Note: Descriptions are shown in the official language in which they were submitted.
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Maize Glutamine Synthetase Gene Promoter
This application claims priority to U.S. Serial No. 60/206,984, filed May 25,
2000,
which is hereby incorporated by reference.
BACKGROUND OF THE INVENTION
In higher plants, the maternal tissues that surround the developing sporophyte
play
vital and dynamic roles in seed development. See Thorne, J.H., Annu. Rev.
Plant Physiol.
36:317-343 (1985). During maize kernel development, the pedicel, the basal
maternal tissue,
acts as a conduit for the transfer of nitrogen and carbon compounds from the
vegetative
portions of the plant to the growing seed and modifies the contents of the
assimilate stream as
it passes through the seed-associated tissues. See Arruda et al.
Phytochemistry, 18:409-410
(1979); Lyznik et al., Maydica, 27:191-198 (1982). Assimilates are unloaded
from the
phloem and traverse the cells of the pedicel tissues via a symplastic route.
See Felker et al.,
Plant Physiol., 65:864-870 (I980). During the transfer from the vascular
tissues to the
endosperm of the developing sporophyte, sucrose is hydrolyzed to hexoses by
the action of
cell wall-associated invertase activities (Shannon et al., Plant PJzysiol.,
49:203-206 (1972))
and transport amino acids are metabolized and re-synthesized in such a manner
as to make
glutamine the main amino acid taken up by the basal endosperm transfer cells.
Glutamine is the predominant amino acid released from the pedicel (Porter et
al.,
Plant Plzysiol., 85:558-565 (1985)). Radiolabeling studies and other
corroborating evidence
suggest that the pedicel is the primary site of glutamine synthesis in the
maize kernel. See
Lyznik et al., Phytochenzistzy, 24:425-430 (1985); Muhitch, M.J.,
Plzytoclzenzistzy, 32:1125-
1130 (1993); Muhitch, M.J., .I Plant Physiol., 143:372-378 (1994); Muhitch,
M.J., Recent
Res. Devel., Phytochezn., 3:63-82 (1999). Consistent with these studies, the
pedicel contains
relatively high levels of glutamine synthetase activity (Enzyme Commission
number 6.3.1.2).
See Muhitch, M.J., PJzysiol. Plant, 74:176-180 (1988).
Glutamine synthetase (GS) catalyzes the assimilation of ammonia into glutamine
and
is a key enzyme in plant nitrogen metabolism. Within the maize kernel pedicel,
glutamine
synthetase consists of two isozymes (Muhitch, M.J., Plant Physiol., 91:868-875
(1989)), one
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of which appears to be unique to the maternally-associated seed tissues
(Muhitch et al., Plant
Playsiol., 107:757-763 (1995)). In higher plants, GS occurs as a family of
isozymes which are
differentially expressed in various organs and tissues. See McGrath et al.,
Plant J., 1:275-280
(1991); Lam et al., Plant Cell, 7:887-898 (1995). In most plants, there
appears to be a single
gene which encodes a plastidic GS, whereas the cytosolic counterparts are
encoded by small
gene families. See Cullimore et al., J. Mol. Appl. Genet., 2:589-599 (1984);
Tingey et al.,
EMBO J., 6:1-9 (1987); Sakabari et al., Plant Cell Playsiol., 33:49-58 (1992);
Li et al., Plant
Mol. Biol., 23:401-407 (1993). One of the cytoplasmic maize GS genes, namely
GS1_2,
recently has been identified as encoding the pedicel-specif c maize GS
protein. See Rastogi
et al., Plant Cell PlZysiol., 39:443-446 (1998).
The metabolic actions occurring within the basal kernel tissues may have
regulatory
implications in seed assimilation and development. For example, there is
indirect evidence to
suggest both that glutamine levels act as an indicator of overall nitrogen
abundance (Muhitch,
Recesat Res. Devel. Phytochem., 3:63-82 (1999)) and that glucose levels
regulate cell
differentiation within the seed itself. See Borisjuk et al., Plant J., 15:583-
591 (1998).
Consistent with the observations of high transport amino acid turnover within
the pedicel,
high GS activities are found in this region. See Lyznik et al.,
Phytochemistfy, 24:425-430
(1985); Muhitch, Physiol. Plant, 74:176-180 (1988). The predominant GS form
within the
pedicel is an unique, tissue-specific isozyme of GS. See Muhitch, Plant
Physiol., 91:868-875
(1989). Immunocytochemical studies, using a monoclonal antibody raised against
the
pedicel-specific GS isozyme, revealed that this isozyme was found not only
within the pedicel
tissues, but also within the lower surrounding pericarp. See Muhitch et al.,
Plant Pl2ysiol.,
107:757-763 (1995).
Due to the putative regulatory role of GS1_2 in pedicel metabolism,
identification of
the transcription regulatory region of GS~_2 would provide an invaluable tool
for affecting
seed assimilation and development. For example, cell wall invertase within
basal kernel
tissues has been shown to play an essential role in maize kernel development.
See Cheng et
al., Plant Cell, 8:971-983 (1996). Also, Borisjuk et al. have demonstrated a.
strong
correlation between glucose levels (a product of invertase) and cell mitotic
index,
differentiation and storage product accumulation in developing cotyledons of
Yicia faba. See
Borisjuk et al., Plant J., 15:583-591 (1998). In addition, pulse chase studies
using
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radioactive amino acids (Muhitch, Phytochenaistfy, 32:I I25-1130, (1993);
Muhitch, J. Plant
Physiol., 143:372-378 (1994)) and evidence that sugars and nitrogen levels
directly effect the
expression of storage protein genes (Grierson et al., Plant J., 5:815-826
(1994); Giroux et al.,
Plant Physiol., 106:X3-722 (1994)) suggest that alterations in the composition
of the
assimilate stream that reaches the developing sporophyte can be used to modify
seed yield
and quality. Such alterations could involve pedicel expression of metabolic
enzymes
involved in carbon and/or nitrogen metabolism (e.g., invertase or amino acid
transaminases)
that would alter the assimilate pools as they pass through the pedicel cells
on their way to the
developing kernel.
Furthermore, identification of the transcription regulatory region of GSI_Z
would
enable tissue-specific expression of antifungal proteins and other disease
resistance genes. For
example, endophytic pathogenic fungi, such as Fusaf-iuna monilifof°me,
grow through the
vegetative tissues and cob and are found in the pedicels of asymptomatic
kernels. These
pedicels appear to serve as both a survival structure for the fungus and as a
ready port of entry
when environmental conditions allow it to invade the embryo and endosperm and
produce
fumonisin mycotoxins (Bacon, et al., 1992). Ingress of pathogenic fungi
external to the seed
has been observed through naturally occurring small ruptures in the attachment
points of the
pedicel bracts (Smart, et al., 1990). Moreover, developing kernels infected by
fungi due to
mechanical or insect damage of the upper pericarp and endosperm, appear to
spread disease
to surrounding kernels by fungal growth down through the pedicel of the
infected kernels,
into the cob and out into the adjacent kernels via their pedicels (Smart, et
al., 1990). The
expression pattern of the GS~_Z gene, with its strong expression in the
pedicel parenchyma, the
subtending bracts and the pericarp, offers an especially appealing vehicle for
attempting to
increase the resistance of maize kernels to cob rotting fungi through tissue-
specific expression
of antifungal proteins and other disease resistance genes. In addition to
preventing yield loss
by pathogenic fungi, this strategy also should reduce the level of seed
mycotoxins, since fungi
appear to have to invade the endosperm and embryo from the surrounding
maternal tissues
before producing these deleterious compounds (Bacon, et al., 1992).
A need therefore exists for the elucidation of the transcription regulatory
region for
the GS ~ _2 gene.
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SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide the
transcription
regulatory region for the maize GS~_2 gene.
In accomplishing this and other objects of the invention, there is provided,
in
accordance with one aspect of the present invention, a DNA sequence which
represents the
transcription regulatory region of the GS1_2 gene. DNA constructs containing
the GS1_z
transcription regulatory region also are provided. Further, host cells
comprising such a
construct, where cells are iya vivo or in vitro, also are contemplated by the
present invention.
In other embodiments, methods of producing proteins are provided. Methods of
producing a plant with reduced mycotoxin levels, comprising transforming a
plant cell with a
recombinant DNA construct comprising a polynucleotide having the sequence of
the
transcription regulatory region of the GS~_Z gene also are provided. In still
other
embodiments, methods of increasing seed yield and quality in a plant of
interest, comprising
transforming a plant cell with a recombinant DNA construct comprising a
polynucleotide
having the sequence of the transcription regulatory region of the GS~_2 gene
are provided.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides the nucleotide sequence of the Maize GS1_2 transcription
regulatory
region, 5' untranslated region, exons 1, 2 and, in part, 3 (underlined in
bold) and
corresponding intronic regions. The 5' deletion start sites up through -664
are indicated with
arrows and are numbered relative to the transcription start site (+1).
Putative transcription
factor binding sites are underline and labeled.
Figure 2 provides the 644 by nucleotide sequence of the Maize GS~_2
transcription
regulatory region. Putative transcription factor binding sites are underline
and labeled.
Figure 3 illustrates the genomic organization of the GS~_Z gene and its
flanking
sequences. Exons are indicated by solid boxes, and the transcription start
site is designated as
+1. The direction and transcription termination of GS~_2 and its flanking
genes (CDC2 and
PGM~) are indicated by arrows.
Figure 4 schematically depicts a series of GS~_2 promoter/reporter gene
constructs.
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Figure 5 shows the normalized luciferase activity from transient expression of
a
deletion series of GSI_2 promoter-reporter gene constructs.
Figure 6 illustrates linker-scanning of the GS~_Z region from -72 to -34,
relative to the
putative transcriptional start site.
Figure 7 shows the histochemical GUS staining of maize kernels from plants
stably
transformed with pGS135 and from kernels from non-transformed control plants.
Figure 8 shows the histochemical GUS staining of maize kernels, leaves and
roots
from plants stably transformed with pGS 135.
Figure 9 provides a vector map of pGS 135.
Figure 10 provides a vector map of pGS 153.
Figure 11 shows the histochemical GUS staining of maize kernels transformed
with
the truncated GS1_2/GUS construct (pGS153) and of kernels from control, non-
transformed
maize plants.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention provides the transcription regulatory region for the
Maize GS~_Z
gene. See SEQ ID NO: 1 in Figure 2. The novel 644 by sequence, located
upstream of the
GS1_2 transcription start site, shares little homology with previously
identified GS promoters.
For example, the disclosed regulatory region has only 37% homology with the
GS3A
regulatory region of Pisum sativum (pea), 39% with the GS2 regulatory region
of Pisunz
sativum (pea), 39% with the GS15 regulatory region of Glycirae max (soybean),
38% with the
GS-gln-a regulatory region of Plaaseolus vulgaf°is (bean) and 36% with
the GS-gln-~3
regulatory region of Phaseolus vulgaris (bean). Moreover, the disclosed GS~_Z
transcription
regulatory region is the first regulatory region known to direct strong
protein expression in the
pedicel region as well as in the other maternal tissues of the corn kernel.
The GSI_2 transcription regulatory region's unique ability to drive maternal,
seed-
associated, tissue-specific expression of an operably linked gene can be used
to enhance seed
assimilation and development. Similarly, the inventive regulatory region can
be used to
increase disease resistance in plants by directing pedicel-specific expression
of antifungal
proteins and other disease resistance genes. Accordingly, the present
invention provides
recombinant DNA constructs and methods for affecting seed development and
disease
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resistance in plants. Methods of producing proteins, as well as transformed
host cells and
plants, also are provided.
In one embodiment of the present invention, the transcription regulatory
region of the
GS1-2 gene is found in a 664 by nucleotide sequence upstream of the
transcription start site
(See Figure 2). In various embodiments of the invention, it may be desirable
to include
additional nucleotide sequences obtained from the GSl-2 promoter recombinant
constructs.
Such additional sequences may include, but are not limited to, sequences
encoding
untranslated leaders of mRNA species, including, but not limited to, the 5'
nontranslated
leader of GS1-2, an intron, including, but limited to, the introns of the
native GSl-2 gene,
targeting sequences that target the gene of interest to the appropriate
subcellular
compartment, and a 3' untranslated sequence such as a polyadenylation signal.
In one such
an embodiment of the invention, the transcription regulatory region of the GS1-
2 gene is
comprised of the nucleotide sequence upstream of the transcription start site,
the native 5'
UTR and at least one of the introns associated with the GS 1-2 gene (see
Figure 1 ).
Heterologous sequences ligated downstream, whose expression will be under the
control of
the GSl-2 regulatory region, may require additional translational control
elements such as an
ATG start site, ribosome binding sites, etc. These can be supplied by the
attached gene of
interest itself in a transcriptional fusion, or, alternatively, the
heterologous sequences may be
ligated in frame in a translational fusion to produce recombinant fusion
proteins.
In other embodiments, truncated versions of the transcription regulatory
region of the
GS~_2 gene are provided. For example, the DNA sequence comprising the 72 by
upstream of
the transcription start site (See Figure 1) has been shown to effectively
direct high levels of
transcription. Recombinant DNA constructs comprising truncated versions of the
transcription regulatory region of the GS 1_2 gene are provided.
Definitions
Definitions are herein provided to facilitate understanding of the invention.
The terms "transcription regulatory region" and "regulatory region" refer to
the
section of DNA which regulates gene transcription. A regulatory region may
include a
variety of cis-acting elements, including, but not limited to, promoters,
enhancers and
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hormone response elements. Also, since introns and 5' UTR have been known to
influence
transcription, a transcription regulatory region can include such sequences.
The terms "gene" and "structural gene" refer to a DNA sequence that is
transcribed
into messenger RNA (mRNA) which is then translated into a sequence of amino
acids
characteristic of a specific polypeptide (protein).
The term "promoter" typically refers to a DNA sequence which directs the
transcription of a structural gene to produce RNA. Typically, a promoter is
located in the 5'
region of a gene, proximal to the transcription start site. If a promoter is
an inducible
promoter, then the rate of transcription increases or decreases in response to
an inducing
agent. In contrast, the rate of transcription is not regulated by an inducing
agent if the
promoter is a constitutive promoter.
The term "enhancer" refers to a genetic element related to transcription. An
enhancer
can increase the efficiency with which a particular gene is transcribed into
mRNA irrespective
of the distance or orientation of the enhancer relative to the start site of
transcription. The
enhancer effect is mediated through sequence-specific DNA binding proteins. An
enhancer
often is referred to as a "response element."
The term "complementary DNA" (cDNA) refers to a single-stranded DNA molecule
that can be formed from an mRNA template by the enzyme reverse transcriptase.
Typically, a
primer complementary to portions of mRNA is employed for the initiation of
reverse
transcription. Those skilled in the art also use the term "cDNA" to refer to a
double-stranded
DNA molecule derived from a single mRNA molecule.
The term "genomic DNA" refers to chromosomal DNA and can include introns. An
intron is an intervening sequence. It is a non-coding sequence of DNA within a
gene that is
transcribed into hnRNA but is then removed by RNA splicing in the nucleus,
leaving a
mature mRNA which is then translated in the cytoplasm. The regions at the ends
of an intron
are self complementary, allowing a hairpin structure to form naturally in the
hraRNA.
The term "expression" refers to the process by which a polypeptide is produced
from a
structural gene. The process involves transcription of the gene into mRNA and
the translation
of such mRNA into polypeptide(s).
The term "cloning vector" refers to a DNA molecule, such as a plasmid, cosmid,
phagemid, or bacteriophage or other virally-derived entity, which typically
has a capability of
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replicating in a host cell and which is used to transform cells for gene
manipulation. Cloning
vectors typically contain one or more restriction endonuclease recognition
sites at which
foreign DNA sequences may be inserted in a determinable fashion without loss
of an essential
function of the vector, as well as a marker gene which is suitable for use in
the identification
and selection of cells transformed with the cloning vector. Appropriate marker
genes
typically include genes that provide various antibiotic or herbicide
resistance. A variety of
markers are available to the skilled artisan.
The term "expression vector" refers to a DNA molecule comprising a cloned
structural gene encoding a foreign protein which provides the expression of
the foreign
protein in a recombinant host. Typically, the expression of the cloned gene is
placed under
the control of (i.e., operably linked to) certain regulatory sequences such as
promoter and
enhancer sequences. Promoter sequences may be either constitutive or
inducible.
The term "recombinant host" refers to a prokaryotic cell or an eukaryotic
cell, such as
a plant cell, which contains either a cloning vector or an expression vector.
This term is also
meant to include those cells that have been genetically engineered to contain
the cloned
genes) in the chromosome or genome of the host cell.
Obtaining the Transcription Regulatory Region of the GSl_z Gene
A y EMBL-3 maize genomic library was screened with a GSI_2 gene-specific probe
using standard hybridization techniques. Eighteen putative positive clones
were isolated.
Detailed restriction mapping and DNA analysis revealed that five clones
contained the 5'
upstream promoter region. Two HindIII fragments, 3 and 4.5 kb, hybridizing to
5' and 3'
end-specific probes respectively, were subcloned from two genomic clones.
Partial
sequencing revealed that these two fragments collectively contained the entire
GS1_2 gene and
that the 3 kb HindIII fragment contained more than 2 kb of sequence 5' to the
translation start
site.
The isolated polynucleotides of the present invention can be obtained in
accordance
with the teachings herein by using (a) synthetic techniques, (b) standard
recombinant
methods, or (c) purification techniques, or combinations thereof, that are
well-known in the
art. For example, the isolated polynucleotides of the present invention can be
prepared by
direct chemical synthesis using the solid phase phosphoramidite triester
method (Beaucage
_g_
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and Caruthers, Tetf°a. Letts. 22(20):1859-1862 (1981)); an automated
synthesizer
(VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984)); or the solid
support method
of U.S. Patent No. 4,458,066. Chemical synthesis generally produces a single
stranded
oligonucleotide. This can be converted into double stranded DNA by
hybridization with a
complementary sequence, or by polymerization with a DNA polymerise using the
single
strand as a template. One of skill in the art will recognize that while
chemical synthesis of
DNA is limited in terms of length, longer sequences may be obtained by the
ligation of
shorter sequences.
Alternatively, the inventive polynucleotides can be obtained by recombinant
methods
using mutually priming long oligonucleotides. See e.g. Ausubel et al., (eds.),
CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1990). Also, see
Wosnick et al., Gene 60:115 (1987); and Ausubel et al. (eds.), SHORT PROTOCOLS
IN
MOLECULAR BIOLOGY, 3rd Edition, (John Wiley & Sons, Inc. 1995). Established
techniques using the polymerise chain reaction provide the ability ' to
synthesize
polynucleotides at least 2 kilobases in length. Adang et al., Plant Molec.
Biol. 21:1131
(1993); Bambot et al., PCR Methods arid Applications 2:266 (1993); Dillon et
al., "Use of the
Polymerise Chain Reaction for the Rapid Construction of Synthetic Genes," in
METHODS
IN MOLECULAR BIOLOGY, Vol. 15: PCR PROTOCOLS: CURRENT METHODS AND
APPLICATIONS, White (ed.), pages 263-268, (Humana Press, Inc. 1993);
Holowachuk et
al., PCR Methods Appl. 4:299 (1995).
Functional Variants
One skilled in the art will recognize that certain changes to the composition
of the
GS~_2 regulatory region will not disrupt its regulatory function. Since
transcription regulation
is limited to a few, discrete sequences within the regulatory region, base
changes in non-
critical sequences will produce minimal changes in gene expression. Functional
variants can
be identified using hybridization assays. Functional variants, for example
fragments, analogs
or derivatives, can be identified by their ability to hybridize to the
complement DNA
sequence of the disclosed regulatory region under stringent conditions.
Suitable hybridization
conditions are discussed below.
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"Hybridization" is used here to denote the pairing of complementary nucleotide
sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base
sequences are those sequences that are related by the base-pairing rules. In
DNA, A pairs
with T and C pairs with G. In RNA, U pairs with A and C pairs with G.
Typically,
nucleotide sequences to be compared by means of hybridization are analyzed
using dot
blotting, slot blotting, Northern or Southern blotting. Southern blotting is
used to determine
the complementarily of DNA sequences. Northern blotting determines
complementarity of
DNA and RNA sequences. Dot and Slot blotting can be used to analyze DNA/DNA or
DNA/RNA complementarity. These techniques axe well-known by those of skill in
the art.
Typical procedures are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
(Ausubel, et al., eds.) (John Wiley & Sons, Inc. 1995).
A probe is biochemically labeled with a radioactive isotope or tagged in other
ways
for ease in identification. A probe is used to identify a gene, a gene product
or a protein.
Thus, a polynucleotide probe can be used to identify complementary nucleotide
sequences.
An mRNA probe will hybridize with its corresponding DNA.
Typically, the following general procedure is used to determine hybridization
under
stringent conditions. A sample polynucleotide is immobilized on a membrane and
a DNA
sequence complementary to the disclosed regulatory region is used as a
"probe." Using
procedures well-known to those skilled in the art, the ability of the probe to
hybridize with the
sample polynucleotide sequence can be analyzed. Conversely, a DNA sequence
complementary to the disclosed regulatory region can be immobilized and a
sample
polynucleotide can be used as a probe.
One of skill in the art will recognize that various factors can influence the
amount and
delectability of the probe bound to the immobilized DNA. The specific activity
of the probe
must be sufficiently high to permit detection. Typically, a specific activity
of at least 108
dpm/~.g is necessary to avoid weak or undetectable hybridization signals when
using a
radioactive hybridization probe. A probe with a specific activity of 10g to
109 dpm/~.g can
detect approximately 0.5 pg of DNA. It is well-known in the art that
sufficient DNA must be
immobilized on the membrane to permit detection. It is desirable to have
excess immobilized
DNA, and spotting 1 O~g of DNA is generally an acceptable amount that will
permit optimum
detection in most circumstances. Adding an inert polymer such as 10% (w/v)
dextran sulfate
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(rnol. wt. 500,000) or PEG 6000 to the hybridization solution can also
increase the sensitivity
of the hybridization. Adding these polymers has been known to increase the
hybridization
signal. See Ausubel et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY
(John Wiley & Sons, Inc. 1995).
To achieve meaningful results from hybridization between a first nucleotide
sequence
immobilized on a membrane and a second nucleotide sequence to be used as a
hybridization
probe, (1) sufficient probe must bind to the immobilized DNA to produce a
detectable signal
(sensitivity) and (2) following the washing procedure, the probe must be
attached only to
those immobilized sequences with the desired degree of complementarity to the
probe
sequence (specificity). "Stringency," as used in this specification, means the
condition with
regard to temperature, ionic strength and the presence of certain organic
compounds, under
which nucleic acid hybridizations are carried out. The higher the stringency
used, the higher
degree of complementarity between the probe and the immobilized DNA.
"Stringent conditions" designates those conditions under which only
polynucleotides
that have a high frequency of complementary base sequences will hybridize with
each other.
Exemplary stringent conditions are (1) 0.75 M dibasic sodium phosphate/0.5 M
monobasic
sodium phosphate/1 mM disodium EDTA/1% sarkosyl at about 42°C for at
least about 30
minutes, (2) 6.0M urea/0.4% sodium laurel sulfate/0.1% SSC at about 42°
C for at least about
30 minutes, (3) O.1X SSC/0.1% SDS at about 68°C for at least about 20
minutes, (4) 1X
SSC/0.1% SDS at about 55°C for about one hour, (5) 1X SSC/0.1% SDS at
about 62°C for
about one hour, (6) 1X SSC/0.1% SDS at about 68°C for about one hour,
(7) 0.2X SSC/0.1%
SDS at about 55°C for about one hour, (8) 0.2X SSC/0.1 % SDS at about
62°C for about one
hour, and (9) 0.2X SSC/0.1% SDS at about 68°C for about one hour. See
Ausubel et al.,
(eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc.
1995); Sambrook et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold
Spring Harbor Press, 1989).
While stringent washes are typically carned out at temperatures from about
42°C to
about 68°C, one of skill in the art will appreciate that other
temperatures may be suitable for
stringent conditions. Maximum hybridization typically occurs at about 20 to
about 25°C
below the Tm for DNA-DNA hybrids. It is well-known in the art that Tm is the
melting
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temperature, or temperature at which two nucleotide sequences dissociate.
Methods for
estimating T", are well-known in the art. See Ausubel et al., (eds.), CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1995). Maximum
hybridization typically occurs at about 10 to about 15°C below the Tm
for DNA-RNA
hybrids.
Functional variants also can be identified by comparing their structural
similarity, or
homology, to the disclosed regulatory region. A DNA fragment possessing 75% or
more
sequence homology, especially 85-95%, to the disclosed regulatory region is
considered a
functional variant and is encompassed by the present invention. Mathematical
algorithms, for
example the Smith-Waterman algorithm, also can be used to determine sequence
homology.
See Smith and Waterman, J. Mol. Biol., 147:195-197 (1981); Pearson, Gehonaics,
11:635-650
(1991). Although any sequence algorithm can be used to identify functional
variants, the
present invention can define functional variants with reference to the Smith-
Waterman
algorithm, where SEQ ID NO: l is used as the reference sequence to define the
percentage of
homolology of polynucleotide homologues over its length. The choice of
parameter values
for matches, mismatches and inserts or deletions is arbitrary, although some
parameter values
have been found to yield more biologically realistic results than others. One
preferred set of
parameter values for the Smith-Waterman algorithm is set forth in the "maximum
similarity
segments" approach, which uses values of 1 for a matched residue and -a for a
mismatched
residue (a residue being a either a single nucleotide or single amino acid)
(Waterman, Bulletifz
ofMathematical Biology 46:473-500 (1984)). Insertions and deletions x, are
weighted as
xk = 1 + k/3,
where k is the number of residues in a given insert or deletion (Id.).
Preferred variant polynucleotides are those having about 75% sequence homology
to
the GS1_a transcription regulatory region using the Smith-Waterman algorithm.
Particularly
preferred variant polynucleotides have at least about 90% sequence homology.
Even more
preferred variant polynucleotides have at least about 95% sequence homology,
and still more
preferred variant polynucleotides have at least 98% sequence homology.
Recombinant DNA Constructs
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In one embodiment of the invention there is provided a recombinant DNA
construct
comprising the transcription regulatory region of the present invention. There
is provided by
the present invention a recombinant DNA construct comprising a polynucleotide
encoding a
protein of interest, and transcriptional and translational termination
regulatory regions,
wherein the polynucleotide encoding the protein of interest is operably linked
to the
transcription regulatory region of the present invention. The constructs also
may comprise
selectable markers, detectable markers and origins of replication. In another
embodiment,
expression cassettes comprising the transcription regulatory region of the
present invention
also are provided. The inventive constructs are useful for directing pedicel-
specific
transcription of the polynucleotide in an intended host.
In one embodiment of the present invention, the recombinant DNA constructs
comprising the transcription regulatory region of the GS 1-2 gene includes a
664 by nucleotide
sequence upstream of the transcription start site (See Figure 2). In another
embodiment,
recombinant DNA constructs comprising the transcription regulatory region of
the GSl-2
gene, wherein the transcription regulatory region comprises the nucleotide
sequence upstream
of the transcription start site, the GS 1-2 5' UTR and at least one of the
introns associated with
the GSI-2 gene, are provided. The nucleotide sequences for the 5' UTR and the
introns
associated with the GS 1-2 gene are provided below.
Intron 1: (SEQ ID NO: 2)
5'GTGCGAATA GATAGAGATC TCCCCGTCTC CGTCTGATGC CCCCCCCCCC CCCCTTTTTT
CCCGTGGTGT CCCTTGGGAT GCTTGCTGTG TTCCATCTTG TGCATGGATT CTCTTTTCCT
CCGTTTCGTG TTTATATTTT ACTAGTACAT GGGAAGCGAG TAGAAGAGAT CGCTCTCTCT
CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCACACACA CACACACACT AGCAGCAATT
TCAAACTGCT GGCGTTTTAA TTCCTTCTCC AGTTCCTCCC TCGATGACCA CGGCATGCCA
TTGCCAGCCA CGTACAACGT ACTACAAGGC ACACTAACCC ACTGCCAAGC ACCTCGTCTG
ATCTGATCTG ATGCTGATGC AG-3'
Intron 2: (SEQ ID NO: 3)
5'GTCAGTAG TAGTACACGC TTTTGTTCAC CTTCAATCTT ATCCTTATCT TGGCAGTGTA
AAAATTTTTT GTACTTTTGT TGGAAGATAG ATAGATAGAT ATATGTGCCT TTGCAAGTGT
GTCTCTTTTC ATGGGCGTCT TCTTCACACG AAGAAAAATG TCAAAGTGCA TGACATCTCA
CCCTGCCTTT TTTTTGGGAG GGTACTCAG-3'
5' UTR: (SEQ ID NO: 4)
5'-GCGAAA GCACACACGG ATCAATCACA CTCACTCGCG GCCATTGTCC TGCCCGTGCG
TGCTCTGCCT TTTCAGGCGA TCGACCAACC AACTTCTCGT CACTGCC-3'
A variety of recombinant DNA constructs comprising the nucleotide sequence
upstream of the regulatory start site, a 5' untranslated region (UTR) and an
intron can be
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prepared using methods well-known in the art in view of the teachings
contained herein. In
one example, the native GSl-2 exons, including the resident ATG translation
start site and
one or both of the introns are eliminated from the nucleotide sequence
provided in Figure 1
and translation begins at the translation start site of the fused gene of
interest. In another
example, the gene of interest can be fused in frame to the first, second or
third GSl-2 exon.
In other embodiments of the invention, recombinant DNA constructs comprising
truncated versions of the transcription regulatory region of the GS~_Z gene
are provided. For
example, the DNA sequence comprising the proximal 72 nucleotides upstream of
the
transcription start site (See Figure 1) has been shown to effectively direct
high levels of
transcription in all kernel tissues as well as in the leaves and roots. Thus,
a recombinant
DNA construct comprising this DNA sequence operably linked to a gene of
interest is useful
for synthesizing large quantities of the desired protein. The DNA sequence of
this 72 by
fragment is provided below.
SEQ ID NO:S 5'-AGCCATTACACCAACCACTCTCGGGCTCTGCTCTATTTATGGAG
GAGCAGCCAGCTACAGGCTACAGCCGTG-3'
The synthesis of recombinant DNA constructs for expressing homologous and
heterologous proteins is well-known to those of ordinary skill in the art. See
Ausubel et al.,
(eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc.
1995). A variety of expression vectors suitable for use in the present
invention also are well-
known in the art. Particularly preferred are expression vectors directing
protein expression in
plant cells. Examples include, but are not limited to the cauliflower virus
(CaMV) 3SS
promoter (Benfey et al., EMBO J., 8:2195-2202; Jefferson et al., EMBO J.,
6:3901-3907
(1987)), the rice actin promoter (McElroy et al., Plant Cell, 2:163-171
(1990)), the maize
ubiquitin-1 promoter (Christensen and Quail, Trarasgenic Reseal°ch,
5:213-218 (1996)) and
the nopaline synthase promoter (Kononowics et al., Plarat Cell, 4:17-27
(1992)).
The constructs of the present invention can be transformed into host cells
using a
variety of methods. Examples include, but are not limited to, microprojectile
bombardment
(Klein et al., Biotechnology, 6:559-563 (1988)), electroporation (Dhalluin et
al., Plant Cell,
4:1495-1505 (1992)), Agf-obacteriZCm-mediated transformation (Ishida et al.,
Nat. Biotechnol.,
14:745-750 (1996)) and polyethylene glycol treatment (Golovkin et al., Plant
Sci., 90:41-52
( 1993)).
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Transformations can be confirmed by, e.g., Southern hybridization. Directed
expression of the protein of interest can be confirmed using a variety of
methodologies,
including, for example, Northern blot analysis, ifZ situ hybridization of
mRNA, histochemical
methods (Muhitch, Plzysiol. Plant., 104:423-430 (1998)), immuno-localization
of the protein
and characterization of the activity of the protein being expressed.
The inventive constructs can be used to express almost any protein of
interest. In a
preferred embodiment, the recombinant DNA constructs of the invention comprise
polynucleotides encoding proteins involved in seed development and metabolism.
Examples
include, but are not limited to, enzymes of nitrogen metabolism, e.g.,
glutamine synthetases
and isozymes thereof, amino acid transaminases, etc., enzymes involved in
carbon
metabolism, e.g., invertases, and enzymes involved in carbon/nitrogen
interaction, e.g., malic
enzyme. In another preferred embodiment, the recombinant DNA constructs of the
invention
comprise polynucleotides encoding antifungal proteins or other disease
resistance peptides.
For example, genes encoding (3-glucanases, chitinases, defensins, ribosomal
inactivation
proteins or thionins can be expressed within the pedicel using the inventive
constructs. In
other examples, the inventive constructs comprise polynucleotides encoding a
mycotoxin
transport protein, a mycotoxin modifying protein or a mycotoxin-resistant host
target protein.
In another embodiment, the inventive constructs can be used to produce
antisense
transcripts in a target tissue. Such constructs comprise the polynucleotide
sequence of the
sense strand of a gene of interest. Thus, when the heterologous gene of the
recombinant DNA
construct is transcribed, an anti-sense transcript is produced. The
synthesized anti-sense
transcript then hybridizes to the transcripts of the endogeneous gene, i. e.
the sense strands,
and prevents the translation of the endogeneous transcript. In this fashion,
the inventive
constructs effectively inhibit the expression of the gene of interest.
In another embodiment, the present invention provides methods of producing
proteins
in plants. Thus, there are provided methods of producing a protein comprising:
(A)
introducing a recombinant DNA construct comprising a polynucleotide having the
sequence
of the transcription regulatory region of the present invention into a host
cell; (B) growing the
cell and isolating the protein. The recombinant DNA construct is transformed
according to
standard methods well-known in the art. Transformation can be verified by
Southern
analysis. Transgenic plants can be analyzed for the presence of the expressed
protein using
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standard immunological methods well-known in the art. The protein can be
purified from the
plant tissues using standard methods well-known in the art.
In another embodiment, there are provided methods of producing a plant with
increased disease resistance, comprising transforming a plant cell with a
recombinant DNA
construct comprising a polynucleotide having the sequence of the transcription
regulatory
region of the present invention. In one example, the recombinant DNA construct
further
comprises an antifungal protein. 'The recombinant DNA construct is transformed
according to
standard methods well-known in the art. Transformation can be verified by
Southern
analysis. Transgenic plants can be analyzed for the presence of the expressed
protein of
interest using standard immunological methods well-known in the art.
In yet another embodiment, the invention provides methods of producing a plant
with
reduced mycotoxin levels, comprising transforming a plant cell with a
recombinant DNA
construct comprising a polynucleotide having the sequence of the transcription
regulatory
region of the present invention. Such recombinant DNA constructs may further
comprise, for
example, a polynucleotide encoding mycotoxin transport protein, a mycotoxin
modifying
protein or a mycotoxin-resistant host target protein. Expression of these
heterologous genes
reduces mycotoxins by altering host target sites, modifying the toxin or
exporting it out of the
plant cell.
In still another embodiment, the present invention provides methods of
increasing
seed yield and quality in a plant of interest, comprising transforming plant
cell with a
recombinant DNA construct comprising a polynucleotide having the sequence of
the
transcription regulatory region of the present invention.
The present invention, thus generally described, will be understood more
readily by
reference to the following examples, which are provided by way of illustration
and are not
intended to be limiting of the present invention.
Examples
1. Generation of 3' Gene-specific Probes
A GS~_2 gene-specific probe was generated by PCR amplification of the GS~_2 3'
UTR
using following primers:
T7 vector primer: 5'-AATACGACTCACTATAGG-3'
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GSi_Z gene-specific primer: S'-TTCTGCGGAGACTGAGCT-'3
PCR amplifications were performed in 100 ~,l reactions, each containing 50
pmoles of
each sense and antisense primers and 20 nmoles of each of the dNTP's, using a
Perkin Elmer
GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, CT). Reaction conditions were
94°C
for 30 seconds for denaturation, 55°C for 30 seconds for primer
annealing and 72°C for 1
minute for synthesis, for a total of 30 cycles. The probe was gel purified and
random-primed
labeled with 32P using the methodology of Feinberg and Vogelstein. See
Feinberg and
Vogelstein, Anal. Biochern., 132:6-13 (1983).
2. Isolation of Genomic Clones
More than 300,000 recombinant plaques from a maize genomic library (Clontech,
Palo Alto, CA) were screened using the random-primed labeled GS~_2 gene-
specific probe
described above. See Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,
(2ND ED) (Cold Spring Harbor Laboratory Press 1989). Filters were washed twice
in O.1X
SSC, 0.1 % SDS at 60°C before autoradiography. Positive plaques were
picked and purified,
and DNA was extracted using a y DNA purification kit. (Stratagene, La Jolla,
CA). Eighteen
putative positive clones were isolated. Detailed restriction mapping and DNA
analysis
revealed that five clones contained the 5' upstream promoter region. Two
HindIII fragments,
3 and 4.5 kb, hybridizing to 5' and 3' end-specifc probes respectively, were
subcloned into
pBluescript from two genomic clones. Genomic inserts were sequenced using the
Taq
DyeDeoxy terminator cycle sequence kit (P.E. Applied Biosystems, Foster City,
CA).
Sequence analysis was performed using DNAMAN (Lynnon BioSoft, Vaudreuil,
Canada).
Partial sequencing revealed that these two fragments collectively contained
the entire GS~_2
gene and that the 3 kb HindIII fragment contained more than 2 kb of sequence
5' to the
translation start site. Approximately 1 kb of the upstream promoter region was
sequenced.
Promoter sequences were compared with previously published sequences in
GenBank using
the Blast program (NCBI).
The transcription regulatory region of the GS ~ _2 gene is provided in Figure
1. The
putative transcription start site (+1, see Figure 1) of the GSI_Z gene, a G
residue 103 by
upstream from the ATG, was deduced from comparing the sequence with that of
the cDNA.
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Sequence analysis of the 5' region identified a TATA-box-like sequence between
-40 and -
45 and a possible CAAT box element between -166 to -163. Partial sequence
analysis of the
coding region indicates the GS~_Z gene contains 13 exons and 12 introns and is
flanked on its
5' end, at only 664 by upstream from the putative transcriptional start site,
by the 3' end of a
CDC2 gene and flanked on its 3' end by a PGMZ gene, oriented in the reverse
direction (see
Figure 3).
3. Construction of Promoter/Ret~orter Gene Cassettes
A 1505 by fragment of the GS~_2 genomic clone extending from 664 by upstream
of
the putative transcription start site to the HindIII restriction site in the
middle of exon III, was
fused in-frame to the GUS reporter gene and the associated 3' NOS terminator
from pBIlOI
(Clontech, Palo Alto, CA). The resultant heterologous gene was subcloned into
the pUCl9
polylinker at the Sall and EcoRl site in a Sall to EcoRl direction. The
resultant plasmid
(pGS135) is provided schematically in Figure 9.
To create a GS~_a promoter deletion series with a luciferase reporter, the
promoter
fragments, along with the 5' UTR and up to the HindIII restriction site in the
middle of exon
III, were obtained by PCR amplification of the 3.0 kb GS~_2 genomic clone
HindIII restriction
fragment. The resulting deletion series was fused in-frame with Luc, derived
from pGEM-luc
(Promega, Madison, WI), containing a NOS terminator, derived from pBI221. The
GS
promoter constructs containing the maize ADH 1 intron were made in the same
manner,
except in this case deleting the GS region from the HindIII site to just
upstream of the ATG to
obtain a series of intronless, exonless GS promoter fragments. The recovered
GS fragments
were ligated to a pUClB-based ADHl/LUC/NOS vector, derived from PCR
amplification of
the ADHl/LUC/NOS from pRLP73 (Dr. Robert Schmidt, University of California at
San
Diego, CA).
A minimal promoter/LUC reporter was used to establish baseline expression in
transient assays. It was made by fusing the 35S CaMV minimal promoter (-72)
from the
pBI221 backbone into the ADH/LUC/NOS vector described above.
A plasmid consisting of the Ubi-1 maize promoter, derived from pAHC25 (Dr.
Peter
Quail, University of California at Berkeley, CA), and the Reriilla luciferase
gene (Promega),
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along with a NOS terminator, was used as an internal control to measure
transformation
efficiency in transient assays.
4. Transient Expression Analysis
Kernels were sterilized in 10% bleach, 0.1 % Tween 20 for 10 min, followed by
washing three times with sterile distilled water. Embryos were excised and
placed on filters
on MS medium plates (~20 embryos per plate). Bombardments were carried out
immediately
as described previously (Muhitch and Shatters, 1998). Twenty ~g of a chimeric
GS promoter
firefly luciferase and 0.2~g of Renilla luciferase reporter (used to determine
transformation
efficiency) were precipitated onto microprojectiles. After the bombardment,
embryos were
incubated on the same media at 28°C for 36 to 48 hours before analysis.
Tissue extracts were
prepared by grinding germinating embryos in passive lysis buffer (Promega,
Madison, WI).
Luciferase activity was assayed according to the Promega Dual Luciferase Assay
manual.
Preliminary transient gene expression experiments, performed by particle
bombardment of a 1.9 kb GS~_2 promoter/GUS construct onto longitudinally
sliced kernel
halves, failed to exhibit GS~_2 driven pedicel-specific gene expression, but
did show reporter
activity in the embryo. As a result, embryo transient gene expression was used
to initially
characterize the GS~_2 promoter. Two GS~_Z promoter deletion sets were made:
1) a
translational LUC fixsion series, each of which included the first two GSI_2
introns, and 2) a
transcriptional fusion series, each containing the maize Adh I intron (see
Figures 4a-4g).
Although the trends were similar with either GS~_2 promoter deletion series,
LUC activity was
consistently higher and more reproducible using the Adh 1 intron-containing
deletion
constructs and therefore only that data is presented. The results (Figure 5)
demonstrate that
GS1_2 promoter activity is unchanged as the 5' end is removed until the
segment between -72
and -34 is deleted, whereupon gene expression drops off abruptly. Analysis of
variance
(ANOVA) revealed that only the -35 construct was significantly different from
the others (p<
0.002, n=4). In additional studies, the vector designated pGS153, containing
the proximal 72
nucleotides of the transcription regulatory region, achieved strong,
ubiquitous, constitutive
expression of the GUS fusion protein when stably transformed into maize
plants. Figures 11 a
and 11 c show kernels from plants transformed with pGS 153, while figures 1 1b
and 11 d show
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control, non-transformed kernels. All kernels were incubated for 16 hours in
GUS
histochemical reaction medium. Figure I O provides a vector map for pGS 153.
The linker-scanning methodology of Gustin and Burke (Biotechfziques, 14:22-23
(1993)) was used to further characterize the promoter. Linker-scanning of the
region from -
72 to -34 showed that disruption of the area of the putative TATA box (-35 to -
40 upstream of
the transcriptional start site) results in the dramatic reduction in promoter
activity (Figure 6).
In Figure 6, the indicated relative activity is the average of four
independent events, and the
symbol "*" denotes statistically differences, as determined by analysis of
variance (p < 0.04),
only between mutant 39-37 or 36-34 and the remaining mutants. That pedicel-
specific
expression could not be demonstrated in kernels in transient expression
experiments is not
surprising given the fact that it is not uncommon for tissue-specific
promoters to fail to show
tissue-specificity in transient gene expression systems. See Russell and
Fromm, Ti°ahsgenic
Res, 6: 157-168 (1997).
5. Tissue-Specific Gene Expression of GUS in Trans~enic Maize Usin tg-he GS1-2
promoter
To assess the capacity of the inventive transcription regulatory region to
direct tissue-
specific gene expression, maize Hi II embryos or embryo-derived callus were
transformed
with the pGS 135 vector (See Figure 9) using the methodology of Klein et al.
(Bioteclziaology,
6:559-563 (1988)). Using this methodology, stable corn transformants were
prepared by
bombarding the embryos with pGS135 and a second vector (provided by Peter H.
Quail)
providing resistance to bialophos. See Christensen and Quail, Traps. Res.,
5:213-218 (1996).
Stable transformations of maize were performed at the Plant Transformation
Facility, Iowa
State University at Ames. Histochemical detection of GUS in maize tissues was
performed as
previously described (Muhitch, Playsiol. Plant., ,104: 423-430 (1998).
Bialophos-resistant
calli were screened by PCR analysis and by histochemical GUS activity
determination. PCR
analysis was performed using standard methods well-known in the art and the
following
primers:
Primer 1: ATGTTACGTCCTGTAGAAACCC
Primer 2: TAGTAACATAGATGACACCGC
Plants from 18 clones were successfully regenerated. Of these, five plants
developed
normally and produced viable seed. Only one regenerated plant clone, however,
exhibited
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GUS activity in both young (23 days after pollination) and old (50 days after
pollination)
kernels. The results are depicted in Figures 7 and 8. Figure 7a shows
histochemical GUS
staining of maize kernels after 16 hours (overnight) for non-transformed
control (left) and
transgenic clone (right). Figure 7b shows the staining of additional kernels
following 16
hours of incubation. Figure 7c shows histochemical staining of control (left)
and transgenic
(right) kernels after four hours of incubation. Figure 7d shows the pericarp
staining of
transgenic kernels incubated in GUS histochemical reaction mixture for 16
hours. Figure 7e
shows the pericarp of a control kernel incubated for 16 hours as in 7d. Figure
8a provides a
close-up view of the pedicel and bracts of a transgenic kernel incubated for
16 hours in GUS
histochemical reaction mixture. Figure 8b is a close-up view of the pedicel
and bracts of a
transgenic kernel incubated for four hours in GUS histochemical reaction
mixture. Figure 8c
shows leaf segments from transgenic maize incubated for 16 hours in GUS
histochemical
reaction mixture, then cleared of chlorophyll with ethanol. Figure 8d shows
root segments
from the same maize clone incubated for 16 hours in GUS histochemical reaction
mixture.
Transgenic kernel halves and slices incubated overnight in GUS histochemical
reaction mixture exhibited strong tissue-specific staining in the maternal
kernel tissues,
including the pericarp, the pedicel and the associated subtending bracts
(Figures 7a-d).
Particularly notable was the GUS staining in the pedicel parenchyma region
which subtends
the basal endosperm transfer cells. This result was more obvious when the
kernels were
incubated for shorter times (Figures 7c, 8b). The pericarp was stained
throughout; however,
it was stronger in the lower parts of the kernel, nearer the pedicel (Figure
7d).
In contrast to the maternal seed tissues, no staining was detected in the
endosperm or
embryo (Figures 7a-c), in the leaves (Figure 8c) or in the roots (Figure 8d).
That the GS1_z
transcription regulatory region did not direct gene expression within the
endosperm or
embryo or in vegetative leaf or root tissues agrees with earlier mRNA analysis
(Rastogi et al.,
Plant Cell Physiol., 39:443-446 (1998)) as well as with immunological and
enzyme activity
studies (Muhitch, Plant Physiol., 91: 868-875 (1989); Muhitch, et al., Plant
Plzysiol.,
107:372-378 (1995)). The results also suggest that this particular GS form has
a very specific
function in the nitrogen assimilation associated with seed development. The
exceptionally
strong gene expression within the pedicel parechemyma subtending the endosperm
is of
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particular interest, as this area is presumably where the majority of the
nitrogen assimilates
pass on their way to the developing endosperm and embryo.
While the above text has discussed certain aspects of the invention, the
skilled person
will be able to make modifications in view of the teachings herein without
departing from the
scope and spirit of the invention.
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