Title of Invention

"AN ISOLATED PROMOTER CAPABLE OF DRIVING AND/OR REGULATING EXPRESSION IN PLANTS"

Abstract The present invention provides several promoters isolated from Oryza sativa, which promoters are capable of driving and/or regulating the expression of an operably linked nucleic acid in a plant. The expression patterns of the promoters according to the present invention have been studied in the Oryza sativa and some of the promoters displayed specific activity in particular cells, tissues or organs of the plant, while other displayed constitutive expression throughout substantially the whole plant as disclosed in figures 1 to 24. Some promoters showed weak expression, while others were strongly active.
Full Text RICE PROMOTERS
The present invention relates to the field of plant molecular biology, more particularly to nucleic
acid sequences useful for driving and/or regulating expression of an operably linked nucleic acid
in plants. The isolati on of these nucleic acid sequences from rice, as well as their use in driving
and/or regulating expression of an operably linked nucleic acid is disclosed. The present invention
therefore concerns promoters, hybrid promote rs, genetic constructs, expression cassettes,
transformation vectors, expression vectors, host cells and transgenic plants comprising the
isolated nucleic acids according to the present invention. The present invention also concerns
methods for driving and/or regulating expression of a nucleic acid and methods for the production
of transgenic plants.
Gene expression is dependent on initiation of transcription, which is mediated via the transcription
initiation complex. Gene expression is also dependent on regulation of transcription, which
regulation determines how strong, when or where a gene is expressed . Said regulation of gene
expression may be mediated via transcriptional control elements, which are generally embedded
in the nucleic acid sequence 5'-flanking or upstream of the expressed gene. This upstream nucleic
acid region is often referred to as a "promoter" since it promotes the binding, formation and/or
activation of the transcription initiation complex and therefore is capable of driving and/or
regulating expression of the 3' downstream nucleic acid sequence.
Genetic engineering of plants with the aim of obtaining a useful plant phenotype, often involves
heterologous gene expression, which is generally mediated by a promoter capable of driving
and/or regulating expression of an operably linked heterologous nucleic acid. The phenotype of
the host plant only depends on the contribution of the heterologous nucleic acid, but also on the
contribution of the specific expression pattern of the chosen promoter determining how, where and
when that heterologous nucleic acid is expressed. Accordingly, the choice of promoter with a
suitable expression pattern is of crucial importance for obtaining the suitable phenotype. A person
skilled in the art will need to have available different promoters, to determine the o ptimal promoter
for a particular nucleic acid. For many different host plants, this availability is rather limited and
there is therefore a continuing need to provide new promoters with various expression profiles.
(a) an isolated nucleic acid as given in any one of SEQ ID NO 1 to 22 or the complement
of any one of SEQ ID N01 to 22; or
(b) an isolated nucleic acid having at least 90% sequence identity with any of the DNA
sequences as given in any one of SEQ ID NO 1 to 22; or
(c) an isolated nucleic acid specifically hybridizing under stringent conditions with any of
the DNA sequences as given in any one of SEQ ID NO 1 to 22; or
(d) an isolated nucleic acid as defined in any one of (a) to (c), which is inte rrupted by an
intervening sequence; or
(e) a fragment of any of the nucleic acids as defined in (a) to (d), which fragment is
capable of driving and/or regulating expression.
Suitable variants of any one of SEQ ID NO 1 to 22 encompass homologues which have in
increasing order of preference at least 90%, 91%. 92%, 93%. 94%, 95%. 96%, 97%, 98% or 99%
sequence identity with any one of the nucleic acids as represented in SEQ ID N01 to 22 .
The percentage of Identity may be calculated using an alignment program. Preferably a pair wise
global alignment program may be used , which implements the algorithm of Needleman -Wunsch
(J. Mol. Biol. 48:443-453,1970). This algorithm maximizes the number of matches and minimizes
the number of gaps. Such programs are for example GAP, Needle (EMBOSS package), stretcher
(EMBOSS package) or Align X (Vector NT) suite 5.5) and may use the standard parameters (for
example gap opening penalty 15 and gap extension penalty 6.66). Alternatively , a local alignment
program implementing the algorithm of Smith -Waterman (Advances in Applied Mathematics 2,
482-489 (1981)) may be used. Such programs are for example Water (EMBOSS package) or
matcher (EMBOSS package). "Sequence identity' as used herein is preferably calculated over the
entire length of the promoters as represented by any one of SEQ ID NO 1 to 22 . The length of
these promoters is presented in Table 2.
Search and identification of homolog ous nucleic acids, would be well within the realm of a person
skilled in the art. Such methods, involve screening sequence databases with the sequences
provided by the present invention, for example any one of SEQ ID NO 1 to 22, preferably in a
computer readable form. Useful sequence databases, include but are not limited to Genbank
(httoMvww.ncbi.nlm.nih.gov/web/Genbank ). the European Molecular Biology Laboratory Nucleic
acid Database (EMBL) (http:yw.ebi.ac.uk/ebi -docs/embl-db.html) or versions thereof, or the MIPS
database (http://mips.gsf.de/). Different search algorithms and software for the alignment and
comparison of sequences are well known in the art. Such software includes , for example GAP,
BESTFIT, BLAST, FASTA and TFASTA. Preferably BLAST software is used, which calculates
percent sequence identity and performs a statistical analysis of the similarity between the
sequences. The suite of programs referred to as BLAST programs has 5 different
implementations: three designed for nucleotide sequence queries (BLASTN, BLASTX, and
TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson,
Trends in Biotechnology: 76 -60,1994; Birren et al., GenomeAnalysis, 1:543,1997). The software
for performing BLAST analysis is publicly available through the National Centre for B iotechnology
Information.
The sequences of the genome of Arabidopsis thaliana and the genome of O/yza sativa are now
available in public databases such as Genbank . Other genomes are currently being sequenced.
Therefore, it is expected that as more sequences of the genomes of other plants become
available, homologous promoters may be identifiable by sequence alignment with any one of SEQ
ID NO 1 to SEQ ID NO 22. The skilled person will readily be able to find homologous promoters
from other plant species, for example from other crop plants, such as maize. Homologous
promoters from other crop plants are especially useful for practising the methods of the present
invention in crop plants.
One example of homologues having at least 90% sequence identity with any one of SEQ ID NO to
22 are allelic variants of any one of SEQ ID NO 1 to 22. Allelic variants are variants of the same
gene occurring in two different individuals of the same species and usually allelic variants differ by
slight sequence changes. Allelic variants may encompass Single Nucleotide Polymorphisms
(SNPs) as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is
usually less than 100 bp. SNPs and INDE Ls form the largest set of sequence variants in naturally
occurring polymorphic strains of most organisms.
Homologues suitable for use in the methods according to the invention may readily be isolated
from their source organism via the te chnique of PCR or hybridization. Their capability of driving
and/or regulating expression may readily be determined, for example, by following the methods
described in the Examples section by simply substituting the sequence used in the actual
Example with the homologue.
Other suitable variants of any one of SEQ ID NO 1 to 22 encompassed by the present invention
are nucleic acids specifically hybridising under stringent conditions to any one of the nucleic acids
of SEQ ID NO 1 to 22. The term "hybridising" means annealing to substantially homologous
complementary nucleolide sequences in a hybridization process. Tools in molecular biology
'relying on such a hybridization process include the polymera se chain reaction (PCR; and all
methods based thereon), subtractive hybridisation, random primer extension, nuclease S1
mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs,
and DNA sequence determination, Northern blotting (RNA blotting), Southern blotting (DNA
blotting). The hybridisation process can also occur with one of the complementary nucleic acids
immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. Tools in
molecular biology relying on such a process include the isolation of poly (A+) mRNA. The
hybridisation process can furthermore occur with one of the complementary nucleic acids
immobilised to a solid support such as a nitro -cellulose or nylon membrane or immobilised by e.g.
photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays
or microarrays or as nucleic acid chips). Tools in molecular biology relying on such a process
include RNA and DNA gel blot analysis, colony hybridisation . plaque hybridisation, in situ
hybridisation and microarray hybridisation. In order to allow hybridisation to occur, the nucleic acid
molecules are generally thermally or chemically denatured to melt a double strand into two single
strands and/or to remove hairpins or other secondary structures from single stranded nucleic
acids. The stringency of hybridisation is influenced by conditions such as temperature, salt
concentration and hybridisation buffer composition. Conventional hybridisation conditions are
described in, for example, Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition
Cold Spring Harbor Laboratory Press, CSH, New York, but the skilled craftsman will appreciate
that numerous different hybridisation conditions can be designed in function of the known or the
expected homology and/or length of the nucleic acid sequence. High stringency conditions for
hybridisation include high temperature and/or low sodium/salt concentration (salts include sodium
as for example in NaCI and Na 3-citrate) and/or the i nclusion of formamide in the hybridisation
buffer and/or lowering the concentration of compounds such as SDS (sodium dodecyl sulphate
detergent) In the hybridisation buffer and/or exclusion of compounds such as dextran sulphate or
polyethylene glycol (promoting molecular crowding) from the hybridisation buffer. Specifically
hybridising under stringent conditions means that the sequences have to be very similar . Specific
hybrisization under stringent conditions is preferably carried out at a temperature of 60°C followed
by washes in 0.1 to 1 XSSC, 0.1XSDS, and 1X SSC, 0.1X SDS.
The invention also relates to a nucleic acid molecule of at least 15 nucleotides in length
hybridizing specifically with any of the nucleic acids of the invent! on. The invention also relates to
a nucleic acid molecule of at least 15 nucleotides in length specifically amplifying a nucleic acid of
the invention by polymerase chain reaction.
Another variant of any of SEQ ID NO 1 to 22 encompassed by the pr esent invention are nucleic
acids corresponding to any one of SEQ ID NO 1 to 22 or variants thereof as described
hereinabove, which are interrupted by an intervening sequence. For example, any of the nucleic
acids as presented in SEQ ID NO 1 to 22 may be interrupted by an intervening sequence. With
"intervening sequences" is meant any nucleic acid or nucleotide, which disrupts another
sequence. Examples of intervening sequences comprise introns, nucleic acid tags, T-DNA and
mobilizable nucleic acids sequences such as transposons or nucleic acids that can be mobilized
via recombination. Examples of particular tra nsposons comprise Ac (activator), Ds (Dissociation),
Spm (suppressor-Mutatorj or En. The introduction of introns Into promoter s is now widely applied.
The methods according to the present i nvenlion may also be practised using a nucleic acid
sequence according to any one of SEQ ID NO 1 to 22 provided with an intron. In case the
intervening sequence is an intron, alternative splice variants of the nucleic acids according to the
invention may arise. The term 'alternative splice variant" as used herein encompasses variants of
a nucleic acid sequence in which intervening introns have been excised, replaced or added. Such
splice variants may be found in nature or may be manmade. Methods fo r making such promoters
with an intron or for making the corresponding splice variants are well known in the art
Variants interrupted by an intervenin g sequence, suitable for use in the methods according to th e
invention may readily be determined for example by following the methods described in the
Examples section by simply substituting the sequence used in the actual Example with the variant.
The variant nucleic acids as described hereinabove may be found in nature (for example allelic
variants or splice variants). Additionally and/or alternatively, variants of any one of SEQ ID N01 to
22 as described hereinabove may be manmade via techniques well known in the art involving for
example mutation, substitution, Insertion, deletions or derivation . T he present Invention also
encompasses such variants, as well as their use in the methods of the present invention .
A "mutation variant" of a nucleic acid may readily be mad e using recombinant DMA manipulation
techniques or nucleotide synthesis. Examples of such techniques include site directed
mutagenesis via M13 mutagenesis, 17-Gen in vitro mutagenesis (USB, Cleveland, OH),
QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR -mediated sitedirected
mutagenesis or other site -directed mutagenesis protocols. Alternatively, the nucleic acid
of the present invention may be randomly mutated.
A "substitutions! variant" refers to those variants in which at least o ne residue in the nucleic acid
sequence has been removed and a different residue inserted in its place. Nucleic acid
substitutions are typically of single residues, but may be clustered depending upon functional
constraints placed upon the nucleic acid sequence; insertions usually are of the order of about 1
to about 10 nucleic acid residues, and deletions can range from about 1 to about 20 residues.
An "insertional variant" of a nucleic acid is a variant in which one or more nucleic acid residues are
introduced into a predetermined site in that nucleic acid. Insertions may comprise 5' -terminal
and/or 3'-terminal fusions as well as intra-sequence insertions of single or multiple nucleotides.
Generally, insertions within the nucleic acid sequence will be smaller than 5'- or 3'-termina!
fusions, of the order of about 1 to 10 residues. Examples of 5' - or 3'-terminal fusions include the
coding sequences of binding domain s or activation domains of a transcriptional activator as used
in the yeast two-hybrid system or yeast one-hybrid system, or of phage coat proteins, (histidine) etag,
glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase,
Tag* 100 epitope, c-myc epitope, FLAG"-epitope, lacZ, CMP (calmodulin -binding peptide), HA
epitope, protein C epitope and VSV epitope.
The term "derivative" of a nucleic acid may comprise substitutions, and/or deletions and/or
additions of naturally and non -naturally occurring nucleic acid residues compared to the natural
nucleic acid. Derivatives may, for example, comprise methylated nucleotides, or artificial
nucleotides.
Also encompassed with in the present invention are promoters, comprising a fragment of any of
the nucleic acids as presented by any one of SEQ ID N01 to 22 or variants thereof as described
hereinabove. A fragment" as used herein means a portion of a nucleic acid sequence. Suitable
fragments useful in the methods of the present invention are functional fragment s, which retain at
least one of the functional parts of the promoter and hence are still capable of driving and/or
regulating expression. Examples of functional fragments of a promoter include the minimal
promoter, the upstream regulatory elements, or any combination thereof.
Suitable fragment? may range from at least about 20 base pairs or about 50,100, 150, 200,250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 100 0 base pairs, up to
about the full length sequence of the invention. T hese base pairs are typically immediately
upstream of the transcription initiation start, but alternatively may be from anywhere in the
promoter sequence.
Suitable fragments useful in the methods of the present invention may be tested for their capability
of driving and/or regulating expression by standard techniques well known to the skilled person, or
by the following method described in the Example section .
The promoters as disclosed in any one of SEQ ID NO 1 to 22 are isolated as nucleic acids of
approximately 1.2kb from the upstream region of particular rice coding sequences (CDS). These
nucleic acids may include typical elements of a promoter, which are presented in Figure 1.
Generally, a promoter may comprises from coding sequence to the upstream direction: (i) an
5'UTR of pre-messenger RNA, (ii) a minimal promoter comprising the transcription initiation
element (INR) and more upstream a TATA box, and (iii) may contain regulatory elements that
determine the specific expression pattern of the promoter.
The term "promoter" as used herein is taken in a broad context and refers to regulatory nucleic
acid sequences capable of effecting (driving and/or regulating) expression of the sequences to
which they are operably linked. A "promoter* encompasses transcriptions! regulatory sequences
derived from a classical genomic gene. Usually a promoter comprises a TATA box, which is
capable of directing the transcription initiation complex to the appropriate transcription initiation
start site. However, some promoters do not have a TATA box (TATA -less promoters), but are still
fully functional for driving and/or regulat ing expression. A promoter may additionally comprise a
CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences or
cis-elements such as enhancers and silencers). A "promoter" may also include the transcriptional
regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box
sequence and/or a -10 box transcriptional regulatory sequences.
"Driving expression" as used herein means promoting the transcription of a nucleic acid.
"Regulating expression" as used herein means influencing the level, time or place of transcription
of a nucleic acid. The promoters of the present Invention may thus be used to increase, decrease
or change in time and/or place transcription of a nucleic acid. For example, t hey may be used to
limit the transcription to certain cell types, tissues or organs, or during a certain period of time, or
in response to certain environmental conditions.
The promoter is preferably a plant-expressible promoter. The term "plant -expressible" means
being capable of regulating expression in a plant, plant cell, plant tissue and/or plant organ.
Accordingly, the inventbn encompasses an isolated nucleic acid as mentioned above, capable of
regulating transcription of an operably linked nucleic acid in a plant or in one or more particular
cells, tissues or organs of a plant.
The expression pattern of the promoters according to the present invention were studied in detail
and it was found that many of them were tissue-specific. Accordingly, the present invention
provides "tissue-specific" promoters. The term "tissue -specific" shall be taken to indicate that
expression is predominantly in a particular tissue, tissue -type, organ or any other pa rt of the
organism, albeit not necessarily exclusively in said tissue, tissue -type, organ or other part.
Accordingly, the invention encompasses an isolated nucleic acid as mentioned above, capable of
driving and/or regulating expression (of an operably linked nucleic acid) in a tissue-specific
manner. Expression may be driven and/or regulated In the seed, embryo, scutellum, aleurone,
endosperm, leaves, flower, calli, meristem, shoot meristem, discriminating centre, shoot, shoot
meristem and root In grasses the shoot meristem is located in the so -called discrimination zone
from where the shoot and the leaves originate.
A tissue-specific promoter is one example of a so -called "regulated promoter*. These promoters
are regulated by endogenous signals such as the presence of certain transcription factors,
metabolites, plant hormones, or exogenous signals, such as ag eing, stresses or nutritional status.
These regulations may have an effect on one or more different levels such spatial specificity or
temporal specificity. Encompassed within the present invention is a nucleic acid as described
hereinabove, which is a "regulated promoter*. Examples of regulated promoters are cell -specific
promoters, tissue-specific promoters, organ-specific promoters, cell cycle-specific promoters,
inducible promoters or young tissue-specific promoters.
Alternatively and/or additionally, some promoters of the present invention display a con stitutive
expression pattern. Accordingly, the present invention provides a promoter as described
hereinabove, which is a constitutive promoter. The term "constitutive" means having no or very
few spatial or temporal regulations. The term "constitutive expression" as used herein refers to a
substantially continuously expression in substantially all tissues of the organism. The skilled
craftsman will understand that a "constitutive promoter" is a promote r that is active during most,
but not necessarily all, phases of growth and development of the organism and throughout most,
but not necessarily all, parts of an organism.
The "expression pattern" of a promoter is not only influenced by the spatial and te mporal aspects,
but also by the level of expression. The level of expression is determined by the so -called
"strength" of a promoter. Depending on the resulting expression level, a distinction is made herein
between "weak" or "strong" promoters. Gen erally by "weak promoter" is meant a promoter that
drives expression of an operably linked nucleic acid at levels of about 1/10 000 transcripts to about
1/100000 transcripts to about 1/500000 transcripts . Generally, by "strong promoter" is meant a
promoter that drives expression at levels of about 1/10 transcripts, to about 1/100 or to about
1/1000 transcripts.
According to a particular embodiment, the invention provides an Isolated promoter as mentioned
hereinabove, which is a hybrid promoter. The term "hybrid promoter" as used herein refers to a
chimeric promoter made, for example, synthetically, for example by genetic engineering. Preferred
hybrid promoters according to the present invention comprise a part, preferably a functional part,
of one of the promoters according to the present invention and at leas t another part, preferably a
functional part of a promoter. The latter part, may be a part of any promoter, including any one of
the promoters according to the present invention and other promoters. One example of a hybrid
promoter comprises regulatory elements) of a promoter according to the present invention
combined with the minimal promoter of another promoter. Another example of a hybrid promoter is
a promoter comprising additional regulatory elements to further enhance its activity and/or to alter
its spatial and/or temporal expression pattern.
The present invention also provides use of a functional fragment of any one of SEQ ID N01 to 22
or variant thereof for changing the expression pattern of a promoter. In such methods , at least part
of any of the nucleic acids according to the present invention are combined with at least one
fragment of another promoter.
Further, the invention provides a genetic constru ctcomprising:
(a) An isolated promoter as defined hereinabove
(b) A heterologous nucleic acid sequence operably linked to isolated promoter of (a), and
optionally
(c) A 3' transcription terminator
The term "genetic construct" as used herein m eans a nucleic acid made by genetic engineering.
The term "operably linked" to a promoter as used herein means that the transcription is driven
and/or regulated by that promoter. A person skilled in the art will understand that being operably
linked to a promoter preferably means that the promoter is positioned upstream fl.e. at the ff-end)
of the operably linked nucleic acid. The distance to the operably linked nucleic acid may be
variable, as long as the promoter of the present invention is capable of driving and/or regulating
the transcription of the operably linked nudeic acid. For example, between the promoter and the
operably linked nucleic acid, there might be a cloning site, an adaptor, a transcription or
translation enhancer.
The operably linked nucleic acid may be any coding or non -coding nucleic acid. The operably
linked nucleic acid may be in the sense or in the anti-sense direction. Typically in the case of
genetic engineering of host cells, the operably linked nucleic acid is to be introduced into the host
cell and is intended to change the phenotype of the host cell. Alternatively, the operably linked
nucleic acid is an endogenous nucleic acid from the host cell.
The term "heterologous" as used herein is intended to be "heterologous to the promoter of the
present invention*. A nucleic add that is heterologous to the promoter of the present invention is
not naturally occurring in the nucleic add sequences flanking the promoter of the present
invention when it is in its biological genomic environment. While the nucleic add may be
heterologous to the promoter of the present invention, it may be h omologous or native or
heterologous or foreign to the plant host cell. The heterologous operably linked nucleic acid may
be any nudeic acid (for example encoding any protein), provided that it comprises or it is flanked
by at least one nucleotide which is normally not flanking the promoter of the present invention.
The term "transcription terminator" as used in (c) refers to a DNA sequence at the end of a
transcriptional unit which signals termination of transcription. Terminators are 3' -non-translated
DNA sequences usually containing a polyadenylation signal, which facilitates the addition of
polyadenylate sequences to the 3' -end of a primary transcript Terminators active in and/or
isolated from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and
have been described in literature. Examples of terminators suitable for use in the genetic
constructs of the present invention include the Agmbacterium tumefaciens nopaline synthase
(NOS) gene terminator, the Agmbacterium tumefaciens octopine synthase (DCS) gene terminator
sequence, the Cauliflower mosa ic virus (CaMV) 358 gene terminator sequence, the Oryza saliva
ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator
sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst
others.
The present invention also provides an expression cassette, a transformation vector or a plant
expression vector comprising a genetic construct as described above.
An "expression cassette" as meant herein refe rs to a minimal genetic construct necessary for
expression of a nucleic acid. A typical expression cassette compris es a promoter-gene-terminator
combination. An expression cassette may additionally comprise cloning sites, for example
GatewayTM recombination sites or restriction enzyme recognition sites , to allow easy cloning of
the operably linked nucleic acid or to allow the easy transfer of the expression cassette into a
vector. An expression cassette may further comprise 5' untranslated regions , 3' untranslated
regions, a selectable marker, transcription enhancers or translation enhancers.
With "transformation vector" is meant a genetic construct, which may be introduced in an
organism by transformation and may be stably maintained in said organism. Some vectors may be
maintened in for example Escherichia coli, A. tumefaciens, Saccharomyces cerevisiae or
Schizosaccharomyces pombe, while others such as phagemids and cosmid vectors, may be
maintained in bacteria and/or viruses. Transformation vectors may be multiplied in their host cell
and may be isolated again therefrom to be transformed into another host cell. Vector sequences
generally comprise a set of unique sites recognized by restriction enzymes, the multiple cloning
site (MCS), wherein one or more non-vector sequence(s) can be inserted. Vector sequences may
further comprise an origin of replication which is required for maintenance and/or replication in a
specific host cell. Examples of origins of replication include, but are not li mited to, the f1 -on and
colE1.
"Expression vectors" form a subset of transformation vectors, which, by virtue of comprising the
appropriate regulatory sequences, enabl e expression of the inserted non -vector sequence(s).
Expression vectors have been described which are suitable for expression in bacteria (e.g. £
coli), fungi (e.g. S. cerevisiae, S. pombe, Fichia pastoris), insect cells (e.g. baculoviral expression
vectors), animal cells (e.g. COS or CHO cells) and plant cells . One suitable expression vector
according to the present invention is a plant expression vector, useful for the transformation of
plant cells, the stable integration in the plant genome, the maintenance in the plant cell and the
expression of the non-vector sequences In the plant cell.
Typically, a plant expression vector according to the present invention comprises a nucleic acid of
any one of SEQ ID NO 1 to 22 or a variant thereof as described hereinabove, optionally operably
linked to a second nucleic acid. Typically, a plant expressible vector according to the present
invention, further comprises T-DNA regions for stable integration into the plant genome (for
example the left border and the right border regions of the Ti plasmid).
The genetic constructs of the invention may further comprise a "selectable marker". As used
herein, the term "selectable marker" includes any gene, which confers a phenotype to a cell in
which it is expressed, to facilitate the identification and/or selection of cells that are transfected or
transformed. Suitable markers may be selected from markers that confer antibiotic or herbicide
resistance. Cells containing the genetic construct will thus survive antibiotics or herbicide
concentrations that kill untransformed cells. Examples of selectable marker genes include genes
conferring resistance to antibiotics (such as nptll encoding neomycin phosphotransferase capable
of phosphorylating neomycin and kanamycin, or h pt encoding hygromycin phosphotransferase
capable of phosphorylating hygromycin), to herbicides (for example bar which provides resistance
to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic
trait (such as manA that allows plants to use mannose as sole carbon source). Visual marker
genes result in the formation of colour (for example beta -glucuronidase, GUS), luminescence
(such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives there of).
Further examples of suitable selectable marker genes include the ampicillin resistance (Ampr),
tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr), phosphinothricin
resistance gene, and the chloramphenicol acetyltransferase (CAT) gene, amongst others.
Furthermore, the present invention encompasses a host cell comprising an isolated promoter, or a
genetic construct, or an expression cassette, or a transformation vector or an expression vector
according to the invention as described hereinabove. In particular embodiments of the invention,
the host cell is selected from bacteria, algae, fungi, yeast, plant s, insect or animal host cells.
In one particular embodiment, the invention provides a transgenic plant cell comprising an isolated
promoter according to the invention, or an isolated nucleic acid, or a genetic construct, or an
eicpression cassette, or a transformation vector or an expression vector according to the invention
as described hereinabove. Preferably said plant cell is a dicot plant cell or a monocot plant cell,
more preferably a cell of any of the plants as mentioned herein. Preferably, in the transgenic plant
cell according to the invention, the promoter or the genetic construct of the invention is stably
integrated into the genome of the plant cell.
The invention also provides a method for the production of a transgenic plant, comprising:
(a) Introducing into a plant cell an isolated promoter, for example any one of
SEQ ID NO 1 to SEQ ID NO 22, or a variant or fragment thereof, or a
genetic construct, or an expression cassette, or a transformation vector or
an expression vector according to the present invention and as described
hereinabove.and
(b) Cultivating said plant cell under conditions promo ting plant growth.
"Introducing" the above mentioned isolated promoter, orgenetic construct, or expression cassette,
or transformation vector or expression vector, into a host cell (e.g. plant cell) is preferably
achieved by transformation. The term "transformation" as used herein encompasses the transfer
of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. In
particular for plants, tissues capable of donal propagation, whether by organogenesis or
embryogenesis, are suitable to be transformed with a genetic construct of the present invention
and a whole plant may be regenerated therefrom. The particular tissue chosen will vary depending
on the clonal propagation systems available for, and best suited t o, the particular plant species
being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons,
hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem,
axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and
hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a plant cell
and may be maintained non -integrated, for example, as a plasmid. Alternatively, it may be
integrated into the plant genome.
Transformation of a plant species is now a fairly routine technique. Advantageously, any of
several transformation methods may be used to introduce the nucleic acid s of the invention into a
suitable ancestor cell. Transformati on methods include the use of liposomes, electroporation,
chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun
bombardment, transformation using viruses or pollen and microprojection. Methods may be
selected from the calcium/polyethylene glycol method for protoplasts (Krens, FA et a/., 1882,
Nature 296. 72-74; Negrutiu I. et a/., June 1987, Plant Mol. Biol. 8. 363 -373); electroporation of
protoplasts (Shillilo R.D. et si.. 1985 Biofiechnol 3,1099-1102); microinjection into plant material
(Crossway A. et at., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle
bombardment (Klein T.M. et al., 1987, Nature 327, 70) infection with (non -integrative) viruses and
the like. A preferred transformation metho d for the production of transgenic plant cells according
to the present invention, is an Agrobacterium mediated transformation method.
Transgenic rice plants comprising any one of the promoters of the present invention are preferably
produced via /\grobactera/m-mediated transformation using any of the well -known methods for
rice transformation, such as the ones described in any of the following: published European patent
application EP1198985 A1, Aldemita and Hodges (Planta, 199,612 -617,1996); Chan etal. (Plant
Mol. Biol. 22 (3) 491 -506,1993); Hiei et al. (Plant J. 6 (2) 271 -282,1994); which disclosures are
incorporated by reference herein as if fully set forth. In the case of com transformation, the
preferred method is as described in either Ishida et al. (Nat. Biotechnol. 1996 Jun; 14(6): 745 -50)
or Frame et al. (Plant Physiol. 2002 May; 129(1): 13 -22), which disclosures are incorporated by
reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are se lected for the presence of one or
more markers which are encoded by plant-expressible genes co -transferred with the gene of
interest (which could be under the control of any of the promoters of the present invention),
following which the transformed materi al may be cultivated under conditions promoting plant
growth.
The resulting transformed plant cell may then be used to regenerate a transformed plant in a
manner known to persons skilled in the ait Accordingly, the method for the production of a
transgenic plant as described hereinabove, may further comprise regenerating a plant from said
plant cell of (a).
The present invention further provides a plant comprising a plant cell as described hereinabove.
The plants may also be able to grow, or even reach maturity including for example fruit production,
seed formation, seed ripening and seed setting.
Furthermore, progeny may be produced from these seeds, which progeny may be fertile.
Alternatively or additionally, the transformed and regenerated plants may also produce progeny by
non-sexual propagation such as cloning, grafting. The generated transformed plants may be
propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
For example, a first generation (or T1) transformed plant may be selfed to give homozygous
second generation (or T2) transformants, and the T2 plants further propagated through classical
breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be
chimeras of transformed cells and non -transformed cells; clonal transformants (e.g., all cells
transformed to contain the expression cassette); grafts of transformed and untransformed tissues
(e.g., in plants, a transformed rootst ock grafted to an untransformed scion).
Following DMA transfer and growth of the transformed cells, putatively transformed plant cells or
plants may be evaluated, for instance using Southern analysis, for the presence of the gene of
interest, copy number and/or genomic organization. Alternatively or additionally, expression levels
or expression patterns of the newly introduced DNA may be undertaken using northern and/or
Western analysis, both techniques being well known to persons having ordinary skill in the art.
The present invention clearly extends to plants obtainable by any of the methods according to the
present invention, which plants comprise any of the isolated promoters or the constructs of the
present invention. The present invention clearly ex tends to any plant parts and propagules of such
plant The present invention extends further to encompass the progeny of a primary transformed
cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods,
the only requirement being that progeny exhibit the same genotypic and/or phenotypic
characteristic^) as those produced in the parent by the methods according to the invention. The
invention also extends to harvestable parts of a plant, such as but not limited to seeds, I eaves,
fruits, flowers, stem cultures, stem, rhizomes, roots, tubers, bulbs and cotton fibers.
The term "plant" or "plants" as used herein encompasses whole plants, ancestors and progeny of
plants and plant parts, including seeds, shoots, stems, roots (i ncluding tubers), and plant cells,
tissues and organs. The term "plant" therefore also encompasses suspension cultures, embryos,
meristernatic regions, callus tissue, gametophytes, sporophytes, pollen, and microspores. Plants
that are particularly useful in the methods of the invention include all plants which belong to the
superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a
fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list
comprising Acacia spp., Acer spp., Actinidia spp.,Aesculus spp., Agatliis australis, Albizia amara,
Alsophlla tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus
deer, Baildaes plurijuga, Betula spp., Brassica spp., Bwguie ra gymnorrhiza, Burkea africana,
Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum
spp., Cassia spp., Centroema pubescens, Chaenomeles spp.,Cinnamomum cassia, Coffea
arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Cnataegus spp.,
Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica,
Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata,
Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp.,
Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp.,
Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa
sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba,
Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma,
Hedysarum spp., Hemarthia attissima, Heteropogon contortus, Hordeum vulgare, Hyp arrhenia
rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,
Lespediza spp., L&ttuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus
spp., Macrotyloma axil/are, Malus spp., Manihot es culenta, Medicago sativa, Metasequoia
glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza
spp., Pettophowm africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp.,
Phoenix canariensis, Phonnium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum
sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Pmsopis
cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp.,
Rhaphiolepsis urn bellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp.,
Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum,
Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor,
Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi
spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla,
Vaccinium spp., Vida spp.Vitis vinif&ra, Watsonia pyramidata, Zantedesc hia aethiopica, Zea
mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot,
cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice,
soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, trees and algae
amongst others. According to a preferred feature of the present invention, the plant is a crop plant
such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, tobacco, squash,
papaya, poplar, leguminosa, flax, lupinus or sorghum. According to another preferred embodiment
of the present invention the plant is a monocotyledonous plant, such as sugarcane, further
preferable a cereal such as rice, maize, wheat, barley, millet, rye or oats.
The invention further provides a method for driving and/or regulating expression of a nucleic acid
in a plant or plant eel I, comprising:
a) Operably linking a nucleic acid to an isolated nucleic acid according to the invention as
described hereinabove, such as to any one of SEQ ID NO 1 to 22 or a variant or
fragment thereof, and
b) Introducing the resultant genetic construct into a plant or plant cell.
Preferably the operably linked nucleic acid of (a) is heterologous to the nucleic acids according to
the present invention.
This method may further comprise cultivating the transformed plant or plant cell under conditions
promoting growth, promoting regeneration and/or promoting maturation.
Furthermore, the expression of the operab ly linked nucleic acid may be driven and/or regulated in
particular cells, tissues or organs of a plant Accordingly, the invention p rovides a method as
described above, wherein the expression is constitutive expression or tissue -specific expression.
For these embodiments, reference is made to the example section where the specific expression
patterns of the promoters according to the invention are described and where different types of
tissue-specific expression are detailed .
The present invention further encompasses the u se of an isolated nucleic acid as defined
hereinabove to drive and/or regulate expression of an operably I inked nucleic acid.
The person skilled in the art will recognize that provision of sequences SEQ ID NO 1 to 22, readily
makes available the tools to isolate related promoters, which may have substantial sequence
identity to any of SEQ ID ID NO 1 to 22. Additionally, provision of sequences SEQ ID NO 23 to 44
(CDS corresponding to the promoters of the present invention, see Table 1) , readily makes
available the tools to isolate related promoters, of which the related CDSs may have substantial
sequence identity to any of SEQ ID NO 23 to 44. Therefore the present invention also
encompasses a method for isolating nucleic acids, capable of driving and/or regulating expression
of an operably linked nucleic acid, comprising screening a nucleic acid sequence database to find
homologues of any of the sequences represented by SEQ ID N01 to 22 or SEQ ID NO 2 3 to 44.
Subsequently these homologues are used to screen a library with genomic DNA, which library is
for example prepared from the organism of origin of the above mentioned homologue. The
screening procedure may for example involve hybridization. Subsequently, the genomic DNA that
matches the homologue, is analysed to identify the transcription initiation site and the translation
initiation site of the gene corresponding to the homologue. Finally, specific primers are designed
for amplification of a nucleic acid located in the region upstream (at the 5' end) of said translation
initiation site.
The present invention exten ds to the Identification of regulatory proteins that are involved in the
regulation of the activity of the promoters according to the present invention. Such Identification
may be achieved using a yeast one-hybrid system. In such a yeast one-hybrid system the
sequences according to any one of SEQ ID NO 1 to 22 are operably linked to the GAL
transcription activator and transformed to a yeast cell culture. That yeast cell culture is again
transformed with a library of constructs encodi ng candidate regulatory factors.
The present invention will now be described with reference to the following figures in which:
Figure 1 shows a general schematic representation of a promoter. Regulatory elements are
sequences that may for example be responsible for special and/or temporal regulation of the
promoter activity. The minimal promoter is the minimal sequence necessary and suff icient to drive
expression. It includes a TATA box, which is necessary to correctly direct the RNA polymerase It
to the transcription initiation site. The transcription initiation element (INR) in dudes the
transcription initiation start site. The 5' untranslated region (5'UTR) is the region that is transcribed
into pre-messenger RNA and eventually in to mRNA, but is not translated into protein. The
translation initiation codon is represented by the startcodon ATG.
Figure 2 is a map of the vector p4581 useful for expression in plants of a p-glucuronidase (GUS)
gene under control of any one of the promoters according to the Invention. Th is binary vector
comprises a Gateway recombination cassette , suitable for the recombination cloning of any of the
promoters of the present invention in front of the Escherichia coli [J-glucuronidase (GUS) gene.
This cassette contains a chloramphenicol resistance gene (CamR) and the ccdB suicide gene for
counter selection of non -recombined plasmids, This GUS expression cassette further comprises
the double terminator sequence T-zein and T-rbcS-deltaGA. This expression cassette is located
within the left border (LB repeat, LB Ti C58) and the right border (RB repeat, RB Ti C58) of the
nopaline Ti plasmid. Cloned within these borders are also selectable marlcer and a screenable
marker genes each under control of a constitutive promoter and a terminator sequence. This
vector also contains an origin of replication (pBR322) for bacterial replication and a bacterial
selectable marker (Spe/SmeR) for bacterial selection .
The following figures show the results of the GUS staining of plants or pla nt parts transformed with
the reporter vector p4581 carrying a promoter according to the present invention operably linked
to the reporter gene GUS. Plants denoted "C plants" are transgenic plants grown to about 5 cm;
Plants denoted "B plants" are grown to about 10 cm; and plants denoted "A plants" are grown to
maturity. These A plants were used to collect different tissue samples from old leaves, young
leaves and seeds.
Figure 3 shows the expression pattern of PRO0110 (RCc3, SEQ ID NO 1). GUS staining is visible
in roots.
Figure 4 shows the expression pattern of PRO0005 ( putative beta-amylase, SEQ ID NO 2). GUS
staining is visible in seeds, more specifically in the embryo or in the scutellum of the embryo .
Figure 5 shows the expression pattern of PRO0009 ( putative cellulose synthetase, SEQ ID NO 3).
GUS staining is visible in roots.
Figure 6 shows the expression pattern of PRO0058 (proteinase inhibitor Rgpi9, SEQ ID NO 4).
GUS staining is visible in the seeds.
Figure 7 shows the expression pattern of PRO0061 (beta expansine EXPB9, SEQ ID NO 5). GUS
staining is visible hi young flowers of A plants (A) and in other young expanding tissues of B plants
(B) and C plants (C).
Figure 8 shows the expression pattern of PRO0063 (putative structural protein, SEQ ID NO 6).
GUS staining is visible In young tissues, for example in the call! (A) or old leaves, young leaves
and seeds of "A plants" (B).
Figure 0 shows the expression pattern of PRO0081 (putative caffeoyl-CoA 3-Omethyltransferase,
SEQ ID NO 7). GUS staining is visible in young tissues, particularly of the
shoot.
Figure 10 shows the expression pattern of PRO0091 ( prolamine RP5, SEQ ID NO 8). GUS
staining is visible in seeds (A), particularly in the endosperm, and in meristem (B).
Figure 11 shows the expression pattern of PRO0095 (putative amino peptidase, SEQ ID NO 9).
GUS staining is visible in seeds, more particularly in the embryo.
Figure 12 shows the expression pattern of PRO0111 (uclacyanin 3-like protein, SEQ ID NO 10).
GUS staining is visible in roots and in meristem.
Figure 13 shows the expression pattern of PRO0116 (26S proteasome regulatory particle non -
ATPase subunit 11, SEQ ID NO 11). GUS staining is weakly visible in the whole plant (weak
constitutive) and is particularly visible in meristem.
Figure 14 shows the expression pattern of PRO0117 (putative 40S ribosomal protein, SEQ ID NO
12). GUS staining is visible in the seeds, more particularly in the endosperm.
. Figure 15 shows the expression pattern of PRO0122 (chlorophyll a/b-binding protein presursor
(Cab27), SEQ ID N013). GUS staining is visible in the shoot.
Figure 16 shows the expression pattern of PRO0123 (putative protochlorophyllide reductase,
SEQ ID NO 14). GUS staining is visible in the shoot (above-ground tissues).
Figure 17 shows the expression pattern of PRO0133 (chitinase Cht-3, SEQ ID NO 15). GUS
staining is visible in the roots and meristem.
Figure 18 shows the expression pattern of PRO01 51 (WSI18, SEQ ID NO 16). GUS staining is
visible in the call! and upper plant parts (A) as well as in the aleurone layer and embryo (B).
Figure 19 shows the expression pattern of PR00169 (aquaporine, SEQ ID N017). GUS staining
is visible in the whole plant (constitutive expression).
Figure 20 shows the expression pattern of PRO0170 (High mobility group protein, SEQ ID NO
18). GUS staining is strongly visible in the whole plant as is illustrated by the "B plants" (A), and
various tissues such as old leaves, young leaves and seeds (B) and call! (C) (constitutive
expression).
Figure 21 shows the expression pattern of PRO01 71 (reversibly glycosylated pro tein RGP1 , SEQ
ID NO 19). GUS staining is visible in all plant parts ( constitutive expression ).
Figure 22 shows the expression pattern of PRO0173 (cytosolic MDH, SEQ ID NO 20). GUS
staining is visible in all p lant parts and particularly in the shoot ( above-ground tissues) and seeds .
Figure 23 shows the expression pattern of PRO0175 ( RAB21, SEQ ID NO 21). GUS staining is
weakly visible in call! (A), meristems and young leaves, and is strongly visible in developing and
maturing seeds (B) more particularly in the embryo .
Figure 24 shows the expression pattern of PRO0177 ( Cdc2-1, SEQ ID NO 22). GUS staining is
weakly visible in meristem and in leaf sheets .
Examples
The promoters according to the present Invention were isolated as DMA regions spanning about
1.2 kb of the sequence upstream of the translation initiation codon (i.e. first ATG, which codon
was excluded) from various rice genes. For determinatio n of their nucleic acid sequence and their
expression pattern, the following procedure was followed: First In silica studies on genomic rice
sequences were performed . However, procedures based on automated prediction programs to
locate promoter-like nucleic acid sequence are highly error prone, even for the localization the
best-characterized promoter control elements such as the TATA box and the transcription initiation
element (INR). Also, in silica determination of expression pattern is extremely speculative.
Therefore, to obtain unambiguous data about the nucleic acid sequence and the expression
pattern of the promoter s, in vivo studies were performed encompassing (i) isolation of the
promoter nucleic acid sequence ; (0) operably linking a reporter gen e to the promoter and
introducing the resulting genetic construct into a host organisms ; (iii) growing the transformed host
cell under conditions allowing expression of the reporter gene, and (iv) determination of the
reporter gene activity in the dilferen t tissues of the host organism. These methods are now
described in more detail.
1. Identification and isolation of the promoters
Identification office ESTs, tlte corresponding genes and their location In the rice genome
Sequence databases, comprising rice sequences, were searched for rice expressed sequence
tags (ESTs). Subsequently an" in silico" Northern -Wot was performed to allow identification of EST
families that are strongly expressed or that are specific for a particular organ. This analysis
included normalization of the numbers of ESTs isolated from different plant organs. The ESTs
families with an interesting distribution among source cDNA libraries were selected for further
analysis and sequence homology searches. After sequence homology searches in combination
with scanning scientific data, the genes that correspond to those families of EST s were identified
from sequence databases and a (putative) function and corresponding gene name was given (see
Table 1). Subsequently , the corresponding promoter region was isolated by the following
procedure. In a first step the TIQR database was searched to find a tentative contig corresponding
to an EST family. Sequence homology was found using standard computer programs , such as
Blast N using standard parameters (typically G Cost to open a gap = 5, E Cost to extend a gap =
2, q Penalty for a mismatch in the blast portion of run = -3, r Reward for a match in the blast
portion of run = 1, e Expectation value = 10.0, W Word size= 11, v Number of one-line
descriptions = 100, b Number of alignments to show = 100, Matrix = BLOSUM62). The TIGR
database (The Institute for Genomic Research), provides Tentative Contigs (TC) which are
sequence predictions based on contig building from all known EST, from all known cDNA and
from reconstructed mRNA. The TCs used for Identification of the promoters of the present
invention are represented in Table 1. In a second step these TCs were used to locate the
corresponding gene on a genomic sequence, which gene comprises the coding region as well as
the promoter region. Generally, these genomic sequences were 6AC clones, which are
represented herein by their Genbank accession number (see Table 1). From these BAC clones
the sequence identity of the promoter region could be determined.
Table 1: list of rice promoters of the present invention. The promoter sequences are represented
herein by their SEQ ID NO and promoter number (PRO). The coding sequences (CDS) naturally
driven by a promoter of the present invention are represented by their name, by SEQ ID NO and
by Tentative contig ( TC) accession number of the TIGR database. The Genomic sequences (BA C
clones or genes) comprising a promoter region of the present invention are represented by their
Genbank accession number.
(Table Removed)
Identification and Isolation of the promoter regions of rice genes
Starting from the sequence information of the gene s and their location in the rice genome, the
promoter regions of these genes were isolated as the DMA region spanning about 1.2 kb
upstream of the translab'on initiation codon (i.e. first ATG), which codon was excluded. When an
intervening sequence such a s an inlron, was present in the 5' untranslated region of the gene, the
isolated DMA region was taken as the region spanning about 1.2 kb plus the length of that
intervening sequence. The promoter regions were isolated from genomic DNA of Oryza sativa
Japonica or exceptionally from Oryza sativa Indica via PCR using specific primers. These specific
primers comprise AttB recombination sites, suitable for recombination cloning of the isolated
25
promoter region These specific primers are herein represented as SEQ ID NO 45 to 88 and are
listed in Table 2. Conditions for PCR were as follows: 1 cycle of 2 min at 94°C, 35 cycles of 1 min
at 94°C, 1 min at 58°C and 2 min at 68°C, and 1 cycle of 5 min at 68"C. The length of the
expected PCR fragment is also indicated in Table 2. The corresponding PCR fragment was
purified from the PCR reaction mix via gele electrophoresis and subsequent purification using
Zymoclean Gel DMA Recovery Kit (Zymo Research, Orange, California).
Table 2: Overview of the primers used to isolate the rice promoters of the present invention and
the length of the rice promoter regions.
(Table Removed)
Example 2. Cloning of promoter-GUS reporter vectors for plant transformation
The purified PCR fragments of Example 1, corresponding to the promoter regions of the present
invention, were cloned into the pDONR201 entry plasmid of the GatewayTM system (Life
Technologies) using the "BP recombination reaction". T he identity and base pair composition of
the cloned insert was confirmed by sequencing and additionally, the resulting plasmid was tested
via restriction digests.
In order to done each of the promoters of the present invention in front of a reporter gene, each
entry clone of Example 1 was subsequently used in an "LR recombination reaction" (Gateway TM)
with the destination vector p4581. This destination vector was designed to operably link each
promoter of the present invention to the Escherichia coli beta-glucuronidase (GUS) gene via the
substitution of the Gateway recombination cassette in front of the GUS gene. Furthermore this
destination vector is suitable for transformation of plants and comprises within the T -DMA left and
right borders the resulting promoter-GUS cassette and selectable marker and screenable marker
cassettes (see Figure 2). The resulting reporter vectors, comprising a promoter of the present
invention operably linked to GUS, are subsequently transformed into Agrobacterium strain
LBA4044 and subsequently into rice plants using standard transformation techniques .
Example 3. Expression pattern s of the promoter-GUS reporter cassette in plants
Growth and harvest of transgenic plants or plant parts at various stages (C plants, B plants
and A plants)
For each promoter-GUS reporter construct, 3 TO transgenic rice plants were generated from
transformed cells. Plant growth was performed under normal conditions. The first transgenic plant
was sacrificed for GUS staining when it had reached a size of about 5 cm, which plant is named
herein "C plant". The second transgenic plant was sacrificed for GUS staining when it had reached
a size of about 10 cm, which plant is named herein "B plant". The third transgenic plant was kept
for seed production and is named herein "A plant". GUS staining was performed on complete C
and B plants. On A plants, GUS staining was performed on leaf pieces, flowers and section of
seeds at various developmental stages. A plants were allowed to set seed, which seeds were
used after harvest for confirmation of the expression pattern in T1 plants.
GUS staining
The sacrificed plants or plant parts were covered with 90% ice-cold acetone and incubated for 30
min at 4 °C. After 3 washes of 5 min with Tris buffer [15,76 g Trizma HCI (Sigma T3253) + 2,922 g
NaCI in 11 bidi, adjusted to pH 7,0 with NaOH], the material was covered by a Tris/ferricyanate/X -
Glue solution [9,8 ml Tris buffer + 0,2 ml ferricyanate stock (0,33 g Potassium ferricyanate (Sigma
P3667) in 10 ml Tris buffer}* 0,2 ml X -Glue stock (26,1 mg X-Gluc (Europa Byproducts ML 113A)
in 500 ul DMSO)]. Vacuum infiltration was applied for 15 to 30 minutes. The plants or plant parts
were incubated for up to 16 hours at 37 °C until development of blue colour was visible. The
samples were washed 3 times for 5 minutes with Tris buffer. Chlorophyll was extracted in ethanol
series of 50%, 70% and 90% (each for 30 minutes).
Expression patterns of the promoters of the present Invention
The expression patterns of the rice promoters of the present invention are summarized in Table 3.
Table 3: expression patterns of the rice promoters of the present invention
(Table Removed)
The following paragraphs describe the observed expression patterns of the promoters of the
present invention in more detail. The observations are based on the visual inspection of the GUS
stained tissues as described above. It is to be understood that for some promoters expression
may be weak and that expression in certain tissues may only be visible with very sensitive
detection methods.
PRO0110 - SEQ ID NO 1-RCc3
1 construct (OS1432), which is a reporter v ector as described in Example 2 comprising PRO0110
was investigated. 25 calli, 14 C, 21 B plants and 21 A plants were analysed. There was no
expression visible in calli, but strong expression in roots of C plants (93%) and of B plants (81%)
was observed. No expression in the shoots of A plants was observed. Therefore the RCc3
promoter PR00110 is suitable for strong expression in root s.
PRO0005 - SEQ ID NO 2 - putative beta -amylase
1 construct (OS1365) was investigated. 28 calli, 24 B plants and 22 A plants were analysed.
Occasional expression in calli (7%) was observed as well as occasional weak expression in root s
(4%) and shoots (12%) of B plants, expression in (he scutellum of embryos of A plants (43%) and
occasional expression in leaves (5%) of A plants. This promoter is therefore suitable for
expression in embryo, more preferably in the scutellum of the embryo. This region of the embryo
is also referred to as the transfer layer of the embryo. This promoter may have some leakiness in
other tissues.
PRO0009 - SEQ ID NO 3- putative cellulose synthase
1 construct (OS1461) was investigated. 20 calli, 20 C, 20 B plants and 20 A plants were analysed.
Occasional expression in calli (20 %) was observed as well as weak expression in roots (55%) of
C plants, occasional expression in young leaves (10%) of C plants and weak expression in the
roots (25%) of B plants. No expression in leaves of A or B plants was observed. Therefore this
promoter is suitable for expression In roots. This promoter may show some leakiness in the
leaves.
PRO0058 . SEQ ID NO 4-protelnase Inhibitor Rgpl9
1 construct (OS1370) was investigated. 13 B plants and 12 A plants were analysed. No
expression was observed in B plants. In A plants, no expression was observed in the leaves, but
there was strong expression in endosperm and embryo (58 -42%). Therefore, this promoter
PRO0058 is suitable for expression in seeds.
PKOOOS1 -SEQ ID NO 5- bate sxpanslne BSPBfl
2 constructs (OS1441 and OS1460) were investigated. 20 calli, 32 C, 32 B plants and 32 A plants
were analysed. Weak expression was observed in the leaves of C and B plants. In A plants
expression in the flowers was observed (44%), more particularly in lemma of young spikelets. It
was concluded that the promoter PRO0061 is suitable for expression in young tissue, more
preferably in young, developing or expanding tissue, more preferably in green tissue.
PRO0063 - SEQ ID NO 6- putative structural protein
1 construct (OS1446) was investigated. 13 calli, 13 C, 13 B plants and 12 A plants were analysed.
In calli, weak expression was detected (92%). In C plants, there was no expression in roots and
there was weak expression in some leaves (46%). In B plants, there was no expression in roots
and weak expression In young tillers (78%) o r young leaves (54%), but no expression in old
leaves. In A plants, there was occasional expression in young leaves (17%) and expression in
embryo and scutellum (42%). Therefore it was concluded that this promoter is active in the aboveground
tissues, such as leaf, stem and seed. These data demonstrate that the promoter is
suitable for expression in calli and in the shoot, and for expression in young tissues and seeds.
PRO0081 • SEQ ID NO 7- putative caffeoyl-CoA 3-O-methyltransferase
1 construct (OS1419) was investigated. 20 calli, 20 C, 20 B plants and 20 A plants were analysed.
No expression was observed in Calli. Expression was observed in C plants, more particularly
weak expression in root cylinder (40%) an d weak expression in young leaves (80%) and in old
leaves. Expression was also observed in B plants, more particularly weak expression in roots
(25%) and weak expression in young leaves (80%). Expression was also observed in young
leaves (50%) of A plants. It was concluded that promoter PRO0081 is suitable for expression in
above-ground tissue s, preferably in the shoot. This promoter may have some leakage of
expression in roots.
PRO0091 - SEQ ID NO 8- prolamlne RPS
1 construct (OS1558) was investigated. 12 C, 12 B plants and 12 A plants were analysed. Weak
expression was observed in the discrimination centre (50%) of C plants and in the discrimination
centre (58%) of B plants. Strong expression was observed in endosperm (55%) of A plants. This
promoter was found to be useful for strong expression in the endosperm, with leakiness in
meristem, preferably the shoot meristem or discrimination centre.
PRO0095 - SEQ ID NO 9- putative meth ionlne amlnopeptidase
1 construct (OS1423) was investigated. 16 calli, 14 C, 14 B plants and 16 A plants were analysed.
Some expression was observed in root-tips (36 %) of C plants and in the embryo (38%) of A
plants, but not in endosperm of A plants. It was concluded that PRO0095 is suitable for
expression in embryo.
PRO0111 - SEQ ID NO 10- uclacyanln 3-//fosprofe/n
1 construct (OS1421) was investigated. 22 calli, 21 C, 22 B pla nts and 21 A plants were analysed.
Weak expression was observed in the discrimination centre and meristems (77%) of B plants. It
was concluded that promoter PRO0111 is suitable for weak expression in the meristem, preferably
in shoot meristem or discrimination centre.
PRO0116 - SEQ ID NO 11-26S proteasome regulatory particle non -ATPase subunit 11
1 construct (OS 1679) was investigated. 13 C, 14 B plants and A plants were analysed. Weak
expression was observed in meristem/discrimination centre of C plants (38%) and of B plants
(71%) and in young leaf sheaths of C plants (77%) and of B plants (21%). It was concluded that
promoter PRO0116 is suitable for expression in meristem, preferably in shoot meristem or
discrimination centre.
PRO0117 - SEQ ID NO 12- putative 40S ribosomal protein
1 construct (OS1425) was investigated. 9 calli, 9 C, 9 B plants and 9 A plants were analysed.
Occasional weak expression was observed in roots (22%) and in young lea f blades (44%) of C
plants. Expression was mainly observed in endosperm (37%) of A plants. Therefore, promoter
PRO117 was found to be suitable for expression in endosperm and may have some leakhess in
young leaves.
PRO0122 - SEQ ID N013- chlorophyll aJb-binding protein presursor (Cab27)
1 construct (OS1675) was investigated . 38 calli, 38 C, 38 B plants and 15 A plants were analysed.
Very weak expression was observed in the discrimination centre and young leaf sheaths of C
plants. It was concluded that this promoter PRO0122 is suitable for weak expression in shoots.
PRO0123 - SEQ ID NO 14-putative protochlorophyllide reductase
1 construct (OS1433) was investigated. 21 calli, 18 C, 19 B plants and 18 A plants were analysed.
Strong expression was observed in shoots (33-68%) of C plants and B plants (63-79%). In B
plants there was also occasional expression in roots. In A plants, again strong expression in
young leaves (73%) was observed, as well as occasional expression in old leaves (39%). It was
concluded that this promoter is suitable for strong expression in shoots, preferably in leaves.
PRO0133 - SEQ ID N015- chltlnase Cht-3
1 construct (OS1687) was investigated. 15 calli, 12 C, 16 B plants and 12 A plants were analysed.
Weak expression was observed in cadi (66%) and in the discrimination centre/meristem (50%) of
B plants. It was concluded that promoter PRO0133 is suitable for weak expression in meristem,
preferably in shoot meristem or discrimination centre.
PKQ0151 - SEQ ID NO IS- WSI18
1 construct (OS1458) was investigated. 22 calli, 16 C, 16 B plants a nd 13 A plants were analysed.
Strong expression was observed in calli (91%) and weak expression in shoot s of C plants (62%).
In A plants there was very strong expression in the aleurone layer and in the embryo (46%). It was
concluded that promoter PRO0151 is suitable for strong expression in calli and in seeds, more
particularly in the aleurone layer and in the embryo of the seeds.

PRO0163 - SEQID N017- aquaporlne
1 construct (OS1911) was investigated . 11 calli, 10 C plants, B plants and A plants were analysed.
Some expression (55%) was observed in calli and in roots (30% ) of C plants. Furthermore, good
expression was observed in shoot tissues (80%) of C plants and in young leaves of B plants. It
was concluded that this promoter is suitable for constituti ve expression, preferably constitutive in
young plants.
PR0170 - SEQ ID NO 18- High mobility group protein
1 construct (OS1434) was investigated. 23 calli, 21 C, 21 B plants and 14 A plants were analysed.
Expression was observed in calli (52%) and in roots (51%) of C plants. Moreover, strong
expression was observed in young leaves (81%) of C plants, in roots (86%) of B plants and in
young leaves (86%) of B plants. In A plants there was strong expression in young leaves (75%),
old leaves (43%), embryo and aleurone but a weaker expression in endosperm (82%). It was
concluded that promoter PRO170 is suitable for strong constitutive expression.
PRO0171 -SEQ ID NO 19-reversibly glycosylated protein RGP1
1 construct (OS1762) was investigated. 18 calli, 11 C and 13 B plants were analysed. Strong
expression was observed in calli (44%) and in all tissues (27%) of C plants. In all tissues of B
plants (16%), expression was somewhat weaker but most pronounced the in discrimination
centres (46%). It was concluded that promoter PRO0171 is suitable for constitutive expression.
PRO0173 - SEQ ID NO 20- cytosollc MDH
1 construct (OS1435) was investigated. 17 calli, 17 C, 17 B plants and 15 A plants were analysed.
Occasional expression (12%) was observed in calli and weak expression was observed in upper
parts (24-69%) of C plants as well as in young leaves (41 %) of B plants. In A plants, expression in
leaves (33%) was observed and strong expression in seeds (38%), but not in the root It was
concluded that the promoter PRO 0173 is suitable for expression in above-ground tissues
especially for constitutive expression in the shoot and especially in the seeds.
PRO017S - SEQ ID NO 21 • RAB21
1 construct (OS1436) was investigated. 16 calli, 12 C, 15 B plants and 15 A plants were analysed.
Expression was observed in some calli (31 %), in the discrimination centre s (42%) of C plants and
in young leaves (25-58%) of C plants and A plants (15 %). Furthermore, very strong expression
was observed in aleurone and embryo (60%) of a plant. It was concluded that promoter PRO0175
Is suitable for strong expression in calli and in seeds, more particularly in developing/maturing
seeds, more particularly in the aleurone layer and in the embryo of the seeds.
PRO0177 - SEQ ID NO 22- CdcZ-1
1 construct (OS1436) was investigated . 16 calli, 12 C, 15 B plants and 15 A plants were analysed.
Expression was observed in some of the call! (31%), in the discrimination centre (42%) of C
plants, in young leaves (25-58%) of C plants and occasionally in young leaves (15 %) of A plants.
Moreover, very strong expression was observed in aleurone and embryo (60%) of seeds from A
plants. It was concluded that this promoter is suitable for specific expression in seeds, more
particularly in developing/maturing seeds.
Example 4. Stability of the expression patterns of the promoters of the present
invention in further generations
The above-mentioned analyses were performed on TO plants originating from the transformed
tissues. The stability of promoter activity in the next generations or progeny plants of the original
TO plant, the so-called T1 and T2 plants, was evaluated as follows. The TO plant transformed with
the reporter constructs as mentioned in the above paragraphs of Example 2, were grown until
maturity (A plants), of which the seeds (T1 seeds) were harvested and sown to generate progeny
T1 plants. These plants were analysed as described above in Example 3 and the A T1 plants
were allowed to reach maturity and to set T2 seeds.
The expression pattern of the promoters of the present invention was studied in TO plants, T1
seeds, T1 plants and T2 seeds and in all the tissues (Including seeds and seed tissues ) as
described in Example 3. The specific expression pattern s as reported from the TO and T1 seeds
and described in Example 3 were confirmed in the following T1 generation and T2 seeds . It is
concluded that the expression pattern of the promoter s of the present are stably inherited in plants
of subsequent generations.
Examples. Stability of expression patterns of the promoters of the present
invention In other plants
The above-mentioned plant analyses were performed on rice plants. This choice was based on
the practical consideration that plant genetic engineering is most profitable for crop plants. Also in
other crop plants, such as for example Zea Mays, the reporter constructs comprising the
promoters according to the present i nvention are introduced and transformed plant are evaluated
as described hereinabove. The expression patterns of the promoters according to the present
invention are conserved among plants. Therefore, the promoters according to the present
invention are also suitable for driving and/or regulating expression of an operably linked nucleic
acid in monocots, such as com.
For many other purposes such as research and horticulture, (small) herbs are being genetically
modified, which involves the use of promoters. Therefore the reporter constructs comprising the
promoters according to the present invention are introduced into other plants species such as for
example Arabidopsis thaliana and transformed plants are evaluated as described herein above.
The expression patterns of the promoters according to the present invention are conserved
among plants. Therefore, the promoters according to the present invention are also suitable for
driving and/or regulating expression of a n operably linked nucleic acid in other plant species such
as for example dicots, such as Arabidopsis.



WE CLAIM:
1. A promoter capable of driving and/or regulating a constitutive expression,
comprising:
(a) a nucleic acid as given in SEQ ID NO 18 or the complement thereof; or
(b) a nucleic acid having at least 90% sequence identity with any of the DNA
sequences as given in SEQ ID NO 18; or
(c) a nucleic acid specifically hybridizing under stringent conditions with any of the
DNA sequences as given in SEQ ID NO 18; or
(d) a nucleic acid as defined in any one of (a) to (c), which is interrupted by an intervening sequence; or
(e) a fragment of any of the nucleic acids as defined in (a) to (d), which fragment is capable of driving and/or regulating expression.
2. A genetic construct comprising:
(a) a promoter as claimed in claim 1; and
(b) a heterologous nucleic acid sequence operably linked to said promoter of (a); and optionally
(c) a 3' transcription terminator.

3. An expression cassette comprising a genetic construct as claimed in claim 2.
4. A transformation vector comprising a genetic construct as claimed in claim 2.
5. An expression vector comprising a genetic construct as claimed in claim 2.

Documents:

3162-DELNP-2005-Abstract(21-1-2008).pdf

3162-DELNP-2005-Abstract-(19-09-2008).pdf

3162-delnp-2005-abstract.pdf

3162-DELNP-2005-Claims(21-1-2008).pdf

3162-DELNP-2005-Claims-(19-09-2008).pdf

3162-delnp-2005-claims.pdf

3162-delnp-2005-Correspondence Others-(26-04-2012).pdf

3162-DELNP-2005-Correspondence-Others(21-1-2008.pdf

3162-delnp-2005-correspondence-others.pdf

3162-delnp-2005-description (complete).pdf

3162-DELNP-2005-Drawings(21-1-2008).pdf

3162-delnp-2005-drawings.pdf

3162-DELNP-2005-Form-1(21-1-2008).pdf

3162-DELNP-2005-Form-1-(19-09-2008).pdf

3162-delnp-2005-form-1.pdf

3162-delnp-2005-form-18.pdf

3162-DELNP-2005-Form-2(21-1-2008).pdf

3162-DELNP-2005-Form-2-(19-09-2008).pdf

3162-delnp-2005-form-2.pdf

3162-DELNP-2005-Form-3(21-1-2008).pdf

3162-delnp-2005-form-3.pdf

3162-delnp-2005-form-5.pdf

3162-DELNP-2005-GPA(21-1-2008).pdf

3162-delnp-2005-GPA-(26-04-2012).pdf

3162-delnp-2005-gpa.pdf

3162-delnp-2005-pct-210.pdf

3162-delnp-2005-pct-237.pdf

3162-delnp-2005-pct-304.pdf

3162-delnp-2005-pct-306.pdf

3162-delnp-2005-pct-308.pdf

3162-delnp-2005-Petition Others-(26-04-2012).pdf

3162-DELNP-2005-Petition-137(21-1-2008).pdf

3162-DELNP-2005-Petition-138(21-1-2008).pdf


Patent Number 224187
Indian Patent Application Number 3162/DELNP/2005
PG Journal Number 44/2008
Publication Date 31-Oct-2008
Grant Date 03-Oct-2008
Date of Filing 18-Jul-2005
Name of Patentee CROPDESIGN N.V.
Applicant Address TECHNOLOGIEPARK 3, B-9052 ZWIJNAARDE, BELGIUM.
Inventors:
# Inventor's Name Inventor's Address
1 YVES HATZFELD 18C RUE DES DONDAINES, F-59000 LILLE, FRANCE.
2 WILLEM BROEKAERT KLUIZENBOSSTRAAT 26, B-1700 DILBEEK, BELGIUM.
PCT International Classification Number C12N 15/82
PCT International Application Number PCT/EP2004/050081
PCT International Filing date 2004-02-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA