Note: Descriptions are shown in the official language in which they were submitted.
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USE OF BIFUNCTIONAL a-AMYLASE SUBTILISIN INHIBITOR PROMOTER SEQUENCE OF BARLEY
TO CONFER
EXPRESSION IN SEEDS
FIELD OF THE 1NVENT10N
The present invention relates generally to genetic sequences which confer
expression
in plant seeds andlor tissues or organs of a plant. In particular, the present
invention
provides a promoter sequence that is capable of conferring expression on a
genetic
sequence to which it is operably connected in the starchy endosperm cells of
an
immature seed andlor the aleurone cells of a mature seed and/or in the
scutellum
and/or in response to the phytohormone abscisic acid. Alternatively or in
addition, the
promoter sequence of the present invention is capable of being repressed or
otherwise
down-regulated in response to the phytohormone gibberellic acid {GA). The
invention
further encompasses transgenic plants carrying genetic constructs expressing a
structural gene, such as a structural gene which encodes a cytotoxin,
antisense,
ribozyme, abzyme, co-suppression, reporter molecule, polypeptide hormone or
other
polypeptide, placed operabiy under the control of the inventive promoter
sequence.
The present invention is particularly useful for expressing desirable
structural genes
in the seeds of plants and in particular, in the starchy endosperm andlor
mature
aleurone cells of plants (collectively referred to as "endosperm"). The
present invention
is further useful in a wide range of applications involving the expression of
desirable
genes in seeds, including the production of seeds having modified protein and
amino
acid composition, modified flour quality for breads, noodles and pasta,
amongst others,
modified malting and brewing characteristics of grains and reduced propensity
of
grains for pre-harvest sprouting.
GENERAL
Those skilled in the art will be aware that the invention described herein is
susceptible
to variations and modifications other than those specifically described. It is
to be
understood that the invention described herein includes all such variations
and
modifications. The invention also includes al! such steps, features,
compositions and
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compounds referred to or indicated in this specification, individually or
collectively, and
any and al! combinations of any two or more of said steps or features.
Throughout this specification, unless the context requires otherwise the word
"comprise", and variations such as "comprises" and "comprising", will be
understood
to imply the inclusion of a stated integer or step or group of integers or
steps but not
the exclusion of any other integer or step or group of integers or steps.
Bibliographic details of the publications referred to by author in this
specification are
collected at the end of the description.
This specification contains nucleotide and amino acid sequence information
(referenced herein by the prefix "SEQ ID NO:<400>"), prepared using the
programme
Patentln Version 2Ø The Sequence Listing is presented herein after the
bibliography.
1S Each nucleotide or amino acid sequence is identified in the Sequence
Listing by the
numeric indicator <210> followed by the sequence identifier (e.g. <210>1,
<210>2,
etc). The length, type of sequence (DNA, protein (PRT), etc) and source
organism for
each nucleotide or amino acrd sequence are indicated by information provided
in the
numeric indicator fields <211 >, <212> and <213>, respectively. Nucleotide and
amino
acid sequences referred to in the specification are defrned by the information
provided
in numeric indicator field <400> followed by the sequence identifier (eg.
<400>1,
<400>2, etc).
The designation of nucleotide residues referred to herein are those
recommended by
the iUPAC-IUB Biochemical Nomenclature Commission, wherein A represents
Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y
represents a pyrimidine residue, R represents a purine residue, M represents
Adenine
or Cytosine, K represents Guanine or Thymine, S represents Guanine or
Cytosine, W
represents Adenine or Thymine, H represents a nucleotide other than Guanine, B
represents a nucleotide other than Adenine, V represents a nucleotide other
than
Thymine, D represents a nucleotide other than Cytosine and N represents any
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nucleotide residue.
Amino acid designations
referred to herein
are listed in Table
1.
TABLE 1
Amino Acid Three-letter One-letter
Abbreviation Symbol
Aianine Ala A
10Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
15Glutamic acid Glu E
Glycirie Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
20Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
25Threonine Thr T
Tryptophan Trp
W
Tyrosine Tyr Y
Valine Val V
Any amino acid as aboveXaa X
30
As used herein, the term "derived from" shall be taken to indicate that a
particular
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integer or group of integers has originated from the species specified, but
has nat
necessarily been obtained directly from the specified source.
BACKGROUND TO THE INVENTION
A major problem in the genetic improvement of agriculturally-important crops,
in
particular those crops producing metabolites (proteins, amino acids, starches
and
secondary metabolites, amongst others), is the manipulation of gene expression
to
produce plants which exhibit novel characteristics. More particularly, the
expression
of novel characteristics is often required to be effected in specific cell
types, tissues or
organs of the plant, or under specific environmental or developments!
conditions.
Advances in biotechnological research have produced an explosion of
information in
relation to the number of genetic sequences identifed which, if appropriately
expressed, are useful to produce improved crop plants, for example plants in
which
reproductive development is controlled, plants having altered shape or size
characteristics, plants capable of rapid regeneration following harvest, or
plants having
improved resistance to pathogens, amongst others.
However, the application of biotechnology to the production of plants
expressing novel
traits is limited by the availability of genetic sequences which are capable
of conferring
appropriate expression patterns upon structural genes. Clearly, in the absence
of
appropriate regulatory sequences to confer expression in a particular cell
type at a
particular stage of development and/or in response to specific environments!
and
hormonal stimuli, the potential of genetic sequences which encode novel
proteins to
express those proteins in plants cannot be realised.
SUMMARY OF THE INVENTION
in work leading up to the present invention the inventors sought to isolate
useful
regulatory sequences which were capable of conferring expression on structural
gene
sequences to which they are operably connected in the seeds of plants in a
developmentally-regulated and/or hormonally-regulated manner. The inventors
isolated
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the bifunctional a-amylase subtilisin inhibitor (BAS)) gene promoter sequence
from a
monocotyledonous plant species and demonstrated that this promoter sequence is
at
least capable of conferring expression on a structural gene sequence in seeds,
in
particular in the endosperm cells of immature seeds andlor the aleurone cells
of
mature seeds andlor in the scutellum.
Accordingly, one aspect of the present invention provides an isolated promoter
sequence derived from a plant cell which is at least capable of conferring,
increasing
or otherwise facilitating the expression of a structural gene in the seeds of
a plant,
wherein said isolated promoter sequence comprises a sequence of nucleotides
which
is at least about 20% identical to the nucleotide sequence set forth in SEQ ID
NO:
<400> 1 or a complementary nucleotide sequence thereto.
Preferably, the promoter sequence of the invention is at least capable of
conferring,
increasing or otherwise facilitating the expression of a structural gene in
the
endosperm and/or aleurone and/or scutellum of a plant, or one or more cells
thereof.
Those skilled in the art will be aware that expression of the BAS~I gene is
also
regulated by the phytohormones abscisic acid (ABA) and giberrellic acid (GA),
Accordingly, an alternative embodiment of the present invention provides an
isolated
BAS/ gene promoter sequence which is capable of regulating or otherwise
modulating
the expression of a structural gene in the seeds of a plant and preferably, in
the
endosperm and/or aleurone and/or scutelium cells of said seeds, in response to
ABA
and/or GA.
The promoter sequence of the invention preferably includes one or more cis-
acting
regulatory sequences selected from the list comprising gibberellic acid
responsive
element (CARE), abscisic acid responsive element (ABRE), Sph element, CA-rich
element, sugar-responsive element (SRE), MYB-transcription factor binding
site,
endosperm box and TT-box, or alternatively or in addition, comprises a
nucleotide
sequence having at least about 20% identity to the BAS/ promoter sequence
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exemplified herein as SEQ ID NO: <400> 1. The invention clearly extends to
isolated
promoter sequences which comprise nucleotide sequences that are complementary
to the nucleotide sequences of said regulatory sequences.
S Preferably, the promoter sequence of the invention comprises at least two of
the
regulatory sequences selected from the list comprising GARE, ABRE, Sph
element,
CA-rich element, SRE, MYB-transcription factor binding site, endosperm box and
TT-
box. More preferably, the promoter sequence of the invention comprises at
least three,
even more preferably at least four, even more preferably at least five, even
more
preferably at least six and even more preferably at least seven of said
regulatory
sequences.
In a particularly preferred embodiment of the invention, there is provided an
isolated
promoter sequence that comprises regulatory sequences selected from the list
comprising GARE, ABRE, Sph element, CA-rich element, SRE, MYB-transcription
factor binding site, endosperm box and TT-box.
!n a further alternative embodiment of the invention, there is provided an
isolated
promoter sequence that is capable of regulating expression in the seeds of a
plant,
wherein said promoter sequence is up-regulated by abscisic acid andlor down-
regulated by gibberellin and comprises a nucleotide sequence selected from the
list
comprising:
(i) a nucleotide sequence having at least about 20% identity to the
nucleotide sequence set forth in SEQ ID NO: <400>1 or a complementary
nucleotide sequence thereto;
(ii) a nucleotide sequence that is capable of hybridising under at least low
stringency, preferably medium stringency, and more preferably under high
stringency hybridisation conditions to the nucleotide sequence set forth in
SEQ
ID NO: <400>1 or a complementary nucleotide sequence thereto; and
(ii) a nucleotide sequence that includes any one of the regulatory sequences
selected from the list comprising GARE, ABRE, Sph element, CA-rich element,
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_ 'j _
SRE, MYB-transcription factor binding site, endosperm box ar.d TT-box.
A second aspect of the present invention is directed to a genetic construct
comprising
the isolated promoter sequence herein described.
A further aspect of the present invention provides a transfected or
transformed cell,
tissue, organ or whole organism which expresses a recombinant polypeptide or a
ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co-
suppression molecule under the control of the promoter sequence described
herein.
I O Preferably, the transfected or transformed cell or tissue is a plant seed
cell or tissue,
more preferably in an endosperm cell or tissue such as aleurone or starchy
endosperm. The organ according to this embodiment is preferably a plant seed
that
comprises cells or tissues, more preferably endosperm cells or tissues such as
aleurone or starchy endosperm, which express the recombinant polypeptide or a
IS ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or
co-
suppression molecule under the control of the promoter sequence of the
invention.
Similarly, the whole plant is preferably a plant that comprises such plant
seed or seed
cells or seed tissues.
20 BRIEF DESCRIPTION OF THE DRA~IINGS
Figure 1 is a schematic representation showing the nucleotide sequence of the
BAST
promoter with the locations and nucleotide sequences of the cis-acting
elements
designated GARE-like, GARE, CA-rich element, SRE, pyrimidine box, Sph element,
ABA- responsive element (ABRE), MYB binding site (myb), Endosperm box and TT-
25 box, in addition to the location and sequence of the TATA box, all
indicated in boldface
type. Nucleotide positions corresponding to the nucleotide positions of SEQ ID
NO:
<400>1 are indicated at the left-hand and right-hand sides of the nucleotide
sequence.
Numbers in brackets refer to nucleotide positions relative to the
transcription start site
(+1 ) of the BASF gene.
Figure 2 is a graphical representation showing the restriction enzyme map of
the BASI
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_g_
promoter region (top) and the location of the amplified 1200 by Scal fragment
(below).
The restriction enzyme sites Pvull, Scal, Dral and EcoRV, in addition to the
location
of the BASI protein-coding region (basi gene) are indicated at the top of the
Figure.
The positions at which the amplification primers GSP1 and GSP2 hybridize to
the BASI
gene are also indicated.
Figure 3 is a schematic representation of plasmid pA17, comprising an
approximately
1200 by Scal fragment containing the BASI gene promoter sequence, including
nucleotides -959 to +74 relative to the transcription start site, inserted
into T-overhangs
of the plasmid vector pGEM-Teasy (Promega). This promoter sequence thus
comprises nucleotides 1 to 1033 of SEQ ID NO: <400>1. The positions of the
ampicillin
resistance gene (Amp-r), Xmnl and Scal restriction sites in plasmid pAl7 are
also
indicated.
Figure 4 is a schematic representation of plasmid pAGN, containing the
structural gfp
gene encoding a red-shifted variant of wild-type green fluorescent protein
(GFP;
Prasher et al., 1992; Chalfse et al., 1994; Inouye and Tsuji, 1994; and
Cormack et al.
(1996), which has been optimised for brighter fluorescence, operably connected
to the
nos gene terminator sequence, in the plasmid vector pGEM3Zf (Promega). The
positions of the ampicillin resistance gene (Amp-r), and the Hindlll, Sphl,
Pstl, EcoRl,
Ncol, Sall, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Scal restriction sites in
plasmid
pAGN are also indicated.
Figure 5 is a schematic representation of plasmid pA57 containing the full-
length BASI
promoter driving gfp gene expression. Piasmid pA57 contains nucleotides 1 to
1033
of SEQ ID NO: <400>1 ( -959basi), which was amplified from plasmid pA17
(Figure 3)
using a first primer BF1 (SEQ ID NO: <400> 20) containing a Hindlll site, and
a second
primer BR (SEQ ID NO: <400> 21 ) containing a BspHl site, to facilitate sub-
cloning of
the amplified DNA into the Hindlll and Ncol sites, respectively, of plasmid
pAGN
(Figure 4), such that the promoter sequence is operably connected to the gfp
gene
coding region and nos terminator sequence therein. The positions of the
ampicillin
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resistance gene (Amp-r), and the Hindlll, EcoRl, Sall, Aval, Xhol, Apal, Kpnl,
Sacl,
Xmnl and Scal restriction sites in plasmid pA57 are also indicated.
Figure 6 is a schematic representation of plasmid pA58. Plasmid pA58 contains
666bp
S of the BASI gene promoter sequence (-592 basi), from position -592 to +74
relative to
the transcription start site (i.e. nucleotides 368 to 1033 of SEQ ID NO:
<400>1 ), which
was amplified from plasmid pA17 (Figure 3) using a first primer BF2 (SEQ ID
NO:
<400> 22) containing a Hindlll site, and a second primer BR (SEQ ID NO: <400>
21 )
containing a BspHl site, to facilitate sub-cloning of the amplified DNA into
the Hindlll
and Ncol sites, respectively, of plasmid: pAGN (Figure 4), such that the
promoter
sequence is operably connected to the gfp gene coding region and nos
terminator
sequence therein. Accordingly, piasmid pA58 contains a truncated BASI promoter
that
comprises the putative SRE (i.e. the pyrimidine box motif}, the putative ABA
responsive element (ABA-RE) or Sph element motif, two putative GARS motifs,
and
IS the putative MYB-binding site, Endosperm box, TT-box and TATA-box motifs,
driving
gfp gene expression. The positions of the ampicillin resistance gene (Amp-r},
and the
Hindlll, EcoRl, Sail, Aval, Xhol, Apal, Kpnl, Sacl, Xmnl and Scal restriction
sites in
plasmid pA58 are also indicated.
Figure 7 is a schematic representation of plasmid pA61. Plasmid pA61 contains
497bp
of the BASI gene promoter sequence (-423 basi) , from position -423 to +74
relative
to the transcription start site (i.e. nucleotides 537 to 1033 of SEQ ID NO:
<400>1},
which was ampl~ed from plasmid pA17 (Figure 3) using a first primer BF3 (SEQ
ID
NO: <400> 23) containing a Hindlll site, and a second primer BR (SEQ ID NO:
<400>
21 ) containing a BspHf site, to facilitate sub-cloning of the amplified DNA
into the
HindIIl and Ncol sites, respectively, of plasmid pAGN (Figure 4), such that
the
promoter sequence is operably connected to the gfp gene coding region and nos
terminator sequence therein. Accordingly, plasmid pA61 contains a truncated
BAS(
promoter that comprises two putative GARE motifs, and the putative MYB-binding
site,
Endosperm box, TT-box and TATA-box motifs, driving gfp gene expression. The
positions of the ampicillin resistance gene (Amp-r), and the Hindlll, EcoRl,
Sall, Aval,
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Xhol, Apal, Kpnl, Sacl, Xmnl and Scal restriction sites in plasmid pA61 are
also
indicated.
Figure 8 is a schematic representation of plasmid pA64. Plasmid pA64 contains
273bp
of the BASI gene promoter sequence (-199 basi), from position -199 to +74
relative to
the transcription start site (i.e. nucleotides 7fi1 to 1033 of SEQ ID NO:
<400>1 ), which
was amplified from plasmid pAl7 (Figure 3) using a first primer BF4 (SEQ ID
NO:
<400> 24) containing a Hindlll site, and a second primer BR (SEQ ID NO: <400>
21 )
containing a BspHl site, to facilitate sub-cloning of the amplified DNA into
the Hindlll
and Ncol sites, respectively, of plasmid pAGN (Figure 4), such that the
promoter
sequence is operably connected to the gfp gene coding region and nos
terminator
sequence therein. Accordingly, plasmid pA64 contains a truncated BASI promoter
that
comprises one putative GARS, and the putative MYB-binding site, Endosperm box,
TT-box and TATA-box motifs, driving gfp gene expression. The positions of the
ampiciilin resistance gene (Amp-r), and the Hindlll, EcoRl, Sail, Aval, Xhol,
Apal, Kpnl,
Sacl, Xmnl and Scal restriction sites in plasmid pA64 are also indicated.
Figure 9 is a schematic representation of plasmid pA67. Plasmid pA67 contains
221 by
of the BASI gene promoter sequence, from position -147 to +74 relative to the
transcription start site (i.e. nucleotides 813 to 1033 of SEQ 1D NO: <400>1 ),
which was
amplified from plasmid pAl7 (Figure 3) using a first primer BF5 (SEQ ID NO:
<400>
24) containing a Hindlll site, and a second primer BR (SEQ ID NO: <400> 21 )
containing a BspHf site, to facilitate sub-cloning of the amplified DNA into
the Hindlll
and Ncol sites, respectively, of plasrnid pAGN (Figure 4), such that the
promoter
sequence is operably connected to the gfp gene coding region and nos
terminator
sequence therein. Accordingly, plasmid pA67 contains a truncated BASF
promoter,
that comprises the putative MYB-binding site, Endosperm box, TT-box and TATA-
box
motifs, driving gfp gene expression. The positions of the ampicillin
resistance gene
(Amp-r), and the Hindlll, EcoRl, Sall, Aval, Xhol, Apal, Kpnl, Sac(, Xmnl and
Scal
restriction sites in piasmid pA67 are also indicated.
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Figure 10 is a schematic representation of piasmid pA70. Plasmid pA70 contains
173bp of the BAS1 gene promoter sequence, from position -99 to +74 relative to
the
transcription start site (i.e. nucleotides 861 to 1033 of SEQ ID NO: <400>1 ),
which was
amplified from plasmid pAl7 (Figure 3) using a first primer BF6 (SEQ ID NO:
<400>
26) containing a Hindlll site, and a second primer BR (SEQ ID NO: <400> 21 )
containing a BspHl site, to facilitate sub-cloning of the amplified DNA into
the Hindlll
and Ncol sites, respectively, of plasmid pAGN (Figure 4), such that the
promoter
sequence is operably connected to the gfp gene coding region and nos
terminator
sequence therein. Accordingly, plasmid pA70 contains a truncated BASI
promoter,
that comprises the TT-box and TATA-box motifs, driving gfp gene expression.
The
positions of the ampicillin resistance gene (Amp-r}, and the Hindlil, EcoRl,
Sall, Aval,
Xhol, Apal, Kpnl, Sacl, Xmnl and Scal restriction sites in plasmid pA70 are
also
indicated.
IS Figure 11 is a schematic representation showing the aligned linear maps of
the
constructs cloned into plasmids pA57, pA58, pA61, pA64, pAfi7 and pA70. The
positions of the TATA box, transcription start site {+1 ), 3'-terminal
nucleotide position
of SEQ ID NO: <400>1 (+74), translation start site (ATG), gfp open reading
frame (gfp}
and NOS terminator sequence (nos} are indicated at the top of the Figure.
Numbering
at the left of the Figure refers to the 5'-terminal nucleotide derived from
the BASI
promoter, relative to the transcription start site, that is present in each
clone.
Figure 12 is a black and white copy of a colour photographic representation
showing
aleurone layers (half grains} of barley observed under a fluorescence
microscope to
detect cells transiently expressing GFP under the control of the Ubiquitin
promoter.
Tissue was co-bombarded with plasmid pA53, containing the Ubiquitin promoter
regulating the expression of green fluorescent protein (gfp) in the aleurone
tissue, and
a control piasmid pDP687, which facilitates the production of red anthocyanin
pigment
(blackigrey spots} in transfected/transformed tissues and was used as a
reporter
construct to normalise variation in results between microparticie
bombardments.
Approximately 24 hr after bombardment of tissue, tissue samples were observed
under
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blue light (490nm) to detect cells expressing GFP (G; white spots in the
Figure) as
indicated by the arrows. Full colour original photographic representations of
this Figure
are available on request.
Figure 13 is a black and white copy of a colour photographic representation
showing
aleurone layers (half grains) of barley observed under a fluorescence
microscope to
detect cells transiently expressing GFP under the control of the Ubiquitin
promoter in
plasmid pA53. Tissue was co-bombarded and incubated as described in the legend
to Figure '12, however tissue was visuaiised under white and blue light to
detect cells
expressing anthocyanin pigment (blacklgrey spots indicated by black-flagged
arrows)
andlor GFP { white spots as indicated by the arrows marked "G"). Full colour
original
photographic representations of this Figure are available on request.
Figure 14 is a black and white copy of a colour photographic representation
showing
leaf tissue of barley observed under a fluorescence microscope to detect cells
transiently expressing GFP and anthocyanin pigment under control of the
Ubiquitin
promoter. Observations were made under blue light {490nm), 24 hr after co-
bombardment with plasmids pA53 and pDP687. The Ubiquitin promoter in plasmid
pA53 is able to direct GFP expression in the leaf tissue, as indicated by the
arrows
marked "G". The plasmid pDP687, which led to the production of red anthocyanin
pigment in both the tissues, was used as a reporter construct to normalise
variation in
results between bombardments. Full colour original photographic
representations of
this Figure are available on request.
Figure 15 is a black and white copy of a colour photographic representation
showing
pericarp tissue of wheat observed under a fluorescence microscope to detect
cells
transiently expressing GFP and anthocyanin pigment under control of the
Ubiquitin
promoter. Observations were made under blue light {490nm), 24 hr after co-
bombardment with plasmids pA53 and pDP687. The Ubiquitin promoter in plasmid
pA53 is able to direct GFP expression in the pericarp tissue, as indicated by
the arrows
marked "G". The plasmid pDP687, which led to the production of red anthocyanin
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pigment in both the tissues, was used as a reporter construct to normalise
variation in
results between bombardments. Full colour original photographic
representations of
this Figure are availabie on request.
Figure 16 is a black and white copy of a colour photographic representatian
showing
aleurone layers (half grains) of barley observed under a fluorescence
microscope to
detect cells transiently expressing GFP and anthocyanin pigment. Observations
were
made under blue light at 490 nm, 24 hr after co-bombardment with plasmids pA57
and
pDP687. The 1033bp BASF promoter present in plasmid pA57 is able to direct GFP
expression in the aleuro~~e tissue, as indicated by the arrows marked "G". The
plasmid
pDP687, which led to the production of red anthocyanin pigment as indicated by
the
flagged arrows marked "A", was used as a reporter construct to normalise
variation in
results between bombardments. Full colour original photographic
representations of
this Figure are available on request.
IS
Figure 17 is a black and white copy of a colour photographic representation
showing
aleurone layers {half grains) of barley observed under a fluorescence
microscope to
detect cells transiently expressing GFP under the control of the BASI promoter
in
plasmid pA57. Tissue was co-bombarded and incubated as described in the legend
to Figure 16, however tissue was visualised under white and blue Light to
detect cells
expressing anthocyanin pigment {blacklgrey spots indicated by flagged arrows
marked
"A") andlor GFP ( white/grey spots as indicated by the arrows marked "G").
Full colour
original photographic representations of this Figure are available on request.
Figure '!8 is a black and white copy of a colour photographic representation
showing
leaf tissue of barley observed under a fluorescence microscope to detect cells
transiently expressing GFP and anthocyanin pigment. Observations were made
under
blue light (490nm), 24 hr after co-bombardment with plasmids pA57 and pDP687.
Data
indicate that the 1033bp BASI promoter present in plasmid pA57 does not confer
detectable expression levels of GFP in the leaf, however anthocyanin pigment
was
detected, as indicated by the arrows marked "A". The piasmid pDP687, which led
to
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_J
the production of red anthocyanin pigment (A}, was used as a reporter
construct to
normalise variation in results between bombardments. Full colour original
photographic
representations of this Figure are available on request.
Figure 19 is a black and white copy of a colour photographic representation
showing
pericarp tissue of wheat observed under a fluorescence microscope to detect
cells
transiently expressing GFP and anthocyanin pigment. Observations were made
under
blue light (490nm), 24 hr after co-bombardment with plasmids pA57 and pDP687.
Data
indicate that the 1033bp BASI promoter present in plasmid pA57 does not confer
detectable expression levels of GFP in the pericarp, however anthocyanin
pigment was
detected, as indicated by the arrow marked "A". The plasmid pDP687, which led
to the
production of red anthocyanin pigment (A), was used as a reporter construct to
normalise variation in results between bombardments. Full colour original
photographic
representations of this Figure are available on request.
Figure 20 is a black and white copy of a colour photographic representation
showing
an immature transgenic rice plantlet in tissue culture, that has been produced
by
bombardment with plasmids pA57 and pG~2. The plantlet was observed under blue
light at 490 nm using a fluorescence microscope to detect cells that stably
express
GFP. The arrows indicate GFP expression in both the immature roots {RG} and
immature shoots (SG) of transformed rice plantlets grown in these conditions,
which
expression is observable in the Figure as brightly-flaring tissue. Full colour
original
photographic representations of this Figure are available on request.
Figure 21 is a black and white copy of a colour photographic representation
showing
the transgenic rice plant depicted in Figure 20, observed under white light.
The arrows
indicate the location of the roots (R) and shoots (S) of transformed rice.
Full colour
original photographic representations of this Figure are available on request.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention provides an isolated promoter sequence
derived
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from a plant cell which is at least capable of conferring, increasing or
otherwise
facilitating the expression of a structural gene to which it is operably
connected in the
seeds of a plant and preferably, in the endosperm cells or tissues of said
seeds.
Reference herein to a "promoter" is to be taken in its broadest context and
includes the
transcriptions! regulatory sequences of a classical genomic gene, including
the TATi4
box which is required for accurate transcription initiation, with or without a
CCAAT box
sequence and additional regulatory elements (i.e. upstream activating
sequences,
enhancers and silencers) which alter gene expression in response to
developmental
and/or hormonal and/or environmental stimuli, or in a tissue-specific or cell-
type-
specific manner. A promoter is usually, but not necessarily, positioned
upstream, or
5', of a structural gene, the expression of which it regulates. Furthermore,
the
regulatory elements comprising a promoter are usually positioned within 2 kb
of the
start site of transcription of the gene.
I5
In the present context, the term "promoter" is also used to describe a
synthetic or
fusion molecule or a derivative of the nucleotide sequence set forth in SEQ ID
NO:
<400>1 or a complementary nucleotide sequence thereto, which possesses the
same
function as the promoter sequence of the invention as described herein.
For the purposes of nomenclature, the nucleotide sequence shown in SEQ 1D NO:
<400>1 comprises a functional promoter sequence derived from the barley
bifunctional
a-amylase subtilisin inhibitor (hereinafter referred to as the "BAS/" gene).
Promoters encompassed by the invention include those promoters that are
derived
from SEQ !D NO: <400>1 or a part thereof or a complementary nucleotide
sequence
thereto and which possess the same function as said promoter sequence or part
or
complement, however comprise additional copies of one or more specific
regulatory
elements, derived from either the exemplified promoter sequence or a
heterologous
promoter sequence, to further enhance expression of a genetic sequence to
which it
is operabiy connected and/or to alter the timing of expression of a genetic
sequence
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to which it is operably connected. For example, chimeric prompter sequences
that
comprise the nucleotide sequence set forth in SEQ ID NO: <400>1 may be
modified
by the inclusion of nucleotide sequences derived from the wheat glutenin gene
promoter region to further enhance expression of a genetic sequence to which
the
promoter of the invention is operably connected in the endosperm of a seed.
The
performance of such embodiments is readily achievable by those skilled in the
art
when provided with the teaching herein.
The terms "operabiy connected", "operably in connection" or similar in the
present
IO context means placing a first genetic sequence, such as a structural gene
that
encodes a polypeptide, under the regulatory control of the promoter sequence
of the
invention by positioning the first genetic sequence such that its expression
is controlled
by the promoter sequence.
I5 Promoters are generally positioned 5' (upstream) to the structural genes
that they
control. In the construction of heterologous promoterlstructural gene
combinations it
is generally preferred to position the promoter at a distance from the gene
transcription
start site that is approximately the same as the distance between that
promoter and
the gene it controls in its natural setting,~i.e., the gene from which the
promoter is
20 derived. As is known in the art, some variation in this distance can be
accommodated
without loss of function. Similarly, the preferred positioning of a regulatory
sequence
element with respect to a heterologous gene to be placed under its control is
defined
by the positioning of the element in its natural setting, i.e., the genes from
which it is
derived. Again, as is known in the art and demonstrated herein with multiple
copies
25 of regulatory elements, some variation in this distance can occur.
As used herein, a "structural gene" shall be taken to refer to that portion of
a gene
comprising a DNA segment encoding a peptide, oligopeptide, polypeptide,
protein or
enzyme or a portion thereof. Accordingly, structural genes may include the
protein-
30 encoding regions of genomic genes, cDNA molecules and other DNA molecules,
including fusion molecules. It is to be understood that the term "structure!
gene" may
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additionally include nucleotide sequences that do not encode a peptide,
oligopeptide,
polypeptide, protein or enzyme or a portion thereof.
Alternatively or in addition, a structural gene may comprise an isolated
nucleic acid
molecule that does not encode a polypeptide, for example an antisense
molecule,
ribozyme, abzyme, co-suppression molecule, gene-silencing molecule or gene=
targetting molecule, amongst others.
The word "expression" as used herein shall be taken to refer to the
transcription of a
particular genetic sequence to produce sense or antisense mRNA, as detectable
by
the appearance of said mRNA andlor by the appearance of a peptide,
polypeptide,
oligopeptide, protein or enzyme molecule encoded by a sense mRNA molecule.
By "conferring, increasing or otherwise facilitating expression" of a
structural gene is
meant that the rate or steady-state level of transcription of mRNA encoded by
said
structural gene placed operabiy under the control of the promoter sequence is
increased andlor the biological activity or steady-state level of a peptide,
polypeptide,
oligopeptide, protein or enzyme molecule encoded by said structural gene is
increased
in comparison to the level of expression that is detectable in the absence of
the
promoter sequence.
Those skilled in the art will be aware of whether expression is conferred,
increased or
otherwise facilitated by the promoter sequence of the invention, without undue
experimentation.
For example, the level of expression of a particular structural gene may be
determined
by polymerise chain reaction (PCR) following reverse transcription of an mRNA
template molecule, essentially as described by McPherson et al. (1991 ).
Alternatively, the expression level of a structural gene may be determined by
northern
hybridisation analysis or dot-blot hybridisation analysis or in situ
hybridisation analysis
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or similar technique, wherein mRNA is transferred to a membrane support and
hybridised to a "probe" molecule which comprises a nucleotide sequence
complementary to the nucleotide sequence of the rnRNA transcript encoded by
the
structural gene, labelled with a suitable reporter molecule such as a
radioactively-
labelled dNTP (eg [a 32P]dCTP or [a-~SjdCTP) or biotinylated dNTP, amongst
others.
Expression of the structural gene may then be determined by detecting the
appearance of a signal produced by the reporter molecule bound to the
hybridised
probe molecule.
Alternatively, the rate of transcription of a particular structural gene rnay
be determined
by nuclear run-on andlor nuclear run-off experiments, wherein nuclei are
isolated from
a particular cell or tissue and the rate of incorporation of rNTPs into
specific mRNA
molecules is determined.
Alternatively, the expression of the structural gene may be determined by
RNase
protection assay, wherein a labelled RNA probe or "riboprobe" which is
complementary
to the nucleotide sequence of mRNA encoded by said structural gene is annealed
to
said mRNA for a time and under conditions sufficient for a double-stranded
mRNA
molecule to form, after which time the sample is subjected to digestion by
RNase to
remove single-stranded RNA molecules and in particular, to remove excess
unhybridised riboprobe.
Those skilled in the art will also be aware of various immunological and
enzymatic
methods for detecting the level of expression of a particular gene at the
protein level,
for example using rocket immunoelectrophoresis, ELISA, radioimmunoassay and
western blot immunoelectrophoresis techniques, amongst others.
Such approaches are described by Sambrook et al. (1989) and Ausubel (1987).
In the present context, the word "seed" shat! be taken to refer to any plant
structure
which is formed by continued differentiation of the ovule of the plant and to
include a
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storage tissue such as a haploid female gametophyte or a triploi:d maternally-
derived
endosperm, an aleurone layer and embryo. As exemplified herein, the promoter
sequence of the invention does not confer expression in the pericarp tissue of
seeds
and, as a consequence, the word "seed" in the context of the invention does
not
include such tissue.
Preferably, the promoter sequence of the invention is a seed-specific
promoter.
As used herein, the term "seed-specific" shall be taken to indicate that the
promoter
sequence is capable of conferring expression on a structural gene sequence
that is
substantially localised to one or more cells or tissues of plant seeds as
defined herein
{i.e. the starchy endosperm andlor aleurone and/or embryo andlor scutellum).
Reference herein to "endosperm" shall be taken as comprising starchy endospem~
and
the aleurone layer{s).
Preferably, the promoter sequence of the invention is at least capable of
conferring
expression on a structural gene sequence in the endosperm tissues of the seed
and
cells derived therefrom or comprising same. Such expression may be in the
starchy
endosperm cells and/or the mature aleurone cells of the endosperm of a plant.
Alternatively or in addition, expression may be conferred in the scutellum.
Even more preferably, the promoter sequence of the invention is capable of
conferring
expression on a structural gene sequence during the coenocytic stage of
endosperm
development of a monocotyledonous plant species.
The term "mature aleurone cell" means those cells of the aleurone layer of a
seed that
have completed the phase of starch accumulation and preferably, a fully-
developed
seed. As will be known to those skilled in the art, a mature aleurone layer
may be
derived from a mature seed of a monocotyledonous plant species, following
imbibing
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of seeds by soaking in a liquid medium such as water.
More preferably, the promoter sequence of the invention is endosperm specific
which
means that the promoter is starchy endosperm-specific andlor aleurone-
specific.
The terms "endosperm-specific" and "aleurone-specific" shall be taken to
indicate that
the promoter sequence is capable of conferring expression on a structural gene
sequence that is substantially localised to the starchy endosperm or aleurone,
respectiveiy.
in a particularly preferred embodiment, the promoter sequence of the invention
is at
least capable of conferring, increasing or otherwise facilitating expression
of a
structural gene sequence in the aleurone cells of a plant seed.
The promoter sequence of the invention is capable of conferring, increasing or
otherwise facilitating expression of a structural gene to which it is operably
connected
in the seeds of any monocotyledonous or dicotyiedonous plant species. Because
the
BASI gene is expressed in plants that produce high pl a-amylases, it is likely
that the
BASI gene promoter exemplified herein may confer expression in any plant
tissue that
produces high pl a-amylases to degrade starch reserves. Accordingly, the
promoter
sequence of the invention may also be operable in any plant species that
produces a
seed containing a starchy endosperm or a plant that produces another starch
storage
organ, such as a tuber.
Preferably, the promoter sequence of the invention is capable of conferring,
increasing
or otherwise facilitating expression of a structural gene in a
monocotyledonous plant
that produces a seed having agronomic importance, for example grain crops such
as
wheat, oats, maize, barley, rice, sorghum, millet or rye, amongst others.
In a particularly preferred embodiment, the promoter sequence of the invention
is at
least capable of conferring expression in the cells, tissues or organs of a
wheat or
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barley plant.
Those skilled in the art will be aware that it is also possible to modify the
level of
structural gene expression andlor the timing of structural gene expression
andlor the
regulation of structural gene expression, by mutation of a regulatory genetic
sequence
(i.e. cis-regulatory region or 5'-non-coding region, etc) within the promoter
sequence'
to which the structural gene is operably connected. In particular, to achieve
such an
objective, the promoter sequence of the present invention may be subjected to
mutagenesis to produce single or multiple nucleotide substitutions, deletions
andlor
additions.
Alternatively, or in addition, the arrangement of specifc regulatory sequences
within
the promoter sequence may be altered, including the deletion therefrom of
certain
regulatory sequences andlor the addition thereto of regulatory sequences
derived from
the same or a different promoter sequence.
Accordingly, the present invention clearly encompasses derivatives of the
nucleotide
sequence set forth in SEQ ID NO: <400>1.
As used herein "derivatives" of the promoter sequence of the invention shall
be taken
to refer to any isolated nucleic acid molecule which comprises at least 10 and
preferably at least 20 contiguous nucleotides, and more preferably at least 30
contiguous nucleotides, derived from the promoter sequence as described herein
according to any embodiment.
As exemplified herein, the present inventors have shown that the minimum
nucleotides
of SECT ID NO: <400>1 required for efficient seed expression of a structural
gene to
which said SEQ 1D NO is operably connected is in the region downstream of
nucleotide position 813. Accordingly, preferred derivatives of SEQ ID NO:
<400>1
comprise nucleotides downstream of position 813 of SEO ID NO: <400> 1.
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In an alternative embodiment, a preferred derivative of the present invention
will
include those nucleic acid molecules which comprise only nucleotides upstream
of
position about 976 of SEQ ID NO: <400>1 and more preferably, nucleotides
upstream
of position about 961, but not including nucleotides downstream of position
977 of SEQ
ID NO: <400>1.
Alternatively or in addition, a derivative of SEQ ID NO: <400> 1 at least
comprises
nucleotides in the region of positions 813 to 961, more preferably, positions
813 to
976.
As will be apparent to those skilled in the art, such derivatives may comprise
one or
more of the functional cis-acting elements present in this region of the BASI
promoter,
operabiy connected to heterologous nucleotide sequences, such as, for example,
the
MYB-binding site and/or endosperm box and/or TT-box depicted in Figure 1, in
combination with a functional TATA-box motif. Additional integers are not
excluded.
Nucleotide insertional derivatives of the promoter sequence of the present
invention
include 5' and 3' terminal fusions as well as intra-sequence insertions of
single or
multiple nucleotides. Insertional nucleotide sequence variants are those in
which one
or more nucleotides are introduced into a predetermined site in the nucleotide
sequence although random insertion is also possible with suitable screening of
the
resulting product. Deletional variants are characterised by the removal of one
or more
nucleotides from the sequence. Substitutional nucleotide variants are those in
which
at least one nucleotide in the sequence has been removed and a different
nucleotide
inserted in its place.
Generally, derivatives of the nucleic acid molecule of the invention are
produced by
synthetic means or alternatively, derived from naturally-occurring sources.
For
example, the nucleotide sequence of the present invention may be subjected to
mutagenesis to produce single or multiple nucleotide substitutions, deletions
and/or
insertions.
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tt _ 23 _
Accordingly, the promoter sequence of the invention may comprise a sequence of
nucleotides or be complementary to a sequence of nucleotides which comprises
one
or more of the following sequences without complete loss of function:
(i) a 5' non-coding region; andlor
(ii) one or more cis-regulatory regions, such as one or more functional
binding sites for a transcriptional regulatory proteins or translational
regulatory
proteins, one or more upstream activator sequences, enhancer elements or
silencer elements; and/or
(iii) a TATA box motif; andlor
(iv) a CCAAT box motif; andlor
(v) an upstream open reading frame (uORF);andlor
(vi) a transcriptionai start site; and/or
(vii) a translational start site; and/or
(viii) a nucleotide sequence which encodes a leader sequence.
i5
As used herein, the term "5' non-coding region" shall be taken in its broadest
context
to include all nucleotide sequences which are derived from the upstream region
of a
seed-expressible gene, preferably derived from the BAS/ gene exemplified
herein,
other than those sequences which encode amino acid residues comprising the
polypeptide product of said gene.
As used herein, the term "uORF" refers to a nucleotide sequence localised
upstream
of a functional translation start site in a gene and generally within the 5'-
transcribed
region (i.e. leader sequence), which encodes an amino acid sequence. Whilst
not
being bound by any theory or mode of action, a uORF functions to prevent over-
expression of a structural gene sequence to which it is operably connected or
alternatively, to reduce or prevent such expression.
As used herein, the term "cis-acting sequence" or "cis-regulatory region" or
similar term
shall be taken to mean any sequence of nucleotides which is derived from a
promoter
sequence wherein the timing, level or regulation of expression conferred by
said
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promoter in a particular cell, tissue or organ is conferred at least in part
by said
sequence of nucleotides. Those skilled in the art will be aware that a cis-
regulatory
region may be capable of activating, silencing, enhancing, repressing or
otherwise
altering the level of expression andlor cell-type-specificity andlor
developmental
specificity of any structural gene sequence to which it is operably connected.
In
general, a single cis-regulatory region may be responsible for conferring one
mode of
regulation on a structural gene sequence to which it is operably connected,
however
the occun-ence of several cis-regulatory regions in operable connection with a
single
structural gene sequence may confer multiple regulatory modes on said
structural
gene, which are not necessarily the mere summation of the individual
regulatory
modes (i.e. there may be interaction between individual cis-regulatory
regions).
Furthermore, such cis-acting regions generally, but not necessarily, comprise
a linear
array of groups of nucleotides which each comprise at least four and
preferably at least
six contiguous nucleotide residues.
Accordingly, the present invention extends to isolated nucleic acid molecules
which
comprise one or more cis-regulatory regions which act to contribute to the
ability of the
promoter sequence described herein to confer, activate or otherwise regulate
expression of a stnrctural gene sequence in a plant seed or a cell or tissue
thereof.
Preferred cis-regulatory regions according to the invention comprise a linear
array of
one or more silencer, enhancer, or upstream activating sequences, not
necessarily
juxtaposed, however in sufficiently close association to be at feast capable
of
conferring, either in concert or independently of each other, one or more
regulated
modes of expression on a structural gene sequence to which they are operably
connected.
Preferred cis-regulatory regions according to the present invention include,
but are not
limited to, one or more of the sequences selected from the list comprising the
GARE,
ABRE, Sph element, CA-rich element, SRE, pyrimidine box, MYB-transcription
factor
binding site, endosperm box and TT-box.
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The invention clearly extends to isolated promoter sequences which comprise
nucleotide sequences that are complementary to the nucleotide sequences of
said
regulatory sequences.
The pyrimidine box, TAACAAA box (set forth in SEQ ID NO: <400>2} and TATCCAC
box (set forth in SEQ lD NO: <400>3) were first described in the context of
the
promoter sequence of the high pl a-amylase gene and are believed to mediate
the
responsiveness of that promoter to GAs (Skriver et al., 1991; Gubler and
Jacobsen,
1992; Gubler ef al., 1995}. There is also evidence that the action of abscisic
acid on
the a-amylase promoter sequence is mediated via the same complex. Analyses of
a
barley low-pi amylase promoter, have shown that GA probably also acts through
similar cis-acting elements, but additional cis-acting elements upstream of
the
pyrimidine box are also important (Lanahan ef al., 1992). A GA-dependent
binding
factor was shown to bind specifically to sequences which coincide with the
TAACAGA
(set forth in SEQ ID NO: <400>4) and TATCCAT (set forth in SEQ ID NO: <400>5)
boxes and proximal sequences in the a-amylase promoter.
Notwithstanding the presence of these sequences in the a-amylase promoter, the
presently-described full-length promoter sequence is inducible by ABA and/or
capable
of repression by GAs, in contrast to the a-amylase promoter which is induced
by GAs
and repressed by ABA. Those skilled in the art will be aware that it may be
possible
to alter the mode of structural gene regulation by the promoter sequence of
the
invention by deleting or otherwise mutating the GARS or ABRE sequences
therein. For
example, by deleting the ABRE sequences, induction of gene expression by ABA
rnay
be lost. Alternatively, by deleting the GARE sequences, repression of gene
expression
by GAs may be lost. The presently described promoter encompasses all such
variants.
As used herein, the term " gibbereliic acid responsive element" or its
abbreviation
"GARS" shall be taken to refer to any sequence of nucleotides that is capable
of
reducing, decreasing or repressing the expression of a structural gene to
which it is
operably connected and which comprises a nucleotide sequence which is
identical to
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one or more of the nucleotide sequences 5'-ATAACTAAGTGGG-3' (set forth in SEQ
ID NO: <400>6) or 5'-ATAGAGTGTA-3' (set forth in SEQ iD NO: <400>7) or 5'-
TATCCA-3'(set forth in SEQ lD NO: <400>8) or 5'-TATAACATTGCTCTG-3' (set forth
in SEQ ID NO: <400>9) or 5'-TCACAAA-3' {set forth in SEQ lD NO: <400>10) or a
complementary nucleotide sequence thereto or a homologue thereof.
As used herein, the term " pyrimidine box" shall be taken to refer to any
sequence of
nucleotides that is capable of regulating, at least in part, the expression of
a structural
gene to which it is operably connected and which comprises a nucleotide
sequence
which is identical to one or more of the nucleotide sequences 5'-TATCCA-3'
(SEQ ID
NO: <400>8) or 5'-TCACAAA-3' (SEQ ID NO: <400>10) or a complementary
nucleotide sequence thereto or a homologue thereof.
As used herein, the term " abscisic acid responsive element" or its
abbreviation
"ABRE" or "ABA-RE" shall be taken to refer to any sequence of nucleotides that
is
capable of conferring, increasing, or enhancing or inducing the expression of
a
structural gene to which it is operably connected and which comprises a
nucleotide
sequence which is identical to the nucleotide sequence 5'-CATGCAT-3'(set forth
in
SEQ ID NO: <400>11) or a complementary nucleotide sequence thereto or a
homologue thereof.
As used herein, the term "Sph element" shall be taken to refer to any sequence
of
nucleotides that is capable of regulating, at least in part, the expression of
a structural
gene to which it is operably connected and which comprises a nucleotide
sequence
which is identical to the nucleotide sequence 5'-CATGCAT-3'(set forth in SEQ
ID NO:
<400>11) or a complementary nucleotide sequence thereto or a homologue
thereof.
As used herein, the term " CA-rich element" shall be taken to refer to any
sequence
of nucleotides that comprises a nucleotide sequence which is identical to the
nucleotide sequence 5'-CATGTCATCAAAATCATC-3' (set forth in SEQ ID NO:
<400>12) or a complementary nucleotide sequence thereto or a homologue
thereof.
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As used herein, the term "sugar responsive element" or its abbreviation "SRE"
shall
be taken to refer to any sequence of nucleotides that is capable of
conferring,
increasing, or enhancing or inducing the expression of a structural gene to
which it is
operably connected under conditions of low intracellular or extracellular
sucrose and
which comprises a nucleotide sequence which is identical to the nucleotide
sequence
5'-TATCCA-3' (SEQ ID NO: <400>10} or a complementary nucleotide sequence
thereto or a homologue thereof.
As used herein, the term "MYB-transcription factor binding site" shall be
taken to refer
to any sequence of nucleotides that is capable of regulating, at least in
part, the
expression of a structural gene to which it is operably connected and which
comprises
a nucleotide sequence that is related to the DNA-binding site of a MYB
transcription
factor, wherein said nucleotide sequence which is identical to the nucleotide
sequence
5'-TTACTG-3' (set forth in SEQ ID NO: <400>13) or a complementary nucleotide
sequence thereto. Preferably, a "MYB-transcription factor binding site" as
used herein
is further capable of binding a transcription factor belonging to the MYB
class of
proteins or a homologue thereof.
As used herein, the term " endosperm box" shall be taken to refer to any
sequence of
nucleotides that is capable of conferring, increasing, or enhancing or
inducing the
expression of a structural gene to which it is operably connected in the
endosperm
and which comprises a nucleotide sequence which is identical to the nucleotide
sequence 5'-TGTAAAGG-3' (set forth in SEQ 1D NO: <400>14) or a complementary
nucleotide sequence thereto or a homologue thereof.
As used herein, the term " TT-box" shall be taken to refer to any sequence of
nucleotides that is capable of regulating, at least in part, the expression of
a structural
gene to which it is operably connected and which comprises a nucleotide
sequence
which is identical to the nucleotide sequence 5'-TTCCAGATCA-3' (set forth in
SEQ
ID NO: <400>'! 5) or a complementary nucleotide sequence thereto or a
homologue
thereof.
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Preferably, the promoter sequence of the invention comprises a sequence of
nucleotides which is identical to at least two of the regulatory sequences
selected from
the list comprising GARS, ABRE, Sph element, CA-rich element, SRE, MYB-
transcription factor binding site, endosperm box and TT-box or a homologue
thereof.
More preferably, the promoter sequence of the invention comprises a nucleotide
sequence which comprises to three, even more preferably at least four, even
more
preferably at least five, even more preferably at least six and even more
preferably at
least seven of said regulatory sequences.
In an even more preferred embodiment of the invention, there is provided an
isolated
promoter sequence that comprises regulatory sequences selected from the list
comprising GARS, ABRE, Sph element, CA-rich element, SRE, MYB-transcription
factor binding site, endosperm box and TT-box or a homologue thereof.
In a particularly preferred embodiment, the isolated promoter sequence of the
invention comprises a nucleotide sequence that at least includes one copy of
one or
more of each of the nucleotide sequences set forth in the Sequence Listing as
SEQ
ID NO: <400>6 to SEQ ID NO: <400>15, as follows:
(i) SEQ ID NO: <400>6: 5'-ATAACTAAGTGGG-3';
(ii) SEQ 1D NO: <400>?: 5'-ATAGAGTGTA-3';
(iii) SEQ ID NO: <400>8: 5'-TATCCA-3';
(iv) SEQ ID NO: <400>9: 5'-TATAACATTGCTCTG-3';
(v) SEQ ID NO: <400>10: 5'-TCACAAA-3';
(vi) SEQ ID NO: <400>11: 5'-CATGCAT-3';
(vii) SEQ 1D NO: <400>12: 5'-CATGTCATCAAAATCATC-3';
(viii) SEQ ID NO: <400>13: 5'-TTACTG-3';
(ix) SEQ ID NO: <400>14: 5'-TGTAAAGG-3'; and
(x) SEQ 1D NO: <400>15: 5'-TTCCAGATCA-3',
or a complementary nucleotide sequence thereto.
Even more preferably, the promoter sequence of the invention includes at least
two or
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three, even more preferably at least four or five, still even more preferably
at least six
or seven, and even still more preferably at least eight or nine or ten, of the
cis-
regulatory regions listed supra.
In a particularly preferred embodiment, a promoter sequence which is capable
of
conferring, activating or otherwise regulating expression of a structural gene
in a plant
seed {including endosperm i.e, starchy endosperm cell andlor a mature aleurone
cell),
developmentally andlor which is regulatable by the phytohormones abscisic acid
{ABA) and gibberellic acid (GA) will comprise at least nucleotide sequence
motifs (viii)
to (x) supra.
Those skilled in the art will recognise that the promoter according to this
embodiment
may require additional sequences for function, for example a TATA box motif,
such as
the TATA-box contained in the promoter sequence of the invention, which TATA
box
motif is set forth herein as SEQ ID NO: <400>16 (i.e. 6'-TATAAATA-3'), amongst
others. Such nucleotide sequences will be readily supplied by persons skilled
in the art
without undue experimentation and the invention according to this embodiment
clearly
encompasses such alternatives.
It is to be understood that modifications may be made to the structural
arrangement
of specific enhancer and promoter elements of the promoter sequence described
herein without destroying the improved enhancing activity of gene expression.
For
example, it is contemplated that a substitution may be made in the choices of
enhancer and promoter elements without significantly affecting the function of
the
recombinant promoter sequence of this invention. Further, it is contemplated
that
nucleotide sequences homologous to the active enhancer elements utilized
herein may
be employed advantageously, either as a substitution or an addition to the
recombinant
promoter construct for improved gene expression in plant seeds, in particular
in
endosperm andlor aleurone and/or scutellum cells. It will be understood that
the
function of the promoter sequence of this invention also potentially results
from the
arrangement, orientation and spacing of the different enhancer elements with
respect
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to one another, and with respect to the position of the TATA box.
The present invention clearly extends to functionally homologous promoters to
the
BASI promoter sequence exemplified herein. Preferred homologues of the BASI
promoter include those promoters that share structural features with the BASI
promoter
that are useful in conferring the seed expression pattern on a structural gene
in plants
that is obtained using the BAS( promoter.
In one embodiment, a homologue of the BASI promoter exemplified herein is
capable
of conferring, increasing or otherwise facilitating the expression of a
structural gene in
the seeds of a plant, wherein said promoter sequence comprises a nucleotide
sequence which is capable of hybridising under at least tow stringency
conditions,
preferably medium stringency conditions, and more preferably under high
stringency
conditions, to a nucleotide sequence selected from the group consisting of:
(i) the nucleotide sequence set forth in SEQ ID NO: <400>1;
(ii) a nucleotide sequence comprising at least 10 contiguous of SEQ ID NO:
<400>1;
(iii) any one of the cfs-acting regulatory sequences selected from the group
consisting of: gibberellic acid responsive element (GARS); abscisic acid
responsive element (ABRE); Sph element; CA-rich element; sugar-responsive
element (SRE); MYB-transcription factor binding site; endosperm box; and TT-
box; and
(iv) a complementary nucleotide sequence to any one or more of (i} to (iii).
For the purposes of defining the level of stringency, those skilled in the art
will be
aware that several different hybridisation conditions may be employed. As used
herein, a low stringency hybridisation may comprise the standard reaction
buffer used
in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to
template
DNA at temperatures in the range 25°C to 37°C or higher, or
alternatively, a standard
DNAIDNA hybridisation and/or wash carried out in a buffer comprising 6xSSC
buffer,
0.1 % (w/v) SDS at ambient temperature, or equivalent annealing/hybridisation
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conditions. in the present context, references to "hybridisation" conditions
clearly refer
to both a standard "Southern" or "northern" type of hybridisation, and to the
conditions
required for annealing of a primer to template nucleic acid in a polymerase
chain
reaction.
As used herein, a medium stringency may comprise the standard reaction buffer
used
in a polymerase chain reaction (PCR) to anneal an oligonucleotide primer to
template
DNA at temperatures in the range 37°C to 42°C or higher, or
alternatively, a standard
DNA/DNA hybridisation andlor wash carried out in 2xSSC buffer, 0.1 % (wlv) SDS
at
a temperature in the range 45°C to 65°C or equivalent
annealinglhybridisation
conditions.
A high stringency may comprise the standard reaction buffer used in a
polymerase
chain reaction {PCR) to anneal an oligonucleotide primer to template DNA at
temperatures higher than 42°C, or alternatively, a standard DNA/DNA
hybridisation
and/or wash can-led out in 0.1 xSSC buffer, 0.1 % {w/v) SDS at a temperature
of at least
65°C, or equivalent annealing/hybridisation conditions.
As will be known to those skilled in the art, the stringency is increased by
reducing the
concentration of SSC buffer, and/or increasing the concentration of SDS in a
standard
hybridisation, andlor increasing the temperature of the
annealing/hybridisation of PCR
or a standard hybridisation, and/or increasing the temperature of the wash in
a
standard hybridisation. Conditions for hybridisations and washes are well
understood
by one normally skilled in the art. For the purposes of clarification of
parameters
affecting hybridisation between nucleic acid molecules, reference is found in
pages
2.10.8 to 2.10.16. of Ausubel et al. {1987), which is herein incorporated by
reference.
As will be known to those skilled in the art, the specificity of PCR may also
be
increased by reducing the number of cycles, or the time per cycle, or by the
use of
specific PCR formats, such as, for example, a nested PCR, a format that is
well-known
to those skilled in the art. For the purposes of clarifcation of the
parameters affecting
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the specificity of PCR, reference is made herein to McPherson et al. (1991 )
which is
incorporated by way of reference.
Alternatively or in addition, a homologue of the BAS/ promoter comprises a
nucleotide
sequence selected from the group consisting of:
(i) a nucleotide sequence having at least about 20% identity to the
nucleotide sequence set forth in SEQ 1D NO: <400>1;
(ii) a nucleotide sequence that includes nucleotide sequences having at
least about 20% identity to any one of the regulatory sequences selected from
the group consisting of: gibberellic acid responsive element (GARS); abscisic
acid responsive element (ABRE); Sph element; CA-rich element; sugar-
responsive element (SRE); MYB-transcription factor binding site; endosperm
box; and TT-box; and
(iii) a complementary nucleotide sequence to (i) or (ii).
The words "identical " or "identity" as used herein includes exact identity
between
compared sequences at the nucleotide level. Where there is non-identity at the
nucleotide level, "similarity" includes differences between sequences that are
nevertheless related to each other at the structural, functional, biochemical
andlor
conformational levels. For example, a cls-acting regulatory sequence present
in the
inventive promoter sequence exemplified herein may be similar to a cis-acting
sequence in a functionally homologous promoter, albeit non precisely conserved
so
as to be considered identical when viewed in context of the consensus sequence
for
that cis-acting sequence (i.e. there are variations from the accepted
consensus), or
even when viewed in the wider context of the minimum promoter required for
function
(i.e. there may be similar sequences and/or different arrangements of
functionally
identical cis-acting elements present in distinct but functionally homologous
promoters). Any number of programs are available to compare nucleotide
sequences.
Preferred programs have regard to an appropriate alignment. One such program
is
Gap which considers all possible alignment and gap positions and creates an
alignment with the largest number of matched bases and the fewest gaps. Gap
uses
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the alignment method of Skriver et al. (1991 ). Gap reads a scoring matrix
that contains
values for every possible GCG symbol match. GAP is available on ANGIS
(Australian
National Genomic Information Service) at website http:l/mel1.angis.org.au.
Alternative percentage identities contemplated by the present invention
include at least
about 34%, at least about 40%, at least about 50%, at least about 60%, at
feast about
70%, at least about 80% and at least about 90% or above compared to a
reference
sequence.
Preferably, the homologous sequence is regulatable by the phytohormones
abscisic
acid (ABA) and gibberellic acid (GA), such that expression of a structural
gene to which
said genetic sequence is operably connected is inducible by ABA and/or is
capable of
being repressed by GAs.
A still further embodiment of the present invention extends to derivatives,
parts,
fragments or analogues of the nucleotide sequence set forth in SEQ ID NO:
<400>1
or a complementary nucleotide sequence thereto which are at least useful as
genetic
probes in the isolation of similar sequences, or primer sequences in the
enzymatic or
chemical synthesis of said promoter sequence or a related promoter sequence.
In a particularly preferred embodiment, the genetic sequence of the present
invention
is employed to identify and isolate similar genetic sequences from other
plants, in
particular grain crops such as wheat, oats, maize, barley, rice, sorghum,
millet or rye,
amongst others.
According to this embodiment, there is contemplated a method for identifying a
related
genetic sequence which is at least capable of conferring, increasing or
otherwise
facilitating the expression of a structural gene in the cells of a plant seed,
in particular
an endosperm cell, such as a starchy endosperm or mature aleurone cell, said
method comprising contacting genomic DNA or parts of fragments thereof, or a
source
thereof, with a hybridisation-effective amount of the nucleotide sequence set
forth in
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SEQ ID NO: <400>1, or a part, analogue or derivative thereof or a
complementary
sequence thereto, and then detecting said hybridisation.
The related genetic sequence may be in a recombinant form, in a virus
particle,
bacteriophage particle, yeast cell, animal cell, or a plant cell.
Preferably, such isolated nucleic acid molecules comprise genomic DNA which is
isolated using polymerase chain reaction or hybridisation approaches, based
upon the
nucleotide information disclosed in SEQ ID NO: <400>1.
In the performance of this embodiment of the invention, the genetic sequence
set forth
in SEQ ID NO: <400>1, or a derivative or analogue thereof, is labelled with a
reporter
molecule capable of producing an identifiable signal (eg. a radioisotope such
as 32P,
or 3~S, or a biotinylated molecule} to facilitate its use as a hybridisation
probe in the
I S isolation of related promoter sequences which are at least capable of
conferring,
activating or otherwise regulating gene expression in seeds.
An alternative method contemplated in the present invention involves
hybridising a
nucleic acid primer molecule of at least 10 nucleotides in length, derived
from SEQ ID
NO: <400>1 or a derivative or analogue thereof, to a nucleic acid "template
molecule",
said template molecule herein defined as genomic DNA, cDNA or RNA, or a
functional
part thereof. Specifrc nucleic acid molecule copies of the template molecule
are
amplified enzymatically in a polymerase chain reaction, a technique that is
well known
to one skilled in the art and described in detail by McPherson et al (1991 },
which is
incorporated herein by reference.
Preferably, the nucleic acid primer molecule or molecule effective in
hybridisation is
contained in an aqueous mixture of other nucleic acid primer molecules. More
preferably, the nucleic acid primer molecule is in a substantially pure form.
The nucleic acid template molecule may be in a recombinant form, in a virus
particle,
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bacteriophage particle, yeast cell, animal cell, or a plant cell.
A second aspect of the present invention is directed to a genetic construct
comprising
a promoter sequence which is at least capable of conferring, increasing or
otherwise
regulating expression of a structural gene to which it is operably connected
in a plant
seed and preferably in the endosperm tissues of the seed, such as the starchy
endosperm andlor the mature aleurone, wherein said promoter sequence
preferably
comprises the nucleotide sequence set forth in SEQ ID N4: <400>1, or a
functional
derivative, part, fragment, or homologue thereof.
The present invention extends to genetic constructs in which the genetic
sequence
of the invention, or a functional derivative, part, fragment, homologue, or
analogue
thereof, is operably linked to a structural gene sequence. The invention is
not to be
limited by the nature of the structural gene sequence contained in such
genetic
constructs.
In one embodiment, the structural gene sequence is a reporter gene, such as
but not
limited to the green fluorescent protein (gfp) gene, the ~3-glucuronidase
gene, the
chloramphenicol acetyl transferase gene, or the firefly luciferase gene,
amongst others.
In a further embodiment, the structural gene encodes a BASI peptide,
polypeptide,
oligopeptide or protein or enzyme. This embodiment of the invention is
particularly
useful for the purpose of over-expressing the BASI protein in the seeds of
developing
grains, in particular wheat grains or other grains which are subject to pre-
harvest
sprouting, with a view to inhibiting inappropriate a-amylase gene expression
leading
to such pre-harvest sprouting. In such applications, it may be necessary to
boost the
activity of the promoter sequence set forth herein by including additional
regulatory
sequences as described supra.
In a further embodiment, the structural gene encodes a peptide, polypeptide,
oligopeptide or protein or enzyme, such as but not limited to enzymes involved
in the
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malting process, for example high pl a-amylase, low pl a-amylase, EII-(1-3,1-
4~~3-
glucanase, Cathepsin ~3-like proteases, a-glucosidase, xylanase and
arabinofuranosidases, amongst others or alternatively, a seed storage protein
selected
from the list comprising glutenins, glutelins, giiadins, hordeins and zeins,
amongst
others, or alternatively, proteins capable of influencing the nutritional
and/or functional
quality of grains, such as those that bind important minerals (eg. hemoglobins
and
ferritins) or are rich in essential amino acids such as lysine, amongst
others.
Structural genes which may conceivably be placed in operable connection with
the
promoter sequence of the invention also include those structural genes that
are
involved in the metabolism of the seed and praduce any product, bi-product or
intermediate of the metabolism of the seed of a plant, including but not
limited to amino
acids, oils (i.e. fatty acids), starch and fibre amongst others.
In a further alternative embodiment, the structural gene sequence may be a
ribozyme,
abzyme, antisense or co-suppression molecule which targets the expression of a
seed-
expressible gene. According to this embodiment, expression of such a
structural gene
under the control of the promoter sequence of the invention will partially or
completely
reduce, delay or inhibit the expression of said structural gene in the seed,
in particular
in the endosperm cells.
Wherein the structural gene being targeted is normally expressed in more than
one cell
type, the expression of said structural gene under control of the promoter
sequence
of the invention may further result in said gene being expressed in a cell-
type or tissue-
type specific pattern, in all cells other than seed cells of the plant.
Accordingly, the
present invention extends to a method of expressing a structural gene in a
cell-type
or tissue-type specific manner, in cells other than seed cells.
The genetic construct according to this aspect of the invention may further
comprise
a transcription termination sequence, placed operably in connection with the
structural
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gene sequence.
In an alternative embodiment, the transcription termination sequence is placed
downstream of the promoter sequence of the invention, optionally spaced
therefrom
by a nucleotide sequence which comprises one or more restriction endonuclease
recognition sites, to facilitate the insertion of a structural gene sequence
a~s
hereinbefore defined between said promoter sequence and said transcription
termination sequence.
The term "terminator" rafers to a DNA sequence at the end of a transcriptional
unit
which signals termination of transcription. Terminators are 3'-non-translated
DNA
sequences containing a polyadenylation signal, which facilitates the addition
of
polyadenylate sequences to the 3'-end of a primary transcript. Terminators
active in
cells derived from viruses, yeasts, moulds, bacteria, insects, birds, mammals
and
plants are known and described in the literature. They may be isolated from
bacteria,
fungi, viruses, animals and/or plants.
Examples of terminators particularly suitable for use in the genetic
constructs of the
present invention include the nopaline synthase (NOS) gene terminator of
Agrobacteriurn tumefaciens, the tumor morphology large (tm~ gene terminator of
Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus
(CaMV) 35S
gene, the ADP-glucose pyrophosphorylase gene terminator (t3'bt2) derived from
Oryza
saliva, the zero gene terminator from Zea mays, the HMW glutenin gene
terminator
derived from Triticum aestivum, the Rubisco small subunit (SSU) gene
terminator
sequences, subclover stunt virus (SCSV) gene sequence terminators, any rho-
independent E. coli terminator, amongst others.
Alternatively or in addition, the BAS/ 3'utr (i.e. 3' untranslated region of
the BAS/
gene; SEQ lD NO: <400> 1 T) may be used, particularly to provide for optimum
stability
of the mRNA encoded by the structural gene placed under control of the
promoter of
the invention. For example, the modulation of expression by phytohormones
under
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control of the BASI promoter may be enhanced by including the BASI 3' ufr
downstream of the translation stop codon of the structural gene, with or
without
additional transcription termination sequences placed downstream thereof.
Those skilled in the art will be aware of additional promoter sequences and
terminator
sequences which may be suitable for use in performing the invention. Such
sequences
may readily be used without any undue experimentation.
The genetic constructs of the invention may further include an origin of
replication
sequence which is required for replication in a specific cell type, for
example a bacterial
cell, when said genetic construct is required to be maintained as an episomal
genetic
element leg. plasmid or cosmid molecule) in said cell.
Preferred origins of replication include, but are not limited to, the f1-on
and colE1
origins of replication.
In a further alternative embodiment, the genetic construct of the invention
further
comprises one or more selectable marker gene or reporter gene sequences,
placed
operably in connection with a suitable promoter sequence which is operable in
a plant
cell and optionally further comprising a transcription termination sequence
placed
downstream of said selectable marker gene or reporter gene sequences.
As used herein, the term "selectable marker gene" includes any gene which
confers
a phenotype on a cell in which it is expressed to facilitate the
identification and/or
selection of cells which are transfected or transformed with a genetic
construct of the
invention or a derivative thereof.
Suitable selectable marker genes contemplated herein include the ampicillin
resistance
(Amp'), tetracycline resistance gene (Tc~), bacterial kanamycin resistance
gene
(Kan'), phosphinothricin resistance gene, neomycin phosphotransferase gene
(nptll ),
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hygrornycin resistance gene (hph), a-glucuronidase (GUS) gene, chioramphenicol
acetyltransferase (CAT) gene and luciferase gene, amongst others.
Those skilled in the art will be aware that the choice of promoter for
expressing a
selectable marker gene or reporter gene sequence may vary depending upon the
level
of expression required andlor the species from which the host cell is derived
and/or the
tissue-specificity or development-specificity of expression which is required.
Examples of further promoters suitable for use in genetic constructs of the
present
invention include promoters derived from the genes of viruses, yeasts, moulds,
bacteria, insects, birds, mammals and plants which are capable of functioning
in
isolated plant cells or whole organisms regenerated therefrom, including whole
plants.
The promoter may regulate the expression of the selectable marker gene or
reporter
gene constitutively, or differentially with respect to the tissue in which
expression
occurs or, with respect to the developmental stage at which expression occurs,
or in
response to external stimuli such as physiological stresses, pathogens, or
metal ions,
amongst others.
Examples of promoters include the CaMV 35S promoter, NOS promoter, octopine
synthase (OGS) promoter, Arabidopsis thaliana SSU gene promoter, napin seed-
specific promoter, P32 promoter, BK5-T imm promoter, lac promoter, tac
promoter,
phage lambda AL or ~ promoters, CMV promoter (U.S. Patent No. 5,168,062), T7
promoter, IacUV5 promoter, SV40 early promoter (U.S. Patent No. 5,118,627),
SV40
late promoter (U.S. Patent No. 5,118,627), adenovirus promoter, baculovirus
P10 or
polyhedrin promoter (U.S. Patent Nos. 5,243,041; 5,242,687; 5,266,317;
4,745,051;
and 5,169,784), and the like. In addition to the specific promoters identified
herein,
cellular promoters for so-called housekeeping genes are useful.
Those skilled in the art will be aware of additional promoter sequences and
terminator
sequences which may be suitable for use in performing the invention. Such
sequences
may readily be used without any undue experimentation.
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A still further embodiment contemplates a genetic construct which further
comprises
one or more integration sequences.
As used herein, the term "integration sequence" shall be taken to refer to a
nucleotide
sequence which facilitates the integration into plant genomic DNA of a
promoter
sequence of the invention with optional other integers referred to herein.
Particularly preferred integration sequences according to this embodiment
include the
left border {LB) and right border (RB) sequences of T-DNA derived from the Ti
plasmid
of Agrobacterium tumefaciens or a functional equivalent thereof.
A further aspect of the present invention provides a transfected or
transformed cell,
tissue, organ or whole organism which expresses a recombinant polypeptide or a
ribozyme, antisense, gene-targetting molecule, gene-silencing molecule or co-
suppression molecule under the control of the promoter sequence herein
described.
The organ according to this embodiment is preferably a plant seed that
comprises cells
or tissues, more preferably endosperm cells or tissues such as aleurone
starchy
endosperm, which express the recombinant polypeptide or a ribozyme, antisense,
gene-targetting molecule, gene-silencing molecule or co-suppression molecule
under
the control of the promoter sequence of the invention. Similarly, the whole
plant is
preferably a plant that comprises such plant seed or seed cells or seed
tissues.
Preferably, the transfected or transformed cell or tissue is a plant seed cell
and more
preferably an endosperm.
This aspect of the invention clearly encompasses a transgenic plant such as a
crop
plant, transformed with a recombinant DNA molecule which comprises at least a
promoter sequence which is at feast 20% identical to SEQ ID NO: <400>1 or
alternatively, a genetic construct comprising said promoter sequence as herein
described.
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A chimeric gene comprising the isolated promoter of the present invention or a
genetic
construct comprising same may be introduced into a cell by various techniques
known
to those skilled in the art. The technique used may vary depending on the
known
successful techniques for that particular organism.
Means for introducing recombinant DNA into bacterial cells, yeast cells, or
plant,
insect, fungal (including mould), avian or mammalian tissue or cells include,
but are not
limited to, transformation using CaCl2 and variations thereof, in particular
the method
described by Hanahan (1983), direct DNA uptake into protoplasts (Krens et al,
1982;
Paszkowski ef al, 1984), PEG-mediated uptake to protoplasts (Armstrong ef al,
1990),
electroporation (Fromm ef al., 1985), microinjection of DNA (Crossway et al.,
1986),
microparticle bombardment of tissue explants or cells (Christou et al, 1988;
Sanford
et al., 1987; Finer and McMullen, 1990; Finer et al., 1992; Sanford et al.,
1993;
Karunaratne et al., 1996; and Abedinia ef at., 1997}, vacuum-infiltration of
tissue with
nucleic acid, or T-DNA-mediated transfer from Agrobacterium to the plant
tissue (An
et al.1985; Herrera-Estrella et at., 1983a; 1983b; 1985).
For the transformation of monocotyledonous plants, microparticle bombardment
of
cells or tissues is preferred, particularly in cases where plant cells are not
amenable
to transformation mediated by A. tumefaciens. In such procedures,
microparticle is
propelled into a cell to produce a transformed cell. Any suitable ballistic
cell
transformation methodology and apparatus can be used in performing the present
invention. Exemplary apparatus and procedures are disclosed by Stomp et al.
(U.S.
Patent No. 5,122,4fi6) and Sanford and Wolf (U.S. Patent No. 4,945,050). When
using
ballistic transformation procedures, the genetic construct may incorporate a
plasmid
capable of replicating in the cell to be transformed.
Examples of microparticles suitable for use in such systems include 0.5 to 5
~cm gold
spheres. The DNA construct may be deposited on the microparticle by any
suitable
technique, such as by precipitation.
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Once introduced into the plant tissue, the expression of a stsvctural gene
under control
of the promoter sequence of the invention may be assayed in a transient
expression
system, or it may be determined after selection for stable integration within
the plant
genome.
Wherein the cell is derived from a multicellular organism and where relevant
technology is available, a whole organism may be regenerated from the
transformed
cell, in accordance with procedures well known in the art.
Plant tissue capable of subsequent clonal propagation, whether by
organogenesis or
embryogenesis, may be transformed with a genetic construct of the present
invention
and a whole plant regenerated therefrom. The particular tissue chosen will
vary
depending on the clonal propagation systems available for, and best suited to,
the
particular species being transformed. Exemplary tissue targets include leaf
disks,
pollen, embryos, scutella, cotyledons, hypocotyls, megagametophytes, callus
tissue
including embryogenic callus, existing meristematic tissue (e.g., apical
meristems,
axillary buds, and root meristems), and induced meristem tissue (e.g.,
cotyledon seed
and hypocotyl seed).
The term "organogenesis", as used herein, means a process by which shoots and
roots are developed sequentially from meristematic centres.
The term "embryogenesis", as used herein, means a process by which shoots and
roots develop together in a concerted fashion (not sequentially), whether from
somatic
cells or gametes.
The regenerated 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 seifed to give homozygous second
generation (or T2) transformant, and the T2 plants further propagated through
classical
breeding techniques.
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The regenerated transformed organisms contemplated herein 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 root stock grafted to an untransformed scion ).
The present invention clearly extends to the progeny plants and plant parts,
in
particular seed, derived from the transformed plants, the only requirement
being that
said progeny plants and plant parts also comprise the introduced promoter
sequence
of the invention.
The present invention is further described by reference to the accompanying
non-
limiting Figures and Examples.
EXAMPLE 1
Isolation of the BAS/ promoter
The BAS/ promoter sequence was isolated from barley leaf tissue using the
Universal
Genome Walker kit supplied by CLONTECH Laboratories, USA, essentially as
described by the manufacturer. Briefly, the procedure involves the digestion
of DNA
with specific enzymes, the addition of adaptors and nested PCR using a first
primer
designed to anneal to the BAS/ gene downstream of the promoter sequence and a
second primer designed to anneal to the adaptor sequence, to amplify the
intervening
promoter sequences.
in particular, genomic DNA was isolated from leaf tissue of Hordeum vulgate cv
Grimmett and digested with restriction enzymes as indicated in Table 2 to
produce
gene fragments of pre-defined size classes following amplifcation. The
following
primers were used to amplify the BAS/ promoter sequence:
GSP1:BASIPROMG: 5'-GCGGTTGGCCGAGAGGACGTAGTAGTTG-3' (set
forth herein as SEQ lD NO: <400> 1$); and
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GSP2:BASIPROMGN 5'-CGCGAGAGGGCGGTGCTGGCCAGAATAAGG-3'(set
forth herein as SEQ 1D NO: <400> 19).
TABLE 2
Restriction PCR fragments (approximate size in base
Enzyme pairs)
Dral 550
EcoRV 400
Stul no band
Scal 1200
Pvul I 1600
The Dral-fragment (Table 2) and the EcoRV-fragment (Table 2) possibly
consisted of
450 by and 300 by respectively of the BASI promoter sequence and may nofi
contain
all the regulatory elements of the promoter. The Pvull-fragment (1600bp) was
amplified in low amounts in the PCR reaction. The low yield of the Pvull-
fragment
would have hindered the subsequent cloning procedures. The Scal-fragment
(1200bp)
was amplified in sufficient amounts and was thus selected for cloning (Figure
2).
The cloning of the Scal-fragment involved ligation of the Scal-fragment into a
suitable
vector and transformation of Escherichia coli DHSa-cells for plasmid
preparation. In
this regard, the Genome walker kit uses Tth as one of the DNA-polymerases in
the
PCR-amplifcation reaction, to preferentially add an extra deoxy-adenosine
nucleotide
at the 3'-end of the PCR-amplified product (i.e. to produce an "A-overhang"),
thereby
facilitating the cloning of fragments with A-overhang, in a suitable vector
having T-
overhangs.
The vector pGEM-Teasy from Promega (pGEM-Teasy kit) was used to ligate the
Scal-
fragment of the BASI promoter. The transformation of E-coli DHSa-cells was
carried
out and a number of white colonies were screened by PCR and by mini-
preparation
of the plasmid. in successful ligation and transformation reactions, it was
expected
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that a 1500 by fragment, including the Scal fragment, would be detected in the
PCR
reaction or alternatively, a recombinant plasmid having a size of 4200bp would
be
obtained from the cloning reaction. However, results of the screening
experiments
indicated that the Scal-fragment was not cloned. This experiment was repeated
a
S number of times with appropriate modifications, and although the control DNA
fragment (supplied in the pGEM-Teasy kit) was cloned the Scal-fragment was not
cloned successfully using this approach.
Proceeding on the basis that the Scat-fragment may not have contained the
requisite
A-overhangs at one or both it's 3'-ends to facilitate ligation into the
vector, an attempt
was made to A-tail the Scat-fragment according to the manufactures protocol.
The A-
tailed Scal-fragment was then successfully cloned into the pGEM-Teasy vector.
A
number of white colonies were screened and three (ID No. P1 C5, P1 C17 and P1
C4)
were found to contain the expected recombinant plasmid containing the Scal-
fragment.
EXAMPLE 2
Nucleotide sequence of the Scat-fragment containing the BASI promoter
Plasmid DNA from the clone P1 C5 described in the preceding Example was used
as
a template for sequencing the BAS! promoter.
Sequencing reactians were carried out using the ABI-big dye kit according to
manufacturers protocol.
The nucleotide sequence of the BASI promoter is set forth in SEQ ID NO:
X400>1.
The sequencing data confirmed that the Scal-fragment contained 56bp of the non-
translatable region of the BASI gene as published by Leah and Mundy (1989),
suggesting that the Scal-fragment contained the linked promoter sequence of
the
BASI gene.
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EXAMPLE 3
Mapping of the BASI promoter sequence for putative DNA-elements
The sequence of the Scal-fragment (1033bp of nucleotide sequence upstream from
the start codon of the BASS gene; SEQ ID NO: <400>1 ) was mapped to identify
the
putative TATA-bax , using WEB-ANGIS to search the Transfec database . A
mapping
programme on WEB ANGIS was also used to identify the location of other
putative cis-
acting DNA-elements. This resulted in the identification of the endosperm-box
and the
gibberellic acid and abscisic acid responsive elements (GARE and ABRE) [Figure
1].
EXAMPLE 4
Preparation of deletion constructs and plasmid maps
A total of six constructs contafining the BASI promoter fused to the green
fluorescent
protein gene {gfp) in the plasmid pGEM3zf(+!-), were prepared using standard
procedures. The construct comprising the full-length promoter sequence was
designated pA57 and tem~inated at position -959 relative to the transcription
start site
of the BASI gene. Five of these constructs comprised 5'-deletions of the
promoter
sequence set forth in SEQ ID NO: <400>1, ending at positions -592 (pA58), -423
(pA61 ), -199 (pA64), -147 {pA67) and -99 (pA70), relative to the
transcription start site
of the BASI gene {Figures 5-11 ).
The sequences of each of the truncated BASI promoters in the deletions were
compared to that of the BASI promoter sequence in pA57 and were found to be
identical. All the six constructs were linearised to verify their size.
The above constructs were prepared such that the ATG of the gfp gene followed
immediately after the nucleotide at position +74 of the BASI promoter. Thus
the
context sequence of the BAST promoter was immediately upstream of the
translation
start codon of the gfp gene.
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EXAMPLE 5
Endosperm-specifc expression of the gfp gene under the control of the BASI
promoter
Particle bombardment of aleurone Payers derived from barley cv. Himalaya, was
carried
out to analyse transient expression of the gfp gene under control of the 1033
by BASI
promoter. Pericarp derived from immature wheat seeds and leaf tissue of barley
(Hordeum vulgare cv. Grimmett) were also transfected as control tissues.
In control experiments, the plasmid pUbi.gfp (pA53) (Christensen and Quail,
199fiJ
containing the Ubiquitin promoter driving gfp gene expression, was also used
to
transfect the same tissues (i.e. leaf, pericarp and aleurone tissue).
The genetic construct pDP687, containing genes of transcription factors
required for
the synthesis of the anthocyanin pigment, was used as reporter construct to
standardise and check the efficiency of each transfection (Bowen; 1992). The
plasmid
pDPfi87 was precipitated onto tungsten particles (1.2 ~cm) in a ratio of 1:2
with all other
constructs.
For the transfection of leaf tissue, barley plants (2 weeks old) growing in
soil, in 50m1
falcon tubes, were placed in the particle bombardment chamber and a defined
area
of the leaf was bombarded. More precise details of the conditions used for
biolistic
bombardments of tissues in transfection assays are presented in Table 3.
Data presented in Figures 12 to 15 indicate that the ubiquitin promoter is
capable of
conferring expression on the gfp structural gene in the aleurone cells of
barley, and
in the leaf and pericarp tissue of wheat seeds.
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Data presented in Figures 18 to 19 indicate that the 1033 by BASI promoter is
capable of conferring expression on the gfp structural gene in the aleurone
cells of
barley, but not in the Leaf, or in the pericarp tissue of wheat seeds.
TABLE 3
Tissue vacuum pressure Distance Distance
Bombarded (kpa) (kpa) from filter from nylon
holder to mesh to
tissue (cms) tissue (cms)
aleurone 91 1650 13 5.5
10leaf 91 1000 13 5.5
pericarp 91 1000 13 5.5
The results of transient expression assays were quantified and data are
presented in
Table 4.
TABLE 4
Construct Transient
bombarded with expression
of green
fluorescent
protein
(GFP)
or anthocyanin
(Antho).
pDP687 (ratio
2~1) aleurone pericarp leaf
GFP Antho GFP Antho GFP Antho
pA53 yes yes yes yes yes yes
pA57 yes yes no yes no yes
pA58 yes yes no yes no yes
pA61 yes yes no yes no yes
pA64 yes yes no yes no yes
pA67 yes yes no yes no yes
pA70 low yes no yes no yes
The data presented in Table 4 confrm the conclusion from histochemical data
present
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in Figures 16 to 19 that the 1033 by BAS) promoter sequence in the genetic
construct
pA57 directs gfp gene expression in the aleurone cells of barley seeds, nut
not in the
leaf tissue of barley or the pericarp tissue of wheat.
The five BASI promoter deletion constructs designated pA58, pA61, pA64, pA67
and
pA70 (Figures 6 to 11 ) were also tested for their ability to confer gfp gene
expression
in barley aleurone tissue, barley leaf tissue, and wheat pericarp tissue. Data
presented
in Table 4 indicate that the plasmids designated pA57, pA58, pA61, pA64, pA67
and
pA70 are able to direct expression of the gfp gene in mature aleurone tissue
of barley,
I O but not the leaf tissue of barley or the pericarp of immature wheat seeds.
However, low
levels of aleurone GFP expression were observed for plasmid pA70 (Table 4).
Accordingly, the elements required for satisfactory aleurone-specific
expression are
present within the region from position -147 to position X74 of the BASI gene
(i.e.
nucleotides 813 to 1033 of SEQ ID NO: <400>1 ).
EXAMPLE 6
Stable expression of the gfp gene directed by the BASI-promoter sequence
I. Transformation of wheat
Wheat is transformed by particle bombardment essentially according to
Karunaratne
et.ai (1996), with the following modifications:
Young caryopses are dissected from spikes of Triticum aestivum L. cv. Hartog,
approximately 12 to 14 days post anthesis and surface sterilised with 10%
Dairy-Chlor
(100gIL available chlorine). immature embryos are isolated and cultured in
dark on MS
medium (Murashige and Skoog, 1962) supplemented with 10 pM 2,4-
dichlorophenoxyacetic acid. After 7 days of culture, the immature embryos are
subjected to particle bombardment.
A plasmid containing a first ubiquitin promoter driving expression of the bar
gene and
the BASI promoter driving expression of the gfp gene is precipitated onto
tungsten
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particles (1.2 Nm) as described by Finer and McMullen (1990) with the
following
modifications. A 500mglml suspension (25 NL) of tungsten particles (1.2 pm) in
distilled water is made in an Eppendorf tube, followed by the stepwise
addition of the
following: 5 pL of plasmid DNA (5 erg); 25 uL of calcium chloride (2.5M}; and
10 pL of
spermidine (0.1 M). The contents in the tube is mixed by finger vortexing and
kept on
ice. After 5 min, 30 NL of the supernatant is discarded and 300 NL ethanol
(90%) is
added, and the suspension is kept on ice after mixing the contents. After 1
min, the
tube is centrifuged and alt the supernatant discarded. This ethanol wash is
repeated
once, and the DNA-coated tungsten is finally suspended in 30 pL of ethanol
(90%).
The DIVA-coated tungsten particles (2 pL) are delivered to target tissue using
a particle
inflow gun {Finer ef al., 1992). The target tissue is placed on a shelf 14 cm
from the
screen of the filter holder, which carries a suspension of plasmid-DNA coated
tungsten
particles. After bombardment, the tissue is transferred to the original medium
and
cultured in the dark for 2 months with fortnightly subculture.
Embryogenesis, leading to plant regeneration, is stimulated by transferring
clumps of
embryogenic callus to MS medium devoid of hormones and containing
Phosphinothricin (PPT) at a concentration of Smg/L. After two weeks, PPT-
resistant
plants and callus are transferred to fresh medium and subcultured weekly. PPT-
resistant plants, 4-5 cm in length, are transferred to soil and kept under
water mist for
two weeks. Plants are then transferred to larger pots and kept in the
glasshouse under
day and night temperature of 22° C and 19° C, respectively.
Transformed plants are analysed to determine the level of expression of the
gfp gene
in the seeds, and the specificity of expression in the aleurone and endosperm
cells.
11. Transformation of barley
Hordeum vuigare cv Golden Promise is transformed by Agrobacterium tumefaciens
mediated transformation, using a binary genetic construct comprising the BAS/
promoter driving gfp gene expression, essentially according to Tingay et al
(1997).
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Expression of GFP under the control of the BASI promoter is analysed in the
leaf, and
aleurone and endosperm cells, to confirm the expression of the gfp gene under
control
of the BASI promoter in the seed, including the immature endosperm and mature
aleurone tissues.
Ill. Transformation of rice.
Rice plants were transformed particle bombardment essentially according to
Abedinia
ef al. (1997).
Briefly, mature caryopses of rice were surface-sterilised using 1 % (v/v)
sodium
hypochlorite solution for 20 min, followed by washing five times in sterile
distilled water.
The sterile caryopses were then incubated on callus induction medium (MSC) for
9 0
days after which the radicle and plumule (formed as a part of the germination
process)
and the swollen embryo axis was discarded. The scutellum with the callus was
separated and cultured for a further twenty days on fresh MSC medium.
The embryogenic calli were bombarded according to Klein et al (1987} using a
particle
inflow gun (Finer et al., 1992). The embryogenic calli were transferred to
medium
containing osrnoticum (MSCO). After four hours on MSCO medium, the embryogenic
calli were subjected to particle bombardment and then left on the same medium
for a
further 16 to 20 hr.
After culture on MSCO medium, callus was transferred to selection medium
(MSCS).
After 15 days on MSCS medium, the surviving callus was transferred to the
proliferation medium (MSP) and cultured for one month with subculture every 10
days.
The surviving calli and somatic embryos from the MSP medium were transferred
to
regeneration medium (MSR). Regenerated plantlets were transferred to 1/2 MS
medium without hormones for additional growth of shoot and roots. Plants, 10
to 15
cms in length, were transferred to soil and kept for a week in the glasshouse
under
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water mist. The plants were later transferred to potting mix and the pots kept
haif
submerged in water.
Putative transformed rice plants that were resistant to hygromycin were tested
for the
presence of the BASI promoter and the gfp gene by PCR essentially according to
Thompson and Henry (1995), using the following primers:
BASIPROMSC: SEQ ID NO: <400> 27:
5'- ATCGGAAGCTTACTGGGCTCGAAACTAAAATAAGAACATG-3'; and
SGFPR1: SEQ ID NO: <400> 28:
5'-GAAGTCGTGCTGCTTCATGTGG-3'.
The priming sites of the primers BASIPROMSCS' and SGFPR1 are in the 1033 by
BAS1 promoter (SEQ lD NO: <400> 1 ) and the gfp gene respectively, both of
which are
present in plasmid pA57. Use of 0.5 NM each of the above primers amplifes a
1300bp
DNA fragment comprising 1033bp of BASI promoter plus 176bp of the gfp gene,
thereby ensuring the detection of both the BASI promoter and the gfp gene.
Amplification reactions were performed using a Perkin Elmer Cetus 9600 under
the
following cycling conditions:
Cycle 1: 94°C for lmin;
Cycles 2-11: 94°C for 30s, followed by 53°C for 30s, followed
by 72°C for 45s
per cycle; and
Cycles12-41: 94°C for 30s, followed by 50°C for 30s, followed
by 72°C for 45s
per cycle.
Amplifcation products were electrophoresed on 1.0% (wlv) agarose gels
containing
ethidium bromide. Data obtained confrm the presence of the BASI promoterlgfp
gene
construct.
The integration of the chimeric the BAS! promoter/gfp gene into the genome of
rice is
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a
-S3-
also confirmed using Southern blot analysis (Sambrook et a1.,1989). High
molecular
weight DNA (at least 90% of total DNA) is digested using four or six base-
cutting
restriction enzymes, preferably a restriction enzyme having no restriction
site
substrates in the introduced plasmid DNA, to provide for the detection of
integration
of the the BASI promoterlgfp gene, as well as for a determination of the
number of
integration events in the rice genome. Preferably, the restriction enzymes
EcoRV or
Nrul or Spel, are used to digest genomic DNA containing the introduced plasmid
pA57.
Digested DNA, transferred onto a nylon Hybond membrane, is hybridised to
radiolabelled probe and the signal detected using standard procedures. Data
confirm
the presence of the introduced transgene.
The transcription of the introduced the BAS! promoter/gfp gene, integrated
into the
genome of rice, is detected using northern blot hybridisation according to
standard
procedures (Sambrook ef al., 1989; Ausubel et al., 1987). Preferably, good
quality
(non-sheared) RNA is produced using the TRI-reagent method (GIBCO-BRL, USA).
RNA (10 to 20 pg) is resolved on a 1 % (wlv) agarose/formaldehyde gel and
transferred
onto nylon membrane, and hybridised using radio-labelled DNA or riboprobe
containing nucleotide sequences complementary to the sense strand of the gfp
structural gene. Data obtained confirm the expression of the gfp gene under
control
of the BASI promoter in transgenic rice seed.
EXAMPLE 7
Hormone-regulated expression of the gfp gene under the control of the
BASI promoter
The regulation of expression of the BASI gene in immature endosperm and mature
aleurone tissue of barley is mediated by the two phytohormones ABA and GA. It
is
known that ABA up-regulates the synthesis of BAS1 protein and GA down
regulates
it in both the endosperm and aleurone tissues.
To demonstrate efficacy of the BASI promoter in modulating the expression of a
structural gene in plants, a genetic construct comprising the full-length BASI
promoter
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operably connected to the gfp structural gene and placed upstream of the BASI
3'utr
is introduced into wheat, barley and rice plants as described in the preceding
examples. The expression of GFP under control of the BAS! promoter is
determined
following the exogenous application of GA alone, ABA alone, or ABA plus GA, in
accordance with standard procedures, such as those described in International
Patent
Application No. PCT/AU96/00383. Results of these experiments confirm the
hormone-
responsiveness of the full-length BASI promoter sequence.
BIBLIOGRAPHY
1. Abedinia et al., (1997) J. Plant Physiol. 24: 133-141.
2. An et aL (1985) EMBO J. 4:277-284.
3. Armstrong, C.L., et al. (1990). Plant Cell Reports 9: 335-339.
4. Ausubel et al. (1987). In: Current Protocols in Molecular Biology. Wiley
lnterscience (ISBN 047150338).
5. Bowen, B. (1992) In: Anthocyanin genes as visual markers in transformed
maize tissues: GUS protocols, Using the GUS gene as a reporter of gene
expression. Academic Press.
6. Chalfie,M. et al (1994) Science 263: 802-805.
7. Christensen A.H. and Quail P.H. (1996) Transgenic Research, 5:213-218.
8. Christou, P., et al. (1988). Plant Physiol 87, 671-674.
9. Cormack, B. et a! (1996) Gene 173: 33-38.
10. Crossway et al. (1986) MoI. Gen. Genet. 202,179-185.
11. Finer and McMullen (1990) Plant Cell Rep. 8: 586-589.
12. Finer et aL (1992) Plant Cell Rep. 11: 323-328.
13. Fromm et aL (1985) Proc. Natl. Acad. Sci. (USA) 82: 5824-5828.
14. Gubler, F. and Jacobsen, J.V. (1992) Plant Cell 4:1435-1441
15. Gubler, F. et al. (1995) Plant Cell l: 1879-1891.
16. Hanahan, D. (1983) J. Molec.Biol. 166: 557-560.
17. Herrera-Estrella et al. (1983a) Nature 303: 209-213.
18. Herrera-Estrella et al. (1983b) EMBO J. 2: 987-995.
19. Herrera-Estrella et al. (1985) In: Plant Genetic Engineering, Cambridge
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University Press, NY, pp 63-93.
20. Inouye, S. and Tsuji, F.I. (1994) FEBS Letts. 341: 277-280.
21. Karunaratne ef al. (1996) Aust. J. Plant Physiol. 23: 429-435.
22. Klein, T.M., et al. (1987) Nature 327(7): 70-73.
23. Krens, F.A., et al. (1982). Nature 296: 72-74.
24. Lanahan, et al.(1992) Plant Cell 4: 203-211.
25. Leah R. & Mundy J. (1989). Plant Molecular Biology 12: 673-682.
26. McPherson, M.J., et al. (1997 ) PCR, A Practical Approach , 1RL Press,
Oxford,
(ISBN 0-19-963196-4).
27. Murashige, T and Skoog, F. (1962). Physiol. Plant 15: 473-497.
28. Pazkowski et al. (1984) EMBO J. 3, 2717-2722.
29. Prasher, D.C. et al. {1992) Gene 111: 229-233.
30. Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Second
edn. New York: Cold Spring Harbor Laboratory Press.
31. Sanford, J.C., et al.(1987) Particulate Sci. Tech. 5: 27-37.
32. Sanford, J.C., ef aJ. (1993) Methods Enzymol. 217: 483-509.
33. Skriver, K., et aJ.(1991 ) Proc. Natl. Acad. Sci. (USA) 88:7266-7270.
34. Thompson, D., and Henry, R. (1995). Biotechniques, 19(3): 394-398.
35. Tingay, S., et aJ. (1997) Plant Journal 11(6): 1369-1376.
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<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 19
cgcgagaggg cggtgctggc cagaataagg 30
<210> 20
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 20
tcggaagctt actgggctcg aaactaaaat aagaacatg 39
<210> 21
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 21
acctattcat gactgaaacc tctgctggag gtcc 3q
<210> 22
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<900> 22
atcggaagct tcataccacc atcgaagatg ccctaag 37
<210> 23
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
CA 02343933 2001-03-23
WO 00/18926 PCTIAU99100823
-6-
<400> 23
atcggaagct tggaggatgt ggaacgcaga tagtgac 37
<210> 24
<211> 38
<212> DNA
<213> Artificial Sequence
<z2o>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 24
atcggaagct tggagtggta ggatataaca ttgctctg 38
<210> 25
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 25
atcggaagct tcggtgtacg cacttactgg atgccac 37
<210> 26
<211> 34
<212> DNA
<2i3> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 26
atcggaagct tcacatcacg caatccacca gaag 34
<210> 27
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 27
atcggaagct tactgggctc gaaactaaaa taagaacatg 40
<210> 28
<211> 22
<2I2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PROBE/PRIMER
<400> 28
gaagtcgtgc tgcttcatgt gg 22
