SEED-COAT PROMOTERS, GENES AND GENE PRODUCTS

PROMOTEURS, GENES ET PRODUITS GENIQUES DE L'ENVELOPPE DE LA SEMENCE

English Abstract

The present invention is directed to isolated genomic sequences that are
differentially expressed within seed-coat tissues. These DNAs
are expressed within the inner integument, thick, or thin, walled parenchyma,
endothelium, palisade, hourglass, or stellate parenchyma cells
of the seed-coat. Furthermore, this invention relates to promoter regions
obtained from genomic sequences that are differentially expressed
in seed-coat tissues, and their use for directing seed-coat specific
expression of genes of interest within transformed plant cells or plants.
Exemplified promoters include those obtained from the differential screening
of a seed-coat library and cryptic promoters obtained using
T-DNA tagging using a promoterless marker gene.

French Abstract

Cette invention se rapporte à des séquences génomiques isolées qui sont exprimées différentiellement dans les tissus de l'enveloppe de la semence. Ces ADN sont exprimés dans les cellules du tégument interne, à paroi mince ou épaisse, du parenchyme, de l'endothélium ou du parenchyme palissadique, en sablier ou étoilé de l'enveloppe de la semence. Cette invention se rapporte en outre à des régions promoteurs obtenues à partir de séquences génomiques qui sont exprimées différentiellement dans les tissus de l'enveloppe de la semence, ainsi qu'à leur utilisation pour diriger dans des cellules végétales ou des plantes transformées l'expression des gènes recherchés, spécifique selon l'enveloppe de la semence. Comme exemples de promoteurs on peut citer ceux obtenus à partir du criblage différentiel d'une bibliothèque d'enveloppes de semence et de promoteurs cryptiques produits au moyen du marquage d'ADN-T à l'aide d'un gène marqueur sans promoteur.

Claims

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WE CLAIM:
1. An isolated nucleotide sequence comprising the nucleic acid sequence
defined by SEQ ID
NO:7, a nucleotide sequence that hybridizes to the nucleic acid sequence of
SEQ ID NO:7, or a
nucleotide sequence that hybridizes to a complement of the nucleotide sequence
of SEQ ID
NO:7, wherein hybridization condition is selected from the group consisting
of:
hybridizing overnight in a solution comprising 4 × SSC at 65°C
and washing one hour in
0.1 × SSC at 65°C;
hybridizing overnight in a solution comprising 4 × SSC at 65°C
and washing 30 minutes
in 0.1 × SSC, 0.1%SDS at 62°C;
hybridizing overnight in a solution comprising 50% formamide, 4 × SSC at
42°C and
washing one hour in 0.1 × SSC at 65°C;
hybridizing overnight in a solution comprising 50% formamide, 4 × SSC at
42°C and
washing 30 minutes in 0.1 × SSC, 0.1 %SDS at 62°C; and
hybridizing overnight in a solution comprising 0.25M Na2PO4 buffer at pH 7.2,
20%
SDS, and 1 mM EDTA, and 0.5% blocking reagent, at 65°C and washing at
22°C in a
solution comprising 20mM Na2HPO4, 1% SDS and 1 mM EDTA, followed by washing
at 68°C in a solution comprising 20mM Na2HPO4, 1% SDS and 1 mM EDTA;
wherein the nucleotide sequence exhibits either promoter activity, imparts a
dull luster to a seed
coat, or both.
2. The isolated nucleotide sequence of claim 1 comprising nucleotides 1-2525
of SEQ ID
NO:7, a nucleotide sequence that hybridizes to nucleotides 1-2525 of SEQ ID
NO:7, or a
nucleotide sequence that hybridizes to a complement of nucleotides 1-2525 of
SEQ ID NO:7,
wherein the nucleotide sequence exhibits promoter activity.
3. A vector comprising a heterologous gene encoding a protein, and the
nucleotide sequence
of claim 2, wherein the heterologous gene is under the control of the
nucleotide sequence.
4. A plant cell which has been transformed with the vector as claimed in claim
3.
5. A transgenic plant cell transformed with the nucleotide sequence as claimed
in claim 2,
operatively linked to a heterologous gene encoding a protein.
6. A method of expressing a protein of interest on a seed coat, the method
comprising:
i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding the protein of interest to produce a transformed
plant;
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing the protein of
interest.
7. A method of modifying storage reserves yields, seed colour, fibre content,
or seed
metabolites in a seed coat, the method comprising:

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i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding a protein of interest having an activity that
modulates either
storage reserves within the seed coat, seed colour, fibre content or seed
metabolites to
produce a transformed plant;
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing the protein of
interest.
8. A method of increasing seed size or decreasing the biomass of a seed coat,
the method
comprising:
i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding a protein of interest having an activity that
increases seed
size or decreases the biomass of a seed coat to produce a transformed plant;
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing the protein of
interest.
9. A method of imparting fungal, insect, viral or bacterial resistance in a
seed, the method
comprising:
i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding a protein of interest having an activity that
imparts fungal,
insect, viral, or bacterial resistance to produce a transformed plant;
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing the protein of
interest.
10. A method of modifying the textural, visual, or chemical properties of a
seed coat, the
method comprising:
i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding a protein of interest that modifies the textural,
visual, or
chemical properties to produce a transformed plant;
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing the protein of
interest.
11. A method of modifying storage reserve yields, seed colour, fibre content,
or seed
metabolites in a seed coat, the method comprising:
i) transforming a desired plant with the vector of claim 3 comprising the
desired
heterologous gene encoding an antisense transcript that modulates either
storage reserves
within the seed coat, seed colour, fibre content or seed metabolites to
produce a
transformed plant;

95
ii) selecting the transformed plant; and
iii) growing the transformed plant to produce seed expressing an antisense
transcript.
12. A method for localizing production of a protein of interest in a seed coat
associated tissue
comprising, providing a plant transformed with the nucleotide sequence of
claim 2 operatively
linked to a heterologous gene encoding the protein of interest, and producing
seed from the plant,
such that the protein of interest is expressed in the seed coat associated
tissue.
13. The method of claim 12, wherein the protein of interest is expressed
within the
membranous endocarp.
14. The method of claim 12, wherein the protein of interest is expressed
within the inner
layer of the pericarp.
15. The method of claim 12, wherein the protein of interest is localized onto
the seed coat.
16. The isolated nucleotide sequence of claim 1 comprising nucleotides 2526-
3368 of SEQ
ID NO:7, a nucleotide sequence that hybridizes to nucleotides 2527-3368 of SEQ
ID NO:7, or a
nucleotide sequence that hybridizes to a complement of nucleotides 2526-3368
of SEQ ID NO:7,
wherein the nucleotide sequence imparts a dull luster to a seed coat.
17. A vector comprising the nucleotide sequence as defined in claim 16 under
the control of a
promoter.
18. A plant cell which has been transformed with the vector as claimed in
claim 17.
19. An isolated protein encoded by the nucleotide sequence of claim 16.
20. The isolated protein of claim 19 further characterized as having a
molecular mass of
about 3.2 kD.
21. A method for modifying luster of a seed coat comprising, transforming a
plant with the
nucleotide sequence of claim 16 under the control of a seed coat promoter, and
producing seed
from the plant, wherein the luster of the seed of the plant, when compared to
a similar plant that
does not comprise the nucleotide sequence, is reduced.
22. A method for imparting a dull luster to a seed comprising, transforming a
plant with the
nucleotide sequence of claim 16 under the control of a seed coat promoter, and
producing seed
from the plant, wherein the luster of the seed of the plant, when compared to
a similar plant that
does not comprise the nucleotide sequence, is dull.

Description

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SEED-COAT PROMOTERS, GENES AND GENE PRODUCTS
Field of Invention
This invention relates to seed-coat promoters, genes and proteins
encoded by these genes. More specifically, it relates to genes and promoters
that are. developmentally regulated and expressed, or activated, within
tissues
comprising the seed-coat, and tissues directly associated with the seed-coat,
of
plants. Furthermore, this invention also relates to proteins encoded by genes
expressed within these tissues are their localization within, or onto, the
seed-
coat.
Background and Prio:r Art
Bacteria from ffie genus Agrobacterium have the ability to transfer
specific segments of DNA (T-DNA) to plant cells, where they stably integrate
into the nuclear chromosomes. Analyses of plants harbouring the T-DNA have
revealed that this genetic element may be integrated at numerous locations,
and
can occasionally be found within genes. One strategy which may be exploited
to identify integration events within genes is to transform plant cells with
specially designed T-DNA vectors which contain a reporter gene, devoid of cis-
acting transcriptional and translational expression signals (i.e.
promoterless),
located at the end of the T-DNA. Upon integration, the initiation codon of the
promoterless gene (reporter gene) will be juxtaposed to plant sequences. The
consequence of T-DNA insertion adjacent to, and downstream of, gene
promoter elements may be the activation of reporter gene expression. The
resulting hybrid genes, referred to as T-DNA-mediated gene fusions, consist of
unknown and thus uncharacterized plant promoters residing at their natural
location within the chromosome, and the coding sequence of a marker gene
located on the inserted T-DNA (Fobert et al., 1991, Plant Mol. Biol. 17,
837-851).

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It has generally been assumed that activation of promoterless or
enhancerless marker genes result from T-DNA insertions within or immediately
adjacent to genes. The recent isolation of several T-DNA insertional mutants
(Koncz et al., 1992, Plant Mol. Biol. 20, 963-976; reviewed in Feldmann,
1991, Plant J. 1, 71-82; Van Lijsebettens et al., 1991, Plant Sci. 80, 27-37;
Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990, Nature 346,
35-39), shows that this is the case for at least some insertions. However,
other
possibilities exist. One of these is that integration of the T-DNA activates
silent regulatory sequences that are not associated with genes. Lindsey et al.
(1993, Transgenic Res. 2, 33-47) referred to such sequences as "pseudo-
promoters" and suggested that they may be responsible for activating marker
genes in some transgenic lines.
Inactive regulatory sequences that are buried in the genome but with the
capability of being fuinctional when positioned adjacent to genes have been
described in a variety of organisms, where they have been called "cryptic
promoters" (Al-Shawi et al., 1991, Mol. Cell. Biol. 11, 4207-4216; Fourel et
al., 1992, Mol. Cell. Biol. 12, 5336-5344; Irniger et al., 1992, Nucleic Acids
Res. 20, 4733-4739; Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-
1244). Cryptic promoters can be found in the introns of genes, such as those
encoding for yeast actin (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-
4739), and a mamma;lian melanoma-associated antigen (Takahashi et al., 1991,
Jpn J. Cancer Res. 82, 1239-1244). It has been suggested that the cryptic
promoter of the yeast actin gene may be a relict of a promoter that was at one
time active but lost function once the coding region was assimilated into the
exon-intron structure of the present-day gene (Irniger et al., 1992, Nucleic
Acids Res. 20, 4733-4739). A cryptic promoter has also been found in an
untranslated region of the second exon of the woodchuck N-myc proto-
oncogene (Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344). This cryptic
promoter is responsible for activation of a N-myc2, a functional processed
gene
which arose from ret:roposition of N-nryc transcript (Fourel et al., 1992,
Mol.

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Cell. Biol. 12, 5336-5344). These types of regulatory sequences have not yet
been isolated from plants.
Weber et al. (1995, Plant Cell 7:1835-1846) disclose a cDNA sequence
of a seed-coat associated invertase. However, all of the cDNA's characterized
were found to be expressed in tissues other than the seed-coat, including
anthers, cotyledon, stem and root. Furthermore, no promoter was isolated,
characterized, or disclosed.
Described herein is the occurrence of seed-coat genes and promoters
that have been obtained as a result of differential screening of seed-coat
genomic libraries, or generated by tagging with a promoterless GUS (P-
glucuronidase) T-DNA vector, or by identification of genes that are highly
expressed in the seed-coat or associated tissues. Expression analysis of these
DNA's reveal that they are spatially and developmentally regulated in seed
coats. Prior to this work, promoters, as well as genes specifically expressed
in
seed coat tissues had riot been isolated or reported. Furthermore, proteins
encoded by genes that are expressed within seed-coat, or associated with seed-
coat tissues, are also disclosed.
Summary of Inventicin
This invention relates to seed-coat promoters and genes. More
specifically, it relates to genes and promoters that are developmentally
regulated and expressed, or activated, within tissues comprising the seed-coat
of plants, and tissues directly associated with the seed-coat, of plants.
Furthermore, this invention also relates to proteins encoded by genes
expressed
within these tissues ar.id their localization within, or onto, the seed-coat..

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A transgenic tobacco plant, T218, contained a 4.7 kb EcoRI fragment
containing the 2.2 kb promoterless GUS-nos gene and 2.5 kb of 5' flanking
tobacco DNA. Deletion of the region approximately between 2.5 and 1.0 kb of
the 5' flanking region did not alter GUS expression, as compared to the entire
4.7 kb GUS fusion. A further deletion to 0.5 kb of the 5' flanking site
resulted
in complete loss of GtJS activity. Thus the region between 1.0 and 0.5 of the
5' flanking region of the tobacco DNA contains the elements essential to gene
activation. This region is contained within a Xbal - SnaBI restriction site
fragment of the flanking tobacco DNA. Furthermore, other promoters have
been identified that are differentially expressed within the seed-coats of
plants,
and that are capable oi= driving expression of heterologous genes that are
operatively linked tliereto.
Thus according to the present invention there is provided an isolated
genomic DNA molecule, differentially expressed in seed coat tissues.
Furthermore, this genomic DNA molecule is differentially expressed within the
outer integument of the seed coat, the inner integument of the seed coat, the
thick walled parenchyma of the seed coat, the thin walled parenchyma of the
seed coat, the endothelium of the seed coat, the hourglass cells of the seed
coat,
the palisade of the seed coat, the stellate parenchyma of the seed coat, or
the
membranous endocarp, or a combination thereof.
This invention is also directed to a seed-coat promoter obtained from the
genomic DNA molecule as described above. Also considered within the scope
of the present invention is a cryptic seed coat promoter. Furthermore, this
invention is directed to a seed coat promoter, as described above, that
controls
the differential expression of a gene associated therewith, within the outer
integument of the seecl coat, the inner integument of the seed coat, the thick
walled parenchyma of the seed coat, the thin walled parenchyma of the seed
coat, the endothelium of the seed coat, the hourglass cells of the seed coat,
the

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palisacle of the seed coat, or the stellate parenchyma the seed coat,
membranous
endocarp, or a combination thereof
This invention also relates to an isolated genomic DNA characterized by
the restriction map selected from the group consisting of Figure 12 (a),
Figure
12 (b), Figure 12 (c) and Figure 12 (d).
According to the present invention, there is also provided an isolated
seed-coat promoter. Furthermore, this seed coat promoter may be obtained
from angiosperms. More specifically, this seed-coat promoter is obtained from
the group consisting of tobacco or soybean.
This invention is also directed to a cloning vector comprising a gene
encoding a protein and an isolated seed-coat promoter, wherein the gene is
under the control of the seed-coat promoter. Furthermore, this invention
includes a plant cell which has been transformed with such a vector.
This invention also provides for a transgenic plant containing a seed-
coat promoter, operatiively linked to a gene encoding a protein.
The present invention is also directed to a seed-coat promoter
comprising at least 10 contiguous nucleotides of nucleotides 1-2526 of SEQ ID
NO:7, or an analogue of the sequence defined by nucleotides 1-2526 of SEQ ID
NO:7, wherein the analogue hybridizes to a nucleic acid defined by nucleotides
1-2526 of SEQ ID NO:7 under stringent hybridization conditions and maintains
seed-coat, or seed-coat associated promoter activity.
This invention also includes a seed-coat promoter comprising at least
10 contiguous nucleotides of nucleotides 1-2450 of SEQ ID NO:8, or an
analogue of the nucleic acid sequence defined by nucleotides 1-2450 of SEQ ID
NO:8õ wherein the analogue hybridizes to a nucleic acid defined by nucleotides

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1-2450 of SEQ ID NO:8 under stringent hybridization conditions and maintains
seed-coat, or seed-coat associated promoter activity.
The present invention also is directed to a seed-coat promoter
comprising at least 10 contiguous nucleotides of nucleotides 1-5514 of SEQ ID
NO:9, or an analogue of the nucleotides sequence defined by nucleotides 1-
5514 of SEQ ID NO:9, wherein the analogue hybridizes to a nucleic acid
defined by nucleotides 1-5514 of SEQ ID NO:9 under stringent hybridization
conditions and maintains seed-coat, or seed-coat associated promoter activity.
Brief Description of the Drawings
Figure 1 depicts the fluorogenic analyses of GUS expression in the plant
T218. Each bar represents the average one standard deviation of three
samples. Nine different tissues were analyzed: leaf (L), stem (S), root (R),
anther (A), petal (P), ovary (0), sepal (Se), seeds 10 days post anthesis (Si)
and seeds 20 days post-anthesis (S2). For all measurements of GUS activity,
the fraction attributed to intrinsic fluorescence, as determined by analysis
of
untransformed tissues., is shaded black on the graph. Absence of a black area
at the bottom of a histogram indicates that the relative contribution of the
background fluorescence is too small to be apparent.
Figure 2 shows the cloning of the GUS fusion in plant T218 (pT218)
and construction of transformation vectors. Plant DNA is indicated by the
solid line and the pror.noterless GUS-nos gene is indicated by the open box.
The transcriptional start site and presumptive TATA box are located by the
closed and open arrow heads respectively. DNA probes #1, 2, 3 and RNA
probe #4 are shown. The EcoRI fragment in pT218 was subcloned in the
pBIN19 polylinker to create pT218-1. Fragments truncated at the Xbal SnaBI
and Xbal sites were also subcloned to create pT218-2, pT218-3 and pT218-4.

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Abbreviations for the endonuclease restriction sites are as follows: EcoRI
(E),
HindI1I (H), XbaI (X), SnaBI (N), SmaI (M), SstI (S).
Figure 3 shows the expression pattern of promoter fusions during seed
development. GUS activity in developing seeds (4-20 days postanthesis (dpa))
of (Fig. 3a) plant T218 (*-*) and (Fig. 3b) plants transformed with vectors
pT218-1 (0-0), pT21.8-2 (0-0), pT218-3 (0-0) and pT218-4 (0-A) which are
illustrated in Figure 2.. The 2 day delay in the peak of GUS activity during
seed development, seen with the pT218-2 transformant, likely reflects
greenhouse variation conditions.
Figure 4 shows GUS activity in 12 dpa seeds of independent
transformants produced with vectors pT218-1 (0), pT218-2 (0), pT218-3 (0)
and pT218-4 (A). The solid markers indicate the plants shown in Figure 3 (b)
and the arrows indicate the average values for plants transformed with pT218-1
or pT218-2.
Figure 5 shows the mapping of the T218 GUS fusion termini and
expression of the region surrounding the insertion site in untransformed
plants.
Figure 5(a) shows the mapping of the GUS mRNA termini in plant T218. The
antisense RNA probe from subclone #4 (Figure 2) was used for hybridization
with total RNA of tissues from untransformed plants (10 g) and from plant
T218 (30 g). Arrowheads indicate the anticipated position of protected
fragments if transcripts were initiated at the same sites as the T218 GUS
fusion.
Figure 5 (b) shows the RNase protection assay using the antisense (relative to
the orientation of the GUS coding region) RNA probe from subclone e (Figure
7) against 30 g total RNA of tissues from untransformed plants. P, untreated
RNA probe; -, control assay using the probe and tRNA only; L, leaves from
untransformed plants; 8, 10, 12, seeds from untransformed plants at 8, 10, and
12 dpa, respectively; T10, seeds of plant T218 at 10 dpa; +, control
hybridization against unlabelled in vitro-synthesized sense RNA from subclone

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c (panel a) or subclorie e (panel b). The two hybridizing bands near the top
of
the gel are end-labelled DNA fragment of 3313 and 1049 bp, included in all
assays to monitor losses during processing. Molecular weight markers are in
number of bases.
Figure 6 proviides the nucleotide sequence of pT218 (top line) (SEQ ID
NO: 1) and pIS-1 (bottom line). Sequence identity is indicated by dashed
lines.
The T-DNA insertion site is indicated by a vertical line after bp 993. This
site
on pT218 is immediately followed by a 12 bp filler DNA, which is followed by
the T-DNA. The first nine amino acids of the GUS gene and the GUS
initiation codon (*) are shown. The major and minor transcriptional start site
is
indicated by a large aind small arrow, respectively. The presumptive TATA
box is identified and is in boldface. Additional putative TATA and CAAT
boxes are marked with boxes. The location of direct (1-5) and indirect (6-8)
repeats are indicated by arrows.
Figure 7 shows the base composition of region surrounding the T218
insertion site cloned fi-om untransformed plants. The site of T-DNA insertion
in plant T218 is indicated by the vertical arrow. The position of the 2
genomic
clones pIS-1 and pIS-2, and of the various RNA probes (a-e) used in RNase
protection assays are indicated beneath the graph.
Figure 8 shows the Southern blot analyses of the insertion site in
Nicotiana species. DNA from N. tomentosiformis (N tom), N. sylvestris (N
syl), and N. tabacum (N tab) were digested with HindIII (H), Xbal (X) and
EcoRI (E) and hybridized using probe #2 (Figure 2). Lambda HindII1 markers
(kb) are indicated.
Figure 9 shows the AT content of 5' non-coding regions of plant genes.
A program was writtein in PASCAL to scan GenBank release 75.0 and to
calculate the AT contents of the 5' non-coding (solid bars) and the coding

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regions (hatched bars) of all plant genes identified as "Magnoliophyta"
(flowering plants). The region -200 to -1 and + 1 to +200 were compared.
Shorter sequences we:re also accepted if they were at least 190 bp long. The
horizontal axis shows the ratio of the AT content (%). The vertical axis shows
the number of the sequences having the specified AT content ratios
Figure 10 shoNvs a Northern analysis of the expression of several of the
genes of the present invention within developing seed coats, embryo, pod,
flower, root, stem ancl leaf tissues. Figure 10 (a) shows the expression of
SC4;
Figure 10 (b) shows the expression of SC20; Figure 10 (c) shows the
expression of SC21, Figure 10 (d) shows the expression of Ep locus peroxidase
within these tissues. Figure 10 (e) shows the expression of HP (hydrophobic
protein) in leaf, flower, pod, seed coat, embryo, stem or root tissues.
Figures
10 (f) and (g), total RNA was isolated from leaf, flower, pod shells, seed
coat,
embryo, stem or root tissue. Equal amounts of RNA (10 g) were vacuum
blotted to nylon and probed with HPS cDNA. Ribosomal RNA (rRNA),
visualized by stainirig with ethidium bromide, is shown as control. Figure 10
(f), RNA from tissues at early (E) mid (M) or late (L) stages of development
were compared for HP gene expression. All samples shown are from dull
seeded phenotype (cv Harosoy 63). Figure 10 (g), RNA from pod tissues of
dull (cv Harosoy 63) and shiny (cv. Williams 82) seeded soybeans were
compared for HP gene expression.
Figure 11 shovvs the restriction maps obtained from Figure 11 (a) SC20;
Figure 11 (b) SC21; Figure 11 (c) HP (hydrophobic protein) genomic region, and
Figure 11 (d) SC4. Included in Figure 11 (c) are restriction enzyme sites for
BamHI, BgIII, HindIII,, and XbaI; the HP ORF; TATA box consensus signals; and
the position of direct repeats of 12 bp or longer.

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Figure 12 shows the morphology of the seed coat of Glycine max.
Figure 12 (a) shows the structures present at six days after anthesis (DAF);
Figure 12 (b), at 12 DAF; and Figure 12 (c) at 18 DAF.
Figure 13 shows in situ hybridization results obtained with seed coats of
Glycine max at different developmental stages, and probed as follows: Figure
13 (a) seed coat at 3 days after anthesis (DAF), probed with SC4; Figure 13
(b) seed coat at 9 DAF, probed with SC20; Figure 13 (c) seed coat at 15 DAF,
probed with SC21; Figure 13 (d) seed coat at 18 days after anthesis, probed
with a soybean peroxidase, corresponding to the Ep locus. Figures 13 (e), (f)
and (g) were obtained from cross sections of developing soybean seeds
(cultivar
Maple Presto, EpEp). Hybridization of 31S-probe to complementary mRNA
appears as bright white signal in these dark field microscopy images. Figure
13 (e) 6 DAF (DPA, days post anthesis), Figure 13 (f) 9 DAF, and Figure 13
(g) 12 DAF. Scale bars are 100 :m. Emb, embryo; F, funiculus; HG,
hourglass cells; PC, pericarp; SC, seed coat.
Figure 14 shows light micrographs of a seed-coat obtained from
soybean. Figure 14 (a) shows a plastic embedded section of the seed-coat near
the hilum at 21 daf and. stained with Toluidine Blue O. Note the association
of
the membranous endocarp with the seed-coat pallisade. Figure 14 (b) shows a
wax-embedded section of a soybean seed-coat as 12 daf probed with 35S-
labelled Hydrophobic Protein (HP) antisense RNA, and counter stained with
Toluidine Blue O. Note strong specific localization of the prbbe within the
membranous endocarp. Pallisade (p), hourglass cells (h), counterpallisade (c),
arial cells (a), stellate parenchyma (s), thin walled parenchyma (n),
thickwalled
parenchyma (k), pod parenchyma (d), and membranous endocarp >. Figures
14 (c) and (d). show localization of HP mRNA transcript by in situ
hybridization. Cross sections of soybean pods containing immature seeds (dull
phenotype, HPS (+), cv Maple Presto). Hybridization of'SS labelled HP probe
to complementary mRNA appears as bright white signal in these dark field

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microscopy images. E, embryo; Ep, inner epidermal layer of endocarp; Ex,
exocarp; F, funiculiis; M, mesocarp; Sc, seed coat; Sm, sclerenchyma layer of
endocarp. Bar = 100 m. Figure 14 (c), Expression at 6 DPA (days post
anthesis). Figure 14 (d), Expression 12 DPA.
Figure 15 shows the Soybean hydrophobic protein (HP) cDNA and
deduced amino acid sequences. Figure 15 (a), the cDNA and amino acid
sequence of HP. The pre-protein signal sequence is underlined. Figure 15 (b)
shows the deduced amino acid sequence of HP pre-protein. Alternate N-
terminal residues are boxed, as determined by peptide microsequence analysis.
Figure 15 (c) shows a Kyle-Doolittle hydrophilicity plot of HP (Lasergene). In
this plot, positive values indicate greater hydrophilic character. Also
represented are the ttiree domains of the HP pre-protein and the length of the
mature peptide. Figu;re 15 (d) shows a schematic comparison of HP domain
structure to three other plant proteins. Bold numbers indicate the length in
amino acid residues for the domain segments. The pattern of spacing between
the eight cysteine residues within the hydrophobic domains is also shown below
each protein. Sequenc:es for tobacco N16 polypeptide (D86629), maize proline
rich hydrophobic protein (PRHP) (X60432), and Arabidopsis lipid transfer
protein 1(LTP1) (M80567) were retrieved from GenBank.
Figure 16 shows scanning electron micrographs of representative 'Dull'
and 'Shiny' seeded soybean cultivars. Scale bars are included in the figures.
The
lowest magnification (x18), Figure 16 (a) is a view of the entire seed. The
large
oval shaped scar on the seed surface is the hilum, corresponding to the point
of
detachment of the mati.ue seed from the funiculus. Figure 16 (b), x100, and
Figure 16 (c) x500, are focused outside of hilum region.
Figure 17 shows a silver stained SDS-PAGE analysis of protein extracts
from seed tissues and surface. Lanes marked 'M' indicate protein standards,
and their corresponding mass in kilodaltons is also provided. Figure 17 (a),

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Soluble protein extracts from the embryo, seed coat, and seed surface of a
dull
phenotype (cv Harosoy 63). Each sample at approximately 1 g of total
protein. Figure 17 (b), Seed surface protein extracts of a dull phenotype (cv
Harosoy 63) with different concentrations of dithiothreitol (DTT) present in
the
sample loading buffer=, as indicated at the top of each lane. Figure 17 (c),
Seed
surface protein extracts of dull (D), shiny (S), and bloom (B).
Figure 18 shows restriction fragment length polymorphisms between
dull and shiny phenotypes. Genomic DNA from dull (cv Harosoy 63) and shiny
(cv Williams 82) soybeans with abundant (+) or trace (-) amounts of HPS on
the seed surface, was digested with restriction enzymes, electrophoretically
separated, blotted, and hybridized to HP cDNA probe. The size of hybridizing
fragments was estimated by comparison with standards and is shown on the
left.
Figure 19 shows the nucleotide sequence and deduced amino acid
sequence of SC4 cDNA, and the sequence comparisons between SC4 protein
and BURP proteins. :Figure 19 (a), 5' and 3' untranslated sequences are in
lowercase lettering. The stop codon is shown with an asterisk and two
polyadenylation signals are underlined. Two copies of a ten amino acid repeat
is also underlined. Concensus sequnences for N-glycosylation (NNT; NSSN;
and NGTV) are also underlined. Figure 19 (b), amino acid alignment of the
carboxyl terminus of the SC4 protein with the BURP domain (A) and the amino
terminus of the SC4 protein with the conserved segments of the second domain
(B) of several BURP domain proteins. Pg1p is not included in panel B as the
second domain of this protein does not contain a conserved segment. Gaps were
introduced to optimize the alignment. Conserved amino acids are shown in bold
face. Amino acids of each protease are numbered from the precursor sequence.
Figure 19 (c) shows the structual similarity between SC4 protein and the BURP
domain proteins.

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Figure 20 shows Northern blot analysis of SC4 and SC20 mRNA
accumulation in seed coat embryo and pod organs of soybean. 10 /.zg total RNA
from seed coat, embryo and pod organs between 6-24 days past anthesis were
hybridized with radiolabled probes. For day 6, total RNA was prepared from
whole seeds. Each blot was hybridized with a SC4 cDNA probe, Figure 20 (a),
a SC20 cDNA probe Figure 20 (b), and an 18S rRNA probe Figure (c).
Figure 21, shows the localization of SC4 mRNA in Seed coat organs of
soybean by in situ hybridization. Transections of seed coats at 3 days past
anthesis (dpa) and 6 cipa. Hybridization to Antisense, Figure 21 (a), and
sense, Figure 21 (b) SC4 labelled RNA probes. Abbreviations, II - inner
integument, 01 outer integument, P pod. Bar equals 100Acm.
Figure 22, shows Southern blot analysis of SC4. Figure 22 (a) shows
Southern analysis of the gene family composition of sc4 in soybean. Figure 22
(b) shows Southern analysis of sc4 in diverse plant species. Hybridized filter
was washed under conditions of low stringency, twice at 52 C for 15 min in 2x
SSC, 0.1 %SSC, 0.1 % SDS and once at 52 C for 30 min in 0. lx SSC, 0.1 %
SDS.
Figure 23 reveals the characterization of sc2O and the SC20 protein.
Figure 23 (a) is a restriction map of sc2O. Figure 23 (b) shows the nucleotide
sequence and deduceci amino acid sequence of sc20 cDNA. The stop codon is
shown with an asterisk and the polyadenylation signal is underlined. The
concensus sequences for N-glycosylation are also underlined. Figure 23 (c)
shows the hydrophobic plot of SC20 protein, where hydrophobic regions
possess a positive sign, and hydrophilic regions possess a negative sign. In
Figure 23 (d), alignment of SC20 protein with other subtilases is shown. D, H
and S regions represent amino acid sequences around the catalytic aspartate,
histidine and serine residues of the subtilases. The catalytic residues are
labelled with an asterisk. N region represents amino acid sequence around the

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conserved asparagine residue, of subtilases. # indicates the conserved
asparagine. AF70, cucumisin, P69B, Ag12, subtilisin BPN', kex2, furin are
from Picea abies, Cucumis melo L., Lycopersicon esculentum, Alnus glutinosa,
Bacillus subtilis, Saccharomyces cerevisiae, and Homo sapiens respectively.
Conserved amino acid,s are shown in boldface. Amino acids of each protease
are numbered from the precursor sequence.
Figure 24 shows localization of SC20 mRNA in seed coats of soybean
by in situ hybridization. Transection of seed coats at 12 days past anthesis
hybridized to Antisense, Fivure 24 (a), and Sense, figure 24 (b), SC20
radiolabelled RNA probes. Abbreviations: H Hilumi, II inner integument, 01
outer integument, * thick walled parenchyma, ** thin walled parenchyma. Bar
equals 100,um.
Figure 25 shows Southern blot analysis of sc2O. Figure 25 (a) and (b),
Southern analysis of the gene family composition of sc2O in soybean under
conditions of medium stringency (twice at 52 C for 15 min in 2x SSC,
0.1 %SDS, and once at 52 C for 30 min in 0. lx SSC, 0.1 % SDS), Figure 25
(a), and high stringency (once at 62 C for 30 min in 0. lx SSC, 0.1 % SDS)
Figure 25 (b). Figure 25 (c) shows Southern analysis of sc4 in diverse plant
species. genomic DNA was digested with EcoRI. Hybridization used a
radiolabelled SC20 cDNA probe. The filter was washed under conditions of
medium stringency, tvvice at 52 C for 15 min in 2x SSC, 0.1 % SDS and once
at 52 C for 30 min in 0.lx SSC, 0.1 % SDS.
Detailed Description of the Preferred Embodiments
T-DNA tagging with a promoterless (3-glucuronidase (GUS) gene
generated a transgenic Nicotiana tabacum plant that expressed GUS activity
only in developing seed coats. Cloning and deletion analysis of the GUS fusion

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revealed that the promoter responsible for seed coat specificity was located
in
the plant DNA proxirr.ial to the GUS gene. Deletion analyses localized the
cryptic promoter to an. approximately 0.5 kb region between a Xbal and a
SnaBI restriction endonuclease site of the 5' flanking tobacco DNA. This
region spans from nucleotide 1 to nucleotide 467 of SEQ ID NO: 1.
Other work, based on the differential screening of seed coat libraries has
led to the identificatioii of several other genes that are differentially
expressed
within, or tissues that are directly associated with, the seed coat of plants.
These genes include SC4 (SEQ ID NO's: 3 and 9, cDNA and genomic
sequences, respectively), SC20 (SEQ ID NO's: 4 and 8, cDNA and genomic
sequences respectively), SC21 (SEQ ID NO: 5, cDNA sequence), and their
associated promoters (see SEQ ID NO 9 and 8 for promoters of SC 4 and
SC20, respectively; also Figure 12). Furthermore, the isolation of genes
encoding highly expressed seed coat proteins led to the identification of a
seed-
coat specific peroxidase from the Ep locus and associated promoter (Ep
genomic sequence, SEQ ID NO:2), as well as a gene encoding a seed-coat
localized hydrophobic protein (HP, cDNA sequence SEQ ID NO:6) and
associated promoter (within genomic sequence, SEQ ID NO:7, also see Figure
11 (c)). Thus, the present invention includes promoters, genes and proteins
isolated from several plant species, that are preferentially expressed, or
specific
to seed-coat tissues, as well as promoters, genes and associated proteins
obtained from tissues associated with the seed-coat.
The term cryptic promoter means a promoter that is not associated with
a gene and thus does not control expression in its native location. These
inactive regulatory sequences are buried in the genome but are capable of
being
functional when positioned adjacent to a gene.
The DNA sequence of an aspect of the present invention includes the
DNA sequence of SEQ! ID NO: 1, the promoter region within SEQ ID NO: 1

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(for example from nucleotide 1 to 476), and analogues thereof. Similarly,
another aspect of this invention includes a DNA sequence of SEQ ID NO:2, the
promoter region of this sequence (nucleotides 1-1532), and analogues thereof.
Another aspect of this invention includes a DNA sequence of SEQ ID NO:7,
the promoter region (nucleotides 1-2526), and analogues thereof, a DNA
sequence of SEQ ID NO 8, the promoter region (nucleotides 1-2450) and
analogues thereof, and a DNA sequence of SEQ ID NO:9, the promoter region
(nucleotides 1-5514) and analogues thereof.
Analogues include those DNA sequences which hybridize under
stringent hybridization conditions (see Maniatis et al., in Molecular Cloning
(A
Laboratory Manual), Cold Spring Harbor Laboratory, 1982, p. 387-389) to the
DNA sequence of SEQ ID NO: 1, 2, 7, 8 or 9 provided that said sequences
maintain the seed coat, or seed-coat associated promoter activity. An example
of one such stringent :hybridization conditions may be hybridization at 4XSSC
at 65 C, followed by washing in 0.1XSSC at 65 C for an hour, or at 62 C for
30 min in 0. lx SSC, 0.1 % SDS. Alternatively an exemplary stringent
hybridization condition could be in 50% formamide, 4XSSC at 42 C. With the
use of Digoxigenin labelled probes, stringent hybridization may include 65 C
in 0.25 M Na2HPO4 (pH 7.2), 20% SDS, 1 mM EDTA and 0.5% blocking
reagent (Boehringer Mannheim) followed by washing at 22 C in 20 mM
Na2HPOa (pH 7.2), 1% SDS and 1 mM EDTA and washes in the same solution
at 68 C. Analogues also include those DNA sequences which hybridize to the
sequence of SEQ ID NO: 1, 2, 7, 8 or 9 under relaxed hybridization conditions
provided that said sequences maintain the seed-coat promoter activity.
Examples of such non-hybridization conditions includes hybridization at 4XSSC
at 50 C or with 30-40% formamide at 42 C. Alternate conditions of medium
stringency include washing the filter twice at 52 C for 15 min in 2x SSC, 0.1
%
SDS and once at 52 C: for 30 min in 0. lx SSC, 0.1 % SDS.

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Furthermore, another aspect of this invention is directed to the
identification and characterization of seed-coat promoters (see Figure 11) and
their corresponding genes of cDNA's (SEQ ID NO's: 3-6), as characterized by
Southern or in situ hybridization analysis of the expression patterns of genes
expressed under the control of seed-coat promoters within developing seed
coats (Figures 13 and 14). Furthermore, restriction maps of the promoter and
downstream regions of several seed-coat genomic clones is presented (Figure
11).
Proteins of interest may be expressed in seed coat tissues by placing a
gene capable of expressing the protein of interest under the control of the
DNA
promoters of this invention. Genes of interest include but are not restricted
to
herbicide resistant genes, genes encoding viral coat proteins, or genes
encoding
proteins conferring biological control of pest or pathogens such as an
insecticidal protein for example B. thuringiensis toxin. Other genes include
those capable of modifying the production of proteins that alter the taste of
the
seed and/or that affecit the nutritive value of the seed.
By "seed-coat'"' it is meant tissues typically found within, and associated
with, the seed-coat of developing or mature angiosperm seeds. With out
wishing to limit the types of tissues found within a seed-coat, this region of
the
seed typically comprises a range of cell types including, and bounded by, an
inner endothelium, and an outer epidermis or palisade cell layer. Within these
inner and out cell layers, there may be found parenchyma-like cells, for
example thin, or thick walled parenchyma, or stellate parenchyma, vascular
tissue, hypodermis, hour-glass cells (osteosclereids), and one or more
integuments, including the inner and outer integuments. However, it is to be
understood that other cell types found within the region between the inner
endothelium and outer epidermis may also be considered to comprise the seed-
coat, for example, but not limited to the hilum, and funiculus comprising
arial
cells. Furthermore, it is to be understood that "seed-coat" also refers to
tissues

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associated with, or adhering to the seed coat, for example the membranous
endocarp (of the inner ovary wall), as this cell type adheres to the seed-coat
and
remains in associationi with the seed coat (see for example Figure 15 (a),
(b),
and Figure 16). Therefore, as used herein, tissues that are associated with,
or
that adhere to, the seed-coat are referred to as "seed-coat associated
tissues", or
"tissues associated with the seed-coat" It is contemplated that other cell
types
may also associate with seed-coat tissues in addition to those disclosed above
and that the tissues identified above should not be considered limiting in any
manner.
By "seed-coat gene" it is meant a gene that is differentially expressed
within the seed-coat as detected under stringent conditions (as defined
above).
Examples of such a gene include, but are not limited to SC4, SC 20, SC 21, or
Ep locus peroxidase. However, the product of the gene may be exported from
the cell to an exterior location of a seed-coat cell, including the surface of
the
seed-coat itself. An e:xample, which is not to be considered limiting in any
manner, of a gene product that is synthesized in within a seed-coat associated
cell, and that is localized onto the surface of the seed coat, is the
hydrophobic
protein (HP; see Figures 14 (a) (b) and Figure 16).
A "seed-coat promoter" is a promoter that is differentially active within
cells of the seed-coat. When operably linked with a gene under its control, a
seed-coat promoter confers expression to a gene within the seed-coat, which
can be detected under stringent conditions (as defined above). Seed coat
associated promoter refers to a promoter that is active in tissue associated
with
the seed coat as defined above.
By "differentially expressed" it is meant the expression of a gene under
the control of a promeRer, as detected by standard means, within a specified
tissue or organ. Such standard means for detecting expression include, but are
not limited to, Northeirns, or in situ hybridizations and the like performed
under

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stringent conditions, or reporter gene expression. For example, a gene that is
differentially expresseci in seed-coat tissues is detectable within seed-coat
tissues, and displays little or no expression in other tissues such as root,
stem.
By "preferentially expressed" it is meant the expression of a gene under
the control of a promoter, as detected by standard means, wherein the majority
of expression is detected within a specified tissue or organ. Such standard
means for detecting expression include, but are not limited to, Northerns, or
in
situ hybridizations and the like performed under stringent conditions, or the
expression of reporter genes. For example, a gene that is preferentially
expressed in seed-coat tissues is detectable within seed-coat tissues, but may
exhibit some expression within other tissues such as root, stem.
By "seed-coat localized" or "localized onto the seed-coat" it is meant a
gene product that, as a result of some property of the amino acid sequence of
the gene product, is targeted within, or onto seed-coat tissues, respectively.
Properties of an amino acid sequence that may direct the targeting of a
protein
within or onto seed-coat tissues include, but are not limited to, signal
sequences
that direct intracellular, and extracellular localization of a protein, and
also
hydrophobic regions within a protein, that results in localization of the
protein
onto the seed coat. An example, which is not to be considered limiting, of a
protein that is localized onto the seed-coat, is the hydrophobic protein (HP).
HP is localized on the outside of the seed-coat following its synthesis within
the
membranous endocarp, and appears to be involved with the adherence of the,
endocarp to the seed-coat (see Figure 16).
Development of the Soybean coat
The seed coat of Glycine max undergoes dramatic changes in the first
two and a half weeks after anthesis (flowering). At six days after anthesis
(DAF; see Figure 12 (a)), the seed coat has a distinct epidermis (10),
consisting

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of thin-walled cuboidal cells; an outer integument (20), consisting of up to a
dozen layers of thin-walled parenchyma, and containing vascular tissue
(recurrent vascular bundle) in the subhilar region; an inner integument (30),
consisting of up to 6 layers of deeply-staining thick-walled parenchyma; and
an
endothelium (40), consisting of thin-walled cuboidal cells.
At 12 days after anthesis (Figure 12 (b)), there is a distinct hypodermis
(15) of thin-walled cuboidal cells directly beneath the epidermis (10); the
outer
integument (20) has differentiated into an upper layer of thin-walled
parenchyma (25), and a lower layer of thick-walled parenchyma (27); the inner
integument (30), while still having very thick, deeply staining cell walls,
has
become stretched, and is compressed to about 3 cells thick; the endothelium
(40) is also retained. Also evident in Figure 12 (b) is the endosperm (50),
and
the developing cotyledons (60).
By 18 days after anthesis (Figure 12 (c)), the epidermal cells have
divided and elongated to form thick-walled macrosciereids, forming a palisade
layer (13). The hypodermis has differentiated into osteosclereids: thick
walled
cells with a characteristic I-shape (hourglass cells; 17). A prominent
vascular
region (70) has developed in the thin-walled parenchyma (25) of the outer
integument which stops before reaching the region of the seed opposite the
hilum; the thick-walled parenchyma (27) is retained. The inner integument
(30) has become completely stretched and crushed, leaving a single, deeply
staining wall layer directly above the endothelium (40). The hilum region
contains a well-developed counter-palisade (80), and a tracheid bar (90). The
seed coat remains attached to the funiculus (100). The sub-hilar region
contains well-develope:d vascular tissue (recurrent vascular bundles; 70) and
stellate parenchyma (110).
At maturity, the seed coat consists of the palisade layer (13), hourglass
cells (17), a partially crushed layer of parenchyma (what remains of the outer

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integument), and an eridothelium (40). The remnant of the inner integument
(30) is often not distinguishable. The tissues of the hilum although
compressed, are retained.
The stages of seed-coat development are also identified in Tables 1 and
2.

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CA 02325819 2000-10-12
WO 99/53067 PCT/CA99/00293
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CA 02325819 2000-10-12
WO 99/53067 PCT/CA99/00293
-24-
~
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CA 02325819 2000-10-12
WO 99/53067 PCT/CA99/00293
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Early development of the tobacco seed coat
.At 6 days after anthesis, the tobacco seed coat consists of an epidermis of
very large, thin walled cells; a layer of parenchyma cells up to 6 cells
thick; and
an endothelium of thin-=walled, cuboidal cells. By 10 days after anthesis, the
inner walls of the epidermis have thickened significantly, with 2-3 layers
discernible; the thin-walled parenchyma has become reduced to 3-4 cells thick
due to stretching of the layer as the seed expands; and the endothelial cells
have
become thinner and elongated. At 22 days after anthesis, the epidermal cells
have stretched and elonigated to accommodate the expanding seed, and the
parenchyma and endottielium have elongated and fused into a crushed layer with
few individual cells distinguishable.
Seed-coat crvntic nromote
There are several lines of evidence that suggest that the seed-coat
expression of GUS activity in the plant T218 is regulated by a cryptic
promoter.
The region surrounding the promoter and transcriptional start site for the GUS
gene are not transcribed in untransformed plants. Transcription was only
observed in plant T218 when T-DNA was inserted in cis. DNA sequence
analysis did not uncover a long open reading frame within the 3.3 kb region
cloned. Moreover, the region is very AT rich and predicted to be noncoding
(data not shown) by the: Fickett algorithm (Fickett, 1982, Nucleic Acids Res.
10,
5303-5318) as implemented in DNASIS 7.0 (Hitachi). Southern blots revealed
that the insertion site is within the N. tomentosiformis genome and is not
conserved among related species as would be expected for a region with an
important gene.
As this is the first report of a cryptic promoter specific to seed-coat
tissues in plants, it is irnpossible to estimate the degree to which cryptic

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promoters may contribute to the high frequencies of promoterless marker gene
activation in plants. It is interesting to note that transcriptional GUS
fusions in
Arabidopsis occur at niuch greater frequencies (54%) than translational
fusions
(1.6%, Kertbundit et al., 1991, Proc. Nati. Acad. Sci. USA 88, 5212-5216).
The possibility that cryptic promoters may account for some fusions was
recognized by Lindsey et al. (1993, Transgenic Res. 2, 33-47).
The results disclosed herewith confirms others (Gheysen et al., 1987,
Proc. Natl. Acad. Sci. USA 84, 6169-6173 and 1991, Genes Dev. 5, 287-297)
that T-DNA may insert into A-T rich regions as do plant transposable elements
(Capel et al., 1993, Nucleic Acids Res. 21, 2369-2373). We illustrate that
promoters of plant genes are also A-T rich raising speculation that gene
insertions into these regions could facilitate the rapid acquisition of new
regulatory elements during gene evolution.
The insertion of functional genes into the nuclear genome and acquisition
of new regulatory sequences has already played a major role in the
diversification
of certain genes and the endosymbiosis of organelles. In plants, most
organellar
proteins are nuclear encoded due to the ongoing transfer of their genes into
the
nucleus (Palmer, 1991õ In Bogorad L and Vasil IK (eds) The Molecular Biology
of Plastids, Academic Press, San Diego, pp 5-53). Recently, it has been shown
that the cox 2 gene of cowpea (Nugent and Palmer, 1991, Cell 66, 473-481) and
soybean (Covello and Gray, 1992, EMBO J. 11, 3815-3820) were transferred
from mitochondria to r.iucleus without promoters by RNA intermediates. The '
results disclosed herewith, with T-DNA-mediated gene fusions reveal the
facility
with which promoters can be acquired by incoming genes. The presence of
cryptic promoters and diverse regulatory elements in the intergenic regions
may
insure that genes rapid:ly achieve the features needed to meet the demands of
complex multicellular organisms.

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Other seed-coat. and seed-coat-associated promoters
Transcripts encoding seed coat specific genes were isolated from seed-
coat cDNA libraries. These cDNA transcripts were then used to identify the
corresponding structural genes and associated promoters from genomic DNA
libraries. These promoters, genes and gene products have been isolated and
characterized Examples of such genes include, but are not limited to, SC4,
SC20,
SC21, a peroxidase cloned from the Ep locus, and HP (hydrophobic protein). It
is to be understood that this seed-coat library comprises tissues typically
found
within the seed-coat and tissues adhering to the seed-coat such as the
membranous endocarp and cells found in the funicular region such as arial
cells
(see above for full defuzition of seed-coat).
Ep locus Peroxidase
The amount of peroxidase activity present in seed coats may vary
substantially among different cultivars. The presence of a single dominant
gene
Ep causes a high seed coat peroxidase phenotype. Homozygous recessive epep
plants are - 100-fold lower in seed coat peroxidase activity which results
from a
reduction in the amount of peroxidase enzyme present, primarily in the
hourglass
cells of the subepidermis (Gijzen et al., 1993). In plants carrying the Ep
gene,
peroxidase is heavily concentrated in the hourglass cells (osteosclereids;
which
forni a highly differentiated cell layer with thick, elongated secondary walls
and
large intercellular spaces).
A seed-coat peroxidase gene, corresponding to the Ep locus, was obtained
from a soybean seed-coat library. The genomic DNA sequence comprises four
exons spanning bp 1533-1752 (exon I), 2383 -2574 (exon 2), 3605-3769 (exon 3)
and 4033-4516 (exon 4) and three introns comprising 1752-2382 (intron 1),
2575-3604 (intron 2) and 3770-4516 (intron 3), of SEQ ID NO:2. Features of
the upstream regulatory region of the genomic DNA include a TATA box

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centred on bp 1487; a cap signa132 bp down stream centred on bp 1520. Also
noted within the genomic sequence are three polyadenylation signals centred on
bp 4520, 4598, 4663 and a polyadenylation site at bp 4700. The promoter
region of the genomic sequence comprises nucleotides 1-1532 of SEQ ID NO:2.
Expression of Ep is first detected at 6 DPA in the thin-walled parenchyma
of the outer integument, adjacent to the thick-walled parenchyma, and flanking
the hilum region. By 9 DPA a thin band of expression extends around the entire
seed coat, at the junction of the thin-and thick-walled parenchyma. Expression
shifts to the hourglass cells as they begin to develop, at 12 DPA (see Figures
13
(e), (f) and (g)).
Expression of a gene under the control of the Ep (peroxidase) promoter
(nucleotides 1-1532 of SEQ ID NO:2,
is observed within the seed-coat from 6 to 18 days after anthesis is
shown in Figures 13 (d) to (g).
Hydrophobic Protein (HP)
Soybean HP is an 8.3 kD protein consisting of 80 amino acids rich in
hydrophobic residues and entirely lacking methionine, phenylalanine,
tryptophan,
lysine and histidine residues (see Figure 15). The amino acid sequence shows
no
significant homology to any known proteins (Odani et al., 1987, Eur J Biochem
162, 485-491).
To determine the composition of proteins deposited on the soybean seed
surface, seeds were washed with a detergent-buffer solution and the extracted
peptides were separated by SDS-PAGE. Protein extracts from the seed coat and

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embryo were also prepared for comparison. These results are shown in Figure 17
(a). The embryo and seed coat extracts contained many proteins covering a wide
molecular mass range. In contrast, extracts from the seed surface were
dominated
by a few low molecular mass proteins. Figure 17 (b) demonstrates that HP
extraction and separation by SDS-PAGE is dependant on dithiothreitol (DTT).
Even though HP is an abundant seed constituent and a potent allergen,
there have been no studies on the expression or localization of the protein or
any
description of the corresponding gene. This is the first report on the
isolation
and characterization of HP cDNA (SEQ ID NO:6) and the corresponding
genomic clone (SEQ ID NO:7), the pattern of gene expression (Figure 10 (e)),
and the localization of the protein (Figure 14 (b)) and its effect on seed
luster
(Figure 16). Figure 14 (c) shows that the presence of surface protein is
related to
the luster, or light reflective properties of the seed surface. Surface
extracts from
shiny seeded phenotypes usually contained far less protein than dull seeded
extracts. Moreover, there were large differences in the amount of protein
present
on the seed surfaces of the two bloom phenotypes examined.
These results also show that the outermost components of the seed coat
are in fact derived from the inner layer of the pod wall (see Figure 14 (a)).
The cDNA and genomic copies of the seed-coat associated HP gene were
obtained from lambda libraries prepared from cultivar Harosoy 63. The genomic
DNA sequence comprises a promoter region from 1-2525 of SEQ ID NO:7.
Within this promoter region are located clustered direct repeats (between 1-
586;
see also Figure 11 (c). and a TATA box located at position 2442-2447. The
ORF for HP is between 2526-2882, with the translational start site at 2526,
followed by a signal sequence from 2526-2642, and the mature protein from
2643-2882. Also noted within the genomic sequence are six polyadenylation
signals and a polyadenylation site at bp 3193.

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Developmental and tissue specific expression patterns for the HP gene
were determined by RNA blot analysis and in situ hybridization. Representative
RNA blots, probed with HP cDNA, are shown in Figures 10 (e) and (f). These
results show that HP is highly expressed in the pod during the mid to late
stages
of seed development. Hybridization signals were also observed in seed coat RNA
samples. No expression was evident in the flower, leaf, embryo, stem, or root.
We also compared HP transcript levels of two different seed luster phenotypes
that differed in the amount of HP present on their seed surfaces. Figure 10
(g)
shows that HP mRNA levels are several fold greater in dull seeded plants that
accumulate large amounts of HP on the seed surface when compared to shiny
seeded plants that have only trace amount of HP on the seed surface. Faint
signals, corresponding to low HP transcript levels, were detectable in shiny
seeded phenotypes after prolonged exposure times (not shown).
Localization of HP gene expression by in situ hybridization is shown in
Figures 14 (b), (c) and (d). At six days post anthesis (DPA) expression of HP
is
limited to the membrarious inner layer of the pericarp. By 12 DPA expression
is
very strong and the inrier epidermis is showing signs of becoming detached
from
the rest of the pericarp and, in places, is adhering to the seed surface.
Tissue
sections from this stage of development also showed strong hybridization
signals
in the sclerenchyma, itidicating that HP expression occurs throughout the
endocarp. Portions of membranous endocarp adhere to the seed during the
course of development (see Figures 14 (a) and 16) and thus constitute a newly
identified component of the seed coat of mature, fully developed soybeans. The
deposition of this material alters the physical properties and the composition
of
the seed surface, as shown by SDS-PAGE analysis (Figures 17 (a), (b) and (c))
and by scanning electron microscopy (Figure 16). A comparison of dull- and
shiny-seeded cultivars reveals that the HP gene controls this phenotypic trait
in
soybeans.

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In Figure 14 (b) can be seen the expression of a gene under the control of
the HP promoter, The promoter (nucleotides 1-2526 of SEQ ID NO:7) is active
within the membranous endocarp associated with the outer seed-coat.
SC 4. 20 and 21
Genes expressin,g specifically in seed coat tissue were isolated from a seed
coat cDNA library obtained from seed coats in later stages of development.
SQ4
The deduced protein sequence from the SC4 cDNA (Figure 19 (a); SEQ
ID NO:3) consists of 289 amino acids and has a molecular mass of 31.9 kDa and
a predicted pI of 7.95. 'Three puatative glycosylation sites are present at
positions
92, 128 and 269. The putative polypeptide encoded by SC4 exhibits similarity
with proteins that comprises a BURP domain (see Figure 19 (b)). The BURP
domain is a long carboxyl terminal domain containing a number of highly
conserved amino acids (Hattori J. et al., 1998. Mol. Gen. Genet. 259: 424-
428).
The genomic sequence of sc4 is provided in SEQ ID NO:9 (also see Restriction
map Figure 11 (d)) and comprises a promoter from nucleotides 1-5514 of SEQ
ID NO:9.
The expression of a gene under control of the SC4 promoter (nucleotides
1-5514 of SEQ ID NO::9) within soybean seed coat at 3 days after anthesis is
shown in Figure 13 (a). The activity of the promoter is localized within the
inner integument (arrow). Other areas of brightness in this figure include the
recurrent vascular bundles in the funiculus, and the trichomes of the pod (the
bright areas are due to the birefringence of crystalline areas in the cell
walls, and
are also present in the negative control; data not shown).

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RNA samples from seed coat, embryo, stem, root, leaf, pod and flower
were hybridized with a radiolabelled SC4 cDNA probe (Figure 10 (a)) to
determine organ specificity of the expression of SC4. The sc4 transcript was
only expressed in the seed coat organ. It was estimated that the size sc4 mRNA
was 1.2 kb (data not shown).
Northern blot arialysis was carried out to determine the temporal
expression pattern of sc4. RNA from seed coat, embryo and pod organs between
6-24 dpa were hybridized with a radiolabelled SC4 cDNA probe. No gene
expression was observed in any of the embryo development stages examined
(Figure 20 (a)). sc4 expression was apparent in the seed by 6 dpa. After 6 dpa
the expression of sc4 in the seed coat increased - 4-fold to its maximum
detected
level between 9-12 dpa. By 15 dpa sc4 expression had decreased by - 2.5-fold
dpa and continued to decline to just detectable levels by 18 dpa (Fig. 3.7).
Expression of sc4 could. only be detected in the seed coat at 21-24 dpa when
the
filter was over-exposed. Gene expression of sc4 in the pod was detected from
12-
21 dpa only after over-exposure of the filter (data not shown).
To analyse the distribution of sc4 expression with respect to cell
differentiation during seed coat development in situ hybridization was
performed
on seed sections from 3-24 dpa seeds. sc4 was expressed throughout the inner
integument of the seed coat at 3 dpa (Figures 13 (a) and 21). By 6 dpa the
expression pattern of se4 had changed, and was localized to the outer
integument
parenchyma but not to the vascular tissue embedded within this layer. sc4
expression in the outer integument was maintained until 18 dpa after which
time
no further expression was detected (see Table 4 in Examples). In concurrence
with northern blot analysis, the in situ hybridization results revealed that
sc4
expression increased to a maximum between 9-12 dpa and decreased thereafter
(Table 4, in Examples). In addition, expression of sc4 was not observed in the
embryo of seed at 3-6 dpa.

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Expression of a gene under the control of the SC4 promoter (1-5514 of
SEQ ID NO:9) is seen in Figures 13 (a) and 21.
Southern blot analysis was carried out to examine the gene family
composition of sc4. Soybean genomic DNA was cleaved with Eco RI, Hind III
and Xba I. which do not: have recognition sites in the SC4c cDNA sequence.
Under conditions of low to high stringency (i.e., from 40-10 C below Tm of the
probe ) the SC4 cDNA probe hybridized to a single band (Figure 22) and
therefore sc4 appears to be a single gene.
Southern blot analysis was also performed to determine the occurrence of
sc4 within the following plant species: pea (Pisum sativum), canola (Brassica
napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum annuum),
Mimosa sp. (Mimosa pudica), black spruce (Picea mariana (Mill) B.S.P.), birch
(Betula pendula Roth). The genomic DNA was digested with Eco RI. Under all
stringency conditions it was observed that the radiolabelled SC4 cDNA probe
hybridized to only soybean genomic DNA (Figure 22 (b)). Further analysis of
more related species to soybean need to be carried out.
SC20
The open reading frame of SC20 encodes a putative protein of 770 amino
acid residues with a calculated molecular mass of 82.688 kDa and a predicted
pI
of 6.93. The predicted protein has ten potential N-glycosylation sites (Figure
23
(b)). The hydropathy profile (Figure 23 (c)) of SC20 protein revealed that the
first 23 amino acids constitute a hydrophobic region typical of an eukaryotic
signal peptide. From noirthern blot analysis, the SC20 cDNA clone hybridizes
to
a-- 2.5 kb transcript.
The genomic sc20 clone is 7235 bp in length (see Figure 23 (a) for
restriction map, and SEQ ID NO:8). Alignment of sc20 genomic and SC20

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cDNA sequences revealed that sc2O contained eight introns of 94 bp, 101 bp,
185 bp, 80 bp, 154 bp, 112 bp, 110 bp and 93 bp respectively (Figure 23 (a)).
A
search of the 5' upstreaun region of sc20 revealed three potential
transcription
start sites at positions 1085, 1156 and 2272. The promoter region of sc2O
spans
nucleotides 1-2450 of SEQ ID NO:8.
Sequence comparisons (Figure 23 (d)) revealed that the putative
polypeptide encoded by SC20 was similar to plant proteins belonging to the
Pyrolysin family in the clan of serine proteases known as the subtilases
(Barrett
A.J. and Rawlings N.D., 1995. Arch. Biochem. Biophys. 318:247-250; Siezen,
R. J. and Leunissen, J. A. M. 1997. Protein Sci. 6: 501-523.). The SC20
protein comprises 3 domains: a signal peptide of 23 residues followed by a
prosequence of 93 resiclues and a mature domain of 654 residues. The predicted
mature domain of SC20 has a calculated molecular weight of 69.918 kDa and an
isoelectric point of 6.34.
Northern blot analysis was carried out to determine specificity of sc20
expression in various soybean organs i.e., seed coat, embryo, stem, root,
leaf,
pod and flower (Figure 10 (b)). sc20 has seed coat-specific expression as its
mRNA was detected ortly in the seed coat organ. The sc20 transcript was
determined to be approximately 2.5 kb (data not shown). Even after prolonged
exposure of the filter, no sc20 transcripts was detected in any of the other
plant
organs.
Northern blot analysis was performed to determine the temporal gene
expression pattern of sc:20 in seed coat, embryo and pod organs of soybean.
Total RNA prepared from organs between 6- 24 dpa were probed with a
radiolabelled SC20 cDNA probe. sc20 expression was detected at 9 dpa and rose
1.5 fold to its maximuin observed level at 12 dpa (Figure 24). By 18 dpa
accumulation of sc20 n:zRNA had decreased 4-fold. Prolonged exposure of the
filter enabled detection of sc20 expression at 6 dpa and 21-24 dpa. No gene

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expression was observed at any stage of embryo or pod development examined
even after prolonged exposure of the filters. This confirmed that sc20
expression
was seed coat-specific.
In situ hybridization was carried out to analyse the spatial gene expression
pattern of sc20 within the seed coat between 3-24 dpa. Seed sections were
hybridized with radiolabelled sense and anti-sense SC20 RNA probes. No
birefringent cell structui-es were evident in the seed sections used (Figure
24).
Gene expression of sc20 was localized to the thick-walled parenchyma of
the outer integument (see Figures 13 (b) and 24). The temporal expression
pattern of 9-21 dpa expression with an observed peak at 12 dpa was almost
identical to that determiined by northern blot analysis (Table 4, in
Examples).
sc20 transcripts were not detected in the embryo between 3-6 dpa. The in situ
hybridization results of the seed sections concur with the northern blot
analysis
that within the seed org~m sc20 is expressed only in the seed coat organs.
Expression of gene under control of the SC20 promoter (1-245 of SEQ
ID NO:8) is seen in Figures 13 (b) and 24.
SouthÃrn blot analysis was performed to ascertain whether sc2O is a single
gene or a member of a gene family. Soybean genomic DNA was cleaved with
Eco RI, Hind III, Xba I and Eco RV which have three, four, two and one
recognition site(s) respectively in the sc20 clone (see Figure 23 (a)). Under
conditions of high stringency to detect genes with at least 90% similarity to
sc20
the probe hybridized to a single band (Figure. 25 (b)). Under medium
stringency
conditions to observe genes with 80% similarity to sc20 it was observed that
the
SC20 probe annealed to 2-3 bands for each digest (Figure 25 (a)). Under
conditions of low stringency i.e., 40 C below Tm the SC20 probe hybridized to
several more bands froni each digest (data not shown). This suggested that
sc20

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is a member of a small gene family composed of 2-3 members and that the
soybean genome contains several genes which are more distantly related to
sc20.
Southern blot arialysis was also performed to determine the distribution of
sc20 among a number of diverse plant species i.e., pea (Pisum sativum), canola
(Brassica napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum
annuum), Mimosa sp. (Mimosa pudica), black spruce (Picea mariana (Mill)
B.S.P.), birch (Betula pendula Roth). The genomic DNA was restricted with Eco
RI. The SC20 cDNA probe hybridized to only the genomic DNA of soybean
(Figure 25 (c)) irrespective of stringency conditions utilized. It is possible
that
the gene may exist in rnore species more closely related to soybean.
SC21
The expression of a gene under the control of SC21 promoter (see Figure
11 (b)) within seed coat tissues at 15 days after anthesis is shown in Figure
13
(c). Note specific localization of the probe in the thin-walled parenchyma of
the
outer integument, including the area immediately surrounding the tracheid bar
(arrow).
The nucleotide sequences of SC21 (SEQ ID NO:5) and SC17 were
identical apart from the position of the poly (A) tail and were just less than
65 %
similar to a Cicer arientinum (chickpea) mRNA for an unknown protein.
The expression of genes under the control of seed-coat promoters of this
invention are shown in Figures 10, 13, 14, 21 and 24.
The results of these and other experiments indicating the expression
patterns of these genes is summarized in Table 4 within the Examples section.

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The promoters of the present invention can be used to control the
expression of any given gene spatially and developmentally within developing
seed coats, or seed-coat associated tissues. Some examples of such uses, which
are not to be considered limiting, include:
1. Modification of storage reserve yields in seed coats, such as starch
by the expression of yeast invertase to mobilize the starch, or
increasing starch levels by increasing the sink strength by
enhancing carbon unloading into seeds, by expressing invertase in
specific seed coat tissues, or reduce starch levels by inhibit starch
biosynthesis through the expression of the antisense transcript of
ADP-glucose pyrophosphorylase.
2. Modification of seed colour contributed by anthocyanin pigments
or condensed tannins in the seed coats by expression of antisense
transcripts of the phenylalanine ammonia lyase or chalcone
synthase genes.
3. Modification of fibre content in seed-derived meal by expression
of antisense transcripts of the caffeic acid-o-methyl transferase or
cinnamoyl alcohol dehydrogenase genes.
4. Inhibition of seed coat maturation by expression of ribonuclease
genes to allow for increased seed size, and to reduce the relative
biomass of seed coats, and to aid in dehulling of seeds.
5. Expression of genes in seed coats coding for insecticidal proteins
such as a: amylase inhibitor or protease inhibitor.
6. Partitioning of seed metabolites such as glucosinolates into seed
coats for fungal or insect resistance.

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7. Production of high value proteins in seed coats for use as
pharmaceuticals or for use in industrial processes.
8. Control af seed borne diseases by expressing antifungal antiviral,
or anti-bacterial proteins within the seed coat
Furthermore, modifications of the nucleotide, or amino acid, sequence of
HP, or the preparation of chimeric gene constructs comprising the regulatory
region of HP associated with a gene of interest will result in:
- alterations in the textural, visual, chemical or other properties of the
seed
coat, including the seed surface;
- the production of plants that are less susceptible to seed borne and pod
diseases by expressing heterologous proteins in tissues of the ovary wall;
- lessening the health hazard of seed dust exposure by genetic selection or
transformation, to produce plants with reduced allergenic protein
expression on the seed surface
Thus this invention is directed to such promoter and gene combinations.
Further this invention is directed to such promoter and gene combinations in a
cloning vector, wherein the gene is under the control of a seed coat specific -
promoter and is capable of being expressed in a plant cell transformed with
the
vector. This invention further relates to transformed plant cells and
transgenic
plants regenerated from such plant cells. The promoter and promoter gene
combination of the present invention can be used to transform any plant cell
for
the production of any transgenic plant. The present invention is not limited
to
any plant species.

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The following list sumrnarises the nucleotide sequence data in the SEQUENCE
LISTING of the present application:
pT218 genomic DNA sequence is found in SEQ ID NO: 1;
Ep genomic DNA sequence is listed in SEQ ID NO:2;
SC4 cDNA sequence is presented in SEQ ID NO:3;
SC20 cDNA sequence is in SEQ ID NO:4;
SC21 cDNA sequence is presented in SEQ ID NO:5;
HP cDNA is listed in SEQ ID NO:6;
HP genomic DNA sequence is found in SEQ ID NO:7;
SC20 genomic I)NA sequence is listed in SEQ ID NO:8; and
SC4 genomic DNA sequence is presented in SEQ ID NO:9.
While this invenition is described in detail with particular reference to
preferred embodiments thereof, said embodiments are offered to illustrate but
not
limit the invention.
EXAMPLES
Characterization of a Seed Coat-Specific GUS Fusion
Transfer of binary constructs to Agrobacterium and leaf disc
transformation of Nicotiana tabacum SRi were performed as described by Fobert
et al. (1991, Plant Molõ Biol. 17, 837-85 1). Plant tissue was maintained on
100
g/ml kanamycin sulfate (Sigma) throughout in vitro culture.
Nine-hundred aiid forty transgenic plants were produced. Several
hundred independent transformants were screened for GUS activity in developing
seeds using the fluorogenic assay. One of these, T218, was chosen for detailed
study because of its unique pattern of GUS expression.

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Fluorogenic and histological GUS assays were performed according to
Jefferson (Plant Mol. Biol. Rep., 1987, 5, 387-405), as modified by Fobert et
al.
(Plant Mol. Biol., 1991, 17, 837-851). For initial screening, leaves were
harvested from in vitro grown plantlets. Later flowers corresponding to
developmental stages 4 and 5 of Koltunow et al. (Plant Cell, 1990, 2,
1201-1224) and beige seeds, approximately 12-16 dpa (Chen et al., 1988, EMBO
J. 7, 297-302), were collected from plants grown in the greenhouse. For
detailed, quantitative analysis of GUS activity, leaf, stem and root tissues
were
collected from kanamycin resistant Fl progeny of the different transgenic
lines
grown in vitro. Floral tissues were harvested at developmental stages 8-10
(Koltunow et al., 1990, Plant Cell 2, 1201-1224) from the original transgenic
plants. Flowers of these plants were also tagged and developing seeds were
collected from capsules at 10 and 20 dpa. In all cases, tissue was weighed,
immediately frozen in liquid nitrogen, and stored at -80 C.
Tissues analyzed by histological assay were at the same developmental
stages as those listed above. Different hand-cut sections were analyzed for
each
organ. :For each plant, histological assays were performed on at least two
different occasions to ensure reproducibility. Except for floral organs, all
tissues
were assayed in phosphate buffer according to Jefferson (1987, Plant Mol.
Biol.
Rep. 5, 387-405), with 1 mM X-Gluc (Sigma) as substrate. Flowers were
assayed in the same buffer containing 20% (v/v) methanol (Kosugi et al., 1990,
Plant Sci. 70, 133-140)õ
Tissue-specific patterns of GUS expression were only found in seeds.
For instance, GUS activity in plant T218 (Figure 1) was localized in seeds
from
9 to 17 days postanthesis (dpa). GUS activity was not detected in seeds at
other
stages of development or in any other tissue analyzed which included leaf,
stem,
root, anther, ovary, petal and sepal (Figure 1). Histological staining with X-
Gluc revealed that GUS expression in seeds at 14 dpa was localized in seed
coats

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but was absent from the embryo, endosperm, vegetative organs and floral organs
(results not shown).
The seed coat-specificity of GUS expression was confirmed with the more
sensitive fluorogenic assay of seeds derived from reciprocal crosses with
untransformed plants. T:he seed coat differentiates from maternal tissues
called
the integuments which do not participate in double fertilization (Esau, 1977,
Anatomy of Seed Plants. New York: John Wiley and Sons). If GUS activity is
strictly regulated, it must originate from GUS fusions transmitted to seeds
maternally and not by pollen. As shown in Table 3, this is indeed the case. As
a
control, GUS fusions expressed in embryo and endosperm, which are the
products of double fertilization, should be transmitted through both gametes.
This is illustrated in Table 3 for GUS expression driven by the napin promoter
(BngNAPI, Baszczynki and Fallis, 1990, Plant Mol. Biol. 14, 633-635) which is
active in both embryo and endosperm (data not shown).

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Table 3. GUS activity in seeds at 14 days post anthesis.
Cross GUS Activity
nmole MU/min/mg Protein
T218 T218 1.09 + 0.39
T218 WTO 3.02 + 0.19
WT T218 0.04 0.005
WT WT 0.04 t 0.005
NAP-5b NAP-5 14.6 t 7.9
NAP-5 WT 3.42 t 1.60
WT NAP-5 2.91 ~ 1.97
a WT, untransformed plants
b Transgenic tobacco plants with the GUS gene fused to the
napin, BngNAP1, promoter (Baszczynski and Fallis, 1990, Plant Mol.
Biol. 14, 633-635).
Cloning and Analysis of the Seed Coat-Specific GUS Fusion
Genomic DNA was isolated from freeze-dried leaves using the protocol of
Sanders et al. (1987, Nucleic Acid Res. 15, 1543-1558). Ten micrograms of
T218 DNA was digestect for several hours with EcoRi using the appropriate
manufacturer-supplied buffer supplemented with 2.5 mM spermidine. After
electrophoresis through a 0.8% TAE agarose gel, the DNA size fraction around
4-6 kb was isolated, purified using the GeneClean kit (BIO 101 Inc., LaJolla,

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CA), ligated to phosphatase-treated EcoRI-digested Lambda GEM-2 arms
(Promega) and packaged in vitro as suggested by the supplier. Approximately
125,000 plaques were transferred to nylon filters (Nytran, Schleicher and
Schuell) and screened by plaque hybridization (Rutledge et al., 1991, Mol.
Gen.
Genet. 229, 31-40), using the 3' (termination signal) of the nos gene as probe
(probe #1, Figure 2). This sequence, contained in a 260 bp SstI/EcoRI
restriction fragment from pPRF-101 (Fobert et al., 1991, Plant Mol. Biol. 17,
837-851), was labelled with [a 32P]-dCTP (NEN) using random priming
(Stratagene). After plaque purification, phage DNA was isolated (Sambrook et
al., 1989, A Laboratory Manual. New York: Cold Spring Harbor Laboratory
Press), mapped and subcloned into pGEM-4Z (Promega). The EcoRI fragment
and deletions shown in Figure 2 were inserted into pBIN19 (Bevan, 1984, Nucl.
Acid Res. 12, 8711-8721). Restriction mapping was used to determine the
orientation of the fusion in pBIN19 and to confirm plasmid integrity. Plants
were transformed with a derivative which contained the 5' end of the GUS gene
distal to the left border repeat. This orientation is the same as that of the
GUS
gene in the binary vector pB1101 (Jefferson, 1987, Plant Mol. Biol. Rep. 5,
387-405).
The GUS fusion in plant T218 was isolated as a 4.7 kb EcoRI fragment
containing the 2.2kb promoterless GUS-nos gene at the T-DNA border of
pPRF120 and 2.5 kb of 5' flanking tobacco DNA (pT218, Figure 2), using the
rnos 3' fragment as probe (probe #1, Figure 2). To confirm the ability of the
flanking DNA to activate the GUS coding region, the entire 4.7 kb fragment was
inserted into the binary transformation vector pBIN19 (Bevan, 1984, Nucl. Acid
Res. 12, 8711-8721), as shown in Figure 2. Several transgenic plants were
produced by Agrobacterium-mediated transformation of leaf discs. Southern
blots indicated that each plant contained 1-4 T-DNA insertions at unique
sites.
The spatial patterns of GUS activity were identical to that of plant T218.
Histologically, GUS staining was restricted to the seed coats of 14 dpa seeds
and
was absent in embryos and 20 dpa seeds (results not shown). Fluorogenic assays
* Trademark

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of GUS activity in developing seeds showed that expression was restricted to
seeds between 10 and 17 dpa, reaching a maximum at 12 dpa (Figure 3 (a) and 3
(b)). The 4.7 kb fragment therefore contained all of the elements required for
the tissue-specific and developmental regulation of GUS expression.
7'o locate regions within the flanking plant DNA responsible for seed
coat-specificity, truncated derivatives of the GUS fusion were generated
(Figure
2) and introduced into tobacco plants. Deletion of the region approximately
between 2.5 and 1.0 kbõ 5' of the insertion site (pT218-2, Figure 2) did not
alter
expression compared with the entire 4.7 kb GUS fusion (Figures 3b and 4).
Further deletion of the DNA, to the SnaBI restriction site approximately 0.5
kb,
5' of the insertion site (pT218-3, Figure 2), resulted in the complete loss of
GUS
activity in developing seeds (Figures 3b and 4). This suggests that the region
approximately between 1.0 and 0.5 kb, 5' of the insertion site contains
elements
essential to gene activation. GUS activity in seeds remained absent with more
extensive deletion of plant DNA (pT218-4, Figures 2, 3b and 4) and was not
found in other organs including leaf, stem, root, anther, petal, ovary or
sepal
from plants transformed with any of the vectors (data not shown).
The transcriptiorial start site for the GUS gene in plant T218 was
determined by RNase protection assays with RNA probe #4 (Figure 2) which
spans the T-DNA/plant DNA junction. For RNase protection assays, various
restriction fragments from pIS-1, pIS-2 and pT218 were subcloned into the
transcription vector pGEM-4Z as shown in Figures 7 and 2, respectively. A
440bp HindIII fragment of the tobacco acetohydroxyacid synthase SURA gene
was used to detect SUR4 and SURI3 mRNA. DNA templates were linearized and
transcribed in vitro withi either T7 or SP6 polymerases to generate strand-
specific
RNA probes using the I'romega transcription kit and [a 32P]CTP as labelled
nucleotide. RNA probes were further processed as described in Ouellet et al.
(1992, Plant J. 2, 321 -330). RNase protection assays were performed as
described in Ouellet et al., (1992, Plant J. 2, 321-330), using 10-30 g of
total

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RNA per assay. Probe digestion was done at 30 C for 15 min using 30 g ml-'
RNase A. (Boehringer Mannheim) and 100 units ml-' RNase Tl (Boehringer
Mannheim). Figure 5 shows that two termini were mapped in the plant DNA.
The major 5' terminus is situated at an adenine residue, 122 bp upstream of
the
T-DNA insertion site (Fiigure 6). The sequence at this transcriptional start
site is
similar to the consensus sequence for plant genes (C/TTC 1 ATCA; Joshi, 1987
Nucleic Acids Res. 15, 6643-6653). A TATA box consensus sequence is present
37 bp upstream of this start site (Figure 6). The second, minor terminus
mapped
254 bp from the insertion site in an area where no obvious consensus motifs
could be identified (Figure 6).
The tobacco DNA upstream of the insertion site is very AT-rich (> 75 %,
see Figure 7). A search for promoter-like motifs and scaffold attachment
regions
(SAR), which are often associated with promoters (Breyne et al., 1992, Plant
Cell 4, 463-471; Gasser and Laemmli, 1986, Cell 46, 521-530), identified
several putative regulatory elements in the first 1.0 kb of tobacco DNA
flanking
the promoterless GUS gene (data not shown). However, the functional
significance of these sequences remains to be determined.
Cloning and Analysis of the Insertion Site from Untransformed Plants
A lambda DASH genomic library was prepared from DNA of
untransformed N. tabacum SRl plants by Stratagene for cloning of the insertion
site corresponding to the gene fusion in plant T218. The screening of 500,000
plaques with probe #2 (Figure 2) yielded a single lambda clone. The EcoRI and
XbaI fragments were subcloned in pGEM-4Z to generate pIS-1 and pIS-2.
Figure 7 shows these two overlapping subclones, pIS-1 (3.0 kb) and pIS-2 (1.1
kb), which contain tobacco DNA spanning the insertion site (marked with a
vertical arrow). DNA sequence analysis (using dideoxy nucleotides in both
directions) revealed that the clones, pT218 and pIS-1, were identical over a

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length of more than 2.5 kb, from the insertion site to their 5' ends, except
for a
12 bp filler DNA insert of unknown origin at the T-DNA border (Figure 6 and
data not shown). The presence of filler DNA is a common feature of T-
DNA/plant DNA junctions (Gheysen et al., 1991, Gene 94, 155-163). Gross
rearrangements that sornetimes accompany T-DNA insertions (Gheysen et al.,
1990, Gene 94, 155-163; and 1991, Genes Dev. 5, 287-297) were not found
(Figure 6) and therefore could not account for the promoter activity
associated
with this region. The region of pIS-1 and pIS-2, 3' of the insertion site is
also
very AT-rich (Figure 7).
To determine whether there was a gene associated with the pT218
promoter, more than 3.3 kb of sequence contained with pIS-1 and pIS-2 was
analyzed for the presence of long open reading frames (ORFs). However, none
were detected in this region (data not shown). To determine whether the region
surrounding the inserticin site was transcribed in untransformed plants,
Northern
blots were performed with RNA from leaf, stem, root, flower and seeds at 4, 8,
12, 14, 16, 20 and 24 dpa. Total RNA from leaves was isolated as described in
Ouellet et al., (1992, Plant J. 2, 321-330). To isolate total RNA from
developing seeds, 0.5 g of frozen tissue was pulverized by grinding with dry
ice
using a mortar and pestle. The powder was homogenized in a 50 ml conical tube
containing 5 ml of buffer (1 M Tris HCI, pH 9.0, 1 % SDS) using a Polytron
homogenizer. After two extractions with equal volumes of
phenol:chloroform:isoamyl alcohol (25:24:1), nucleic acids were collected by
ethanol precipitation and resuspended in water. The RNA was precipitated
overnight in 2M LiCI alt 0 C, collected by centrifugation, washed in 70%
ethanol
and resuspended in water. Northern blot hybridization was performed as
described in Gottlob-McHugh et al. (1992, Plant Physiol. 100, 820-825). Probe
#3 (Figure 2) which spans the entire region of pT218 5' of the insertion did
not
detect hybridizing RNA. bands (data not shown). To extend the sensitivity of
RNA detection and to include the region 3' of the insertion site within the
analysis, RNase protection assays were performed with 10 different RNA probes

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that spanned both strands of pIS-1 and pIS-2 (Figure 7). Even after lengthy
exposures, protected fragments could not be detected with RNA from 8, 10, 12
dpa seeds or leaves of untransformed plants (see Figure 5 for examples with
two
of the probes tested). The specific conditions used allowed the resolution of
protected RNA fragments as small as 10 bases (data not shown). Failure to
detect protected fragments was not due to problems of RNA quality, as control
experiments using the same samples detected acetohydroxyacid synthase (AHAS)
SURA and SURB mRNA which are expressed at relatively low abundance (data
not shown). Conditions used in the present work were estimated to be sensitive
enough to detect low-abundance messages representing 0.001-0.01 % of total
mRNA levels (Ouellet et al., 1992, Plant J. 2, 321-330). Therefore, the region
flanking the site of T-DNA insertion does not appear to be transcribed in
untransformed plants.
Genomic Origins of the Insertion Site
Southern blots were performed to determine if the insertion site is
conserved among Nicaticzna species. Genomic DNA (5 jig) was isolated,
digested and separated b;y agarose gel electrophoresis as described above.
After
capillary transfer on to nylon filters, DNA was hybridized, and probes were
labelled, essentially as described in Rutledge et al. (1991, Mol. Gen. Genet.
229,
31-40). High-stringency washes were in 0.2 x SSC at 65 C while low-
stringency washes were in 2 x SSC at room temperature. In Figure 8, DNA of
the allotetraploid species N. tabacum and the presumptive progenitor diploid
species N. tomentosiformis and N. sylvestris (Okamuro and Goldberg, 1985, Mol.
Gen. Genet., 198, 290-298) were hybridized with probe #2 (Figure 2). Single
hybridizing fragments of identical size were detected in N. tabacum and N.
tomentosiformis DNA digested with HindIII, XbaI and EcoRI, but not in N.
sylvestris. Hybridizations with pIS-2 (Figure 8) which spans the same region
but
includes DNA 3' of the insertion site yielded the same results. They did not
reveal hybridizing bands, even under conditions of reduced stringency, in

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additional Nicotiana species including N. rustica, N. glutinosa, N.
megalosiphon
and N. debneyi (data not shown). Probe #3 (Figure 2) revealed the presence of
moderately repetitive DNA specific to the N. tomentosiformis genome (data not
shown). These results suggest that the region flanking the insertion site is
unique
to the N. tomentosiformis genome and is not conserved among related species as
might be expected for regions that encode essential genes.
Cloning of seed-coat genes from Soybean:
a) Isolation of seed-coat cDNA clones
A seed coat cDNA library was constructed in Lambda GEM-4 from
poly(A)+ mRNA isolated from soybean [Glycine max (L.) Merrill] seed coats. A
sample of the total ampl:ified library was used to sub-clone inserts from the
original lambda vector iinto pBK-CMV (Stratagene). Random clones were
selected from this mass excision for plasmid purification and single-run DNA
sequencing to construct an expressed sequence tag (EST) database.
For differential screening, an additional cDNA library was constructed
from cultivar Maple Presto (EpEp) seed coats. The seed coats were harvested
from seeds of four fresh weight groups: <50 mg, 50-100 mg, 150-250 mg and
> 250 mg, to represent all developmental stages. Total RNA was isolated from
the seed coats using Trizole reagent (BRL) from which poly (A) + RNA was
isolated using Oligotex resin (Qiagen). First and second strand cDNAs were
synthesized using the Ri.boclone cDNA synthesis kit and then cloned into a
lambda GEM-4 vector (Promega). This seed coat library was differentially
screened with positive and negative cDNA probes to identify genes
preferentially
expressed in the seed coat. The positive probe was derived from poly (A)+
mRNA isolated from seed coat tissues while the negative probe was made from
poly (A)+ mRNA from, seedling, flower bud, leaf, pod and root tissue. The
cDNA library was screened with cDNA synthesized from RNA using oligo(dT)15

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primer, and hybridizations were carried out in Denhardt's solution (Sambrook
et
al. (1989) Molecular Cloning, Second Edition) at 65 C; wash 4 x 30 minutes
0.1X SSC 0.1% SDS at 65 C.
T'wenty-one positive clones were identified after plaque purification. The
Lambda vector GEM-4 contains a complete pGEM1 plasmid. During the cloning
procedure the cDNA is inserted into the Lambda vector at the multicioning site
of this plasmid. The ent:ire pGEM1 plasmid, containing the cDNA insert, can be
removed from the Lambda vector by digestion with Spel and then can be
relegated to form a functional plasmid. Except for SC 11 and SC 19, the insert
was removed from pGE:M1 by digestion with Xbal and EcoRl and ligated into
an alternative plasmid vector pGEM4-Z. Following this protoco121 seed coat
clones were used to tran.sform E. coli DH5a. No transformants were obtained
with seed coat clones SC:7 and SC10 and so these clones were not processed
further.
Seed surface pro'teins were obtained from soybean. A single seed was
placed in a 2 mL plastic capped test tube and surface proteins were extracted
by
adding 0.5 mL of a buffer-detergent solution containing 10 mM Tris-Cl (pH 7.5)
0.5 % SDS, and 20 mM DTT, and placing the tube in a boiling water bath for 2
min. The contents of the tube were mixed and an aliquot was withdrawn and
centrifuged for 5 min at 14,000 g. Freshly prepared loading buffer containing
20
mM DTT was added to the sample and proteins were electrophoretically
separated on 15 % acrylarnide gels in the presence of SDS (see Figure 17)
using a
modified Laemmli system, as described by Fling and Gregerson (1986, Anal
Biochem 155:83-88). Fixation and visualization of the proteins by silver
staining
followed the method of Blum et al., (1987 Electrophoresis 8: 93-99). The amino
terminal of the major peptides (indicated as HPS in Figure 17 (b)) were micro-
sequenced from the blotted proteins according to Moos et al., (1988 J. Biol.
Chem 263: 6005-6008). The resulting amino acid sequences were identical and
matched existing sequer.ices in the GenBank protein database for HP (Odani et

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al., 1987 Eur J. Biocheni 162, 485-491). Both peptides had alternative N-
terminal residues of Ala or Ile, as has been previously noted for HP. The
different electrophoretic mobilities of the two peptides could not be
accounted for
from the microsequencing analysis, but may be due to differences in
glycosylation.
Several different soybean varieties were also compared by SDS-PAGE
analysis (see Figure 17 (c)).
To obtain the cD]!VA transcript of HP, sequences in the seed coat
expressed sequence tag database were searched for reading frames corresponding
to the HP amino acid sequence. Using this strategy, several identical cDNA
transcripts were isolated from the cDNA library obtained from Harosoy 63 seeds
described above that included in their reading frames peptide sequences
exactly
matching HP. The encoded products of these DNA sequences were identified
using the BLASTX program at the NCBI site.
b) Characterization of cDNA clones
Sequence analysis
Agarose gel electrophoresis of Xbal /EcoRl digests of the 19 remaining
plasmid clones indicated that the inserts ranged in size from approximately
350bp
to 1600bp, including the poly A tail. Inserts of the seed-coat clones were
characterized by double stranded dideoxy sequencing of the 5' and 3' ends of
the
clones. A preliminary classification of the seed coat cDNA clones was made on
the basis of sequence homology in the 3' and 5' ends of the clones. Based on
sequence similarity with each other these 19 clones were grouped into 7 groups
of clones. Sequence siniilarity was found between four of these groups and
GenBank (with proline rich protein and three peroxidase groups). The three
remaining groups had no sequence similarity with GenBank. SC4, SEQ ID

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NO:3 (found to be the same as SCl), SC20, SEQ ID NO:4 (found to be the same
as SC15), and SC21, SEQ ID NO:5, each represent one clone of each of the
three groups which did riot exhibit similarity with anything in the GenBank
database.
SC4
The 1119 bp nuclleotide sequence of SC4 (SEQ ID 3, Figure 19 (a); also
see Restriction Map Figure 11 (d)) does not represent the full-length cDNA
clone
as it does not contain an ATG codon for translation initiation. Two typical
polyadenylation signals (AATAAA) are located at positions 1096 and 1102. The
deduced protein sequence from the SC4 cDNA (Figure 19 (a)) consists of 289
amino acids and has a molecular mass of 31.9 kDa and a predicted pI of 7.95.
Three puatative glycosylation sites are present at positions 92, 128 and 269.
The putative polypeptide encoded by SC4 exhibits similarity with proteins
that comprises a BURP domain (e.g. RD22, an Arabidopsis thaliana
dehydration-responsive protein (Yamaguchi-Shinozaki K. and Shinozaki, K.
1993. Mol. Gen. Genet., 238: 17-25); PG1(3, a Lycopersicon esculentum
polygalacturonase isoenzyme 1(3 subunit (Zheng L. et al., 1992. Plant Cell. 4:
1147-1156); Sali3-2, a Glycine max L aluminium-induced protein (Ragland M.
and Soliman, K.M. 1997. Plant Physiol. 114: 395); USP, a Viciafaba unknown
seed protein (Baumlein H. et al., 1991. Mol. Gen. Genet. 225: 459-467) and
ADR6, a Glycine max L auxin-induced protein (Datta N. et al., 1993. Plant Mol.
Biol. 21: 859-869); see Figure 19 (b)). The BURP domain is a long carboxyl
terminal domain containing a number of highly conserved amino acids (Hattori
J.
et al., 1998. Mol. Gen. Genet. 259: 424-428). The carboxyl terminal of the
conceptual SC4 protein sequence contains the following conserved amino acids
which are typical of the BURP domain proteins: two phenylalanine residues, two
cysteine residues and four cysteine-histidine motifs which are also in the
conserved alignment of CHX10CHX25-27CHX25-26 CH, where X is any amino

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acid (Figure 19 (b)). This BURP domain proteins also share a similar
structural
make-up of 3-4 domains (Figure 19 (c)) i.e., an amino-terminal domain
containing a hydrophobic sequence, a second domain which may or may not be
conserved, a third domain consisting of tandem repeats of a short amino acid
sequence (not all BURP domain proteins have this domain) and a long carboxyl-
terminal BURP domain (Hattori J. et al.,1998. Mol. Gen. Genet. 259: 424-428).
The tandem repeats which make up the third domain do not appear to have a
common concensus sequence between the different BURP domain proteins. In
addition to the BURP domain, the putative SC4 protein shares sequence
similarity between its am.ino terminus and the conserved segment of the second
domain possessed by several of the BURP domain proteins (Figure 19 (b)). It
was also determined that the SC4 protein has a region containing two copies of
the repeated sequence ESRSIXXYAG where X is any amino acid (Figure 19 (a))
which is similar to the staructural organization of the third domain of
several
BURP domain proteins. :Due to the extent of structural and sequence similarity
between the SC4 protein and the BURP domain proteins it is likely that SC4
also
contains a hydrophobic amino terminal if it was full-length.
SC20
The SC20 cDNA clone was sequenced (Figure. 23 (b)) and found to
consist of 2447 bp with one 2310 bp open reading frame starting at nucleotide
position 13 and ending at 2322. The TAG stop codon may be leaky as plants
have tRNAs capable of niisreading it. However, any readthrough will be
terminated by a second stop codon TGA which is immediately adjacent to UAG.
The 3' untranslated region contains one putative polyadenylation signal
(AATAAA) located 21 nt after the stop codon.
The open reading frame of SC20 encodes a putative protein of 770 amino
acid residues with a calculated molecular mass of 82.688 kDa and a predicted
pI
of 6.93. The predicted protein has ten potential N-glycosylation sites (Figure
23

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(b)). The hydropathy profile (Figure 23 (c)) of SC20 protein revealed that the
first 23 amino acids constitute a hydrophobic region typical of an eukaryotic
signal peptide. From northern blot analysis, the SC20 cDNA clone hybridizes to
a-- 2.5 kb transcript (data not shown). SC20 was used to obtain the genomic
clone which was from a soybean cv. Harovinton genomic library.
Sequence comparisons (Figure 23 (d)) revealed that the putative
polypeptide encoded by SC20 was similar to a Picea abies (black spruce) AF70
protein (Sabala et al., 1997. Physiol. Plant. 99: 316-322); cucumisin,
(Yamagata
Y. et al. 1994 J. Biol. Chem. 269: 32725-32731) from Cucumis melo L. (musk
melon); a pathogen-induced protein, P69B, (Tornero P. et al., 1997 J. Biol.
Chem. 272: 14412-12219) from Lycopersicon esculentum (tomato) and a nodule-
specific protein, Ag12, (Ribeiro A. et al., 1995 Plant Cell. 7: 785-794) from
Alnus glutinosa (black alder). These plant proteins belong to the Pyrolysin
family
in the clan of serine proteases known as the subtilases (Barrett A.J. and
Rawlings
N.D., 1995. Arch. Biochem. Biophys. 318:247-250; Siezen, R. J. and
Leunissen, J. A. M. 1997. Protein Sci. 6: 501-523.). The SC20 protein contains
the conserved catalytic residues aspartate, histidine and serine as well as
the
highly conserved asparagine residue which is involved in stabilizing substrate
binding. Moreover, the order of these four conserved residues in the SC20
protein is also a characteristic feature of subtilases. The SC20 protein also
has a
large sequence insertion between the conserved asparagine and serine residues
found in plant subtilases but not in other subtilase members such as
subtilisin
BPN'(Power S.D. et al., 1986 PNAS. 83:3096-3100), kex2 (Mizuno K. et al.',
1988 Biochem. Biophys. Res. Comm. 156: 246-254) or furin (van de Ven
W.J.M. et al.,1990 Mol. Biol. Rep. 14: 265-275). Based on sequence similarity
between the N-termini of mature plant subtilases and the SC20 protein (data
not
shown) it was predicted that the SC20 protein had a mature domain starting
from
residue 1.17. Therefore the SC20 protein appears to be composed of 3 domains:
a
signal peptide of 23 residues followed by a prosequence of 93 residues and a

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mature domain of 654 residues. The predicted mature domain of SC20 has a
calculated molecular weight of 69.918 kDa and an isoelectric point of 6.34.
HE
The cDNA sequence for HP (pHPScDNAl) is (SEQ ID NO:6) shown in
Figure 15. The 700 bp transcript includes 30 bp of 5' untranslated region
(UTR),
an open reading frame (ORF) of 119 amino acids, and 313 bp of 3' UTR.
Several polyadenylation signals were identified in the 3' UTR. The final 80
residues of deduced amino acid sequence exactly match the peptide sequence
reported for the hydrophobic protein (Odani et al., 1987, Eur J Biochem 162,
485-491). Thus, the HP cDNA transcript indicates that hydrophobic protein is
translated with a leader sequence of 39 amino acids.
Northern blot analysis
Northern analysis, using the cDNA inserts of each clone as a probe, was
performed to investigate: the expression pattern of the 19 seed coat clones.
RNA isolation from leaf, stem, pod and flower tissue was optimized
based on a protocol adapted from Tripure Isolation reagent kit (Boehringer
Mannheim). Plant tissue was frozen in liquid nitrogen and homogenized with the
Tripure reagent (a monophasic solution of phenol and guanidine thiocyanate).
After the addition of chloroform the sample is centrifuged so that it
separates into
three phases. RNA is recovered from the upper aqueous phase by isopropanol
precipitation. Due to the problem of polysaccharide contamination which
increases with seed maturity, the isopropanol precipitation step was carried
out in
the presence of high salt which effectively maintains the polysaccharides in a
soluble form whilst the RNA is precipitated.

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Total RNA from seed-coat, embryo and root tissue was isolated as
described by Fobert et al. (Plant J. 1994 6:567-577). Plant tissue was frozen
in
liquid nitrogen and homogenized in 1M Tris-HCI, pH9, 1 % SDS buffer. The
sample was extracted twiice with equal volume phenol:chloroform:isoamyl
alcohol (25:24:1), nucleic acids were collected by ethanol precipitation,
collected
by ethanol precipitation and resuspended in water. The RNA was precipitated
overnight in 2M LiCl at 0 C, collected by centrifugation and resuspended in
water.
RNA was denatured and size fractionated by formaldehyde gel
electrophoresis and transferred onto nylon filters. Northern hybridization was
carried out using radioactively labeled cDNA probes with hybridization in
modified Church's buffer (Church and Gilbert (1994) PNAS USA 81:
1991-1995) at 65 C; wash 2 x 30 minutes 0.1X SSC 0.1 % SDS at 65 C. From
this analysis, it was observed that SC4, and SC20 have seed coat specific
expression. Ep locus peroxidase has preferential expression within seed-coat
tissues, and SC21 was only expressed in seed coat, stem, root and flower
tissues.
The results are shown in Figure 10 (a) - (d).
HE
For the analysis of HP expression, total RNA was isolated from roots,
stems, leaves, flowers, pods, seed coats, and embryos dissected from soybean
plants at various stages of development, according to published methods (Warig
and Vodkin (1994) Plant Mol Biol Rep 12, 132-145). The RNA samples were
quantitated by measuring absorbence at 260 nm, and by electrophoretic
separation in formaldehyde gels and comparison to known standards. Samples of
total RNA (10 g each) were blotted to nylon membrane using a vacuum
manifold apparatus and fixed by UV cross-linking. Digoxigenin-labelled cDNA
was prepared according to instructions of the manufacturer (Boehringer) and
used
to probe the RNA blots,. Results, Figures 10 (e) and (f) show that the HP gene
is

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highly expressed in the pod tissues during the later stages of development.
Hybridization signals were also noted in RNA samples derived from seed coat
tissue, but not in RNA samples from the leaf, flower, embryo, stem or root.
These results, together with the data from the in situ hybridizations (see
below)
and the scanning electron microscopy analysis, indicate the HP gene is
specifically expressed iri the endocarp of the ovary wall. Pieces of this
tissue
detach from the pod wa;ll and adhere to the seed surface during development,
thus becoming a component of the mature seed coat (see Figure 14 (a).
RNA samples from seed coat, embryo, stem, root, leaf, pod and flower
were hybridized with a radiolabelled SC4 cDNA probe (Figure 10 (a)) to
determine organ specificity of the expression of SC4. The sc4 transcript was
only expressed in the seed coat organ. It was estimated that the size sc4 mRNA
was 1.2 kb (data not shown).
Northern blot analysis was carried out to determine the temporal
expression pattern of sc4. RNA from seed coat, embryo and pod organs between
6-24 dpa were hybridized with a radiolabelled SC4 cDNA probe. At 6 dpa the
seed is too small to separate the seed coat and embryo organs and so total RNA
was isolated from an enitire seed. sc4 expression was already apparent in the
seed by 6 dpa. No gene: expression was observed in any of the embryo
development stages examined (Figure 20 (a)). sc4 mRNA transcripts were not
observed in the embryo of 3-6 dpa seed sections examined by in situ
hybridization using a radiolabelled SC4 antisense RNA probe (data not shown).
Therefore the sc4 expre:ssion observed at 6 dpa in the seed tissue is likely
to be
seed coat derived. After 6 dpa the expression of sc4 in the seed coat
increased
- 4-fold to its maximuin detected level between 9-12 dpa. By 15 dpa sc4
expression had decreased by - 2.5-fold dpa and continued to decline to just
detectable levels by 18 dpa (Fig. 3.7). Expression of sc4 could only be
detected

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in the seed coat at 21-24 dpa when the filter was over-exposed. Gene
expression
of sc4 in the pod was detected from 12-21 dpa only after over-exposure of the
filter (data not shown).
SC20
Northern blot analysis was carried out to determine specificity of sc20
expression in various soybean organs i.e., seed coat, embryo, stem, root,
leaf,
pod and flower (Figure 10 (b)). sc20 has seed coat-specific expression as its
mRNA was detected only in the seed coat organ. The sc20 transcript was
determined to be approximately 2.5 kb (data not shown). Even after prolonged
exposure of the filter, no sc20 transcripts was detected in any of the other
plant
organs.
Northern blot analysis was performed to determine the temporal gene
expression pattern of sc20 in seed coat, embryo and pod organs of soybean.
Total RNA prepared from organs between 6- 24 dpa were probed with a
radiolabelled SC20 cDNA probe. sc20 expression was detected at 9 dpa and rose
1.5 fold to its maximum observed level at 12 dpa (Figure 24). By 18 dpa
accumulation of sc20 mRNA had decreased 4-fold. Prolonged exposure of the
filter enabled detection of sc20 expression at 6 dpa and 21-24 dpa. No gene
expression was observed at any stage of embryo or pod development examined
even after prolonged exposure of the filters. This confirmed that sc20
expression
was seed coat-specific.
b) In situ hybridization
To analyze the distribution of the clones mRNA expression with respect
to cell differentiation during development, in situ hybridization, on sections
from
3, 6, 9, 12, 15, 18, 21 and 24 DAF seeds was used. Seeds or parts of seeds
were
fixed in. FAA fixative (50% ethanol, 5% acetic acid and 3.7 % formaldehyde),

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dehydrated in an ethanol./ tertiary butyl alcohol series and infiltrated and
embedded in paraplast plus. Sections (5-l0 m) were cut on a microtome,
transferred onto Superfrost slides which are positively charged to allow
better
adherence of the sections to the slide surface. Prior to in situ hybridization
the
samples were dewaxed in a xylene/ethanol series. In situ hybridization was
carried out with 35S-labelled cDNA sense and anti-sense probes following the
method of Cox and Goldberg (1998).
For the in situ analysis of Ep expression, flowers were tagged on days of
full anthesis when the banner petal was fully extended and harvested at three
day
intervals from 1-30 days post anthesis (DPA), and at 45 DPA [19]. Tissue
samples were fixed in a solution of 3.7% formaldehyde, 50% ethanol, and 5%
acetic acid for 3 h at room temperature, dehydrated in an ethanol series (50,
60,
70, 80, 90, 95, 100%) then infiltrated with t-butyl alcohol (TBA) in ethanol
in a
stepwise series (25, 50, 75, and 100%), followed by infiltration with
Paraplast
and several changes of pure melted Paraplast at 57 C. After infiltration,
samples
were placed in blocks and allowed to harden. Sections of 8-10 :m were cut on a
rotary microtome and affixed to glass slides. Prior to hybridization, sections
were de-waxed in xylene, and re-hydrated in an ethanol series (100, 95, 85,
70,
50, 30, 15, 0% ethanol in distilled RNAse free water).
Localization of RNA was performed with 35S-labelled RNA probes
generated from Ep cDNA clones. The prehybridization, hybridization, and wash
conditions followed published methods (Cox K.H., and Goldberg R.B. 1988,
Analysis of plant gene expression. In Shaw CH (ed), Plant Molecular Biology:
A Practical Approach, pp. 1-35. IRL Press, Oxford). Briefly, sections were
treated with Proteinase K and acetylated with acetic anhydride in
triethanolamine.
The sections were then hybridized with 35S-RNA probes overnight at 42 C,
washed, and dehydrated in an ethanol series before application of Kodak NTB-2

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track emulsion. After 1 week at 4 C, slides were developed in Kodak D-19
developer, fixed in Kodak Fix, and stained in Toluidine Blue O(0.05 % in 50
mM Acetate buffer, pH 4.5). Slides were then dehydrated in an ethanol and
xylene series, and mounted in Permount TM. Slides were photographed on Kodak
EPL 400 slide film, using dark field optics .
The expression of a gene under the control of the Ep (peroxidase)
promoter (nucleotides 1-1532 of SEQ ID NO:2,
is localized within the hourglass cells (arrow; Figure
13(d)) within the seed-coat at 18 days after anthesis. Expression of Ep is
first
detected at 6 DPA in the thin-walled parenchyma of the outer integument,
adjacent to the thick-walled parenchyma, and flanking the hilum region (Figure
13 (e)). By 9 DPA a thin band of expression extends around the entire seed
coat,
at the junction of the thin-and thick-walled parenchyma (Figure 13 (f)).
Expression shifts to the hourglass cells as they begin to develop, at 12 DPA
(Figure 13 (g)).
HP
For the analysis of HP (Figures 14 (c) and (d)), tissue samples were fixed
in a solution of 50 % ethanol, 5 % acetic acid, 3.7 % formaldehyde for 3 h at
room temperature, dehydrated in an ethanol series (50, 60, 70, 80, 90, 95, 100
%) then infiltrated with t-butyl alcohol (TBA) in a stepwise series (25, 50,
75;
and 100 % TBA in ethanol), followed by infiltration with Paraplast by gradual
addition of increasing amounts of Paraplast to 100 % TBA, followed by several
changes of pure melted Paraplast at 57 C. After infiltration, samples were
placed
in blocks and allowed to harden. Sections of 8 to 10 m were cut on a rotary
microtome and affixed to glass slides. Prior to hybridization, sections were
de-
waxed in xyiene, and re-hydrated in an ethanol series (100, 95, 85, 70, 50,
30,
15, 0 % ethanol in distilled RNAse free water). Sections were then treated
with

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Proteinase K and acetylated with acetic anhydride in triethanolamine. Sections
were hybridized with -5S-RNA probes overnight at 42 C, then washed and
dehydrated in an ethanol series before application of Kodak NTB-2 track
emulsion. After 1 week at 4 C, slides were developed in Kodak D-19 developer,
fixed in Kodak Fix, and briefly stained in Toluidine Blue 0 before dehydrating
in
an ethanol and xylene series, then mounting in Permount. Slides were
photographed on Kodak EPL 400 slide film, using dark field optics.
The expression of a gene under the control of the HP promoter
(nucleotides 1-2526 of SEQ ID NO:7) is localized within the membranous
endocarp (arrow, Figure 14 (b)) at 12 days after anthesis. At six days post
anthesis (DPA) expression of HPS is limited to the membranous inner layer of
the pericarp. By 12 DPA expression is very strong and the inner epidermis is
showing signs of becomrng detached from the rest of the pericarp and, in
places,
is adhering to the seed surface. Tissue sections from this stage of
development
also showed strong hybridization signals in the scierenchyma, indicating that
HP
expression occurs throughout the endocarp.
SC4
To analyse the distribution of sc4 expression with respect to cell
differentiation during seed coat development in situ hybridization was
performed
on seed sections from 3-24 dpa seeds. The seed sections were hybridized with
radiolabelled sense and antisense SC4 RNA probes which were detected by
exposure of the sections, to photographic emulsion. Within the seed sections
the
antisense or sense RNA probes can be localized by observing the accumulation
of
silver grains (produced in the emulsion by the emitted (3-particles) under
dark-
field illumination with a light microscope. Cell walls of some plant
structures can
be birefringent (i.e., reflect light) under dark-field illumination. Two
birefringent
areas can be observed in both the hilum and the funiculus of the seed sections
in

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Figure 21 therefore any expression or lack there-of by sc4 will be masked in
these locations.
sc4 was expressed throughout the inner integument of the seed coat at 3
dpa (Figure 21). By 6 dpa the expression pattern of sc4 had changed, and was
localized to the outer integument parenchyma but not to the vascular tissue
embedded within this layer. sc4 expression in the outer integument was
maintained until 18 dpa after which time no further expression was detected
(see
Table 4 below). In concurrence with northern blot analysis, the in situ
hybridization results revealed that sc4 expression increased to a maximum
between 9-12 dpa and decreased thereafter (Table 4). In addition, expression
of
sc4 was not observed in the embryo of seed at 3-6 dpa.
The expression of a gene under control of the SC4 promoter (nucleotides
1-5514 SEQ ID NO:9) within soybean seed coat at 3 days after anthesis is also
shown in Figure 13 (a). Expression is localized within the inner integument
(arrow; Figure 13 (a)). Other areas of brightness in this figure include the
recurrent vascular bundles in the funiculus, and the trichomes of the pod (the
bright areas are due to the birefringence of crystalline areas in the cell
walls, and
are also present in the negative control; data not shown).
SC20
In situ hybridization was carried out to analyse the spatial gene expression
pattern of sc20 within the seed coat between 3-24 dpa. Seed sections were
hybridized with radiolabelled sense and anti-sense SC20 RNA probes. No
birefringent cell structures were evident in the seed sections used (Figure
24).
Gene expression of sc20 was localized to the thick-walled parenchyma of
the outer integument (see Figures 13 (b) and 24). The temporal expression
pattern of 9-21 dpa expression with an observed peak at 12 dpa was almost

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identical to that determined by northern blot analysis (Table 4, in Examples).
sc20 transcripts were not. detected in the embryo between 3-6 dpa. The in situ
hybridization results of the seed sections concur with the northern blot
analysis
that within the seed orga:n sc20 is expressed only in the seed coat organs.
Expression of gene under control of the SC20 promoter (1-2450 of SEQ
ID NO:8) is seen in Figures 13 (b) and 24.
SC21
The expression of a gene under the control of SC21 (see Figure 11 (b))
within seed coat tissues at 15 days after anthesis is localized in the thin-
walled
parenchyma of the outer integument, including the area immediately surrounding
the tracheid bar (arrow; Figure 13 (c)).
c) Construction of genomic libraries
In order to characterise the gene corresponding to seed coat cDNA
clone(s), several genomic libraries were constructed in A. vectors from total
DNA
isolated from etiolated seedlings of various soybean cultivars. Two soybean
genomic libraries were constructed in 1Lambda FixII (Stratagene, La Jolla, CA)
from the total DNA isolated from etiolated seedlings of soybean [Glycine max
(L.) Merrill ] cvs. Harosoy 63 and Harovinton. The DNA was partially digested
with Bgl JI prior to ligation into the cloning vector.
Genomic clones corresponding to the cDNA clone SC4 and SC20 were
obtained. Lambda DNA was isolated from each plaque. An -- 8 kb Xba I
fragment from the SC201ambda clone and an - 8 kb Sac I fragment from the
SC4 lambda clone, identified by southern blotting, were ligated into
pBlueScript-
SK (Stratagene, La Jolla, CA) and transformed into E. coli TOP 10 cells.

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Southern blot analysis of genomic soybean DNA, was carried out with 7
seed coat cDNA probes to determine similarities between clones and whether the
clones represent a single gene or a gene family. Southerns were also performed
to determine the occurrence of the seed-coat specific genes within other
dicotyledonous and monocotyledonous plant species. Soybean genomic DNA
was cleaved with several restriction enzymes and the resulting DNA fragments
were size fractionated using agarose gel electrophoresis, denatured and
transferred to nylon filters. Hybridization was carried out with radiolabelled
cDNA probes.
Isolation of genomic clones
Initially, soybear.i genomic libraries were screened for the presence of the
seed coat clone using the polymerase chain reaction with primers specifically
designed from each cDNA sequence. This helped to target potential libraries
for
the isolation of genomic clones. The chosen genomic library was then screened
using nucleic acid hybridization with cDNA probes. For genomic library
screening hybridization conditions involved using modified Church's buffer
(Church and Gilbert (1994) PNAS USA 81: 1991-1995) at 65 C; wash 0.1X
SSC 0.1 % SDS at 52-55 C. Probes were random primed in presence of
32PdCTP using standard protocols.
A seed-coat peroxidase gene, corresponding to the Ep locus, was obtained
from a soybean seed-coat library. The genomic DNA sequence comprises four
exons spanning bp 1533-1752 (exon I), 2383 -2574 (exon 2), 3605-3769 (exon 3)
and 4033-4516 (exon 4) and three introns comprising 1752-2382 (intron 1),
2575-3604 (intron 2) and 3770-4516 (intron 3), of SEQ ID NO:2. Features of
the upstream regulatory region of the genomic DNA include a TATA box
centred on bp 1487; a cap signal 32 bp down stream centred on bp 1520. Also

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noted within the genomic sequence are three polyadenylation signals centred on
bp 4520, 4598, 4663 and a polyadenylation site at bp 4700. The promoter
region of the genomic sequence comprises nucleotides 1-1532 of SEQ ID NO:2
(see co-pending US patent application serial No. 08/723,414 and 08/939,905,
both of which are incorporated by reference).
HE
For the isolation of the genomic HP gene, a genomic library was
constructed from DNA isolated from the soybean cultivar Harosoy 63. The DNA
was purified and partially digested with Bgl H prior to ligation into the
cloning
vector lambda FixII (Stratagene). The resulting library was amplified and
screened with the hydrophobic protein cDNA probe (pHPScDNAl). A positive
clone was identified, purified, and found to contain a 14 kb insert. The
entire
insert was sub-cloned into pBluescript KS(+) and named pHPS1. The HP gene
was determined by PCR analysis to lie near one end of the 14 kb Bgl II
fragment
(for restriction map see Figure 11 (c)). This region of the pHPS1 insert was
sequenced by primer wa:lking, and 3368 bp of this sequence data is disclosed
here (SEQ ID NO:7). Aside from the polyadenylation site, the cDNA sequence
(pHPScDNAl) exactly matches a stretch of sequence encoded on the genomic
clone (pHPS1), indicating that this gene contains no introns. Additionally, a
TATA box consensus signal was identified 81 bp upstream from the ATG
translation start site.
SC4
A genomic clone corresponding to SC4 cDNA clone was isolated from
the soybean genomic library Harosoy 63 (Bgl II digest). The genomic sc4 clone
is 8310 bp in length (SEQ ID NO:9). The promoter region is found between
nucleotides 1-5514 of SEQ ID NO:9. The restriction map is provided in Figure
11 (d).

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SC20
A genomic clone corresponding to SC20 cDNA clone was isolated from
soybean genomic library prepared from cv Harovinton (GigapackGold
packaging). The genomic sc20 clone is 7235 bp in length (see Figure 23 (a),
SEQ ID NO:8). Alignment of sc20 genomic and SC20:2 cDNA sequences
revealed that sc20 contained eight introns of 94 bp, 101 bp, 185 bp, 80 bp,
154
bp, 112 bp, 110 bp and 93 bp respectively (Figure 23 (a)). A search
(www.hgc.lbl.gov/cgi-bin/promoter.pl) of the 5' upstream region of sc20
revealed three potential transcription start sites at positions 1085, 1156 and
2272.
The promoter region is found between nucleotides 1-2450 of SEQ ID NO:8. The
restriction map of SC20 is presented in Figure 11 (a) and 23(a).
SC21
A genomic clone corresponding to SC21 cDNA clone was isolated from
the soybean genomic libr-ary prepared from Harosoy 63 (EcoRl digest). The
DNA of the SC21 genomic clone was digested with several restriction enzymes,
fractionated by agarose gel electrophoresis and transferred onto nylon
membrane.
Hybridizations were carried out using radiolabelled cDNA. A restriction map of
this clone is presented in Figure 11 (b).
Southern analysis
SL4
Southern blot analysis was carried out to examine the gene family
composition of sc4. Soybean genomic DNA was cleaved with Eco RI, Hind III
and Xba I. which do not have recognition sites in the SC4c cDNA sequence.
Under conditions of low to high stringency (i.e., from 40-10 C below Tm of the

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probe ) the SC4 cDNA probe hybridized to a single band (Figure 22) and
therefore sc4 appears to be a single gene.
SC20
Southern blot analysis was performed to ascertain whether sc20 is a single
gene or a member of a gene family. Soybean genomic DNA was cleaved with
Eco RI, Hind III, Xba I and Eco RV which have three, four, two and one
recognition site(s) respectively in the sc20 clone (see Figure 23 (a)).
Hybridization was carried out with radiolabelled SC20 cDNA probe which could
anneal from the middle of exon 6 to the Eco RI site on exon 9. For each digest
the probe was expected bind to only one of the resulting sc20 restriction
fragments. Under conditions of high stringency to detect genes with at least
90%
similarity to sc20 the probe hybridized to a single band (Figure. 25 (b)).
Under
medium stringency conditions to observe genes with 80% similarity to sc20 it
was observed that the SC20 probe annealed to 2-3 bands for each digest (Figure
(a)). Under conditions of low stringency i.e., 40 C below Tm the SC20 probe
hybridized to several more bands from each digest (data not shown). This
suggested that sc20 is a:member of a small gene family composed of 2-3
20 members and that the soybean genome contains several genes which are more
distantly related to sc20.
Southern blot analysis was performed to determine the occurrence of the
seed-coat genes within the following plant species: pea (Pisum sativum),
canola
25 (Brassica napus), oat (Avena sativa), onion (Allium cepa), pepper (Capsicum
annuum), mimosa (Mimosa pudica), black spruce (Picea mariana (Mill B.S.P.),
birch (Betula pendula Roth). Genomic DNA was cleaved with EcoRI and the
resulting DNA fragments were fractionated using agarose gel electrophoresis,
denatured and transferred to nylon filters. Hybridization was carried out with
radiolabelled SC4 (Figure.22 (b)), SC20 (Figure 25 (c)), SC21, Ep locus
peroxidase, and HP cDNA probes, using modified Church's buffer at 65 C. The

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filters were washed with 2XSSC, 0.1 %SDS at 42 C for 30 minutes, followed by
0.1XSSC, 0.1%SDS at 65 C for 30 minutes. SC4, SC20 and Ep locus
peroxidase cDNA hybridized to the genomic DNA of soybean only. SC21
cDNA hybridized to the genomic DNA of both soybean and oat. HP cDNA
hybridized to the genomic DNA of soybean.
Analysis of promoter activity
The developmental expression of genes under the control of SC4, SC20
SC21 and the peroxidase: promoter were further characterized during
development of the seed coat by in situ hybridization as described above. The
results are summarized i:n Table 4.
Developmental analysis of SC20 indicates that the promoter is highly
active at 12 DAF within the outer integument and thick walled parenchyma,
however, activity of the SC20 promoter is detectable from about 9 DAF (as per
Figure 13 (b)) to about 1.8 DAF, and is partially detected at 21 DAF.
The SC4 promoter is active from about 3 daf (also see Figure 13 (a)) to
about 6 DAF within the inner integument, and then is highly active at 9 DAF
within the outer integument and stellate parenchyma, and strongly active at 12
DAF in these same tissues. The SC4 promoter is still active within the outer
integument up to 18 DAF.
The SC21 promoter is active throughout seed coat development during all
stages examined, from 3 about DAF to about 24 DAF, with strongest activity
noted from about 9 DAF to about 15 DAF (also see Figure 14 (c)). The gene
under the control of the SC21 promoter is expressed primarily within the outer
integument and derived tissues.

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The EP (peroxidase) promoter is active from about 6 DAF to about 24 DAF.
Expression of the
peroxidase gene, from about 12 DAF to about 24 DAF, is predominantly within
cells of the outer integument, and the hourglass cells (see also Figure 13
(d)).
The HP promoter is active from about 9 daf through to about 24 daf. The
promoter is active within the membranous endocarp throughout this period of
time (see also Figure 14 (b)).

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Seed Surface Analysis of Dull and Shiny Soybean Varieties
Seed surface proteins of several different soybean varieties were
compared by SDS-PAGE analysis. A single seed was placed in a 2 mL plastic
capped test tube and surface proteins were extracted by adding 0.5 mL of a
buffer-detergent solution (10 mM Tris-Cl (pH 7.5) 0.5% SDS, 20 mM DTT)
and placing the tube in a boiling water bath for 2 min. The contents of the
tube
were mixed and a sample was withdrawn and centrifuged for 5 min at 14,000 g.
The proteins in the supernatant were electrophoretically separated on 15 %
acrylamide gels in the presence of SDS (Fling and Gregerson (1986) Anal
Biochem 155, 83-88) and detected by silver staining. This analysis revealed
that
the 8.3 kD hydrophobic protein is by far the most abundant protein molecule
occurring on the seed surface of 'Dull' seeded varieties. Only trace amounts
of
hydrophobic protein was detected on the surface of 'Shiny' seeded soybean
varieties (results not shown).
Analysis of seed coat tissues using light microscopy indicated that the
membranous endocarp of the pod wall remains in association with the seed-coat
(Figure 14 (a). Scanning electron microscopy (SEM) of the seed surface of
soybeans also showed obvious differences between 'Dull' (e.g.cultivar Clark)
and 'Shiny' (e.g. cultivar Williams 82) varieties (see Figure 16). Whole seeds
were sputter coated with gold and examined by SEM at several magnifications.
When viewed with the naked eye, 'Dull' varieties present a surface with a
powder-like coating whereas 'Shiny' types appear to have a smoother and more
light-reflective surface. Examination by SEM at low magnification (18 X)
reveals that the surface of 'Dull' types is uniformly covered with small,
dimple-
like indentations and bits; of adhering material. These indentations are also
visible on 'Shiny' types, but the surface is virtually free of adhering
material. At
higher SEM magnifications, the surface of 'Dull' types appears rough and
ragged whereas the 'Shiny' seeded soybeans have a relatively smooth and
undulating surface.

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Without wishing to be bound by theory, it appears that the adhering
material on the 'Dull' seeded types are remnants of the membranous endocarp
tissue and is rich in hydrophobic protein. The expression of the hydrophobic
protein in the endocarp causes bits of this tissue to stick to the seed
surface,
resulting in the 'Dull' phenotype. Lack of expression similarly may result in
the
'Shiny' phenotype. The hydrophobic protein may be involved in the adherence
of the endocarp to the seed surface.
Analysis of 'Dull' and 'Shiny' Seeded Varieties
Total genomic DNA was extracted from 'Dull' or 'Shiny' seeded
varieties and amplified by PCR using primers targeted to the HP gene. The
resulting amplification products were clearly polymorphic between the two
genotypes. Good amplification of target segments of DNA were regularly
observed when template DNA was from 'Dull' types whereas DNA from
'Shiny' types produced multiple products or products that were shorter or
longer
than expected, depending on the primer combination. These results indicate
that
different alleles the HP gene occurs in 'Dull' and 'Shiny' types of soybean.
This
allelic variation causes profound differences in seed surface morphology and
composition.
To compare HP gene structure in two different seed luster phenotypes
that were also different i:n the amount of HP present on the seed surfaces, we
hybridized genomic DNA blots with probes derived from the HP cDNA
sequence under high stringency conditions.
Soybean genomic DNA was isolated from frozen, lyophilized tissue
according to the method of Dellaporta et al., (1983). Restriction enzyme
digestion of 30 tcg DNAõ separation on 0.5 % agarose gels and blotting to
nylon
membranes followed staiidard protocols (Sambrook et al., 1989). Digoxigenin
labelled cDNA was prepared and used to probe DNA blots according to the
instructions provided by the manufacturer (Boehringer Mannheim).

CA 02325819 2005-07-29
WO 99/53067 PCT/CA99/00293
-72-
Hybridization was carried out at 65 C for 16 h in 0.25 M Na2HPO4 (pH 7.2),
20% SDS, 1 mM EDTA and 0.5% blocking reagent (Boehringer Mannheim).
Filters were then washed 4 x 15 min at 22 C in high stringency wash solution
(20 mM Na2HPO4 (pH 7.2), 1% SDS and 1 mM EDTA), followed by 3 x 15
min washes in the same solution at 68 C.
A typical result from such a Southern analysis is shown in Figure 18.
Genomic DNA blots from cultivars that accumulated large amounts of HP on the
seed surface produced strong hybridization signals. These intensely
hybridizing
fragments are not present in genomic DNA from plants that have only trace
amounts of HP on the seed surface. However, several fainter signals are also
present in DNA blots from both types of plants. These results indicate that
sequences related to the HP cDNA are prevalent in the soybean genome, and
that the HP gene structure is polymorphic among soybean cultivars. Soybean
types that accumulate large amounts of HP on the seed surface possess
additional copies of this gene.
The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of
variations and modifications can be made without departing form the scope of
the invention as described in the following claims.

CA 02325819 2007-06-15
73
SEQUENCE LISTING
<110> Miki Dr., Brian L
Gizen Dr., Matk
Miller Dr., Shea
Boutilier Dr., Kim
Hu., Ming
Bowman, LuAnn
Batchelor, Anthea
<120> Seed-Coat Promoters, Genes and Gene Poducts
<130> 08-869947W0
<140> 2,325,819
<141> 1999-04-13
<150> 09/059,090
<151> 1998-04-13
<160> 12
<170> PatentIn Ver. 2.0
<210> 1
<211> 1070
<212> DNA
<213> Nicotiana tabacum
<400> 1
tctagacttg tcttttcttt acataatcct cttcttcttt tttttgttag tttcttctgt 60
tttatccaaa aaacgaatta ttgattaaga aatacaccag acaagttttt tacttctttt 120
tctttttttt tttgtggtaa aaaattacac ctggacaagt ttatcacgaa aatgaaaatt 180
gctatttaag ggatgtagtt ccggactatt tggaagataa gtgttaacaa aataaataaa 240
taaaaagttt atacagttag atctctctat aacagtcatc cttatttata acaatacttt 300
actataaccg tcaaatttat tttgaaacaa aattttcatg ttatgttact ataacagtat 360
tttattatag caaccaaaaa atatcgaaac agatacgatt gttatagagc gatttgattg 420
tatcattatc cacatatttt cgtaagccca attactcctc ctacgtacga tgaaagtaaa 480
ccaatttaaa gttgcaaaaa tccaatagat ttcaatactt cttcaactgg cgttatgtta 540
ggtaatgact cctttttaac ttttcatctt taatttgaag tttctttcat taaaagaaag 600
tttctagaag agaagtgttt taacacttct agctctacta ttatctgtgt ttctagaaga 660
aaaatagaaa atgtgtccac ctcaaaaaca actaaaggtg ggcaaatctc cacctattta 720

CA 02325819 2007-06-15
74
ttttattttg gattaattaa gatatagtaa agatcagtta taaacggagt tttgagttga 780
tacagtgaat tttaagatgt gtaccgattt aactttattt acatttatgt ttcgcacata 840
taagaagtcc gatttggaaa tactagattt tgtcaatcag gcaattcatg tggttgaaga 900
atttaagtta tatacaatga tgatataaag aatttttata ctattagtgc aaattaatcg 960
attactaaaa attattattc tattaattta tgctatcgtg cctccccaac ccgtcgaccg 1020
cggtacccgg tggtcagtcc cttatgttac gtcctgtaga aaccccaacc 1070
<210> 2
<211> 4700
<212> DNA
<213> Glycine max
<400> 2
tagataaaaa aatgggatat aatttttctc agatgttgtt tatactgttt ttttaatcag 60
aattaaaatt cctctttaat tatcgacata attttttttg gtgaatatta tcgacataat 120
tatttaatac aaatttttat tgtacataga agtgatactt caattttaat attggagaac 180
agtacgaaaa cataaaaaaa ctgttattag aagaaaaaaa tatatggaaa aggttagcta 240
catatattag ctaaattagt tgttctaatt ggctatataa accctattgt actctttgta 300
atctcacctt tttcatttaa atacatttct actttttaag ttctatattt tctctcaatt 360
ttcttcgata aaccatgaaa tttaacatgg tatatcagcg ataccaccca ctttgaaagc 420
catgtatggc tagtatgggc agccaaaatt tgccctggtt caagcaaagc aagtgtttat 480
atagatgtga cttttgttga ggaactcatg ccaatggtac tgattgtgaa actgagaaaa 540
ctaatttgga gaatttgaat tatgatcatt aaatactcct ctcctgacta ccttcgtccc 600
tcaaatttgt accatcatta tttcccaaaa atttgattac aatgcactaa ttaatgaatg 660
tttcttacat tatcatatta tcatatctga cattttgttt ttacttttta taataattat 720
tttaaaaagt catacatgca aataattttt taatagttta cagttaaatt tttacagtaa 780
aaatgcatga aaattaaact ttatttttcc aagtcatcat ttagtcaaat cccaaaacaa 840
tgattatttt ttgcaaatga atgtttattg aacatttaaa tgtagcctaa ttaattctgg 900
ttatggtgtc aatgttccaa aacctaatgc aagatcttag caagtacata catagatcta 960
attttaaact tatctttacg caagagatat aaagattata catctagttt taaacattaa 1020
cttttgtttt tgtgttaaaa aacagtaaca ttttcttaat tttgtagagt gacgtgctcc 1080
aaccatatta acgaagattt taattggtat tcaagttcat gaacttagta aataagtttt 1140
ggtcttcagt tttcaatttt cattacaaca tttatgtaaa atatcaacgt tttctgaaat 1200
ttgttgcttg tgtgctccaa ccacatttaa gagattatag aaattaattt tcaagaagat 1260
aatgattcct actcttgctg gccctaccat agtacaataa atccactcat aaatcaacaa 1320
gtcgtcgtca taggcaattg ggcatcatat cataaacaat acgtacgtga tattatctag 1380
tgtctctcag tttactttat gagaaattat ttttctttaa aaaaagttaa ttaataaaaa 1440
catttgcgat accgtgagtt acaagaaatc cgccgaattc atctctataa ataaaaggat 1500
ctatatgaga ggtaaaatca tattaactca aaatgggttc catgcgtcta ttagtagtgg 1560
cattgttgtg tgcatttgct atgcatgcag gtttttcagt ctcttatgct cagcttactc 1620
ctacgttcta cagagaaaca tgtccaaatc tgttccctat tgtgtttgga gtaatcttcg 1680
atgcttcttt caccgatccc cgaatcgggg ccagtctcat gaggcttcat tttcatgatt 1740

CA 02325819 2007-06-15
gctttgttca agtacgtact tttttttttc cttccaaaat gccctgcata tttaacaaga 1800
ttgctttgtt cacctagaaa aatgtgtttt tttcaacgat cttacgtacg tttgtttggt 1860
ttgaaaaata aatcagaaag agatcaagaa aatagctaga aagaaagcaa cgttttttta 1920
aaaggtattt agtgtgagaa aaatattaaa actgaagaga aagaaattaa ataagctttt 1980
cttgaatgat atttacatgt cttattaact taaagtcacc ttttttcttt aagttgtgct 2040
tgaagaaaaa agatgtcttt cagtttagtt ttgattaatg ctaattatat ttttaattaa 2100
ttaattaata ctatatatct atttaccata ttaattatta ctatatttca tgatgacaac 2160
agacaagtat tctaaagagg tatcggtaga tgattaattt ttttataaaa aaatcttttg 2220
cgtgtataga tattctttta taattggtgc agaaacttgt aatgctaatt gcaattaatc 2280
ttacattgat taactaatag ctataatcaa tatttaggtt aggtatagga gacaaatcaa 2340
gtgatctgaa caaattaagt tgttatattt gcattgtgac agggttgtga tggatcagtt 2400
ttgctgaaca acactgatac aatagaaagc gagcaagatg cacttccaaa tatcaactca 2460
ataagaggat tggacgttgt caatgacatc aagacagcgg tggaaaatag ttgtccagac 2520
acagtttctt gtgctgatat tcttgctatt gcagctgaaa tagcttctgt tctggtaatt 2580
aataactcct aattaattcc caaccattaa aaagttgcat gattggattc aaaattctat 2640
ggtattgggg ttctgatata aatttgtaat taaattgcac taaaaaaaat tatcatatac 2700
ttttaataaa aaaaatttat ctaatttaat ttattattaa aactattttt aaaattcaat 2760
cctaactctt ttttaatcgg agcatgtaag ctggcaccca ccgtatatcg ttggaagatg 2820
ctataaaacc atttaattaa tggatggaat cagtcaaaac atttaattca aaatactctt 2880
aattgtgatt agtaatcatg ttcgggcaag ttacgttgtg tataattaat ttgacttaat 2940
cagataaaaa aacaaatgga cgcaagccgg ttggtataga tatcactggc ctgtagaata 3000
tgtggttttt cacgtttaaa taaaagctag ctactatatt atatttagtc tttttttttc 3060
ttaaacccat ttaacgtgat ttattgactg tgaaacatgt ttccacacac aggcttagaa 3120
actcctcgca actaacatct ccaaaatttg actatttatt tatgaagata attcatctat 3180
gatgttcaac tctattatat atatgtatca tcgcagtatt aagaattata atagtcaaat 3240
atagaagtat atcgggtaaa tgtagttgca tgtgcgacct gtttcgtgta aaatgcttat 3300
tctatatagc tttttttatt ggaaaataac gatgaactaa aaacgaaagg gtatcatata 3360
gtttgacttt tatgttagag agagacatct taatttggtc atatgttaaa taattaatta 3420
caatgcatac acaaatattt atgccatatc taaaaaatga taaaatatca taggtatact 3480
caactatatg atatccccat aacagaaatt gtacttttct tcaggcaatg aacttaacat 3540
ttctgtttgc taaaaacaaa catccactta aagtggttca acatatttat gtaataattt 3600
acagggagga ggtccaggat ggccagttcc attaggaaga agggacagct taacagcaaa 3660
ccgaaccctt gcaaatcaaa accttccagc acctttcttc aacctcactc aacttaaagc 3720
ttcctttgct gttcaaggtc tcaacaccct tgatttagtt acactctcag gtatacataa 3780
tcaatttttt atttgctatt agctagcaat aaaaagtctc tgatacagac atatttagat 3840
aaattaattt ctccataaac atttataata aaattatcaa tttatgtact taaaaattat 3900
ggattgaagc tcttttcatc caacttttac taaagttaag gtgcatataa tataaaataa 3960
actatctctt gtttcttata aaaagattga agataagtta aagtctactt ataaatcatt 4020
aatatatgta taggtggtca tacgtttgga agagctcggt gcagtacatt cataaaccga 4080
ttatacaact tcagcaacac tggaaaccct gatccaactc tgaacacaac atacttagaa 4140
gtattgcgtg caagatgccc ccagaatgca actggggata acctcaccaa tttggacctg 4200
agcacacctg atcaatttga caacagatac tactccaatc ttctgcagct caatggctta 4260

CA 02325819 2007-06-15
76
cttcagagtg accaagaact tttctccact cctggtgctg ataccattcc cattgtcaat 4320
agcttcagca gtaaccagaa tactttcttt tccaacttta gagtttcaat gataaaaatg 4380
ggtaatattg gagtgctgac tggggatgaa ggagaaattc gcttgcaatg taattttgtg 4440
aatggagact cgtttggatt agctagtgtg gcgtccaaag atgctaaaca aaagcttgtt 4500
gctcaatcta aataaaccaa taattaatgg ggatgtgcat gctagctagc atgtaaaggc 4560
aaattaggtt gtaaacctct ttgctagcta tattgaaata aaccaaagga gtagtgtgca 4620
tgtcaattcg attttgccat gtacctcttg gaatattatg taataattat ttgaatctct 4680
ttaaggtact taattaatca 4700
<210> 3
<211> 1119
<212> DNA
<213> Glycine max
<400> 3
caatgctgcg ttaactccta gacattactg ggaaacgatg cttccaagaa ctcccttgcc 60
gaaagcaatc acagagctac taagccttga aagtaggtcc atatttgaat atgccgggaa 120
tgatgaccag tcagaaagta ggtccatatt aggatacgct ggctataatc aagacgagga 180
tgatgtgagc aaacacaata tacaaatctt caacaggttg tttttcttgg aagaggacct 240
gcgtgctggc aaaatattca acatgaagtt cgtcaacaac acaaaagcca cagtcccgtt 300
gctaccgcgc caaatttcga aacaaatacc gttctcagaa gataaaaaga agcaagtgtt 360
ggcgatgctt ggcgtggaag cgaactcaag caacgccaag atcatagcgg agaccattgg 420
tctttgccaa gagcctgcaa cggagggaga aaggaaacac tgcgcgactt cgttggagtc 480
catggttgat ttcgtcgttt ccgcgctcgg gaagaacgtt ggtgctttct caacagagaa 540
agaaagggaa actgagtctg gaaagtttgt agtggtgaaa aatggggtga ggaagttggg 600
agatgataag gttattgcct gtcatccaat gagttaccct tatgttgtgt ttgggtgtca 660
tctagtgcca aggagtagcg ggtatttggt gcgcttgaag ggagaagatg gggttcgagt 720
gaaagcagtt gttgcgtgcc acagagacac gtcaaagtgg gaccataatc atggggcatt 780
caaagtgctc aatcttaagc ctgggaatgg tacagtatgc catgtcttca ctgaggggaa 840
tcttctttgg cttccaaatt agattaatta ccatatacat atttgtcctt gttctatcct 900
taaataagtg gaatcacctg aagaattgtg cgtaatgagt tgtttgtctt tgtggaaatt 960
gttatctgtc ttgcatcacc aaataggtat atataaaata acaggagcgt ggtatttgtt 1020
gcacaaaaat ggatttcaac cgatcaaaaa aatatagcct ttaccaatta gaagggtttg 1080
gctttgttag caaataataa aaataaaata tcttgatgg 1119
<210> 4
<211> 2447
<212> DNA
<213> Glycine max
<400> 4
caaagtttta acatgaaagg caataataca cttttgttgc atttattcta cactactctc 60

CA 02325819 2007-06-15
77
ttcctgtttc ttgtagtgtc aagttcatct tcaacaggga atgaaagtaa cgatgacact 120
aacagtaaag aagtttatat cgtgtacatg ggagctgcag attcaacaaa agcttctctt 180
aaaaatgagc acgctcagat tctgaattca gtgctaagaa ggaatgagaa tgccctagta 240
cggaactaca agcatggttt ctctgggttc gcagctcgtc tatcaaaaga ggaggcaaac 300
tcaattgctc agaaacctgg tgtggtgtct gttttccctg accccattct gaagctccac 360
actacacgtt catgggattt cctcaaaagc caaactcgtg tcaatatcga caccaaacca 420
aatacgctgt ccggttcttc tttttcttca tcagacgtca ttcttggcgt cttagacaca 480
ggcatatggc cagaggcggc gagttttagc gacaagggtt tcggtcctgt tccatcccga 540
tggaaaggca cctgcatgac atcaaaagac ttcaattcct cttgttgtaa caggaagata 600
attggcgcga ggttttaccc taacccagag gagaaaacgg caagggattt caacggacat 660
gggactcacg tttcgtcgac ggcagtgggc gtgccggtga gtggcgcatc gttctatggt 720
ctggcggcgg ggacggcaag gggtgggtcc cctgagtcaa ggttggcggt ttacaaagtg 780
tgtggggctt ttgggtcatg tcctgggtcg gccattcttg cggggtttga cgatgccatt 840
cacgacggag tggatatctt gtcgctgtcg ctcggtggat tcggtggaac caaaaccgat 900
ttgaccaccg acccgattgc gattggagca ttccactccg tccagcgcgg catcctggtg 960
gtctgcgccg ccgggaacga cggagaacca ttcaccgttc tcaacgacgc accttggatt 1020
ttaaccgttg cagcttccac catcgaccgt gatcttcaat ccgacgtggt cttgggtaat 1080
aaccaagtcg tcaagggaag agccataaat ttctcccctc ttttaaattc tcccgattat 1140
ccaatgatat atgctgagtc tgctgccagg gcaaatatct ccaacataac tgatgcaaga 1200
caatgccacc cagattcatt agatccaaaa aaagtcatag ggaagattgt ggtttgtgat 1260
ggaaaaaatg acatttatta ttcaactgat gagaaaattg tcatagtgaa ggcgttggga 1320
ggaataggtc tggttcatat tactgatcaa tctggatcag tagcatttta ttatgtggac 1380
ttcccagtaa cagaggtaaa atcaaaacat ggcgacgcaa tcctccagta catcaactca 1440
actagccatc cagtgggaac aatactagca acagttacaa ttcctgatta taagcctgct 1500
ccccgggtgg gttatttttc atcaagaggg ccttcattga ttacaagcaa tgttctcaag 1560
cctgatattg cagccccggg agttaacatt ctcgctgcat ggtttggaaa tgacacatca 1620
gaggttccaa aaggaagaaa gccctcacta tatcgcatac tctcaggaac ttccatggct 1680
actccacatg tttcagggct tgcatgcagt gtcaaaagaa aaaaccccac ttggagtgcc 1740
tccgcaatca aatctgccat catgacttca gcaattcaaa atgacaattt gaagggtccc 1800
ataacaacgg attcagggtt gatagccaca ccttatgact atggagcagg ggcaattaca 1860
acatctgaac cattgcaacc ggggctagtt tatgaaacca acaacgttga ctacttgaac 1920
tatttgtgtt acaatggact taacataacc atgataaagg tcatctccgg aactgtcccc 1980
gagaatttca attgtcccaa ggattcgagc tctgatctca tctccagcat caactaccct 2040
tccatagcag taaacttcac tggcaaagca gacgcggtcg tgagtagaac tgtcacaaac 2100
gttgacgaag aagatgaaac agtgtacttc cccgttgttg aagctcctag tgaagtaatt 2160
gtcacactct ttccatataa tcttgagttt acgacaagta ttaaaaaaca aagctacaat 2220
attactttca gaccgaagac ctccttgaag aaagatttgt ttggatctat cacttggagt 2280
aacgacaaat atatggttcg aattcctttt gtattaacta aatagtgaaa ttaaaaagta 2340
gcgatgaata aatgcaagct aagttcttcg tggtgcctac actcgagtcc tgattattta 2400
ttattcatat gccttctgtt ttaatttatt tattatactt tcagcct 2447
<210> 5

CA 02325819 2007-06-15
78
<211> 829
<212> DNA
<213> Glycine max
<400> 5
gccttaaggc aacgacagcg agttcttctg ttgttcgttg actccaagga cggggtctta 60
gttggtggct tcgtggtttc cttctttggt ggcttcgtgg ttgttgtctt tgtcatcctt 120
gttgttgatg tcttcttcgg ttcggtctcc tcggatttct tcaacgtcaa ctctggctcc 180
tccaccactg tttcctctgt ctccttctct tcggtcgtcg tcttctcttt agtctcctct 240
tttgacttct tcactttctt cttcgtcttc tcggatttct ttgatgactt tgtctttgtc 300
ttagtcgtgg tcgtcgttgg tgtctccttc tcttgtttgg tcgactctgt caccttcggg 360
gtcatctcca aggacaactc ttttgacttc gaatctgaat acctgtcaca ctcactcttt 420
ttgttcaaat ttttaccagg atcaacaccc ctagtaatcc agatggtaaa acgtacagta 480
caacttttcg aaaaaaaaaa taaataaaga aatcaaatga aataataatt atataataat 540
aatatactac caattcagaa accaacataa tacctcccat atggatgcac actcgtgttg 600
tgaaccgagg tgtagtcgca cgaatggtgc accactgtac tctttaagat cctcaacaca 660
taacttcact cgttcgacga cgaccacgac ctcaaatccc tactcccaaa taaaataacc 720
gtcccaatgt actcaaacac tactacacaa taacacaaag aaatccaaac ttatttacta 780
taaatgaaat tttttttttt ttttttagat ctagctggcg tcgggttga 829
<210> 6
<211> 700
<212> DNA
<213> Glycine max
<400> 6
taagctttca agagacaaac tgctttgaaa aatgggatcc aaggttgttg catccgttgc 60
ccttctcctc tccatcaaca ttcttttcat ttccatggtt agctccagca gccactacga 120
tccacagccc caaccttctc acgtcactgc tcttattaca cgacctagtt gtccggatct 180
gagtatttgc ctcaatattt taggcgggtc tctaggaacc gtggatgatt gttgtgccct 240
catcggtggt cttggtgaca ttgaagccat tgtgtgcctt tgcatccaac tcagggccct 300
cggaatatta aaccttaacc gtaatttgca gttaatatta aactcctgtg gacgaagcta 360
cccgtcaaac gccacttgcc cccgaaccta agaacagaat atgtatggca ctaattacca 420
tattacttcg tatcatggtg tttgtttgtt tgtctgtgtt taaagttaag gatgttatac 480
ccttcgtgcc tgctacatat atatagtggg cactataata ttaccaataa attaacgtcc 540
atatataaga ataataataa ataaataaat atttctatac aaataaaggt tacgtaatgt 600
tgttgttctc gtggatgggg atcttatctt cctcctcgct atctttgttt atcgtatttc 660
agtgaaagtt gttcaataaa agtcctttgt tcaacaagta 700
<210> 7
<211> 3368
<212> DNA

CA 02325819 2007-06-15
79
<213> Glycine max
<400> 7
tctttcgatc aatactaata aagtcttatt tgccttccag agacaattga gtccgttggc 60
acgcagagac aaattatggt aatttgcccc tttttgaaga cttcaatgtc tttcgatcaa 120
gactattaaa gtcttctttg ccttctagag acaaattatg gtcatctgat tctttttgaa 180
tacttcaatg tctttcaatc aagacaatca aagttttttc gaatacttca aagtcttctt 240
tgccttccgg agacaattaa gtctgttgga acgcagagac aaattatgat catctacccc 300
atttcgaaga cttcaatgtc tttcgataaa gactattaaa gtcttctttg ccttccggag 360
acaatcaagt tcattggcac gtagagacaa attatggtca tctgcctctt ttcgaatact 420
tcaatgtctt tcgattaaga ctatcaaagt cttcttttcc ttccggagac aatcaggttc 480
tttggcacgc agagacaaat tatgttcatc tgcctctttt cgaagacttc aatgtctttc 540
gatcaagact atcaaagtct tctttgcctt ccgaagacaa tcaagtctgt tggcacgcaa 600
agtttgagga aaaattggac gaagatcggg acaaatggac cgtatggttt gacggagcgt 660
caaacattct aggccatggc attggggcag tattggtctc tccggacaat caatgtgtac 720
ctttcacaac caggctagga ttcgactgca ccaacaacga tcttcggtgc aattactcaa 780
atcggggaac taaggtggaa tgaagttgtc gtcattttgg gcaaggacaa gggagttgtt 840
ggagctaacc atggcatagc aaaggagatt gagggagagg ataattaggg catcaacacc 900
catggaaccc atttaatttc ccaacataga ttgatagaaa tattattgca gtctctcttg 960
atagcttaaa tattgatgag caagtgctct tgcttgtggt ttctagctga actttacagg 1020
tacgagtata agattactaa acttgtttcg atcctgaacc cgaactcgcc tgtcacttaa 1080
aatttttaaa atttttgcat aatttaatca aaaggcataa aatttttatt actagttaat 1140
ttttttttta gaatttttac ataatttaat attattttct taactatttt ttagatacac 1200
gcgctataat aaatttattt atatatatgt agttaaaaat aaatgtttaa taatcaattt 1260
attttttcta aaatcaaatt tttaatattt ttttacaaaa aaatattttt ctaagttgaa 1320
tcgtgtatgg gacggggtca gggatacccg atatctgacg ggtacgagga tgagacaata 1380
aacttaaatc cgtcgagtat tggatacgag tatgggaata tgttggggag tcggggtaag 1440
gaattgagga aacaatatcc atacccaccc gccctattgt catgtctaga cactacaaag 1500
aagggttaaa gaaacctaag ttaaaatagt agattatatg acatttagtc ctgtaaaaag 1560
aagaagagaa agatgtagaa aattttcaag aaagatatca agttaaataa tatttttcaa 1620
agtttgattt ttaattatat caaacaacgt agtgtgattc atgtaattgg tgacttacct 1680
actagtataa aaatttgttc tctttgttgt tgttgcatgt atggaatgaa ttttaaaaaa 1740
atcataaata taatttgaaa tcattttaaa attatgtaaa atcatttcga attattgatc 1800
tagattaaac aattacttag tgtaacaaga gaatttttgc ttagatttaa actttaatct 1860
ggctagcacc tagagattta tttttgtaat gatccatgac aatatcataa ttatgataat 1920
atatgtcata atttaaattt gtattcatct ttctttaaaa aatatacttg aaagtgttaa 1980
attgtacttc aaagatttag catattagtt tagttctgga taataaatta aaattattat 2040
tctcaaaaat gagataattc tttcatgtac aattcttcat acatagtatc aaatgtcttc 2100
ttcattttat gacacatgcc ttttaatttt tatattaata aaattaattt tttattaaat 2160
taaataatat tttaatctct ttaatgcttg aattaatata ttttttttta aaaaactaag 2220
catgacaagg tatttacaat ttactctaga aataatatac actaattaac acaagaataa 2280
gtatttttca aaatattttt tttttcatac aaaccacaag tatctgcaac aaaacttcct 2340

CA 02325819 2007-06-15
ttgagtgttt aagagagtta catacccaaa acagaaatgt gggaccgttg atcatcacac 2400
caattcaatt tattcagacg ctcgctttgt ggtaattggc ctataaattg tatcccaaac 2460
ttcagttaga caacaaaagc acttgttcac caattaagct ttcaagagac aaactgcttt 2520
gaaaaatggg atccaaggtt gttgcatccg ttgcccttct cctctccatc aacattcttt 2580
tcatttccat ggttagctcc agcagccact acgatccaca gccccaacct tctcacgtca 2640
ctgctcttat tacacgacct agttgtccgg atctgagtat ttgcctcaat attttaggcg 2700
ggtctctagg aaccgtggat gattgttgtg ccctcatcgg tggtcttggt gacattgaag 2760
ccattgtgtg cctttgcatc caactcaggg ccctcggaat attaaacctt aaccgtaatt 2820
tgcagttaat attaaactcc tgtggacgaa gctacccgtc aaacgccact tgcccccgaa 2880
cctaagaaca gaatatgtat ggcactaatt accatattac ttcgtatcat ggtgtttgtt 2940
tgtttgtctg tgtttaaagt taaggatgtt atacccttcg tgcctgctac atatatatag 3000
tgggcactat aatattacca ataaattaac gtccatatat aagaataata ataaataaat 3060
aaatatttct atacaaataa aggttacgta atgttgttgt tctcgtggat ggggatctta 3120
tcttcctcct cgctatcttt gtttatcgta tttcagtgaa agttgttcaa taaaagtcct 3180
ttgttcaaca agtgattcct tctctctctg tctttctttt cactttcgta ttttctttag 3240
gtataaggtg gcaaaaatag acaggaatat cgatcttgtg ataaaattaa aatcggtttg 3300
ctgatgtttt aattagttag aaaaaagaag acatatattt atcgtaattc ctgttcatga 3360
ttataaga 3368
<210> 8
<211> 7235
<212> DNA
<213> Glycine max
<400> 8
gtcgactcga tctcaaattt tatttcattt aaaataaaac ataatttaat tttcgtctct 60
cttccttatt gtatcattat aaaagtagga aaacaaatat aaattagaac aaacataata 120
ttaattaata agaataattt gtttgttgct ttgaattttc tattctaata acattaggta 180
gtaataaaat taagttgagt ttcatttttt tgaaagaatt aacttaataa ttgtatattt 240
ttgtttaagt ataatatttt agataatgta ttatcacatt aaaaatttag agtgatagac 300
aaattatgtt tattaatcaa tattatgttt atttaattgt ttgttttaag ttaggtttgt 360
tttcatattt tttttagtgt ttttattata atgagaaaaa aaatggagga taaaagataa 420
aaattatatt atattttact cttaaataaa acaaaatttg gagtcctaaa aattagttta 480
atgaaaactt gttgacatag gtctaatcta ttcaataatc atgttagctt atttgtgctc 540
ttggactcct tcattaacgg taatagatga atgaatttat tgcatttctt tcacttttga 600
ttactaaaat ttaaattttc atttttcgaa aatgaattcg ttactatttt atacatttaa 660
aaataaaaat gattatttca tcttataatt atcaaataat atgcctgtga tgatgatttt 720
tttcaaaaat tgaaaatgtc ttattgccta gttaggctat agaatctttt tggctcatct 780
caatcgcatg gcgacatggt taccgcgttc attgggtaat tatatttatg tatttaaatt 840
aattctatta aaaccaattc tattaaagct gaaaactaaa atgacactta atttctgtaa 900
gagtcgtgta attagcgagc cagagtaatg caaccaaaga gcttctttcc ccaccttaat 960
tcctttataa tgaatttgat tgataccata catagctaag cttttttttc tttcgattta 1020

CA 02325819 2007-06-15
81
catagccaag ctttagctgc tatatataat atgttgttta tataattttg acatgttgca 1080
ttacatgtta ctaactggtt gcaacct.cca gttttggtta tcgaatatat gctggccggc 1140
accaactaca aaaaatttct gtatggctgg ctactaatac tatatcttca aatgtctcac 1200
tttacatgga ctgagtgcat aacgggcaat tgcagtgctc ataaggaata cgcatgcaat 1260
cattttatca tcgcagattg ttgcttttcc ttcactgtaa aaaaaaataa aaataacaat 1320
taattgagca ccgaatgaac tagaccgatt gcatgcatta taatacatga agaaaattat 1380
ttatactaat cttttgtttc aagttttaaa gttgactttt ttgttttaca agcacaataa 1440
caatcaaata accaataatt taatcattaa aaagataaac aaactcatta gataaaataa 1500
taattatgat aaaatatgtt taagtcctct atatttaagt tgaattttgt ttttagtctt 1560
tcaaattgtg cattttagtc tctgaaattt tattttaaaa aaatagtctt tgggcttact 1620
ctccaaaatt tgcacatttt aattttaggt tattgattaa agattaaaaa catttttttt 1680
tatcaaattt agggaccaaa ttacttgata taaatactca agaactaaaa acaaatttca 1740
ataaaaatat aagggattaa aaatatattt tattctgata actaacaatc atcaaatttt 1800
atgacaatca tatgtaacct tttataatat aattgtaaga ataattaatt ttaataaata 1860
aataaataaa tgagacttat aatagtaaat acatatgttt gcctaggtaa taacaatgat 1920
aacaagtaat agaattatca ttcttattgt atctattaat taatatatta ttagatgtat 1980
taattagtat atatattttt tactataaat agtatatatt attaattatt atacatttat 2040
taaagtatat tcattaaaga ttttgaggga ggggagctga agccactaac ccccgtaaat 2100
ccgtccttgc acaaaacacg acatgagaat ggttttgtat actccacaat ttaatatcca 2160
ataaaataat ttctttttta ttttaattag gaaactccaa gattgcttta acttatacaa 2220
aatctgaata acacaaaaaa ataaaaaatc ggaattaaat gttgcccgat tgttgactaa 2280
cttatacaaa atcttattta aatgcttaaa atcgtgtcat aatataatga atatatattt 2340
gcaaatatat atttattata taattgcaaa tatatattct aattttgagt ataaataaca 2400
gcatgtgagg gtgcagcaaa acacacactg agtgcaacaa agttttaaca tgaaaggcaa 2460
taatacactt ttgttgcatt tattctacac tactctcttc ctgtttcttg tagtgtcaag 2520
ttcatcttca acagggaatg aaagtaacga tgacactaac agtaaagaag tttatatcgt 2580
gtacatggga gctgcagatt caacaaaagc ttctcttaaa aatgagcacg ctcagattct 2640
gaattcagtg ctaagaaggt acgtataatt acataatatt attattatat gggcaccaat 2700
taattaattt gatgattgat gtgtttacat attttgtgtg aatgaattga aggaatgaga 2760
atgccctagt acggaactac aagcatggtt tctctgggtt cgcagctcgt ctatcaaaag 2820
aggaggcaaa ctcaattgct cagaaacctg gtgtggtgtc tgttttccct gaccccattc 2880
tgaagctcca cactacacgt tcatgggatt tcctcaaaag ccaaactcgt gtcaatatcg 2940
acaccaaacc aaatacgctg tccggttctt ctttttcttc atcagacgtc attcttggcg 3000
tcttagacac aggttgtcca taatcaaaaa aaaaaaaaaa acatgatata tatgtgtgtg 3060
tttcattttt taaaaatgtt aataataata tatacaaaaa tggaatattt caggcatatg 3120
gccagaggcg gcgagtttta gcgacaaggg tttcggtcct gttccatccc gatggaaagg 3180
cacctgcatg acatcaaaag acttcaattc ctcttgttgt aacaggtaaa ctaaaatgtg 3240
aaaccataat aataataata ataataataa taaatatata aaggcgaacg ttattaatta 3300
ttaattatta ttagaaaaaa ggtgatttca gcttgctgtt taagaaggtt tggaatgaat 3360
cctatttaat taggtagtgg atggaataac ggttaggttt gtatttatag gaagataatt 3420
ggcgcgaggt tttaccctaa cccagaggag aaaacggcaa gggatttcaa cggacatggg 3480
actcacgttt cgtcgacggc agtgggcgtg ccggtgagtg gcgcatcgtt ctatggtctg 3540

CA 02325819 2007-06-15
82
gcggcgggga cggcaagggg tgggtcccct gagtcaaggt tggcggttta caaagtgtgt 3600
ggggcttttg ggtcatgtcc tgggtcggcc attcttgcgg ggtttgacga tgccattcac 3660
gacggagtgg atatcttgtc gctgtcgctc ggtggattcg gtggaaccaa aaccgatttg 3720
accaccgacc cgattgcgat tggagcattc cactccgtcc agcgcggcat cctggtggtc 3780
tgcgccgccg ggaacgacgg agaaccattc accgttctca acgacgcacc ttggatttta 3840
accgttgcag cttccaccat cgaccgtgat cttcaatccg acgtggtctt gggtaataac 3900
caagtcgtca aggtacctac atattctact ttaaatcggt gcagtgcaac taatgtcatc 3960
ttttctcatc gttgataatt attaaacttc agggaagagc cataaatttc tcccctcttt 4020
taaattctcc cgattatcca atgatatatg ctgagtctgc tgccagggca aatatctcca 4080
acataactga tgcaaggtac gtactctaaa aaccatttgt cgtttcgtat tggacaaact 4140
tcaaatcaag caatcaacta agcaataaca aacaagtgtt tcatcaccaa ttatatgtaa 4200
tactcatata taacctctta gcaaatgatt aaatcatttg tcacatgcag acaatgccac 4260
ccagattcat tagatccaaa aaaagtcata gggaagattg tggtttgtga tggaaaaaat 4320
gacatttatt attcaactga tgagaaaatt gtcatagtga aggcgttggg aggaataggt 4380
ctggttcata ttactgatca atctggatca gtagcatttt attatgtgga cttcccagta 4440
acagaggtaa aatcaaaaca tggcgacgca atcctccagt acatcaactc aactaggtaa 4500
ggatattata tagcacttga aagaagcaac attcttgatt aattttagaa tttgctttga 4560
tcacgagtta ttttctttta attctttgtg catatatgta atataaagcc atccagtggg 4620
aacaatacta gcaacagtta caattcctga ttataagcct gctccccggg tgggttattt 4680
ttcatcaaga gggccttcat tgattacaag caatgttctc aaggtatgat atgacgatcg 4740
atagaattat acatatcaat catcatcctc aatatgctca ttgctcaaac actaaacaga 4800
acattcattc tttctttctt tctttctttc tagcctgata ttgcagcccc gggagttaac 4860
attctcgctg catggtttgg aaatgacaca tcagaggttc caaaaggaag aaagccctca 4920
ctatatcgca tactctcagg aacttccatg gctactccac atgtttcagg gcttgcatgc 4980
agtgtcaaaa gaaaaaaccc cacttggagt gcctccgcaa tcaaatctgc catcatgact 5040
tcaggtcacc catttgataa tgtgatctaa gtaagtaatg tgatccagca aaatgtacca 5100
taccaactca tatcattcta taaattaata tgtatgcagc aattcaaaat gacaatttga 5160
agggtcccat aacaacggat tcagggttga tagccacacc ttatgactat ggagcagggg 5220
caattacaac atctgaacca ttgcaaccgg ggctagttta tgaaaccaac aacgttgact 5280
acttgaacta tttgtgttac aatggactta acataaccat gataaaggtc atctccggaa 5340
ctgtccccga gaatttcaat tgtcccaagg attcgagctc tgatctcatc tccagcatca 5400
actacccttc catagcagta aacttcactg gcaaagcaga cgcggtcgtg agtagaactg 5460
tcacaaacgt tgacgaagaa gatgaaacag tgtacttccc cgttgttgaa gctcctagtg 5520
aagtaattgt cacactcttt ccatataatc ttgagtttac gacaagtatt aaaaaacaaa 5580
gctacaatat tactttcaga ccgaagacct ccttgaagaa agatttgttt ggatctatca 5640
cttggagtaa cgacaaatat atggttcgaa ttccttttgt attaactaaa tagtgaaatt 5700
aaaaagtagc gatgaataaa tgcaagctaa gttcttcgtg gtgcctacac tcgagtcctg 5760
attatttatt attcatatgc cttctgtttt aatttaattt attatacttt cagcctctct 5820
aatatgtttt tttcttttgc aaatatataa gctgacttac tatttacact caaaattagt 5880
tccaacttat tcactagccg tttgccctca gcttaattaa aaaaaagaaa tgtgatttaa 5940
ttacattaat tatagctgga tcgtagtaac ctcggatttt tacacgggtt ggtaattcaa 6000
catcaatttc atgcttcaaa tgcaaactcc tcaaaagtag ttgcagacta aaatgatgaa 6060

CA 02325819 2007-06-15
83
tttttaacaa aacttgtaca aaggtaaggg ggaactaggg aagtgagctc ataaaataag 6120
gaaactgttt cgactgagtt ttgagtaagg tgtggctgaa ttttggcttg agttgtggtg 6180
agctgtagca gagtttcgac tttgttgtgg taagctatga cttagttttg acgaattgtg 6240
gtgagttgtg gtcgaatgga atcttggatc tcctaatccg gtgtaggaga agtacctaca 6300
aaaaggactc caacaatcaa ctcaattgga tccgagatac ttatgtatcg atgtatgaaa 6360
tgattaaaac ataccttgtt gtgttttatt tatgtcaata tatacatatt cgacattaag 6420
gaggttacac taactagata gtcctctaag cgttgcgtga tcgtgaaaag ttagtgatca 6480
tcactatcga gttcataggt tgtacggtac ataaatccca ctaatcgagt aactatcaag 6540
ttcatggggg ttgtacggta cataagtctg tcagattccc atgatgggta ttgctaagtt 6600
gaataaatcg ggcatataca ttacacgagt ttaagatgat ttaattttcc ttatatatca 6660
ttattttata ctgggtgagt gttttctttg aaaaactgag gtgtggatcc aacctcttga 6720
gaagtgcttt taaaaaaatg aggtttggat cctatctctt gagaggtgct tgaaaaagct 6780
tatcaaaata gttattggca ttcaatgttg ttttcaaccg agggcaatta aacacacctt 6840
tgattagtgg gcaattgaag ttagaaagaa gcttataaag gatagtatta ttatactatt 6900
acaaccaagc aaacaatgtt tacttcaaag ttgatacctt attgatataa tgtattattt 6960
actatgagtt aaaatgtaat ttgaaaaaaa aaatgtaact gaaaatgttc ggatgatttt 7020
catattttgt aaaaaaaaaa aaaaaagaaa aaaaagttat taggctaatt tttgcaaagt 7080
acattttggt atgaaaaacc aaaaacagaa gtaatgcatt ttgtgccatg gcagaatgca 7140
gagtatctac ccaggattca ttatgaacaa cttaatgcta agcatcatca gttaaccccc 7200
caaattaatc ttgaaagcta caattctatt ctaga 7235
<210> 9
<211> 8310
<212> DNA
<213> Glycine max
<400> 9
gagctccgcg aaatttgtta tggccatact cttccttgcg agccctcttg gtctcttgtt 60
caagggctct tgcggtagtt gcattctctt cccgtaattt ggcacactcc ttccggatgt 120
gtgtagcggc taacttgaac ttctccttgg caagtttcgc ctttcctaac tcgtttttga 180
gagcttggac ttcttcgtct tcttccggtg cttcgaaact gtctttgctg acgactttta 240
acttggcgag ccaatctaaa cctcgtattt gaactttcag ccattcatga taaccaccaa 300
tgatgccatt acgaatgccc ctaagttctt gatctttcct taacggggtt tcccatgcct 360
tatggattct ttgtatagcc ttgaaatttt gcatgccgaa atctctcaca aggaaaggag 420
aaatcctttc ttccatcggt gttcccctca tggggtaccc tagttgtctt atagcgagcg 480
cgggattgta gttgatacaa cccctcgttc ttatcagtgg aatgtttggg taccctccac 540
atgagaaaag gactccctcc tttccttcct tccatcgggg gaaccaacta attgttctac 600
ctcctatctc ggccaagagt tggtcccaat ctattcctct cttttcagca cacgagtgat 660
ggctttggag cggacatgga tgcctcgtgt tttgctggaa caggtgtgaa accaaccaaa 720
cacagagagc gggcaagcaa cagatgatcc atgcgctact cttctcgcac cttcggtcaa 780
atgtgtcaaa taaatctgcc aagacagcta ccaccgggct ttcctgtttg gaaagaggac 840
gaccccaaag attagcaatg ctaacacatc cataaacggg acccaatctt cttgatttgc 900

CA 02325819 2007-06-15
84
catatccctc gccttgtctt ctaggtactt ccgtggtagg cccgctatgc cgtttcgagt 960
ttgttttacg cggtccaaac ctcttgctga atctttgacc acggtcgcaa ttctgctcaa 1020
agaggggaga cacccggaga aaagatatga ttttcttccc ccgagaggac atcctagaat 1080
ctcctcaaat tcttcaatgg tcggtaccaa ttggaagtct ccgaacgtga agcatctcaa 1140
aggctggtcg tagtattggg tgagtgacgc aatggcctct atggatacct ctggtatggt 1200
caaatctaag atcgatcttg gattgttgtt ggttttgttg aagtattttc taaccttttg 1260
aatatgttac taatgtcaaa tttattattt gttataaaat ctttttgtct gggtttattt 1320
tctagggttt tctttaattt ttccagaaaa actttctttt cctggggatc aggtatagaa 1380
ttgattgcct caaacaagag atcgatctct ccgaaattgc acacagtggt caatgtctca 1440
gcatatctgt tttatagttg tggattctat aagtcaattt agattattgt taattaccta 1500
agttattgtt ttaaattcat acataattaa ctttgtctta acaacaacag acatctgact 1560
gagacaagta tgtgagcggg gaattctgat gtgtatggat tcctcgagcc acagtctatt 1620
cagagatctg ggcaatcgca gtttgaatca gaaagttaca tcaagagttg gatgcagagt 1680
tcaaaacgcg atgtctacct tggagcctac ctgaatgggt aagtcaaata aaacaactaa 1740
atttaaataa tatataatac tacaataacc catattcatc tccactgcag cggacacagg 1800
aaaatggtct ttattctgcc taaggaaaac cttgttgtct ggttttgttc cttgcataac 1860
aggtcagaca actaccttaa gggaataatt aacaggtcag tgttgttttt aatacatttg 1920
cattagcata actcaacaac atcaatattt taatgttcct cgtattcaac agtgctttga 1980
aaggacttga tgatactcca caatctaaat ccaaggctgg tgctaggtgg atcgttgtta 2040
aggtacgtga tttaaataaa acttctactt atatatactg cttatgtgtg tgtacactaa 2100
ttgtagttaa cgtttaatta atatccaaat ttcattatgt atttagtgta atagacaaaa 2160
aggaagcact gagtgcggct attacatcat acactagatg tccatgataa tcttaggaac 2220
ttttaggaat aattgggaaa ccgtaattgt ttatttcaaa caaagtttat tttcttgtca 2280
ttggtattac attattaact tatgttttat ttgatcatgc agtattttaa cgatgttaaa 2340
ccattggaag caaagagatt gaaggtgctt cgcatccagt gggcacaatt ttatctcaaa 2400
gttacaaatc aaagttagga tgttagggaa ctatttcatt ttaggttact taattagttt 2460
actaccttgt tttacatttt tgccaattgc tatgccattt gaacattgaa tattcttaat 2520
tgatagttat taataaatca tttattcata gttatcaatt gatagtttac tatagcttta 2580
tactaaaaac cgtttaaaag cagaatgcaa atgatatatg ctgtgatcta tggtctgatt 2640
tcaattttac aggttcaatt ttgagttttt ttgtaaaaac aaaaagtgta ttaaaaaaaa 2700
ctaaaaacaa aatcggtttt ttacaaaaat cgatgttaac atacaactta acatcggttt 2760
ctcaaaaaac cgatgtttgt gtatattaac attgattttt gtaaaaaacc aatattaacc 2820
tatgaaaatt taacattggt ttttatacaa aaccgatgtt aacttataga ttaacatcag 2880
ttttgtataa aaacttatgt taacgtttct aacttaacat caatttttat agaaaaccga 2940
tgttaatata tgagttaaca tcgattttga tacaaaactg atgttaatct ataagctaac 3000
atcggttttg tagataaccg atattttaaa ttttacgtta aactgatgtt aacgataata 3060
ctttcaacat cgattaaaaa tcgatgtaga aagtcgtaaa taaccgatgt agaaaatcta 3120
ttttctaata gtgtccatct caatatatgt ttggatagtg tcggcattac cctggtttga 3180
ctaatgcttt tgcactcaaa ttgtcacacc ataaaatcgg tgttctgaga atgggcaagc 3240
taatcttctt gatccattga gaacctaatt ttcattattt ttttacctaa acataaaaac 3300
acaaaaatat aagaaaataa aataaaatcc ttatctaaat tcagaacata aaataaataa 3360
catctttcat gttaatattt taaaattcat tattttgaag ccaaaatggg ttaaaattta 3420

CA 02325819 2007-06-15
taacaagtga cttaaaatta aaatttccaa aaataagtag aaaattagat aatttaaaaa 3480
ttaccaaact cattttatcc ttcatgactg ttatgtactg atgcagtgta attacccaca 3540
ttataaatgg taattacaaa ctagtactac cttataaatt aattaccccc attctcctat 3600
cttacaaata aaattaaaaa tacggacaaa catggcagtg gtgtggtttc gctttaaaaa 3660
taaaaatata caaacatgaa agtgatgtgt tttcgttgtt atatataata atgctcacga 3720
tcgagactca actaaatgat cacgcaaatg tttttcttag gatgaagcct gttgctaatt 3780
tcttcatctc aaaatccggt cattacgcaa tttatagatt tgtcgagatt ttggcttctt 3840
ctttaaattt gcgcataaaa acttatcaat gtatacctta tggctgcatg aaattaccta 3900
ttcttatggt tgcatattaa atgttcatac tttttatatt attttattta gattaaattg 3960
taattttgat ccctattttt aaattaaggc attccattaa tggaccattt atgtgacaaa 4020
agattaaata acttaaaaat acaaaaaggt cttagtataa attaaattta aaatatttta 4080
tataaagata aaataataaa accataaaat acttacttta tttagttaat tttataaata 4140
ctattttaca tgtatcatta attagtccag aattattaat ttttttaatt ataaaaattt 4200
ataaattgaa ataaaatatt taatatttaa atatatttaa catttaattt tacatgtact 4260
tatttttctt ataaaatttt attttaaaat aaatcaacta aaatttgttg ttattttagt 4320
ataattttaa aaaaatacat aaactaatat atagattatt ttaaaattat tttatctaat 4380
ttatgaaaat taagtagatt taaaataaca tttaactaat ctaaaattta aaatttatta 4440
cttgttataa ttaaaataaa ctttttcatt atttatatat aaaaataaat gtagataatt 4500
ttatcaatta cataaaataa tttatatata actaatttac atattaattt atttaatttt 4560
cctcatttat ataattgata ttgaaaaact tatttactaa atatttttta tagttaaaat 4620
aaaaatatta acatgttttt ttcctaaaaa cattaaatat tcaaatttta ttcatttatt 4680
taattaaaat aaaggtaatt tacatattaa aataaattac ataatttgta taatttatat 4740
attaataaaa aaatatataa attaatttaa attagtcaaa taaaaataaa aatatataaa 4800
ctattcaata tatatatata tatatatata tatatatata tatatatata taatataatg 4860
acatacaaga aaattaaaag gatttataaa aaggatttat atattaaata ttttttaatt 4920
cataaatttt tataatttaa aaaattaact catacataaa taatatttaa aaaattagtt 4980
aaaaaaataa atatcttaat agtttgttat tttaatttta aataagagat tataagttta 5040
attcttatta ttttttttca taattttaac tcttaccaag tatttttttc actttgttta 5100
atctctattc tctctctctt tatatatata tatatatata tatataataa atttctaatc 5160
aactaaatag tgtatatttt acatgcagag tacataaaaa ttaaaattgg gtataatagt 5220
tattgtacgt catctcactc actcttcctc ttagagtcta tatatatgca atatctctgg 5280
tctccctatc accttctctt ctctaagcac tttacttttt ttttcagcca tggagtttca 5340
ttgccttcca atatttcttt atctcaatgt gagcataaac cttccttttt catctcgttt 5400
tttaacctgt attgcgtgaa gttggatttc ttcattaatt atcattttaa ttatagcaat 5460
aagtatcaag tgatgggttt ttgcatgaat tatgtagttg atgttgatga cagccaatgc 5520
tgcgttaact cctagacatt actgggaaac gatgcttcca agaactccct tgccgaaagc 5580
aatcacagag ctactaagcc ttggtgagta agaaatcaaa ttgaaataaa ataagcaaca 5640
cttttgtaat taaatctgaa acgataaatg tacgtaaaca aaaaatagaa ttagaaacca 5700
atgcaaacag ctataagcct ttttttgtaa tagctaatct gccttaatta aaccattgaa 5760
tgactcaata tgttcatctt tttttccttt tagtagaata ctgcccttct tcatattcga 5820
tatactattg gcttccttag tgaattctta ttctaatggt tgcatagctg tgttctcact 5880
aagcctcctt tcccatgtct ttgattctat gaaacgaaac tttcaaaact tggaatcacc 5940

CA 02325819 2007-06-15
86
tattaagtac aataaagaaa taattaattt tatttttatc ctttaattat tttatgaatt 6000
tggttccttt attttatttg atatattttt tacaaatttg gttttctcat tattttaatt 6060
gtttattttt tgttcatctc ttaacttaat atccaacatt tcataaatat gctgacataa 6120
tactagatta ttatatcacc tgtggggata ttcatgttgc acaattagtg gaggctcaaa 6180
ttcgtgaaaa aataaataaa atttggagag accatattca ccaaatcaga atagtataca 6240
gagacaaaaa aaaaaaaaaa aaacaaatta tgcaacgaat gaaaagttaa ccattatatt 6300
tagtattgag taatttgaag tcagtgtgag acatggcaac gtagttaact tactttaatt 6360
aaaaatatga ataattaaaa taggaatata taacggattc ggattctctc cagcaatgat 6420
gaggacgtag gattaatttt atggctgaga gtaaatccaa aaagatacgc aaaccacgta 6480
tcaccgctac tgcttgacca aatggaagcg acttcaaacc aaaattagga caaagaaaat 6540
aaaaatacaa atgtcaataa actcaatata caaagacaga gtgtaagttt tactaaatgg 6600
aaagctggaa ttgaagtaaa agctttacat atatattgct tgctaaacaa ttttgaattt 6660
tttttatctt ggattcttgc taaacaatta attatatcta ttaatatttc tctaataata 6720
tgggatacac ttattattat tattattatt attattatta ttattattat tatttgattt 6780
aaatataatt ttgatatttg actaatatta aatttatttt aaacaatcac aaatttcgaa 6840
taattaatga taatattaag ttattttcat atttgactag tatcaaaaag ttatttacac 6900
atttgactta ctgttatttt attttagttc tctagttttt aaaagtataa taagttagtt 6960
ctttcatttc ttttctattt tccattttaa ttaaaaaaaa ttcaattttt attttatttt 7020
gtaacaatgg gctgaaaata gctactaaaa ttttggctgt gtttttcttt accaatcaga 7080
aagtaggtcc atatttgaat atgccgggaa tgatgaccag tcagaaagta ggtccatatt 7140
aggatacgct ggctataatc aagacgagga tgatgtgagc aaacacaata tacaaatctt 7200
caacaggttg tttttcttgg aagaggacct gcgtgctggc aaaatattca acatgaagtt 7260
cgtcaacaac acaaaagcca cagtcccgtt gctaccgcgc caaatttcga aacaaatacc 7320
gttctcagaa gataaaaaga agcaagtgtt ggcgatgctt ggcgtggaag cgaactcaag 7380
caacgccaag atcatagcgg agaccattgg tctttgccaa gagcctgcaa cggagggaga 7440
aaggaaacac tgcgcgactt cgttggagtc catggttgat ttcgtcgttt ccgcgctcgg 7500
gaagaacgtt ggtgctttct caacagagaa agaaagggaa actgagtctg gaaagtttgt 7560
agtggtgaaa aatggggtga ggaagttggg agatgataag gttattgcct gtcatccaat 7620
gagttaccct tatgttgtgt ttgggtgtca tctagtgcca aggagtagcg ggtatttggt 7680
gcgcttgaag ggagaagatg gggttcgagt gaaagcagtt gttgcgtgcc acagagacac 7740
gtcaaagtgg gaccataatc atggggcatt caaagtgctc aatcttaagc ctgggaatgg 7800
tacagtatgc catgtcttca ctgaggggaa tcttctttgg cttccaaatt agattaatta 7860
ccatatacat atttgtcctt gttctatcct taaataagtg gaatcacctg aagaattgtg 7920
cgtaatgagt tgtttgtctt tgtggaaatt gttatctgtc ttgcatcacc aaataggtat 7980
atataaaata acaggagcgt ggtatttgtt gcacaaaaat ggatttcaac cgatcaaaaa 8040
aatatagcct ttaccaatta gaagggtttg gctttgttag caaataataa aaataaaata 8100
tcttgatgga taaatggttg ctaagttgat taagattgtg gcagaatacc aagtcaatga 8160
atagtccatc acaagcatca aaagaaaaga taatgattcc ttgaaaatag aaagcacttt 8220
gtgttttgaa ttcaaaatgc acactggaga gttgttggag gttacaagcc agatcgagtc 8280
gactcccttt agtgagggtt aattgagctc 8310
<210> 10

CA 02325819 2007-06-15
87
<211> 119
<212> PRT
<213> Glycine max
<400> 10
Met Gly Ser Lys Val Val Ala Ser Val Ala Leu Leu Leu Ser Ile Asn
1 5 10 15
Ile Leu Phe Ile Ser Met Val Ser Ser Ser Ser His Tyr Asp Pro Gln
20 25 30
Pro Gln Pro Ser His Val Thr Ala Leu Ile Thr Arg Pro Ser Cys Pro
35 40 45
Asp Leu Ser Ile Cys Leu Asn Ile Leu Gly Gly Ser Leu Gly Thr Val
50 55 60
Asp Asp Cys Cys Ala Leu Ile Gly Gly Leu Gly Asp Ile Glu Ala Ile
65 70 75 80
Val Cys Leu Cys Ile Gln Leu Arg Ala Leu Gly Ile Leu Asn Leu Asn
85 90 95
Arg Asn Leu Gln Leu Ile Leu Asn Ser Cys Gly Arg Ser Tyr Pro Ser
100 105 110
Asn Ala Thr Cys Pro Arg Thr
115
<210> 11
<211> 286
<212> PRT
<213> Glycine max
<400> 11
Asn Ala Ala Leu Thr Pro Arg His Tyr Trp Glu Thr Met Leu Pro Arg
1 5 10 15
Thr Pro Leu Pro Lys Ala Ile Thr Glu Leu Leu Ser Leu Glu Ser Arg
20 25 30

CA 02325819 2007-06-15
88
Ser Ile Phe Glu Tyr Ala Gly Asn Asp Asp Gln Ser Glu Ser Arg Ser
35 40 45
Ile Leu Gly Tyr Ala Gly Tyr Asn Gln Asp Glu Asp Asp Val Ser Lys
50 55 60
His Asn Ile Gln Ile Phe Asn Arg Leu Phe Phe Leu Glu Glu Asp Leu
65 70 75 80
Arg Ala Gly Lys Ile Phe Asn Met Lys Phe Val Asn Asn Thr Lys Ala
85 90 95
Thr Val Pro Leu Leu Pro Arg Gln Ile Ser Lys Gin Ile Pro Phe Ser
100 105 110
Glu Asp Lys Lys Lys Gln Val Leu Ala Met Leu Gly Val Glu Ala Asn
115 120 125
Ser Ser Asn Ala Lys Ile Ile Ala Glu Thr Ile Gly Leu Cys Gln Glu
130 135 140
Pro Ala Thr Glu Gly Glu Arg Lys His Cys Ala Thr Ser Leu Glu Ser
145 150 155 160
Met Val Asp Phe Val Val Ser Ala Leu Gly Lys Asn Val Gly Ala Phe
165 170 175
Ser Thr Glu Lys Glu Arg Glu Thr Glu Ser Gly Lys Phe Val Val Val
180 185 190
Lys Asn Gly Val Arg Lys Leu Gly Asp Asp Lys Val Ile Ala Cys His
195 200 205
Pro Met Ser Tyr Pro Tyr Val Val Phe Gly Cys His Leu Val Pro Arg
210 215 220
Ser Ser Gly Tyr Leu Val Arg Leu Lys Gly Giu Asp Gly Val Arg Val
225 230 235 240
Lys Ala Val Val Ala Cys His Arg Asp Thr Ser Lys Trp Asp His Asn
245 250 255

CA 02325819 2007-06-15
89
His Gly Ala Phe Lys Val Leu Asn Leu Lys Pro Gly Asn Gly Thr Val
260 265 270
Cys His Val Phe Thr Glu Gly Asn Leu Leu Trp Leu Pro Asn
275 280 285
<210> 12
<211> 770
<212> PRT
<213> Glycine max
<400> 12
Met Lys Gly Asn Asn Thr Leu Leu Leu His Leu Phe Tyr Thr Thr Leu
1 5 10 15
Phe Leu Phe Leu Val Val Ser Ser Ser Ser Ser Thr Gly Asn Glu Ser
20 25 30
Asn Asp Asp Thr Asn Ser Lys Glu Val Tyr Ile Val Tyr Met Gly Ala
35 40 45
Ala Asp Ser Thr Lys Ala Ser Leu Lys Asn Glu His Ala Gln Ile Leu
50 55 60
Asn Ser Val Leu Arg Arg Asn Glu Asn Ala Leu Val Arg Asn Tyr Lys
65 70 75 80
His Gly Phe Ser Gly Phe Ala Ala Arg Leu Ser Lys Glu Glu Ala Asn
85 90 95
Ser Ile Ala Gln Lys Pro Gly Val Val Ser Val Phe Pro Asp Pro Ile
100 105 110
Leu Lys Leu His Thr Thr Arg Ser Trp Asp Phe Leu Lys Ser Gln Thr
115 120 125
Arg Val Asn Ile Asp Thr Lys Pro Asn Thr Leu Ser Gly Ser Ser Phe
130 135 140
Ser Ser Ser Asp Val Ile Leu Gly Val Leu Asp Thr Gly Ile Trp Pro
145 150 155 160

CA 02325819 2007-06-15
Glu Ala Ala Ser Phe Ser Asp Lys Gly Phe Gly Pro Val Pro Ser Arg
165 170 175
Trp Lys Gly Thr Cys Met Thr Ser Lys Asp Phe Asn Ser Ser Cys Cys
180 185 190
Asn Arg Lys Ile Ile Gly Ala Arg Phe Tyr Pro Asn Pro Glu Glu Lys
195 200 205
Thr Ala Arg Asp Phe Asn Gly His Gly Thr His Val Ser Ser Thr Ala
210 215 220
Val Gly Val Pro Val Ser Gly Ala Ser Phe Tyr Gly Leu Ala Ala Gly
225 230 235 240
Thr Ala Arg Gly Gly Ser Pro Glu Ser Arg Leu Ala Val Tyr Lys Val
245 250 255
Cys Gly Ala Phe Gly Ser Cys Pro Gly Ser Ala Ile Leu Ala Gly Phe
260 265 270
Asp Asp Ala Ile His Asp Gly Val Asp Ile Leu Ser Leu Ser Leu Gly
275 280 285
Gly Phe Gly Gly Thr Lys Thr Asp Leu Thr Thr Asp Pro Ile Ala Ile
290 295 300
Gly Ala Phe His Ser Val Gln Arg Gly Ile Leu Val Val Cys Ala Ala
305 310 315 320
Gly Asn Asp Gly Glu Pro Phe Thr Val Leu Asn Asp Ala Pro Trp Ile
325 330 335
Leu Thr Val Ala Ala Ser Thr Ile Asp Arg Asp Leu Gln Ser Asp Val
340 345 350
Val Leu Gly Asn Asn Gln Val Val Lys Gly Arg Ala Ile Asn Phe Ser
355 360 365
Pro Leu Leu Asn Ser Pro Asp Tyr Pro Met Ile Tyr Ala Glu Ser Ala
370 375 380

CA 02325819 2007-06-15
91
Ala Arg Ala Asn Ile Ser Asn Ile Thr Asp Ala Arg Gln Cys His Pro
385 390 395 400
Asp Ser Leu Asp Pro Lys Lys Val Ile Gly Lys Ile Val Val Cys Asp
405 410 415
Gly Lys Asn Asp Ile Tyr Tyr Ser Thr Asp Glu Lys Ile Val Ile Val
420 425 430
Lys Ala Leu Gly Gly Ile Gly Leu Val His Ile Thr Asp Gln Ser Gly
435 440 445
Ser Val Ala Phe Tyr Tyr Val Asp Phe Pro Val Thr Glu Val Lys Ser
450 455 460
Lys His Gly Asp Ala Ile Leu Gln Tyr Ile Asn Ser Thr Ser His Pro
465 470 475 480
Val Gly Thr Ile Leu Ala Thr Val Thr Ile Pro Asp Tyr Lys Pro Ala
485 490 495
Pro Arg Val Gly Tyr Phe Ser Ser Arg Gly Pro Ser Leu Ile Thr Ser
500 505 510
Asn Val Leu Lys Pro Asp Ile Ala Ala Pro Gly Val Asn Ile Leu Ala
515 520 525
Ala Trp Phe Gly Asn Asp Thr Ser Glu Val Pro Lys Gly Arg Lys Pro
530 535 540
Ser Leu Tyr Arg Ile Leu Ser Gly Thr Ser Met Ala Thr Pro His Val
545 550 555 560
Ser Gly Leu Ala Cys Ser Val Lys Arg Lys Asn Pro Thr Trp Ser Ala
565 570 575
Ser Ala Ile Lys Ser Ala Ile Met Thr Ser Ala Ile Gln Asn Asp Asn
580 585 590
Leu Lys Gly Pro Ile Thr Thr Asp Ser Gly Leu Ile Ala Thr Pro Tyr
595 600 605

CA 02325819 2007-06-15
92
Asp Tyr Gly Ala Gly Ala Ile Thr Thr Ser Glu Pro Leu Gln Pro Gly
610 615 620
Leu Val Tyr Glu Thr Asn Asn Val Asp Tyr Leu Asn Tyr Leu Cys Tyr
625 630 635 640
Asn Gly Leu Asn Ile Thr Met Ile Lys Val Ile Ser Gly Thr Val Pro
645 650 655
Glu Asn Phe Asn Cys Pro Lys Asp Ser Ser Ser Asp Leu Ile Ser Ser
660 665 670
Ile Asn Tyr Pro Ser Ile Ala Val Asn Phe Thr Gly Lys Ala Asp Ala
675 680 685
Val Val Ser Arg Thr Val Thr Asn Val Asp Glu Glu Asp Glu Thr Val
690 695 700
Tyr Phe Pro Val Val Glu Ala Pro Ser Glu Val Ile Val Thr Leu Phe
705 710 715 720
Pro Tyr Asn Leu Glu Phe Thr Thr Ser Ile Lys Lys Gln Ser Tyr Asn
725 730 735
Ile Thr Phe Arg Pro Lys Thr Ser Leu Lys Lys Asp Leu Phe Gly Ser
740 745 750
Ile Thr Trp Ser Asn Asp Lys Tyr Met Val Arg Ile Pro Phe Val Leu
755 760 765
Thr Lys
770