Note: Descriptions are shown in the official language in which they were submitted.
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
REGULATION OF EMBRYONIC TRANSCRIPTION IN PLANTS
FIELD OF THE INVENTION
The invention is in the field of nucleic acid sequences capable of regulating
transcription, particularly sequences that may promote transcription during
embryogenesis in
plants.
BACKGROUND OF THE INVENTION
Most of the information about seed-specific gene expression comes from studies
of
genes encoding seed storage proteins like napin, a major protein in the seeds
of Brassica
napus, or conglycinin of soybean. Upstream DNA sequences directing strong
embryo-specific
expression of these storage proteins have been used successfully in transgenic
plants to
manipulate seed lipid composition and accumulation (Voelker et al.. 1996).
However.
expression of storage protein genes begins fairly late in embryogenesis. Thus.
promoters of
seed storage protein genes may not be ideal for all seed-specific
applications. For example,
storage oil accumulation commences significantly before the highest level of
expression of
either napin (Stalberg et al., 1996) or conglycinin (Chen et al., 1988) is
achieved. It is,
therefore of interest to identify other promoters which may modulate
expression of genes in
developing plant embryos.
A variety of transcriptional regulatory regions that may be active during
plant
embryogenesis are known, as disclosed for example in: U.S. Patent No.
5,792.922 issued 11
August 1998 to Moloney; U.S. Patent No. 5.623.067 issued 22 April 1997 to
Vandekerckhove
et al.: International Patent Publication W09845461 published 15 October 1998.
There remains
a need for alternative transcriptional regulatory regions.
15 FATTY ACID ELONGATION! (FAE1) genes encode condensing enzymes
involved in
plant very long chain fatty acid biosynthesis. The FAE1 condensing enzyme is
thought to be
localized in the endoplasmic reticulum where it catalyzes the sequential
elongation of C18
fatty acyl chains to C22 in length (Kunst et al., 1992). FAEI genes have been
cloned and
described recently by James et al. (1995), International Patent Publication WO
96/13582.
SUMMARY OF THE INVENTION
In one aspect, the invention provides transcriptional regulatory regions
derived from
FAEI genes. The transcriptional regulatory regions of the invention may be
useful in
- 1 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
promoting early seed-specific transcription of heteroloaous sequences to which
they are
operably linked. The transcriptional regulatory regions of the invention may
be used in a wide
variety of plants. including Brassica sp., Arabidopsis and other plant
species. DNA constructs
comprising the transcriptional regulatory sequences of the invention may be
active during
fatty acid or lipid biosynthesis in the plant embryo. Certain embodiments of
the constructs of
the invention may be used in transgenic plants to promote expression of
heterologous
sequences in developing seeds. In various embodiments, the constructs of the
invention may
be used to mediate gene expression that affects seed lipid metabolism, or seed
protein
composition or seed carbohydrate composition, or seed development. In
alternative
embodiments, the transcriptional regulatory regions of the invention may also
be useful for the
production of modified seeds containing novel recombinant proteins which have
pharmaceutical. industrial or nutritional value.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a 934 bp DNA sequence comprising the Arabidopsis thaliana FAE1
transcription regulatory sequence.
Figure 2 shows a 1588 bp DNA sequence comprising the Brassica napus FAE1
transcription regulatory sequence.
Figure 3 shows a 1069 bp DNA sequence comprising the Lunaria annua FAE1
transcription regulatory sequence.
Figure 4 shows an alignment of the Arabidopsis thaliana (A. t.), Lunaria annua
(L.a.)
and Brassica napus (B.n.) transcription regulatory sequences. Asterisks below
the sequences
indicate identical nucleotides in each of the three sequences. A number of
putative cis-acting
sequence motifs are identified in the A. thaliana sequence: an EMI ABA box at -
44bp to -
36bp having the sequence ACATCTCAT, for which the published consensus sequence
is
ACGTGTCAT (Rowley, D.L. and Herman. E.M. (1997), Biochimica et Biophysica Acta
1345:1-4); an A-300 box at -51bp to -46bp having the sequence TGCAAT, for
which the
published consensus sequence is TG(T/A/C)AAA(G/T) (Morton et al. (1994) in
Seed
Development and Germination (Kigel, J. and Gallili. G., eds.) pp. 103-138.
Marcel Dekker.
New York); G-box 1 at -105 to -100 bp having the sequence CACATG, for which is
the
consensus sequence is CACCTG, and G-box 2 at -164 to -159 bp having the
sequence
CAACTT, for which the consensus sequence is CAACTG (Kawogoe, Y. and Murai, N.
(1992) Plant J. 2:927-936; CE I element at -226 to -218 bp having the sequence
- 2 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
TTCCATCGA. for which the consensus sequence is TGCCACCGG. and a CE3 element at
-
381bp to -369 bp having the sequence ACACATTCCCTC, for which the consensus
sequence
is ACGCGTGTCCTC (Shen et al., (1996) Plant Cell 8:1107-1119). Not highlighted
is a
putative RY repeat motif at -53bp to -47bp having the sequence CATGCAA, for
which the
consensus sequence is CATGCAT (Dickinson et al. (1988) Nucleic Acid Res.
16:371;
Lelievre et al. (1992) Plant Physiol. 98:387-391). Also shown, as Con. 4, is a
consensus
sequence. wherein R=G or A, Y=T/U or C, M=A or C, K=G or T/U, S=G or C, W=A or
T/U.
B=G or C or T/U, D=A or G or T/U, H=A or C or T/U, V=A or G or C and N=A or G
or C or
T/U.
Figure 5 shows an alignment of the Arabidopsis thaliana (A.t.) and Lunaria
annua
(L.a.) transcription regulatory sequences. Asterisks below the sequences
indicate identical
nucleotides in each of the two sequences. The base at position -400 in the
A.t. sequence is
highlighted. The alignment of sequences in both Figure 4 and Figure 5 was
accomplished
using the CLUSTALW program (version 1.74) for multiple sequence alignments,
using a trap
open penalty of 15, a gap extension penalty of 6.66 and an IUB DNA weight
matrix. Also
shown, as Con. 5, is a consensus sequence, wherein R=G or A. Y=T/1J or C, M=A
or C, K=G
or T/U, S=G or C, W=A or T/U, B=G or C or T/U, D=A or G or T/U, H=A or C or
T/U, V=A
or G or C and N=A or G or C or T/U.
Figure 6 includes two bar graphs illustrating hydroxy fatty acid content of A)
FAE1-
FAH12 and B) napin-FAH12 transgenic seeds, expressed as percentage of total
seed fatty
acids. Figure 7 shows an alignment of the Brassica napus (B.n.) and Latnaria
annua (L.a.)
FEA1 transcription regulatory sequences. Asterisks below the sequences
indicate identical
nucleotides in each of the two sequences.
Figure 8 shows an alignment of the Brassica napus (B.n.) and Arabidopsis
thaliana
(A.I.) FEA1 transcription regulatory sequences. Asterisks below the sequences
indicate
identical nucleotides in each of the two sequences.
DETAILED DESCRIPTION OF THE INVENTION
The recombinant nucleic acid molecules of the invention may comprise a
heterologous
promoter sequence operably linked to a nucleic acid sequence, wherein the
promoter
sequence comprises a transcriptional regulatory region capable of mediating
seed-specific
expression in Arabidopsis. The transcriptional regulatory region may be
obtainable from a
- 3 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
plant FAE1 gene. Altemtively, The transcriptional regulatory region may
hybridize under
stringent conditions to a 5' region of the plant FAEI gene. In further
alternative
embodiments, The transcriptional regulatory region may be at least 70%
identical when
optimally aligned to the 5' region of the plant FAE1 gene.
In alternative embodiments, the invention provides isolated nucleic acids
comprising
the transcriptional regulatory regions of the invention. By isolated, it is
meant that the
isolated substance has been substantially separated or purified away from
other biological
components with which it would otherwise be associated, for example in vivo.
The term
'isolated' therefore includes substances purified by standard purification
methods, as well as
substances prepared by recombinant expression in a host, as well as chemically
synthesized
substances.
In the context of the present invention, "transcriptional regulatory region"
means a
nucleotide sequence capable of mediating or modulating transcription of a
nucleotide
sequence of interest, when the transcriptional regulatory region is operably
linked to the
sequence of interest. Conversely, a transcriptional regulatory region and a
sequence of interest
are "operably linked" when the sequences are functionally connected so as to
permit
transcription of the sequence of interest to be mediated or modulated by the
transcriptional
regulatory region. In some embodiments, to be operably linked, a
transcriptional regulatory
region may be located on the same strand as the sequence of interest. The
transcriptional
regulatory region may in some embodiments be located 5' of the sequence of
interest. In such
embodiments, the transcriptional regulatory region may be directly 5' of the
sequence of
interest or there may be intervening sequences between these regions. The
operable linkage of
the transcriptional regulatory region and the sequence of interest may require
appropriate
molecules (such as transcriptional activator proteins) to be bound to the
transcriptional
regulatory region, the invention therefore encompasses embodiments in which
such molecules
are provided, either in vitro or in vivo.
The term "recombinant" means that something has been recombined. so that when
made in reference to a nucleic acid molecule the term refers to a molecule
that is comprised of
nucleic acid sequences that are joined together by means of molecular
biological techniques.
The term "recombinant" when made in reference to a protein or a polypeptide
refers to a
protein molecule which is expressed using a recombinant nucleic acid molecule.
The term
"heterologous" when made in reference to a nucleic acid sequence refers to a
nucleotide
sequence which is ligated to, or is manipulated to become ligated to, a
nucleic acid sequence
- 4 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
to which it is not ligated in nature, or to which it is ligated at a different
location in nature. The
term "heterologous" therefore indicates that the nucleic acid molecule has
been manipulated
using genetic engineering, i.e. by human intervention.
Sequences may be derived or obtainable from plant FAE1 genes by deduction and
synthesis based upon the wild-type FAE1 gene sequences. Derived sequences may
be
identified in different organisms. for example by isolation using as probes
the nucleic acid
sequences of the invention. Alternative transcriptional regulatory regions may
be derived
through mutagenesis or substitution of wild-type sequences, such as the
sequence disclosed
herein. Derived nucleic acids of the invention may be obtained by chemical
synthesis,
isolation, or cloning from genomic DNAs using techniques known in the art,
such as the
Polymerase Chain Reaction (PCR). Consensus sequences, such as those
illustrated in Figures
4 and 5 are alternative embodiments of the nucleic acids of the invention,
derived from the
disclose wild-type FAE1 gene sequences. Nucleic acids of the present invention
may be used
to design alternative primers (probes) suitable for use as PCR primers to
amplify particular
regions of an FAE1 gene. Such PCR primers may for example comprise a sequence
of 15-20
consecutive nucleotides of the sequences of the invention. To enhance
amplification
specificity, primers of 20-30 nucleotides in length may also be used. Methods
and conditions
for PCR amplification are described in Innis et al. (1990); Sambrook et al.
(1989); and
Ausubel et al. (1995). As used herein, the term "probe" when made in reference
to an
oligonucleotide refers to an oligonucleotide which is capable of hybridizing
to another
oligonucleotide of interest. A probe may be single-stranded or double-
stranded. Probes are. for
example, useful in the detection, identification. amplification and isolation
of particular gene
sequences. Oligonucleotide probes may be labelled with a "reporter molecule."
so that the
probe is detectable using a detection system, such as enzymatic, fluorescent.
radioactive or
luminescent detection systems.
Derived nucleic acids of the invention may also be identified by
hybridization, such as
Southern or Northern analysis. Southern analysis is a method by which the
presence of DNA
sequences in a target nucleic acid mixture are identified by hybridization to
a labeled probe.
comprising an oligonucleotide or DNA fragment of a nucleic acid of the
invention. Probes for
Southern analysis may for example be at least 15 nucleotides in length.
Southern analysis
typically involves electrophoretic separation of DNA digests on agarose gels,
denaturation of
the DNA after electrophoretic separation, and transfer of the DNA to
nitrocellulose, nylon, or
another suitable membrane support for analysis with a radiolabeled,
biotinylated. or enzyme-
- 5 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
labeled probe as described in Sambrook et al. (1989). Similarly. Northern
analysis may be
used to identify RNAs that hybridize to a known probe such as an
oligonucleotide. DNA
fragment, cDNA or fragment thereof, or RNA fragment of a nucleic acid of the
invention or a
known FAE1 sequence. The probe may be labeled with a radioisotope such as 32P,
by
biotinylation or with an enzyme. The RNA to be analyzed may be
electrophoretically
separated on an agarose or polyacrylamide gel, transferred to nitrocellulose.
nylon, or other
suitable membrane, and hybridized with the probe, using standard techniques
well known in
the art such as described in Sambrook et al. (1989).
In alternative embodiments, a transcriptional regulatory region of the
invention may
be at least 70% identical when optimally aligned to the 5' region of a plant
FAEI gene, such
as the Arabidopsis FAE1 gene. In alternative embodiments, the degree of
identity may be
between 50% and 100%, such as 60%, 80%, 90%, 95% or 99%. When a position in
the
compared sequence is occupied by the same nucleotide or amino acid. following
optimal
alignment of the sequences. the molecules are considered to have identity at
that position. The
degree of identity between sequences is a function of the number of matching
positions shared
by the sequences. In terms of percentage, identity is the sum of identical
positions. divided by
the total length over which the sequences are aligned, multiplied by 100.
Various aspects of the present invention encompass nucleic acid or amino acid
sequences that are homologous to other sequences. As the term is used herein,
an amino acid
or nucleic acid sequence is "homologous" to another sequence if the two
sequences are
substantially identical and the functional activity of the sequences is
conserved (for example.
both sequences function as or encode a FAE1 enzyme: as used herein, the term
'homologous'
does not infer evolutionary relatedness). Nucleic acid sequences may also be
homologous if
they encode substantially identical amino acid sequences. even if the nucleic
acid sequences
are not themselves substantially identical, a circumstance that may for
example arise as a
result of the degeneracy of the genetic code.
Two amino acid or nucleic acid sequences are considered substantially
identical if.
when optimally aligned (with gaps permitted), they share at least about 50%
sequence
similarity or identity, or if the sequences share defined functional motifs.
In alternative
embodiments, sequence similarity in optimally aligned substantially identical
sequences may
be at least 60%, 70%, 80%, 90% or 95%. As used herein, a given percentage of
homology
between sequences denotes the degree of sequence identity in optimally aligned
sequences.
- 6 -
CA 02345028 2009-06-26
Optimal alignment of sequences for comparisons of similarity may be automated
using a variety of algorithms, such as the local homology algorithm of Smith
and Waterman
(1981) Adv. AppL Math 2: 482, the homology alignment algorithm of Needleman
and
Wunsch (1970) J. MoL Biol. 48: 443, the search for similarity method of
Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized
implementations
of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, Madison, WI, U. S. A.).
Sequence
similarity may also be determined using the BLAST algorithm, described in
Altschul et al.
(1990), J. Mol. Biol. 215: 403-10 (using the published default settings). The
BLAST
algorithm involves first identifying high scoring sequence pairs (HSPs) by
identifying short
words of length W in the query sequence that either match or satisfy some
positive valued
threshold score T when aligned with a word of the same length in a database
(reference)
sequence. T is referred to as the neighborhood word score threshold. Initial
neighborhood
word hits act as seeds for initiating searches to find longer HSPs. The word
hits are extended
in both directions along each sequence for as far as the cumulative alignment
score can be
increased. Extension of the word hits in each direction is halted when the
following
parameters are met: the cumulative alignment score falls off by the quantity X
from its
maximum achieved value ; the cumulative score goes to zero or below, due to
the
accumulation of one or more negative-scoring residue alionments : or the end
of either
sequence is reached. The BLAST algorithm parameters W. T and X determine the
sensitivity
and speed of the alignment. The BLAST program may use as defaults a word
length (W) of
11, the BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl.
Acad. Sci.
USA 89: 10915-10919), a gap existence cost of 11, a per residue gap cost of 1,
a lambda ratio
of 0.85, alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a
comparison of both
strands. One measure of the statistical similarity between two sequences using
the BLAST
algorithm is the smallest sum probability (P(N)), which provides an indication
of the
probability by which a match between two nucleotide or amino acid sequences
would occur
by chance. In alternative embodiments of the invention, nucleotide or amino
acid sequences
are considered substantially identical if the smallest sum probability in a
comparison of the
test sequences is less than about 1, preferably less than about 0.1, more
preferably less than
- 7 -
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
about 0.01, and most preferably less than about 0.001. In the PSI-BLAST
implementation of
the BLAST algorithm, an expect value for inclusion in PSI-BLAST iteration may
be 0.001
(Altschul et al. (1997), Nucleic Acids Res. 25:3389-3402). Searching
parameters may be
varied to obtain potentially homologous sequences from database searches.
An alternative indication that two nucleic acid sequences are substantially
identical is
that the two sequences hybridize to each other under moderately stringent, or
preferably
stringent, conditions. Hybridization to filter-bound sequences under
moderately stringent
conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl
sulfate
(SDS), 1 mM EDTA at 65EC, and washing in 0.2 x SSC/0.1% SDS at 42EC (see
Ausubel. et
al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green
Publishing Associates.
Inc., and John Wiley & Sons. Inc., New York, at p. 2.10.3). Alternatively,
hybridization to
filter-bound sequences under stringent conditions may, for example, be
performed in 0.5 M
NaHPO4, 7% SDS, 1 niM EDTA at 65EC, and washing in 0.1 x SSC/0.1% SDS at 68EC
(see
Ausubel. et al. (eds), 1989, supra). Hybridization conditions may be modified
in accordance
with known methods depending on the sequence of interest (see Tijssen. 1993,
Laboratory
Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic
Acid
Probes, Part I, Chapter 2 "Overview of principles of hybridization and the
strategy of nucleic
acid probe assays", Elsevier, New York). Generally, stringent conditions are
selected to be
about SEC lower than the thermal melting point for the specific sequence at a
defined ionic
strength and pH.
A FAEI promoter is any naturally occurring transcriptional regulatory region
that
mediates or modulates the expression of a plant FAE1 condensing enzyme. Plant
FAE1
condensing enzymes are proteins that are homologous to known FAE1 condensing
enzymes.
such as those cloned and described in International Patent Publication WO
96/13582.
Heterologous DNA sequences may for example be introduced into a host cell by
transformation. Such heterologous molecules may include sequences derived from
the host
cell species. which have been isolated and reintroduced into cells of the host
species.
Heterologous nucleic acid sequences may become integrated into a host cell
genome. either as
a result of the original transformation of the host cells, or as the result of
subsequent
recombination events. Transformation techniques that may be employed include
plant cell
membrane disruption by electroporation, microinjection and polyethylene glycol
based
transformation (such as are disclosed in Paszkowski etal. EMBO J. 3:2717
(1984); Fromm et
at., Proc. Natl. Acad. Sci. USA 82:5824 (1985); Rogers et at., Methods
Enzymol. 118:627
- 8
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
(1986); and in U.S. Patent Nos. 4,684,611; 4,801,540; 4,743,548 and
5.231.019), biolistic
transformation such as DNA particle bombardment (for example as disclosed in
Klein, et al.,
Nature 327: 70 (1987); Gordon-Kamm, etal. "The Plant Cell" 2:603 (1990); and
in U.S.
Patent Nos. 4,945,050; 5,015,580; 5,149,655 and 5,466,587); Agrobacterium-
mediated
transformation methods (such as those disclosed in Horsch etal. Science 233:
496 (1984);
Fraley et aL, Proc. Nat'l Acad. ScL USA 80:4803 (1983); and U.S. Patent Nos.
4,940,838 and
5,464,763).
Standard methods are available for the preparation of constructs for use in
identifying
and characterizing transcriptional regulatory regions useful in various
embodiments of the
invention. General molecular techniques may for example be performed by
procedures
generally described by Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG,
Smith
JA, Stuhl K. (1995) Current Protocols in Molecular Biology, Vols 1, 2 and 3.
Alternative
equivalent methods or variations thereof may be used in accordance with the
general
knowledge of those skilled in this art and the functional requirements of the
present invention.
In some aspects of the invention, transformed plant cells may be cultured to
regenerate
whole plants having a transformed genotype and displaying a desired phenotype,
as for
example modified by the expression of a heterologous protein mediated by a
transcriptional
regulatory region of the invention. A variety of plant culture techniques may
be used to
regenerate whole plants, such as are described in Gamborg and Phillips, "Plant
Cell. Tissue
and Organ Culture, Fundamental Methods", Springer Berlin, 1995); Evans et aL
"Protoplasts
Isolation and Culture". Handbook of Plant Cell Culture, Macmillian Publishing
Company,
New York. 1983: or Binding, 'Regeneration of Plants. Plant Protoplasts". CRC
Press. Boca
Raton. 1985: or in Klee et al., Ann. Rev. of Plant Phys. 38:467 (1987). A
cell. tissue. organ, or
organism into which has been introduced a foreign nucleic acid, is considered -
transformed-.
-transfected". or -transgenic". A transgenic or transformed cell or organism
also includes
progeny of the cell or organism and progeny produced from a breeding program
employing a
transgenic plant as a parent in a cross and exhibiting an altered phenotype
resulting from the
presence of a recombinant nucleic acid construct. A transgenic plant is
therefore a plant that
has been transformed with a heterologous nucleic acid, or the progeny of such
a plant that
includes the transgene. The invention provides vectors, such as vectors for
transforming plants
or plant cells. The term "vector" in reference to nucleic acid molecule
generally refers to a
molecule that may be used to transfer a nucleic acid segment(s) from one cell
to another. One
of skill will recognize that after the nucleic acid is stably incorporated in
transgenic plants and
- 9 -
CA 02345028 2001-03-30
WO 01/11061 PCT/CA00/00907
confirmed to be operable, it can be introduced into other plants by sexual
crossing. Any of a
number of standard breeding techniques may be used, depending upon the species
to be
crossed.
In various embodiments, the invention comprises plants transformed with the
nucleic
acids of the invention. In some embodiments, such plants will exhibit altered
fatty acid
content in one or more tissues. These aspects of the invention relate to all
higher plants,
including monocots and dicots, such as species from the genera Fragaria.
Lotus, Medicago,
Onobrychis, Triforium, Trigonelia, Wgna, Citrus, Linum. Geranium, Manihot,
Caucus,
Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Hyoscyamus.
Lycopersicon,
Nicotiana, Solanum. Petunia, Digitalis, Majorana. Cichorium, Helianthus,
Lactuca, Bromus,
Asparagus, Antirrhinum, Heterocatlis, Nemesia, Pelargonium, Panicum,
Penniserum.
Ranunculus, Senecio, Salpiglossis. Cucarnis, Browallia, Glycine, Lolium., Zea,
Triticum,
Sorghum. and Datura. Such plants may include maize, wheat. rice, barley.
soybean. beans.
rapeseed. canola. alfalfa, flax, sunflower, cotton, clover, lettuce. tomato
cucurbits. potato
carrot, radish, pea lentils, cabbage, broccoli, brussei sprouts. peppers.
apple, pear, peach.
apricot, carnations and roses. More specifically, in alternative embodiments,
plants for which
the invention may be used in modifying fatty acid content include oil crops of
the Cruciferae
family: canola, rapeseed (Brassica spp.), crambe (Crambe spp.), honesty
(Lunaria spp.)
lesquerella (Lesquerela spp.), and others; the Composirae family: sunflower
(Helianthus
spp.), safflower (Carthamus spp.), niger (Guizotia spp.) and others; the
Palmae family: palm
(Elaeis spp.), coconut (Cocos spp.) and others; the Leguminosae family: peanut
(Arachis
spp.), soybean (Glycine spp.) and others: and plants of other families such as
maize (Zea spp.).
cotton (Gossvpiun sp.), jojoba (Simonasia sp.), flax (Linum sp.). sesame
(Sesamum spp.),
castor bean (Ricinus spp.), olive (Olea spp.), poppy (Papaver spp.), spurge
(Euphorbia. spp.),
meadowfoam (Limnanthes spp.), mustard (Sinapis spp.) and cuphea (Cuphea spp.).
Nucleic acids of the invention may also be used as a plant breeding tool. as
molecular
markers to aid in plant breeding programs. Such techniques would include using
the gene
itself as a molecular probe or using the DNA sequence to design PCR primers to
use PCR
based screening techniques in plant breeding programs.
Deletion or insertion constructs may be useful for domain mapping to determine
the
functional domains or motifs of a transcriptional regulatory region derived
from a FAE1 gene.
An aspect of the invention is the construction and testing of such constructs,
as described
below for the 5' deletion construct of the A. thaliana FAEI 5' region. One
aspect of the
-10-
CA 02345028 2001-03-30
WO 01/11061 PCT/CA00/00907
invention comprises transcriptional regulatory regions that are derived from
functionally
important regions of a FAEI promoter. As outlined above, the functionally
important regions
of a FAEI promoter may be determined through routine assays. Alternatively,
randomly
selected portions of a a FAEI promoter may be selected for use in routine
assays to determine
whether the selected region is capable of functioning as a transcriptional
regulatory region in
the context of the present invention. In various embodiments, regions of the
Arabidopsis
thaliana. Brassica napus or Lunaria annua promoters may be used. For example,
the
following motifs in the A. t. FAEI promoter may be used alone or in
combination in novel
transcriptional regulatory regions (see Figure 4): the CE-like elements (CEI
and CE3), the RY
repeat motif, the G-boxes (G-boxl and G-box2), the A-300 box, the EMI ABA box,
or the
CTA IIIIG element. Constructs of the invention comprising such motifs,
deletions or
insertions may be assayed for activity as transcriptional regulatory regions
of the invention by
testing for strong seed-specific activity providing expression of a sequence
of interest (such as
a reporter sequence) before the torpedo stage and persisting throughout embryo
development.
in accordance with standard testing methods that may be adapted from the
methods disclosed
herein.
Alternative embodiments of the transcriptional regulatory regions of the
invention may
be identified using information available through NCBI databases at
http://www.ncbi.nih.gov.
In various embodiments, transcriptional regulatory regions derived from plant
FAEI
genes are shown to be capable of directing expression of desired genes at an
early stage of
development in a seed-specific manner in disparate plant species. In
particular embodiments.
the transcriptional regulatory regions of the invention may be used in a wide
variety of
dicotyledonous plants for modification of the seed phenotype. For example. new
seed
phenotypes may include:
(1) altered seed fatty acid composition or seed oil composition and
accumulation
(2) altered seed protein or carbohydrate composition or accumulation
(3) enhanced production of desirable endogenous seed products
(4) suppression of production of undesirable gene products using antisense. co-
suppression or
ribozyme technologies
(5) production of novel recombinant proteins for pharmaceutical. industrial or
nutritional
purposes
Isolation of a seed-specific promoter from A. thaliana
-11-
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
Using the sequence information of the A. thaliana genome sequencing project.
synthetic oligonucletide primers were designed to amplify the FAEI 5'
untranslated region, to
isolate it by PCR. As shown in Figure 1, the upstream primer 5%
CTAGTAGATTGGTTGGTTGGTTTCC-3' (AtproFW) in combination with the downstream
primer 5'-TGCTCTGTTTGTGTCGGAAAATAATGG-3' (AtproRV) were used, and resulted
in the synthesis of a fragment of the correct size (934 bp). The amplified
product was
subcloned in the Hindi site of the plasmid pT7T3-18U (Pharmacia) to produce
plasmid
pT7T3-18U/proFAE900, followed by complete sequence determination of both
strands to
verify the fragment identity. A BLAST search of the A. thaliana Database
identified a single
BAC clone T4L20 (GenBank ATFIOM6) 125,179 bp long, which contains the complete
FAE1 gene.
Functional analysis of the F.4E1 5' upstream region
5' upstream fragments of the FAEI gene were shown to confer seed-specific and
temporally regulated gene expression in plants. A translational fusion was
made between the
FAEI 5' region and the coding region of the reporter gene 13-glucuronidase
(GUS). The
chimeric gene (pFAE900-GUS or pFAE400-GUS) was transferred into Arabidopsis
and
tobacco and GUS activity was monitored in various tissue of transgenic plants.
Construction of the vectors pFAE900-GUS and pFAE400-GUS, and transformation of
Arabidopsis and tobacco, was as follows. The insert was cleaved out of pT7T3-
18U vector
with HindIII and Xbal and directionally subcloned into the corresponding sites
of the binary
Ti plasmid pBI101 (Clontech), which contains a promoterless GUS gene
(Jefferson et al.
1987), to obtain the vector pFAE900-GUS. Another construct. pFAE400-GUS.
containing
only 393 bp of the 5 FAEI region directly upstream of the ATG initiation codon
fused to the
GUS coding sequence was also generated. For that. the pT7T3-18U/proFAE900
vector was
digested with BglII and Psd, the sticky ends were filled in using T4 DNA
polymerase.
followed by re-ligation to obtain pT7T3-18U/proFAE400. The 393 bp 5" FAE1
upstream
fragment was then excised with HindIII and Xbal and cloned into the binary
vector pBI101 to
obtain the plasmid pFAE400-GUS. The pFAE400-GUS and pFAE900-GUS fusion
constructs
in pBI101 were introduced into Agrobacterium tumefaciens strain GV3101 (Koncz
and
Schell. 1986) by heat-shock and selected for resistance to kanamycin (50
ig/m1). A. thaliana
(L.) Heynh. ecotype Columbia was transformed with the pFAE400-GUS and pFAE900-
GUS
constructs using floral dip method (Clough and Bent, 1998). Screening for
transformed seed
- 12 -
CA 02345028 2009-06-26
was done on 501.tg/mL kanamycin as described previously (Katavie et al.,
1994).
Approximately 100 transgenic lines were generated for each construct.
For transformation of tobacco, A. tumefaciens harbouring the pFAE900-GUS
construct was co-cultivated with leaf pieces of Nicotiana tabacum SRI and
transformants
were selected with kanamycin (100)tg/mL) on solid medium (Lee and Douglas,
1996).
Histochemical localization of GUS activity and analysis of transgenic plants
was as
follows. Tissue sections were placed in 100 mM NaPO4 (p1-17) and 1 mM
spermidine for 15
min, then incubated at 37 C in 0.5 K3 [Fe(CN)6], 0.01 % Triton X100TM, 1mM
EDTA, 10
mM 13-mercaptoethanol, 5-bromo-4-chloro-3-indoly1-13-D-glucuronide in 100 mM
NaPO4
(pH7), until a blue color appeared (after approximately 1 hr). Following
incubation with the
substrate, chlorophyll was removed from the sections using a graded ethanol
series.
Using this assay, five independent transgenic Arabidopsis lines were examined
for the
embryo-specific expression of the GUS gene. In addition, leaf, stem and
siliques were
histochemically stained for p-glucuronidase activity. The results indicate
that the reporter
gene fused to the transcriptional regulatory region of the invention is not
expressed in
vegetative tissues, whereas it is highly expressed in developing seeds
(embryos). Both the
934 bp and the 393 bp transcriptional regulatory regions derived from the Al.
FAEI gene
caused the appearance of GUS activity by the torpedo stage embryo (6 days
after flowering).
GUS activity in all five lines persisted throughout subsequent embryo
development.
Leaves, stems, pods and seeds of three regenerated tobacco lines transformed
with the
pFAE900-GUS construct were also assayed for p-glucuronidase activity. The
results
obtained indicate that the 934 bp FAE1 promoter fragment contains sufficient
information to
direct seed-specific expression of a reporter gene in transgenic tobacco. Thus
the
transcriptional regulatory regions of the invention may be used for seed-
specific expression
of foreign genes in transgenic plants.
The in vivo activity of a FAEI promoter of the invention was compared to the
activity
of the napin promoter by expressing the castor bean hydroxylase gene FAHI2
(Broun and
Somerville. 1997) behind either the FAE/-promoter (a transcriptional
regulatory region of
approximately 1 kb) or the napin promoter in an Arabidopsis fad2/fael double
mutant. This
mutant accumulates as a proportion of fatty acids about 85% of the 18:1 acyl
group, which is
the substrate for the hydroxylase. The levels of hydroxylated fatty acids
accumulating in a
large number of independent transgenic lines were used to estimate the
relative strength of
- -
CA 02345028 2001-03-30
WO 01/11061 PCT/CA00/00907
each promoter. As shown in Figure 6, the two populations of transgenic plants
accumulated
levels of hydroxylated fatty acids, ranging from 0.2% to about 11-12% of total
fatty acids,
with the levels being on average slightly higher in FAE1-FAH12 lines.
Similarly, the best
FAEl-FAH12 plant accumulated just over 12% of hydroxylated fatty acids (w/w of
total FAs),
whereas the best napin-FAH12 plant produced 10.8% of hydroxylated fatty acids
(w/w of total
FAs). These results indicate that the FAE1 promoter is highly active in
transgenic .4rabidopsis
and that its in vivo activity may be superior to napin in Arabidopsis seeds.
Sequence elements or motifs that confer both tissue specificity and
developmental
regulation of transcription reside within 393 bp of the AUG translation
initiation codon in the
At. FAE1 gene. The seed-specific expression conferred by the transcriptional
regulatory
regions of the invention is independent of the native terminator of the FAE1
gene 3' end. For
example, in the exemplified constructs disclosed herein, a terminator flerived
from the
Agrobacterium nopaline synthase gene was used.
Lunaria annua and Brassica napus F.4EI 5'regulatorv regions
Two sequences originating from B. napus and L. annua were isolated and
characterized to demonstrate that regulatory regions conferring seed-specific
transcription
early in embryo development can also be found upstream of other plant F.4E1
genes.
Sequences were cloned using the technique of polymerase chain reaction (PCR)
walking on
uncloned plant genomic DNA (Devic et al., 1997). Approximately 5 g of genomic
DNA
from 1 g of fresh tissue was used for the construction of 5 different
libraries by digesting DNA
with a series of enzymes that produce blunt end fragments to which special
adaptors are
ligated. The adaptor molecules consist of a long upper strand, which contains
successive
sequences common to the adaptor primers. AP 1 and AP2. annealed at its 3' end
to a shorter
strand lacking the API sequence. However, this short strand posseses an amine
group at its 3'
end to prevent filling in by the DNA polymerses during the first PCR
amplification step and
generation of the AP 1 binding site. This suppression PCR effect prevents
exponential
amplification of molecules containing the adaptor at each end, and the adaptor
primer binding
sites are only produced when a strand complementary to the upper strand of the
adaptor is
synthesized by extension from a gene specific primer. The first PCR reaction
is performed
using an adaptor primer API and a gene specific primer. An aliquot of the
first PCR product is
used a template in a second PCR amplification using the nested gene specific
primer and AP2.
In order to isolate the regulatory regions upstream of the B. napus F.4E1
coding
sequence. genomic DNA was prepared from developing leaves and digested with 5
blunt-end
-14-
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
cutting restriction enzymes (DraL EcoRV, Hpal, PITH and Scal) to generate a
series of DNA
libraries. After ligation of adapter molecules, individual libraries were used
as templates in a
two step PCR. In the first PCR amplification using the API primer 5'-
GGATCCTAATACGACTCACTATAGGGC-3' and the FAEI gene specific primer 5'-
AAAGAGTGGAGCGATGGTTATGAGG-3' (Bnwalk 1 ), multiple DNA fragments were
amplified from all five library templates. After a second round of PCR, using
the AP2 primer
5'-CTATAGGGCTCGAGCGGC-3' and the nested FAEI specific primer 5' -
CGGAAAGAAGCAAAGGTTGAAAAGG-3' (Bnwalk2), the longest single fragment of 1.6
kb was obtained from the Hpal library template. This fragment was inserted
into the pCR2.1
plasmid (Invitrogen) and sequenced. The sequence is shown in Figure 2.
For the PCR walking experiment to isolate the L. annua 5' regulatory region,
in
adition to the standard API and AP2 primers, the following FAEI specific
primers were used:
5'-GATCGTTTGTGGTAAGACGAGAGC-3' (Lawalkl) and
5.-
GTCAGTGGGAAGAAACAGAGGTTG-3' (Lawalk2). In the first PCR reaction. the DraI,
EcoRr Pvull, Scal and Ssp/ library templates were used. In a second PCR
amplification the
longest single fragment 1.1 kb in length was synthesized using the EcoRV
library template.
This fragment was inserted into the Hindi site of the pT7T3-18U vector
(Promega),
sequenced on both strands and analyzed (Figure 3).
Using the sequence data obtained for the 5' regulatory regions generated by
PCR
walking, specific primers were generated for the amplification of the L. annua
and B. napus
FAEI promoter fragments. For the PCR-amplification of B. napus promoter
fragment the
upstream primer was 5' -CTGACTTCACCAAAGAAACAACTCG-3' (BnproFW) in
combination with the downstream primer 5.-CGGAATTCCGTTTTTTTTTTTAGGCG-3'
(BnproRV). The synthesized fragment was ligated into the Smal site of pGEM-7Zf
(Promega), then excised with XbaI/BamHI and cloned into the equivalent sites
of the pBI101
binary vector (Clontech). L. annua 5' regulatory region was amplified using
the 5%
CAGCTTAACCGGTAAAATTGGCC-3' (LaproFW) upstream primer together with the 5.-
TGTTCAGTTTTGTGTCGGAGAGG-3' (LaproRV) downstream primer and inserted in the
HincH site of pT7T3-18U (Promega) plasmid. In order to clone the L. annua
promoter
fragment into the pBI101 binary vector, an Xbal site was added by subcloning
the Pstl/Kpnl
fragment released from the pT7T3-18U vector into pBluescript II KS+
(Stratagene). The
fragment was then excised and cloned in the Xbal site of the pBI101 vector.
- 15 -
CA 02345028 2009-06-26
The resulting vectors pBnFAEI-GUS and pLaFAEI-GUS in pB1101 were then
introduced into A. turnefaciens strain GV3101 by heat-shock, and used to
transform
Arabidopsis as described above. Transformants were selected on agar-solidified
medium
containing kanamycin (50 gg/ml). More than 100 transformants were generated
for each
construct. The activity of the L. annua and B. napus FAEI promoters was
determined by
GUS expression assays on the developing seeds and also on non-reproductive
plant tissues
as controls. Consistent seed-specific GUS expression was obtained for both
promoter
constructs in independent transgenic lines. In contrast, there was no
detectable GUS activity
in leaf, stem and silique samples.
References
Ausubel FM. Brent R, Kingston RE, Moore DD. Seidman JG. Smith JA. Stuhl
K.(1995) Current Protocols in Molecular Biology, Vols 1,2 and 3.
244,174- 181.Benfey, P. N. and Chua, N.-H. (1989) Regulated genes in
transgenic plants. Science
Bevan, M. Shufflebottom, D., Edwards, K., Jefferson, R. and Schuch. W. (1989)
Tissue-and cell-specific activity of a phenylalanine ammonia-lyase promoter in
transgenic
plants. EMBO. J. 8,1899-1906.
Broun, P. and Somerville, C. (1997) Accumulation of ricinoleic. lesquerolic.
and
densipolic acids in seeds of transgenic Arabidopsis plants that express a
fatty acyl
hydroxylase cDNA from castor bean. Plant Physiol. 113,933-942.
Chen. Z. L., Pan, N. S., and Beachy, R. N. (1988) A DNA sequence element that
confers seed-specific enhancement to a constitutive promoter. EMBO J 6: 3559-
3564.
Clough, S. J. and Bent, A. F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation ofArabdiopsis thaliana. Plant J. 16: 735-
743.
Devic, M., Albert, S., Delseny, M., and Roscoe. T. J. (1997) Efficient PCR
walking
on plant genomic DNA. Plant Physiol. Biochem. 35: 331-339.
James. D. W.. Jr., Lim, E., Keller, J., Plooy, I., Ralston, E., and Dooner. H.
K. (1995)
Directed tagging of the Arabidopsis FATTY ACID LONGATION (FAEI) gene with the
maize
transposon Activator. Plant Cell 7 : 309-319.
-16-
CA 02345028 2001-03-30
WO 01/11061
PCT/CA00/00907
Jefferson, R.A., Kavanaugh, T. and Bevan, M.W. (1987) GUS fusions: 13-
glucuronidase as a sensitive and versatile gene fusion marker system in higher
plants. EMBO
J. 6: 3901-3907.
Katavic, V., Haughn, G.W., Reed, D., Martin, M., and Kunst, L. (1994) In
planta
transformation of Arabidopsis thaliana. Mol.Gen.Genet. 245: 363-370.
Koacz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5 controls the
tissue-
specific expression of chimaeric genes carried by a novel type of
Agrobacterium binary
vector. Mol. Gen. Genet. 204: 383-396.
Kunst, L., Taylor, D.C.. and Underhill, E.W. (1992) Fatty acid elongation in
developing seeds of Arabidopsis thaliana. Plant PhysioL Biochem. 30: 425-434.
Lee D., and Douglas C.J. (1996) Manipulation of plant gene expression using
antisense RNA. In: Plant Biochemistry/Molecular Biology LaboratoryManual, pp.
423-439.
Dashek. W.V. , ed., CRC Press. Inc.. Boca Raton.
Murphy, D.J., Cummins. I., and Ryan, A.J. (1989) Immunocrochemical and
biochemical study of the biosynthesis and mobilisation of the major seed
storage proteins of
Brassica napus. Plant Physiol. Biochem. 27, 647-657.
Stalberg, K., Ellerstoem, M., Ezcurra, I., Ablov, S. , and Rask, L. (1996)
Disruption of
an overlapping e-box-ABRE motif abolished high transcription of the napA
storage-protein
promoter in transgenic Brassica napus seeds. Planta 199: 515-519.
Voelker, T.A., Hayes, T.R., Cranrner, A.M.. Turner. J.C., and Davies H.M.
(1996)
Genetic engineering of a quantitative trait: Metabolic and genetic parameters
influencing the
accumulation of laurate in rapeseed. Plant 9: 229-241.
- 17-
