaattt aaaagaagaa aaataaattg attgctattg ttataaacat atgattcctg 180
agagataaaa gaaataaagc gaatcgtagt gaatcatttt aatcaaataa aaacaaactc 240
gtagataaat gttccacaaa gataaaatat tacgattttt ttcataagcc cttcaatatt 300
cagtgaatcg atttcttcta tcaacgtttt ttcagttgat ttgtcacaac ggagttgctc 360
aaagcagctt ttatatgatt tgcaagtaaa tgcacatgag caatttatcg gcgggcaccc 420
gaagaatagc ttacccattt atttttttaa aaaaagatta agtacaatac catgatgtgg 480
attgtaagtt gtgctcaaca agtacaaata attaatcgac accaaataat ggacagtatt 540
tgttaagcct acacatatct caacttttaa atattaattt tatcaatttt tcacaaccaa 600
aagaaatgaa caaacaacat tcttgcaagt caacaaataa tcgattcaaa gtttagaaat 660
aggtgagtca agcaaatgtg tatgaatagt ttatgacttc ccattatctc aaaaccaacc 720
ttagtggaac agcattaacc aaaaaacggc tgatatgttt ggattattta atttctaatt 780
tatcaaatca catgttagtt atgttatata aagtcctaat tcctccattc taaaacacac 840
aagaaaaaga aaaacaaaga tatatctagc cttagaatcc aattcttctc c 891
CA 02350186 2001-05-08
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<210> 6
<211> 575
<212> DNA
<213> Lycopersicon esculentum
<400> 6
tctatcaacg ttttttcagt tgatttgtca caacggagtt gctcaaagca gcttttatat 60
gatttgcaag taaatgcaca tgagcaattt atcggcgggc acccgaagaa tagcttaccc 120
atttattttt ttaaaaaaag attaagtaca ataccatgat gtggattgta agttgtgctc 180
aacaagtaca aataattaat cgacaccaaa taatggacag tatttgttaa gcctacacat 240
atctcaactt ttaaatatta attttatcaa tttttcacaa ccaaaagaaa tgaacaaaca 300
acattcttgc aagtcaacaa ataatcgatt caaagtttag aaataggtga gtcaagcaaa 360
tgtgtatgaa tagtttatga cttcccatta tctcaaaacc aaccttagtg gaacagcatt 420
aaccaaaaaa cggctgatat gtttggatta tttaatttct aatttatcaa atcacatgtt 480
agttatgtta tataaagtcc taattcctcc attctaaaac acacaagaaa aagaaaaaca 540
aagatatatc tagccttaga atccaattct tctcc 575
<210> 7
<211> 1420
<212> DNA
<213> Lycopersicon esculentum
<220>
<221> CDS
<222> (256)~.(1197)
<400> 7
taaacaaaga tatatctagc cttagaatcc aattcttctc aatgggaagc caaataaaga 60
agcaatggct tcaatttgct tgtcttttga catttttctt gattgccaca tgctctatgg 120
catattcacc ttacaattct tatgaatcat cagactcaac atataataaa gtaccaacca 180
cagtagtcaa aagtgaagac ttcaaggtac cctcagagtc ggaaaaggaa tataagtcgt 240
catttttgcc aaaaa atg att act ata aga agc cat caa ttt cag agg ata 291
Met Ile Thr Ile Arg Ser His Gln Phe Gln Arg Ile
1 5 10
actataaga aagtatcat ttgttcccg aacatgaat cattcctgc caa 339
ThrIleArg LysTyrHis LeuPhePro AsnMetAsn HisSerCys Gln
15 20 25
agaatgact actacaaga agccattat tttcggaag ataactaca aga 387
ArgMetThr ThrThrArg SerHisTyr PheArgLys IleThrThr Arg
30 35 40
aggagtcat atgttcaag aggtaccct cgaaggcta aaccagaat ata 935
ArgSerHis MetPheLys ArgTyrPro ArgArgLeu AsnGlnAsn Ile
45 50 55 60
aggagtcat ttttttcaa aatttgact acttcaaga agccatcat ttt 483
ArgSerHis PhePheGln AsnLeuThr ThrSerArg SerHisHis Phe
65 70 75
tcgaagaca actacaaga acgacgtca tatgttcca aaggtaccc tcg 531
SerLysThr ThrThrArg ThrThrSer TyrValPro LysValPro Ser
80 85 90
CA 02350186 2001-05-08
519
atg get aaa cca gaa tat aag gag tca ttt ttt cca aaa ttt gac tac 579
Met Ala Lys Pro Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe Asp Tyr
95 100 105
tttaagaagcca tcagtt tcagaagat aactacaag aagacgtca tat 627
PheLysLysPro 5erVal SerGluAsp AsnTyrLys LysThrSer Tyr
110 115 120
gtttcagaggtg ccctcg atggetaaa ccagaatat aaggagtca ttt 675
ValSerGluVal ProSer MetAlaLys ProGluTyr LysGluSer Phe
125 130 135 140
tttccaaaattt gactac tttaagaag tcattaget cctgaagat aaa 723
PheProLysPhe AspTyr PheLysLys SerLeuAla ProGluAsp Lys
145 150 155
tacaagaaggcg ccatat gttccagag gtatccaca gagcctaaa ccg 771
TyrLysLysAla ProTyr ValProGlu ValSerThr GluProLys Pro
160 165 170
gaa tac aag gta cca tct ttg cca aag aat gac tac tat aag aag cca 819
Glu Tyr Lys Val Pro Ser Leu Pro Lys Asn Asp Tyr Tyr Lys Lys Pro
175 180 185
aca att cca gaa gat aac tat aaa aag gtg tca tat gtt tca aag gtg 867
Thr Ile Pro Glu Asp Asn Tyr Lys Lys Val Ser Tyr Val Ser Lys Val
190 195 200
ccc tca gtg cct aaa gaa gaa tac aag gca cct act ttg cca aag aat 915
Pro Ser Val Pro Lys Glu Glu Tyr Lys Ala Pro Thr Leu Pro Lys Asn
205 210 215 220
gat tac tac aag aag cca tca gtt caa gaa gaa aac tac aaa aag gta 963
Asp Tyr Tyr Lys Lys Pro Ser Val Gln Glu Glu Asn Tyr Lys Lys Val
225 230 235
cca ctt att tca aag ctg ccc tca gtg cct aaa gaa gaa tac aag gtg 1011
Pro Leu Ile Ser Lys Leu Pro Ser Val Pro Lys Glu Glu Tyr Lys Val
240 245 250
cct tct ttg tca aaa aaa gac tac tac aag aag cca tta gtt tct gaa 1059
Pro Ser Leu Ser Lys Lys Asp Tyr Tyr Lys Lys Pro Leu Val Ser Glu
255 260 265
gat aac tac aaa aag gtt tca tat gtt cca aag gtg ccc tcc gtg cct 1107
Asp Asn Tyr Lys Lys Val Ser Tyr Val Pro Lys Val Pro Ser Val Pro
270 275 280
aaa gaa gaa tac aag gcc cct tct ttg tca aag aat gac tac tac aag 1155
Lys Glu Glu Tyr Lys Ala Pro Ser Leu Ser Lys Asn Asp Tyr Tyr Lys
285 290 295 300
aag tca tcg cct tct cca tca cca cca cca cct cca tat tat 1197
Lys 5er Ser Pro Ser Pro Ser Pro Pro Pro Pro Pro Tyr Tyr
305 310
taaatttctt ttcatgttaa gcatggctgt ttaattaaag tccttctcca gctcaacatt 1257
tataactcat ggagttctca tattatgagt tagaaggcat gatataaaaa tttgcattta 1317
CA 02350186 2001-05-08
6/9
tttatttatt tttccttttc ctaccttggt ttgtaattga tttgtcattt aatttctata 1377
ttttgtatga gaaatttatt tcaattgaat acaagttttt tta 1420
<210> B
<211> 314
<212> PRT
<213> Lycopersicon esculentum
<400> 8
Met Ile Thr Ile Arg Ser His Gln Phe Gln Arg Ile Thr Ile Arg Lys
1 5 10 15
Tyr His Leu Phe Pro Asn Met Asn His Ser Cys Gln Arg Met Thr Thr
20 25 30
Thr Arg Ser His Tyr Phe Arg Lys Ile Thr Thr Arg Arg Ser His Met
35 40 45
Phe Lys Arg Tyr Pro Arg Arg Leu Asn Gln Asn Ile Arg Ser His Phe
50 55 60
Phe Gln Asn Leu Thr Thr Ser Arg Ser His His Phe Ser Lys Thr Thr
65 70 75 80
Thr Arg Thr Thr Ser Tyr Val Pro Lys Val Pro Ser Met Ala Lys Pro
85 90 95
Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe Asp Tyr Phe Lys Lys Pro
100 105 110
Ser Val Ser Glu Asp Asn Tyr Lys Lys Thr Ser Tyr Val Ser Glu Val
115 120 125
Pro Ser Met Ala Lys Pro Glu Tyr Lys Glu Ser Phe Phe Pro Lys Phe
130 135 140
Asp Tyr Phe Lys Lys 5er Leu Ala Pro Glu Asp Lys Tyr Lys Lys Ala
145 150 155 160
Pro Tyr Val Pro Glu Val Ser Thr Glu Pro Lys Pro Glu Tyr Lys Val
165 170 175
Pro Ser Leu Pro Lys Asn Asp Tyr Tyr Lys Lys Pro Thr Ile Pro Glu
180 185 190
Asp Asn Tyr Lys Lys Val Ser Tyr Val Ser Lys Val Pro Ser Val Pro
195 200 205
Lys Glu Glu Tyr Lys Ala Pro Thr Leu Pro Lys Asn Asp Tyr Tyr Lys
210 215 220
Lys Pro Ser Val Gln Glu Glu Asn Tyr Lys Lys Val Pro Leu Ile Ser
225 230 235 240
Lys Leu Pro Ser Val Pro Lys Glu Glu Tyr Lys Val Pro Ser Leu Ser
245 250 255
Lys Lys Asp Tyr Tyr Lys Lys Pro Leu Val Ser Glu Asp Asn Tyr Lys
CA 02350186 2001-05-08
7I9
260 265 270
Lys Val Ser Tyr Val Pro Lys Val Pro Ser Val Pro Lys Glu Glu Tyr
275 280 285
Lys Ala Pro Ser Leu Ser Lys Asn Asp Tyr Tyr Lys Lys Ser Ser Pro
290 295 300
Ser Pro Ser Pro Pro Pro Pro Pro Tyr Tyr
305 310
<210> 9
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: artificial
<400> 9
gagagtcgac gatatcgggc gaattgggta cc 32
<210> 10
<211> 35
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: artificial
<400> 10
gagatctaga ggtaccggac tttatataac ataac 35
<210> 11
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: artificial
<400> 11
gagatctaga ccatggagaa gaattgg 27
<210> 12
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> Description of the artificial sequence: artificial
<400> 12
gagagtcgac gggcgaattg ggtaccg 27
CA 02350186 2001-05-08
8/9
<210> 13
<211> 3259
<212> DNA
<213> Lycopersicon esculentum
<400> 13
ggtaccattg tttctccatc aggccccttt ttcataggaa tagtaggacc atctaagact 60
acactcaaga gatcaggatt ttctcctatc agatgatcca tcaagcgatt cttccaccat 120
ccatagtatt ttccattgaa caatgggggc cgagtttgag aagctccttc ttggggggta 180
ggtggtgctg ccatttaatt ttcaagatgt taatctttta gtgaaaagat cctgctctga 240
taccaattga agaaacctta cccctaaatc aagaaccagg ttcttgattg tttgcctaag 300
aaaagagtta attaaaacaa tatactttaa ctcaaacctt cctatctcaa cgaaggaaaa 360
actcaacttc gttttattac tttttcttac tcaataataa ttttcgatta actctaatcg 420
aattctctaa atttacaaaa agccaaaccc acttcttgca aacttactca ctaatctcct 480
ctctccacaa gaagccaaac ccacttcttg tttacaatca cacaagctta agatcttact 540
aagaacaaag ctcacaagtt aacaaaatac acagaactta gaaaaacgct ctttgctgcc 600
tctctctttg tcttacctca ccctttcttt cattgcaaag aactgctctc tttgtcttga 660
aatctctgtt tatatagact tttgaacaag agttatttcc tctctttgaa gttgataata 720
attagatttc tgtatccttc tttgatatga aaaagaattg ctttcttttt tctgtttata 780
cgcaaccttt tttgaaaagg ttttgtcttc atataaagta gacaactttt tcagaaaaga 840
ttttatctta tttatttatg gaaataatta ttatattcat ttttttcatc aatacatttg 900
tgccttaata tttgtccttc gcctcttttc aattagaacg acaactgctt taataaatga 960
gcctggttca taagtgagtc aacatcaaaa gtatttgtca ttatcaatga acctggttca 1020
tgattgatga catcaaaagt atttgtcatt ataaaaagtt attaacacaa cttccatcac 1080
attaaagaca tcatttgata acattcatgt cagcatactt atactaaagt tgacgggctt 1140
caatataaaa ttaataagga taaattttaa gaatcacata gtttttaact atgaaattac 1200
aatctattcg tctttagttt gtaattacat taatacctta aaaagcaata aaaatcgatt 1260
atattaatat gcagcttgaa tacattaaag agtatctcag atacattaca atataaatcg 1320
ggtaaataat atgtagctcg gatacattat attaataagt agcttatata caataaagaa 1380
ataagagatt ttagaggttt atagaaatag aagggaatat tgaaaataag gaaaagaaag 1490
gtgtgtattt aagtattttt tccaattaag aatacatttt tcacgaaaga ggtgttcaag 1500
ccaaatgaaa gaagattggg ccacccaaaa cccaataaaa gagtctaaaa agctgaacgc 1560
tttaagtcaa gagttcctat tttaaaagtt tcctagttta aaaagtttgt tagtttaaaa 1620
agtttcctag ttaaagtttt tcagctttct tagtttaatg tttcctagtt taaaagtttt 1680
cataaaagta ggatttaaat cgaggaaagg ctcaaaataa tctctcatct ttaggttaag 1740
actcaaagtg atccttaagt tttcacatgg agcactagta atctatcatg tttgcaatat 1800
tggtacactt ttggtcctaa aaaaactcta acatacttca aaaataggta actaagaata 1860
atactattga tatattacgc aaaataatcg atacaatttg tctaatctgt ttgaatttaa 1920
aagaagaaaa ataaattgat tgttatagtt ataaacatat gattcctgag agataaaaga 1980
aacaaaagga atcgtagtga atcattttaa tcaaataaaa agaaaatcga agataaatgt 2040
tccacaaaga taaaatatca cgattttttt cataagccct tcaatattaa tcatttagtg 2100
attgtttagg tgtatttgat ttatattgca gaagttttaa gctctaagta tgaatttttt 2160
taatatcgga acttctaaaa tattttcaga aaattttcaa aataatttga tctactcaat 2220
ttttttccgg tggggttcga tattcgggaa tgaagtaata tctcatacaa cacttttttc 2280
tcaagactag atcatacggg acacaaattt gaattattat aattaataac caaaaatatg 2340
aaaatatgac attaaaaaca atagcgtcta attattcccg ttggtatcat taactaatca 2400
aacccaaaat attgtcattc ttggtgatga aaagtatgta tggtactcgc aagggtacgc 2460
aaaataatcg atacaatttg tctaatctgt ttgaatttaa aagaagaaaa ataaattgat 2520
tgctattgtt ataaacatat gattcctgag agataaaaga aataaagcga atcgtagtga 2580
atcattttaa tcaaataaaa acaaactcgt agataaatgt tccacaaaga taaaatatta 2640
cgattttttt cataagccct tcaatattca gtgaatcgat ttcttctatc aacgtttttt 2700
cagttgattt gtcacaacgg agttgctcaa agcagctttt atatgatttg caagtaaatg 2760
cacatgagca atttatcggc gggcacccga agaatagctt acccatttat ttttttaaaa 2820
aaagattaag tacaatacca tgatgtggat tgtaagttgt gctcaacaag tacaaataat 2880
taatcgacac caaataatgg acagtatttg ttaagcctac acatatctca acttttaaat 2940
attaatttta tcaatttttc acaaccaaaa gaaatgaaca aacaacattc ttgcaagtca 3000
acaaataatc gattcaaagt ttagaaatag gtgagtcaag caaatgtgta tgaatagttt 3060
atgacttccc attatctcaa aaccaacctt agtggaacag cattaaccaa aaaacggctg 3120
atatgtttgg attatttaat ttctaattta tcaaatcaca tgttagttat gttatataaa 3180
CA 02350186 2001-05-08
919
gtcctaattc ctccattcta aaacacacaa gaaaaagaaa aacaaagata tatctagcct 3240
tagaatccaa ttcttctca 3259
