SEED COAT SPECIFIC DNA REGULATORY REGION AND PEROXIDASE

PEROXYDASE ET REGION DE REGULATION DE L'ADN SPECIFIQUES DU TEGUMENT

English Abstract

A novel seed coat specific peroxidase genomic sequence is characterized and
presented. Adjacent DNA regulatory regions have also been characterized. The
seed
coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa
comprising a 26 amino acid signal sequence which when cleaved results in a 35
kDa
protein. Plants containing a dominant Ep allele accumulate large amounts of
peroxidase in the hourglass cells of the subepidermis. Homozygous recessive
epep
genotypes do not accumulate peroxidase in the hourglass cells and are much
reduced
in total seed coat peroxidase activity. Probes derived from the cDNA, or
genomic
DNA can be used to detect polymorphisms that distinguished EpEp and epep
genotypes. Cosegregation of the polymorphisms in an F2 population from a cross
of
EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase
protein. Comparison of Ep and ep alleles indicates that the recessive gene
lacks 87 bp
of sequence encompassing the translation start codon. The heterologous
expression,
as well as vectors and hosts to be used for the expression of the seed coat
peroxidase,
are also disclosed. The seed-specific DNA regulatory region may be used to
control
expression of genes of interest such as i) genes encoding herbicide
resistance, or ii)
biological control of insects or pathogens (e.g. B. thuringiensis), or iii)
viral coat
proteins to protect against viral infections, or iv) proteins of commercial
interest (e.g.
pharmaceutical), and v) proteins that alter the nutritive value, taste, or
processing of
seeds.

Claims

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THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMS ARE DEFINED AS FOLLOWS:
1. An isolated DNA molecule comprising the nucleotide sequence of SEQ ID
NO:1.
2. An isolated DNA molecule comprising at least 24 contiguous nucleotides
selected from nucleotides 1-1532 of SEQ ID NO:2, wherein the DNA
molecule controls expression of a DNA sequence that is operably linked
thereto.
3. An isolated DNA molecule comprising a nucleotide sequence of
nucleotides 1533-4700 of SEQ ID NO:2
4. An isolated DNA molecule comprising a nucleotide sequence of
nucleotides 1-4700 of SEQ ID NO:2.
5. An isolated DNA molecule comprising a nucleotide sequence that
hybridizes to nucleotides 1-1532 of SEQ ID NO:2 or a complement
thereof, under hybridization conditions comprising hybridization in
6xSSC, 20mM NazHPO4, 0.4% SDS, 500 µg salmon sperm DNA at 65°C
for 20 hours, followed by two 30 minute washes at 65°C with 0.1x SSC,
0.5% SDS, wherein the nucleotide sequence controls expression of a DNA
sequence that is operably linked thereto.
6. An isolated DNA molecule comprising the nucleotide sequence of
nucleotides 1-1532 of SEQ ID NO:2.
7. A vector which comprises the DNA molecule of claim 1.
8. A vector which comprises the DNA molecule of claim 2, wherein the
DNA molecule is operably linked to the DNA sequence, and the DNA
sequence encodes a protein of interest.

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9. A vector which comprises the DNA molecule of claim 3.
10. The vector of claim 8 wherein the DNA sequence is a heterologous gene
of interest.
11. A host cell expressing the DNA molecule within the vector of claim 7.
12. A host cell expressing the DNA molecule within the vector of claim 8.
13. A host cell expressing the DNA molecule within the vector of claim 9.
14. A host cell expressing the DNA molecule within the vector of claim 10.
15. A method for the production of soybean seed coat peroxidase in a host cell
comprising:
i) transforming the host cell with a vector comprising an isolated
DNA molecule selected from the group consisting of SEQ ID
NO:1, and SEQ ID NO:2; and
ii) culturing the host cell under conditions to allow expression of the
soybean seed coat peroxidase.
16. A process for producing a heterologous protein of interest comprising
transforming a plant with the vector of claim 10.
17. A vector comprising the DNA molecule of claim 5.
18. A process for producing a heterologous protein of interest in a transgenic
plant comprising,
- transforming a plant with a vector comprising the DNA molecule of
claim 5, to obtain a transgenic plant, the DNA molecule operably linked to the
DNA sequence, and the DNA sequence encodes the heterologous protein of
interest, and

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- growing the transgenic plant under conditions to produce the
heterologous protein of interest.
19. An isolated DNA molecule comprising at least 20 contiguous nucleotides
selected from nucleotides 1524-1610 of SEQ ID NO:2 for annealing with a
nucleic acid sequence from a plant carrying an Ep allele.
20. The isolated DNA molecule of claim 19 comprising nucleotides 1524-
1610 of SEQ ID NO:2.
21. The use of the isolated DNA molecule of claim 19 as a marker for
selecting soybean plants carrying an Ep allele.
22. The use of the isolated DNA molecule of claim 19 as a marker to select
soybean plants comprising a deletion of nucleotides 1524-1610 of SEQ ID
NO:2 in a peroxidase gene.
23. A method of selecting between an EpEp and an epep plant genotype
comprising the steps of
a) preparing genomic DNA, or cDNA from a plant;
b) fragmenting the genomic DNA or cDNA to produce DNA
fragments;
c) separating the DNA fragments;
d) hybridizing the fragments with a labeled nucleotide sequence,
where the nucleotide sequence is the isolated DNA molecule
defined in claim 19, to produce a hybridization pattern; and
e) determining whether the hybridization pattern is
representative of an EpEp or an epep genotype.

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24. A method of selecting between an EpEp and an epep plant genotype
comprising the steps of
a) preparing genomic DNA, or cDNA from a plant;
b) fragmenting the genomic DNA, or cDNA to produce DNA
fragments;
c) amplifying the DNA fragments using at least one primer, the
at least one primer comprising the isolated DNA molecule
defined in claim 19 to produce an amplified product; and
d) determining whether the amplified produce is representative
of an EPEP or epep genotype.

Description

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SEED COAT SPECIFIC DNA REGULATORY REGION AND PEROXIDASE
The present invention relates to a novel DNA molecule comprising a plant seed
coat specific DNA regulatory region and a novel structural gene encoding a
peroxidase.
The seed-coat specific DNA regulatory region may also be used to control the
expression of other genes of interest within the seed coat.
BACKGROUND OF THE INVENTION
Full citations for references appear at the end of the Examples section.
Peroxidases are enzymes catalyzing oxidative reactions that use H202 as an
electron acceptor. These enzymes are widespread and occur ubiquitously in
plants as
isozymes that may be distinguished by their isoelectric points. Plant
peroxidases
contribute to the structural integrity of cell walls by functioning in lignin
biosynthesis
and suberization, and by forming covalent cross-linkages between extension,
cellulose,
pectin and other cell wall constituents (Campa, 1991). Peroxidases are also
associated
with plant defence responses and resistance to pathogens (Bowles, 1990;
Moerschbacher 1992). Soybeans contain 3 anionic isozymes of peroxidase with a
minimum Mr of 37 kDa (Sessa and Anderson, 1981). Recently one peroxidase
isozyme, localised within the seed coat of soybean, has been characterized
with a Mr
of 37 kDa (Glikin and Graham, 1991).

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In an analysis of soybean seeds, Buttery and Buzzell (1968) showed that 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 (Buzzell and Buttery, 1969). Homozygous recessive epep
plants
are - 100-fold lower in seed coat peroxidase activity. This 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). These cells form
a highly
differentiated cell layer with thick, elongated secondary walls and large
intercellular
spaces (Baker et al., 1987). Hourglass cells develop between the epidermal
macrosclereids and the underlying articulated parenchyma, and are a prominent
feature
of seed coat anatomy at full maturity. The cytoplasm exudes from the hourglass
cells
upon imbibition with water and a distinct peroxidase isozyme constitutes five
to 10%
of the total soluble protein in EpEp seed coats. It is not known why the
hourglass cells
accumulate large amounts of peroxidase, but the sheer abundance and relative
purity
of the enzyme in soybean seed coats is significant because peroxidases are
versatile
enzymes with many commercial and industrial applications. Studies of soybean
seed
coat peroxidase have shown this enzyme to have useful catalytic properties and
a high
degree of thermal stability even at extremes of pH (McEldoon et al., 1995).
These
properties result in the preferred use of soybean peroxidase, over that of
horseradish
peroxidase, in diagnostic assays as an enzyme label for antigens, antibodies,
oligonucleotide probes, and within staining techniques. Johnson et al report
on the use
of soybean peroxidase for the deinking of printed waste paper (U.S. 5,270,770;

CA 02211.019 2002-08-28
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December 6, 1994) and for the biocatalytic oxidation of prunary alcohols (U.S.
5,391,488; February 13, 1996). Soybean peroxidase has also been used as a
replacement for chlorine in the pulp and paper industry, or as formaldehyde
replacement (Freiberg, 1995).
An anionic soybean peroxidase from seed coats has been purified (Gill.ikin and
Graham, 1991). This protein has a pI of 4.1 and Mr of 37 kDa. A method for the
bulk extraction of peroxidase from seed hulls of soybean using a freeze thaw
technique
has also been reported (U.S. 5,491,085, February 13, 1996, Pokara and
Johnson).
Lagrimini et al ((1987) Molecular cloning of complementary DNA encoding the
lignin-forming peroxidase from tobacco: Molecular analysis and tissue specific
expression. Proc. Natl. Acad. Sci. USA 84:7542-46) disclose the cloning of a
ubiquitous
anionic peroxidase in tobacco encoding a protein of MT of 36 kDa. This
peroxidase has
also been over expressed in -transgenic tobacco plants (Lagrimini et al 1990)
and
Maliyakal discloses the expression of this gene in cotton (WO 95/08914).
Huangpu et al (GENEMBL, Accession number: U41657; 1985) reported the
partial closing of a soybean anionic seed coat peroxidase. The 1031 bp
sequence
contained an open reading frame of 849 bp encoding a 283 amino acid protein
with a Mr
of 30,577. The M, of this peroxidase is 7 kDa less than what one would expect
for a
soybean seed coat peroxidase as reported by Gillikin and Graham (1991) and
possibly
represents another peroxidase isozyme within the seed coat.

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The upstream promoter sequences for two poplar peroxidases have been
described by Osakabe et al (1995). A number of characteristic regulatory sites
were
identified from comparison of these sequences to existing promoter elements.
Additionally, a cryptic promoter with apparent specificity for seed coat
tissues was
isolated from tobacco by a promoter trapping strategy (Fobert et al. (1994) T-
DNA
tagging of seed coat-specific cryptic promoter in tobacco. Plant J. 6(4):567-
77). The
upstream regulatory sequences associated with the Ep gene in soybean are
distinct from
these and other previously characterized promoters. The soybean Ep promoter
drives
high-level expression in a cell and tissue specific manner. The peroxidase
protein
encoded by the Ep gene accumulates in the seed coat tissues, especially in the
hour glass
cells of the subepidermis. Minimal expression of the gene is detected in root
tissues.
One problem arising from the desired use of soybean seed coat peroxidase is
that there is variability between soybean varieties regarding peroxidase
production
(Buttery and Buzzell, 1968; Freiberg, 1995). Due to the commercial interest in
the use
of soybean seed coat peroxidase new methods of producing this enzyme are
required.
Therefore, the gene responsible for the expression of the 37 kDa isozyme in
soybean
seed coat was isolated and characterized.
Furthermore, novel regulatory regions obtained from the genomic DNA of
soybean seed coat peroxidase have been isolated and characterized and are
useful in
directing the expression of genes of interest in seed coat tissues.

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SUMIVIA.RY OF THE IIWENTION
The present invention relates to a DNA molecule that encodes a soybean seed
coat peroxidase and associated DNA regulatory regions.
This invention also embraces isolated DNA molecules comprising the nucleotide
sequence of either SEQ ID NO:I (the cDNA encoding soybean seed coat
peroxidase)
SEQ ID No:2 (the genomic sequence).
This invention also provides for a chimeric DNA molecule comprising a seed
coat-specific regulatory region having nucleotides 1-1532 of SEQ ID NO:2 and a
gene
of interest under control of this DNA regulatory region. Also included within
this
invention are chimeric DNA molecules comprising genomic DNA sequences
exemplified by nucleotides 1752-2382, 2575-3604 or 3770-4032 of SEQ ID NO:2.
Furthermore, this invention is directed to isolated DNA molecules comprising
at least
1) 24 contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID
NO:2;
2) 32 contiguous nucleotides selected from nucleotides 1752-2382 of SEQ
ID NO:2;
3) 23 contiguous nucleotides selected from nucleotides 2575-3604 of SEQ
ID NO:2; or
4) 22 contiguous nucleotides selected from nucleotides 3770-4032 of SEQ
ID NO:2.

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The present invention also provides for vectors which comprise DNA molecules
encoding soybean seed coat peroxidase. Such a construct may include the DNA
regulatory region from SEQ ID NO:2, including nucleotides 1-1532, or at
least.24
contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2 in
conjunction with the seed coat peroxidase gene, or the seed coat peroxidase
gene under
the. control of any suitable constitutive or inducible promoter of interest.
This invention is also directed towards vectors which comprise a gene of
interest placed under the control of a DNA regulatory element derived from the
genomic sequence encoding soybean seed coat peroxidase. Such a regulatory
element
includes nucleotides 1-1532 of SEQ ID NO:2, or at least 24 contiguous
nucleotides
selected from nucleotides 1-1532 of SEQ ID NO:2. Elements comprising
nucleotides
1752-2382, 2575-3604 or 3770-4032 of SEQ ID NO:2, or 32 contiguous nucleotides
selected from nucleotides~ 1752-2382 of SEQ ID NO:2, 23 contiguous nucleotides
selected from nucleotides 2575-3604 of SEQ ID NO:2, or 22 contiguous
nucleotides
selected from nucleotides 3770-4032 of SEQ ID NO:2 may also be used.
This invention also embraces prokaryotic and eukaryotic cells comprising the
vectors identified above. Such cells may include bacterial, insect, mammalian,
and
plant cell cultures.
This invention also provides for transgenic plants comprising the seed coat
peroxidase gene under control of constitutive or inducible promoters.
Furthermore,

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this invention also relates to transgenic plants comprising the DNA regulatory
regions
of nucleotides 1-1532 of SEQ ID NO:2 controlling a gene of interest, or
comprising
genes of interest in functional association with genomic DNA sequences
exemplified
by nucleotides 1752-2382, 2575-3604 or 3770-4032 of SEQ ID NO:2. Also embraced
by this invention are transgenic plants having regulatory regions comprising
at least 24
contiguous nucleotides selected from nucleotides 1-1532 of SEQ ID NO:2, 32
contiguous nucleotides selected from nucleotides 1752-2382 of SEQ ID NO:2, 23
contiguous nucleotides selected from nucleotides 2575-3604 of SEQ ID NO:2, or
22
contiguous nucleotides selected from nucleotides 3770-4032 of SEQ ID NO:2.
This invention is also directed to a method for the production of soybean seed
coat peroxidase in a host cell comprising:
i) transforming the host cell with a vector comprising an oligonucleotide
sequence that encodes soybean seed coat peroxidase; and
ii) culturing the host cell under conditions to allow expression of -the
soybean seed coat peroxidase.
This invention also provides for a process for producing a heterologous gene
of interest within seed coats of a transformed plant, comprising propagating a
plant
transformed with a- vector comprising a gene of interest under the control of
.
nucleotides 1-1532 of SEQ ID NO:2. Furthermore; this invention embraces a
process
for producing a heterologous gene of interest within seed coats of a
transformed plant,
comprising propagating a plant transformed with a vector comprising a gene of
interest

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under the control of a regulatory region comprising at least 24 nucleotides
selected
from nucleotides 1-1532 of SEQ ID NO:2.
Although the present invention is exemplified by a soybean seed coat
peroxidase
and adjacent DNA regulatory regions, in practice any gene of interest can be
placed
downstream from the DNA regulatory region for seed coat specific expression.

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BRIEF DESCRIPTION OF Z'HE DRAWINGS
These and other features of the invention will become more apparent from the
following description in which reference is made to the appended drawings
wherein:
Figure I is the cDNA and deduced amin.o acid sequence of soybean seed coat
peroxidase (SEQ ID NO:1). Nucleotides are numbered by assigning +1 to the
first base of the ATG start codon; amino acids are numbered by assigning +1 to
the N-terminal Gln residue after cleavage of the putative signal sequence. The
N-terminal signal sequence, the region of the active site, and the heme-
binding
domain are underlined. The numerals I, II and III placed directly above single
nucleotide gaps in the sequence indicate the three intron splice positions.
The
target site and direction of five different PCR primers are shown with dotted
lines
above the nucleotide sequence. An asterisk (*) marks the translation stop
codon.
Figure 2 is the genomic DNA sequence of the Soybean seed coat peroxidase
(commencing at nucleotide 1342 of SEQ ID NO:2).
Figure 3 is a comparison of soybean seed coat peroxidase with other closely
related
plant peroxidases. The GenBank accession numbers are provided next.to the
name of the plant from which the peroxidase was isolated. The accession
number for the soybean sequence is L78163. (A) A comparison of the nucleic
acid sequences; (B) A comparison of the amino acid sequences. (L78163
nucleotide sequence SEQ ID NO:1; L78163 amino acid sequence SEQ ID NO: 1,
U41657 nucleotide sequence SEQ ID NO:10; U41657 amino acid sequence SEQ
ID NO:15; X90693 nucleotide sequence SEQ ID NO:11; X90693 amino acid
sequence SEQ ID NO: 16; X90694 nucleotide sequence SEQ ID NO:12; X90694
amino acid sequence SEQ ID NO:17; L36156 nucleotide sequence SEQ ID
NO:13; L36156 amino acid sequence SEQ ID NO:18; X90692 nucleotide
sequence SEQ ID NO:14; X90692 amino acid sequence SEQ ID NO:19).

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Figure 4 is a restriction fragment length polymorphisms between EpEp and epep
genotypes using the seed coat peroxidase cDNA as probe. Genomic DNA of
soybean lines OX312 (epep) and OX347 (EpEp) was digested with restriction
enzyme, separated by electrophoresis in a 0.5% agarose gel, transferred to
nylon, and hybridized with 32P-labelled cDNA encoding the seed coat
peroxidase. The size of the hybridizing fragments was estimated by comparison
to standards and is indicated on the right.
Figure 5 exhibits the structure of the Ep Locus. A 17 kb fragment including
the Ep
locus is illustrated schematically. A 3.3 kb portion of the gene is enlarged
and
exons and introns are represented by shaded and open boxes, respectively. The
final enlargement of the 5' region shows the location and DNA sequence
around the 87 bp deletion occurring in the ep allele of soybean line OX312.
Nucleotides are numbered by assigning + 1 to the first base of the ATG start
codon (OX347 (Ep) sequence defined by nucleotides 1513-1621 of SEQ ID
NO:2; OX342 (ep) sequence defined by nucleotides 1513-1621 of SEQ ID NO:2
but with deletion of nucleotides 1524-16 10).
Figure 6 displays PCR analysis of EpEp and epep genotypes using primers
derived
from the seed coat peroxidase cDNA. Genomic DNA from soybean lines
OX312 (epep) and OX347 (EpEp) was used as template for PCR analysis with
four different primer sets. Amplification products were separated by
electrophoresis through a 0. 8% agarose gel and visualized under UV light
after
staining with ethidium bromide. Genotype and primer combinations are

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indicated at the top of the figure. The size in base pairs of the amplified
DNA
fragments are indicated on the right.
Figure 7 exhibits PCR analysis of an F2 population from a cross of EpEp and
epep
genotypes. Genomic DNA was used as template for PCR analysis of the
parents (P) and 30 F2 individuals. The cross was derived from the soybean
lines
OX312 (epep) and OX347 (EpEp). Plants were self pollinated and seeds were
collected and scored for seed coat peroxidase activity. The symbols (-) and
(+)
indicate low and high seed coat peroxidase activity, respectively. Primers
prx9+ and prx10- were used in the amplification reactions. Products were
separated by electrophoresis through a 0. 8% agarose gel and visualized under
UV light after staining with ethidium bromide. The migration of molecular
markers and their corresponding size in kb is also shown (lanes M).
Figure 8 displays PCR analysis of six different soybean cultivars with primers
derived
from the seed coat peroxidase cDNA sequence. Genomic DNA was used as
template for PCR analysis of three EpEp cultivars and three epep cultivars.
Primers used in the amplification reactions and the size of the DNA product is
indicated on the left. Products were separated by electrophoresis through a
0. 8% agarose gel and visualized under UV light after staining with ethidium.
bromide.
(A) Forward and reverse primers are downstream from deletion
(B) Forward primer anneals to site within deletion

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(C) Primers span deletion
Figure 9 shows the accumulation of peroxidase RNA in tissues of GEp and epep
plants. Figure 9(A): A comparison of peroxidase transcript abundance
in cultivars Harosoy 63 (Ep) or Marathon (ep). Seed and pod tissues
were sampled at a late stage of development corresponding to a whole
seed fresh weight of 250 mg. Root and leaf tissue was from six week
old plants. Autoradiograph exposed for 96 h. Figure 9(B):
Developmental expression of peroxidase in cultivar Harosoy 63 (Ep).
Flowers were sampled immediately after opening. Seed coat tissues
were sampled at four stages of development corresponding to a whole
seed fresh weight of: lane 1, 50 mg; lane 2, 100 mg; lane 3, 200 mg;
lane 4, 250 mg. Autoradiograph exposed for 20 h.

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DESCRIPTION OF PREFERRED EMBODIIVIENT
The present invention is directed to a novel oligonucleotide sequence encoding
a seed coat peroxidase and associated DNA regulatory regions.
According to the present invention DNA sequences that are "substantially
homologous" includes sequences that are identified under conditions of high
stringency. "High stringency" refers to Southern hybridization conditions
employing
washes at 65 C with 0.1 x SSC, 0.5 % SDS.
By "DNA regulatory region" it is meant any region within a genomic sequence
that has the property of controlling the expression of a DNA sequence that is
operably
linked with the regulatory region. Such regulatory regions may include
promoter or
enhancer regions, and other regulatory elements recognized by one of skill in
the art.
A segment of the DNA regulatory region is exemplified in this invention,
however, as
is understood by one of skill in the art, this region may be used as a probe
to identify
surrounding regions involved in the regulation of adjacent DNA, and such
surrounding
regions are also included within the scope of this invention.
In the context of this disclosure, the term "promoter" or "promoter region"
refers to a sequence of DNA, usually upstream (5) to the coding sequence of a
structural gene, which controls the expression of the coding region by
providing the
recognition for RNA polymerase and/or other factors required for transcription
to start
at the correct site.

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There are generally two types of promoters, inducible and constitutive. An
"inducible promoter" is a promoter that is capable of directly or indirectly
activating
transcription of one or more DNA sequences or genes in response to an inducer.
In
the absence of an inducer the DNA sequences or genes will not be transcribed.
Typically the protein factor, that binds specifically to an inducible promoter
to activate
transcription, is present in an inactive form which is then directly or
indirectly
converted to the active form by the inducer. The inducer can be a chemical
agent such
as a protein, metabolite, growth regulator, herbicide or phenolic compound or
a
physiological stress imposed directly by heat, cold, salt, or toxic elements
or indirectly
through the action of a pathogen or disease agent such as a virus. A plant
cell
containing an inducible promoter may be exposed to an inducer by externally
applying
the inducer to the cell or plant such as by spraying, watering, heating or
similar
methods.
By "constitutive promoter" it is meant a promoter that directs the expression
of a gene throughout the various parts of a plant and continuously throughout
plant
development. Examples of known constitutive promoters include those associated
with
the CaMV 35S transcript and Agrobacterium Ti plasmid nopaline synthase gene.
The chimeric gene constructs of the present invention can further comprise a
3' untranslated region. A 3' untranslated region refers to that portion of a
gene
comprising a DNA segment that contains a polyadenylation signal and any other
regulatory signals capable of effecting mRNA processing or gene expression.
The

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polyadenylation signal is usually characterized by effecting the addition of
polyadenylic
acid tracks to the 3' end of the mRNA precursor. Polyadenylation signals are
commonly recognized by the presence of homology to the canonical form 5'
AATAAA-
3' although variations are not uncommon.
Examples of suitable 3' regions are the 3' transcribed non-translated regions
containing a polyadenylation signal of Agrobacterium tumour inducing (Ti)
plasmid
genes, such as the nopaline synthase (Nos gene) and plant genes such as the
soybean
storage protein genes and the small subunit of the ribulose-1, 5-bisphosphate
carboxylase (ssRUBISCO) gene. The 3' untranslated region from the structural
gene
of the present construct can therefore be used to construct chimeric genes for
expression in plants.
The chimeric gene construct of the present invention can also include further
enhancers, either translation or transcription enhancers, as may be required.
These
enhancer regions are well known to persons skilled in the art, and can include
the ATG
initiation codon and adjacent sequences. The initiation codon must be in phase
with
the reading frame of the coding sequence to ensure translation of the entire
sequence.
The translation control signals and initiation codons can be from a variety of
origins,
both natural and synthetic. Translational initiation regions may be provided
from the
source of the transcriptional initiation region, or from the structural gene.
The
sequence can also be derived from the promoter selected to express the gene,
and can
be specifically modified so as to increase translation of the mRNA.

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To aid in identification of transformed plant cells, the constructs of this
invention may be further manipulated to include plant selectable markers.
Useful
selectable markers include enzymes which provide for resistance to an
antibiotic such
as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes
providing
for production of a compound identifiable by colour change such as GUS
((3-glucuronidase), or luminescence, such as luciferase are useful.
Also considered part of this invention are transgenic plants containing the
chimeric gene construct of the present invention. Methods of regenerating
whole
plants from plant cells are known in the art, and the method of obtaining
transformed
and regenerated plants is not critical to this invention. In general,
transformed plant
cells are cultured in an appropriate medium, which may contain selective
agents such
as antibiotics, where selectable markers are used to facilitate identification
of
transformed plant cells. Once callus forms, shoot formation can be encouraged
by
employing the appropriate plant hormones in accordance with known methods and
the
shoots transferred to rooting medium for regeneration of plants. The plants
may then
be used to establish repetitive generations, either from seeds or using
vegetative
propagation techniques.
The constructs of the present invention can be introduced into plant cells
using
Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation,
micro-
injection, electroporation, etc. For reviews of such techniques see for
example

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Weissbach and Weissbach (1988) and Geierson and Corey (1988). The present
invention further includes a suitable vector comprising the chimeric gene
construct.
Buttery and Buzzell (1968) showed that 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
(Buzzell
and Buttery, 1969). Homozygous recessive epep plants are - 100-fold lower in
seed
coat peroxidase activity. This 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). These cells form a highly differentiated
cell layer with
thick, elongated secondary walls and large intercellular spaces (Baker et al.,
1987).
Screening a seed coat cDNA library prepared from EpEp plants with a
degenerate primer derived from the active site domain of plant peroxidase
resulted in
a high frequency of positive clones. Many of these clones encode identical
cDNA
molecules and indicate that the corresponding mRNA is an abundant transcript
in
developing seed coat tissues. The sequence of the cDNA is shown in Figure 1.
Previous studies on soybean seed coat peroxidase indicated that this enzyme is
heavily glycosylated and that carbohydrate contributes 18% of the mass of the
apo-
enzyme (Gray et al., 1996). The seven potential glycosylation sites identified
from the
amino acid sequence of the seed cost peroxidase (Figure 1) would accommodate
the

CA 02211018 2002-08-28
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five or six N-linked glycosylation sites proposed by Gray et al. (1996). The
heme-
binding domain encompasses residues Asp161 to Phe171 and the acid-base
catalysis
region from G1y33 to Cys44. The two regions are highly conserved among plant
peroxidases and are centred around functional histidine residues, His169 and
His40.
There are eight conserved cysteine residues in the mature protein that provide
for four
. di-sulfide bridges found in other plant peroxidases and predicted from the
crystal
structure of peanut peroxidase (Welinder,. 1992; SchuU.er et al., 1996). Other
conserved areas include residues Cys91 to A1a105 and Va1119 to Leu127 that
occur in,
or around helix D. The most divergent aspects of the seed coat peroxidase
protein
sequence are the carboxy- and amino-terminal regions. These sequences probably
provide special targeting signals for the proper processing and delivery of
the. peptide
chain. It is possible the carboxy-terminal extension of the seed coat
peroxidase is
removed at maturity, as has been shown for certain barley and horseradish
peroxidases
(Welinder, 1992).
The molecular mass of the enzyme has been determined by denaturing gel
electrophoresis to be 37 kDa (Sessa and Anderson, 1981; Gillikin and Graham,
1991)
or 43 kDa (Gijzen et al., 1993). Analysis by mass spectrometry indicated a
mass of
40,622 Da for the apo-enzyme and 33,250 Da after deglycosylation (Gray et al.,
1996). These values are in good agreement with the mass of 35,377 Da
calculated from
the predicted amino acid sequence for the mature apo-protein prior to
glycosylation and
other modifications. Huangpu et al (GENEMBL, Accession number: U41657; 1995)
reported an anionic seed coat peroxidase having a Mr of 30,577 Da and
characterized a
partial eDNA encoding this protein.

CA 02211018 2002-08-28
-19-
This 1031 bp cDNA contained an open reading frame of 849 bp encoding a 283
amino
acid protein. There are several differences between this reported sequence and
the
sequence of this invention that are manifest at the amino acid level (see
Figure 3 for
sequence comparison). The enzyme encoded by the gene reported by Huangpu et al
is different from that of this invention as the peroxidase of this invention
has a M, of
35,377 Da.
Genomic DNA blots probed with the seed coat peroxidase cDNA produced two
or three hybridizing fragments of varying intensity with, most restriction
enzyme
digestions, despite that several peroxidase isozymes are present in soybean.
The results
indicate that this seed coat peroxidase is present as a single gene that does
not share
sufficient homology with most other peroxidase genes to anneal under
conditions of
high stringency.
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 3770-4032
(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 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.

CA 02211018 1997-09-19
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This =
promoter is considered seed coat specific since the peroxidase protein
encoded by the Ep gene accumulates in the seed coat tissues, especially in the
hourglass
cells of the subepidermis, and is not expressed in other tissues, aside from a
marginal
expression of peroxidase in the root tissues. This is also true at the
transcriptional
level (see Figure 9). The DNA regulatory regions of the genomic sequence of
Figure
2 are used to control the expression of the adjacent peroxidase gene in seed
coat tissue.
Such regulatory regions include nucleotides 1-1532. Other regions of interest
include
nucleotides 1752-2382, 2575-3604 and/or 3770-4032 of SEQ ID NO:2. Therefore
other 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
regulatory
elements 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 the
production
of proteins that alter the taste of the seed and/or that affect the nutritive
value of the
soybean.
A modified DNA regulatory sequence may be obtained by introducing changes
into the natural sequence. Such modifications can be done through techniques
known
to one of skill in the art such as site-directed mutagenesis, reducing the
length of the
regulatory region using endonucleases or exonucleases, increasing the length
through
the insertion of linkers or other sequences of interest. Reducing the size of
DNA
regulatory region may be achieved by removing 3' or 5' regions of the
regulatory

CA 02211018 1997-09-19
-21-
region of the natural sequence by using a endonuclease such as BAL 31
(Sambrook et
al 1989). However, any such DNA regulatory region must still fnnction as a
seed coat
specific DNA regulatory region.
It may be readily determined if such modified DNA regulatory elements are
capable of acting in a seed coat specific manner transforming plant cells with
such
regulatory elements controlling the expression of a suitable marker gene,
culturing
these plants and determining the expression of the marker gene within the seed
coat as
outlined above. One may also analyze the efficacy of DNA regulatory elements
by
introducing constructs comprising a DNA regulatory element of interest
operably
linked with an appropriate marker into seed coat tissues by using particle
bombardment
directed to seed coat tissue and determining the degree of expression of the
regulatory
region as is known to one of skill in the art.
Two tandemly arranged genes encoding anionic peroxidase expressed in stems
of Populus kitakamiensis, prxA3a and prxA4a have been cloned and characterized
(Osakabe et al, 1995). Both of these genomic sequences contained four exons
and
three introns and encoded proteins of 347 and 343 amino acids, respectively.
The two
genes encode distinct isozymes with deduced Mrs of 33.9 and 34.6 kDa.
Furthermore, a 532 bp promoter derived from the peroxidase gene of Armoracia
rusticana has also been reported (Toyobo KK, JP 4,126,088, April 27, 1992).
However, a search using GenBank revealed no substantial similarity between the
promoter region, or introns 1, 2 and 3 of this invention and those within the
literature.

CA 02211018 1997-09-19
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Digestion of the genomic DNA with BamHI or SacI revealed restriction
fragment length polymorphisms that distinguished EpEp and epep genotypes.
Although
the XbaI digestion did not produce a readily detectable polymorphism, the size
of the
hybridizing fragment in both genotypes was - 14 kb. Thus, a 0.3 kb size
difference is
outside of the resolving power of the separation for fragments this large.
Sequence
analysis of EpEp and epep genotypes indicates that the mutant ep allele is
missing 87
bp of sequence at the 5' end of the structural gene. This would account for
the
drastically reduced amounts of peroxidase enzyme present in seed coats of epep
plants
since the deletion includes the translation start codon and the entire N-
terminal signal
sequence. However, the 87 bp deletion cannot account for the differences
observed in
the RFLP analysis since the missing fragment does not include a BamHI site and
is
much smaller than the 0.3 kb polymorphism detected in the SacI digestion.
Thus,
other genetic rearrangements must occur in the vicinity of the ep locus that
lead to
these polymorphisms.
The results shown here indicate that the mutation causing low seed coat
peroxidase activity occurs in the structural gene encoding the enzyme. This
mutation
is an 87 bp deletion in the 5' region of the gene encompassing the translation
start site.
Several different low peroxidase cultivars share a similar mutation in the
same area,
suggesting that the recessive ep alleles have a common origin or that the
region is
prone to spontaneous deletions or rearrangements.

CA 02211018 1997-09-19
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Due to the industrial interest in soybean seed coat peroxidase, alternate
sources
for the production of this enzyme are needed. The DNA of this invention,
encoding
the seed coat soybean peroxidase under the control of a suitable promoter and
expressed within a host of interest, can be used for the preparation of
recombinant
soybean seed coat peroxidase enzyme.
Soybean seed coat peroxidase has been characterized as a lignin-type
peroxidase
that has industrially significant properties ie: high activity and stability
under acidic
conditions; exhibits wide substrate specificity; equivalent catalytic
properties to that
of Phanerochaete chrysosporium ligin peroxidase (the currently preferred
enzyme used
for treatment of industrial waste waters (Wick 1995) but is at least 150-fold
more
stable; more stable than horseradish peroxidase which is also used in
industrial effluent
treatments and medical diagnostic kits (McEldoon et al., 1995). These
properties are
useful within industrial applications for the degradation of natural aromatic
polymers
including lignin and coal (McEldoon et al, 1995), and the preferred use of
soybean
peroxidase, over that of horseradish peroxidase, in medical diagnostic tests
as an
enzyme label for antigens, antibodies, oligonucleotide probes, and within
staining
techniques (Wick 1995). Soybean peroxidase is also used in the deinking of
printed
waste paper (Johnson et al., U.S. 5,270,770; December 6, 1994) and for the
biocatalytic oxidation of primary alcohols (Johnson et al., U.S. 5,391,488;
February
13, 1996). Soybean peroxidase has also been used as a replacement for chlorine
in
the pulp and paper industry, in order to remove chlorine, phenolic or aromatic
amine
containing pollutants from industrial waste waters (Wick 1995), or as
formaldehyde

CA 02211018 1997-09-19
-24-
replacement (Freiberg, 1995) for use in adhesives, abrasives, and protective
coatings
(e.g. varnish and resins, Wick 1995).
Furthermore, the seed coat peroxidase gene may be expressed in an organ or
tissue specific manner within a plant. For example, the quality and strength
of cotton
fibber can be improved through the over-expression of cotton or horseradish
peroxidase
placed under the control of a fibre-specific promoter (Maliyakal, WO 95/08914;
April
6, 1995).
Similarly, seed-specific DNA regulatory regions of this invention may be used
to control expression of genes of interest such as:
i) genes encoding herbicide resistance, or
ii) biological control of insects or pathogens (e.g. B. thuringiensis), or
iii) viral coat proteins to protect against viral infections, or
iv) proteins of commercial interest (e.g. pharmaceutical), and
v) proteins that alter the nutritive value, taste, or processing of seeds
within the seed coat of plants.
While this invention is described in detail with particular reference to
preferred
embodiments thereof, said embodiments are offered to illustrate but not to
limit the
invention.

CA 02211018 2002-08-28
- 25 -
EXAMPLES
Plant material
All soybean (Glycine max [L.] Merr) cultivars and breeding lines were from the
collection at Agriculture Canada, Harrow, Ontario.
Seed Coat cDNA library Construction and Screening
High seed coat peroxidase (EpEp) soybean cultivar Harosoy 63 plants were
grown in field plots outdoors. Pods were harvested 35 days after flowering and
seeds
in the mid-to-late developmental stage were excised. The average fresh mass
was 250
mg per seed. Seed coats were dissected and immediately frozen in liquid
nitrogen. The
frozen tissue was lyophilized and total RNA extracted in 100 mM Tris-HCl pH
9.0,
mM EDTA, 4%(w/v) sarkosyl, 200 mM NaCI, and 16 mM DTT, and precipitated
15 with LiCI using the standard phenol/chloroform method described by Wang and
Vodkin (1994). The poly(A)' RNA was purified on oligo(dT) cellulose columns
prior
to cDNA synthesis, size selection, ligation into the IZAPTMExpress vector, and
packaging according to instructions (Stratagene). A degenerate oligonucleotide
with the
5' to 3' sequence of TT(C/T)CA(C/T)GA(C/T)TG(C/T)TT(C/T)GT (SEQ ID NO:3) was
20 5' end labelled to high specific activity and used as a probe to isolate
peroxidase cDNA
clones (Sambrook et aL, 1989). Duplicate plaque lifts were made to nylon
filters
(Amersham), UV fixed, and prehybridized at 36 C for 3 h in 6 x SSC, 20 mM
NaZHP04
(pH6.8),

CA 02211018 2002-08-28
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x Denhardt's, 0.4 % SDS, and 500 jcg/mL salmon sperm DNA. Hybridization was
in the same buffer, without Denhardt's, at 36 C for 16 h. Filters were washed
quickly
with several changes of 6 x SSC and 0.1 % SDS, first at room temperature and
finally
at 40 C, prior to autoradiography for 16 h at 70 C with an intensifying
screen.
5 Genomic DNA Isolation, Library Construction, and DNA Blot Analysis
Soybean genomic DNA was isolated from leaves of greenhouse grown plants
or from eti.olated seedlings grown in vermiculite. Plant tissue was frozen in
liquid
nitrogen and lyophilized before extraction and purification of DNA according
to the
method of Dellaporta et al. (1983). Restriction enzyme digestion of 30 g DNA,
separation on 0.5 % agarose gels and blotting to nylon membranes followed
standard
protocols (Sambrook et al., 1989). For construction of the genomic library,
DNA
purified from Harosoy 631eaf tissue was partially digested with BamHI and
ligated into
the ), FIXM II vector (Stratagene). GigapackTM XL packaging extract
(Stratagene) was used
to select for inserts of 9 to 22 kb. After library amplification, duplicate
plaque lifts
were hybridized to cDNA probe.
Blots or filter lifts were prehybridized for 2 h at 65 C in 6 x SSC, 5 x
Denhardt's, 0.5 % SDS, and 100 ug/mL salmon sperm DNA. Radiolabelled cDNA
probe (20 to 50 ng) was prepared using the Ready-to-GoTM labelling kit
(Pharmacia) and
32P-dCTP (Amersham). Unincorporated2 P-dCTP was removed by spin column
chromatography before adding radiolabelled cDNA to the hybridization buffer

CA 02211018 2002-08-28
-27-
(identical to prehybridization buffer without Denhardt's). Hybridization was
for 20 h
at 65 C. Membranes were washed twice for 15 min at room temperature with 2 x
SSC,
0.5 % SDS, followed by two 30 min washes at 65 C with 0.1 x SSC, 0.5 % SDS.
Autoradiography was for 20 h at -70 C using an intensifying screen and X-
OMATTM film
(Kodak).
DNA Sequencing
Sequencing of DNA was performed using dye-labelled terminators and Taq-FSTM
DNA polymerase (Perkin Elmer). The PCR protocol consisted of 25 cycles of a 30
sec
melt at 96 C, 15 sec annealing at 50 C, and 4 min extension at 60 C. Samples
were
analyzed on an Applied Biosystems 373A StretchTM automated DNA sequencer.
Polymerase Chain Reaction
PCR amplifications contained 1 ng template DNA, 5 pmol each primer, 1.5
mM MgC12, 0.15 mM deoxynucleotide triphosphates mix, 10 mM Tris-HCI; 50 mM
KC1, pH 8.3, and 1 unit of TaqTM polymerase (Gibco BRL) in a total volume of
25 L.
Reactions were performed in a Perlcin Elmer 480 thermal cycler. After an
initial 2 min
denaturation at 94 C, there were 35 cycles of 1 min denaturation at 94 C, .1
min
annealing at 52 C, and 2 min extension at 72 C.' A final 7 min extension at 72
C
completed the program. The following primers were used for PCR analysis of
genomic
DNA:

CA 02211018 2002-08-28
-28-
prx2+ CTTCCAAATATCAACTCAAT (SEQ ID NO:4)
prx6- TAAAGTTGGA.AAAGAAAGTA (SEQ ID NO:5)
prx9 ATGCATGCAGGTTTTTCAGT (SEQ ID NO:6)
prxlO- TTGCTCGCTTTCTATTGTAT (SEQ ID NO:7)
prx12+ TCTTCGATGCTTCTTTCACC (SEQ ID NO:8)
prx29+ CATAAACAATACGTACGTGAT (SEQ ID NO:9)
RNA Isolation
For isolation of RNA, tissue was harvested from greenhouse grown plants,
dissected, frozen in liquid nitrogen, and lyophilized prior to extraction.
Total RNA was
purified from seed coats, embryos, pods, leaves, and flowers using standard
phenol/chloroform method (Sambrook et al., 1989). This method did not afford
good
yields of RNA from roots, therefore this tissue was extracted with TriazoleT"i
reagent
(GibcoBRL) and total RNA purified according to manufacturers' instructions
with an
additional phenol-chloroform extraction step. The amount of RNA was estimated
by
measuring absorbance at 260 and 280 nm, and by electrophoretic separation in
formaldehyde gels followed by staining with ethidium bromide and comparison to
known standards. Total RNA (10 /.cg per sample) was prepared, subject to
electrophoresis through a 1 % agarose gel containing formaldehyde, and then
stained
with ethidium bromide to ensure equal loading of samples. The gel was blotted
to
nylon (Hybond''Tt, Amersham) according to standard methods and the RNA was
fixed
to the membrane by UV cross linking.

CA 02211018 1997-09-19
- 29 -
Seed Coat Peroxidase Assays
The F3 seed was measured for peroxidase activity to score the phenotype of the
F2 population because the seed testa is derived from maternal tissue. The
seeds were
briefly soaked in water and the seed coat was dissected from the embryo and
placed in
a vial. Ten drops (-500 ,uL) of 0.5% guaiacol was added and the sample was
left to
stand for 10 min before adding one drop (-50 ,uL) of 0.1 % H202. An immediate
change in colour of the solution, from clear to red, indicates a positive
result and high
seed coat peroxidase activity.
Example 1: The Seed Coat Peroxidase cDNA and genomic DNA sequences
To isolate the seed coat peroxidase transcript, a cDNA library was constructed
from developing seed coat tissue of the EpEp cultivar Harosoy 63. The primary
library contained 106 recombinant plaque forming units and was amplified prior
to
screening. A degenerate 17-mer oligonucleotide corresponding to the conserved
active
site domain of plant peroxidases was used to probe the library. In screening
10,000
plaque forming units, 12 positive clones were identified. The cDNA insert size
of the
clones ranged from 0.5 to 2.5 kb, but six clones shared a common insert size
of 1.3
kb. These six clones (soyprxO3, soyprx05, soyprxO6, soyprxll, soyprxl2, and
soyprxl4) were chosen for further characterization since the 1.3 kb insert
size matched
the expected peroxidase transcript size. Sequence analysis of the six clones
showed that
they contained identical'cDNA transcripts encoding a peroxidase and that each
resulted

CA 02211018 2002-08-28
-30-
from an independent cloning event since the junction between the cloning
vector and
the transcript was different in all cases.
Since it was not clear that the entire 5' end of the cDNA transcript was
complete in any of the cDNA clones isolated, the structural gene corresponding
to the
seed coat peroxidase was isolated from a Harosoy 63 genomic library . A
partial BamHI
digest of genomic DNA was used to construct the library and more than 106
plaque
forming units were screened using the cDNA probe. A positive clone, G25-2-1-1-
1,
containing a 17 kb insert was identified and a 4.7 kb region encoding the
peroxidase
was sequenced SEQ ID NO:2. This region includes 1532 nucleotides of the 5'
region
of the peroxidase gene.
The genomic sequence matched the cDNA sequence except for three introns
encoded within the gene. The genomic sequence also revealed two additional
translation start codons, beginning one bp and 10 bp upstream from the 5' end
of the
longest cDNA transcript isolated. Figure 1(SEQ ID NO:1) shows the deduced cDNA
sequence. The open reading frame of 1056 bp encodes a 352 amino acid protein
of
38,106 Da. A heme-binding domain, a peroxidase active site signature sequence,
and
seven potential N-glycosylation sites were identified from the deduced amino
acid
sequence. The first 26 amino acid residues conform to a membrane spanning
domain.
Cleavage of this putative signal sequence releases a mature protein of 326
residues with
a mass of 35,377 Da and an estimated pI of 4.4.

CA 02211018 2002-08-28
-31-
Relevant features of the genomic fragment (Figure 2) include four exons at bp
192-411 (exon 1; 1533-1751 of SEQ ID NO:2), 1042 -1233 (exon 2; 2383-2574 of
SEQ ID NO:2), 2263-2429 (exon 3; 4033-4516 fo SEQ ID NO:2) and 2692-3174
(exon 4; 1752-2382 of SEQ ID NO:2) and three introns at bp 412-1041 (intron 1;
1752-2382 of SEQ ID NO:2), 1234-2263 (intron 2; 2575-3604 of SEQ ID NO:2) and
2430-2691 (intron 3; 3770-4032 of SEQ ID NO:2). The 1532 bp regulatory region
of
the genomic DNA include a TATA box centred on bp 1487 and a cap signal 32 bp
down stream centred at bp 1520 of SEQ ID NO:2. Also noted within the genomic
sequence are three polyadenylation signals centred on bp 4520, 4598, 4700 and
a
polyadenylation site at bp 4700 of SEQ ID NO:2.
Figure 3 (SEQ ID Nos:10-19) illustrates the relationship between the soybean
seed coat peroxidase and other selected plant peroxidases. Th-6 soybean
sequence is most
closely related to four peroxidase cDNAs isolated from aYfalfa, (see Figure 3)
sharing
from 65 to 67% identity at the amino acid level with the alfalfa proteins
(X90693,
X90694, X90692, el-Turk et al 1996; L36156, Abrahams et al 1994). When
compared
with other plant peroxidases, soybean seed coat peroxidase exhibits from 60 to
65%
identity with poplar (D30653 and D30652, Osakabe et al 1994)) and flax (L0554,
Omann
and Tyson 1995); 50 to 60% identity with horseradish (M37156, Fujiyama et al.
1988),
tobacco (D 11396, Osakabe et al 1993), and cucumber (M91373, Rasmussen et
a1.1992);
and 49% identity with barely (L36093, Scott-Craig et al. 1994), wheat (X85228,
Baga
et al 1995) and tobacco (L02124, Diaz-De-Leon et al 1993) peroxidases.

CA 02211018 1997-09-19
-32-
A comparison of the promoter region, 1-1532 of SEQ ID NO:2, indicates that
there are no similar sequences present within the GENBANK database.
Example 2: DNA Blot Analysis Using the Seed Coat Peroxidase cDNA Probe
Reveals Restriction Fragment Length Polymorphisms Between EpEp and epep
Genotypes
Genomic DNA blots of OX347 (EpEp) and OX312 (epep) plants were
hybridized with 32P-labelled cDNA to estimate the copy number of the seed coat
peroxidase gene and to determine if this locus is polymorphic between the two
genotypes. Figure 4 shows the hybridization patterns after digestion with
BamHI, XbaI,
and Sacl. Restriction fragment length polymorphisms are clearly visible in the
BamHI
and SacI digestions. The BamHI digestion produced a strongly hybridizing 17 kb
fragment and a faint 3.4 kb fragment in the EpEp genotype. The 3.4 kb BamHI
fragment is visible in the epep genotype but the 17 kb fragment has been
replaced by
a signal at > 20 kb. The SacI digestion resulted in detection of three
fragments in EpEp
and epep plants. At least two fragments were expected here since the cDNA
sequence
has a SacI site within the open reading frame. However, the smallest and most
strongly
hybridizing of these fragments is 5.2 kb in EpEp plants and 4.9 kb in epep
plants.
Digestion with Xbal produced hybridizing fragments of - 14 kb and 7.8 kb for
both
genotypes, with the larger fragment showing a stronger signal.

CA 02211018 1997-09-19
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Example 3: A Deletion Mutation Occurs in the Recessive ep Locus
The structural gene encoding the seed coat peroxidase is schematically
illustrated in Figure 5. The 17 kb BamHI fragment encompassing the gene
includes 191
bp of sequence upstream from the translation start codon, three introns of 631
bp, 1030
bp, and 263 bp, and 13 kb of sequence downstream from the polyadenylation
site. The
arrangement of four exons and three introns and the placement of introns
within the
sequence is similar to that described for other plant peroxidases (Simon,
1992; Osakabe
et al. 1995).
Primers were designed from the DNA sequence to compare EpEp and epep
genotypes by PCR analysis. Figure 6 shows PCR amplification products from four
different primer combinations using OX312 (epep) and OX347 (EpEp) genomic DNA
as template. The primer annealing site for prx29 + begins 182 bp upstream from
the
ATG start codon; the remaining primer sites are shown in Figure 1.
Amplification with
primers prx2+ and prx6-, and with prx12+ and prxlO- produced the expected
products of 1.9 kb and 860 bp, respectively, regardless of the Eplep genotype
of the
template DNA. However, PCR amplification with primers prx9 + and prx10-, and
with
prx29+ and prxlO- generated the expected products only when template DNA was
from plants carrying the dominant Ep allele. When template DNA was from an
epep
genotype, no product was detected using primers prx9+ and prx10- and a smaller
product was amplified with primers prx29 + and prx10-. The products resulting
from
amplification of OX312 or OX347 template DNA with primers prx29+ and prxlO-

CA 02211018 2002-08-28
-34-
were directly sequenced and compared. The polymorphism is due to an 87 bp
deletion
occurring within this DNA fragment in OX312 plants, as shown in Figure 5
(deletion of
nucleotides 1524-1610 of SEQ ID NO:2). This deletion begins nine bp upstream
from
the translation start codon and includes 78 bp of sequence at the 5' end of
the open
reading frame, including the prx9 + primer annealing site.
To test whether this deletion mutation cosegregates with the seed coat
peroxidase phenotype, genomic DNA from an F2 population segregating at the Ep
locus
was amplified using primers prx9 + and prx10- and F3 seed was tested for seed
coat
peroxidase activity. Figure 7 shows the results from this analysis. Of the 30
F2
individuals tested, a1123 that were high in seed coat peroxidase activity
produced the
expected 860 bp PCR amplification product. The remaining seven FZ's with low
seed
coat peroxidase activity produced no detectable PCR amplification products.
Finally, to determin.e if the OX312(epep) and OX347(EpEp) breeding lines are
representative of soybean cultivars that differ in seed coat peroxidase
activity, several
cultivars were tested by PCR analysis using primer combinations targeted to
the Ep
locus. Figure 8 shows results from this analysis of six different soybean
cultivars, thiree
each of the homozygous dominant EpEp and recessive epep genotypes. As observed
with OX3 IZ and OX347, amplification products of the expected size were
produced
with primers prxl2+ and prxlO- regardless of the genotype, whereas epep
genotypes
yielded no product with primers prx9 and prxlO- or a smaller fragment with
primers
prx29+ and prx10-.

CA 02211018 2002-08-28
-35-
Example 4 Developmental Pattern of Expression of the tp gene
The seed coat peroxidase mRNA levels were determined by hybridizing RNA. gel
blots
with radio labelled cDNA probe. Figure 9 illustrates the transcript abundance
in
various tissues of epep and EpEp plants. The mRNA accumulated to high levels
in seed
coat tissues of EpEp plants, especially in the later stages development when
whole seed
fresh weight exceeded 50 mg. Low levels of transcript could also be. detected
in root
tissues but not in the flower, embryo, pod or leaf. The transcript could also
be detected
in seed coat and root tissues epep plants but in drastically reduced amounts
compared
to the EpEp genotype. The reduced amounts of peroxidase mRNA present in seed
coats
of epep plants , indicates that the transcriptional process and/or the
stability of the
resulting naRNA is severely affected. The Ep gene has a TATA box and a 5' cap
signal
beginning 47 bp and 15 bp, respectively, upstream from the translation start
codon.
The 87 bp deletion in the ep allele extends into the 5' cap signal and
therefore could
interfere with transcript processing. Regardless, any resulting transcript
will not be
properly translated since the AUG initiation codon and the entire amino-
terminal
signal sequence is deleted from the ep allele. Not wishing to be bound by
theory, the
lack of peroxidase accumulation in seed coats of epep plants appears to be due
to at
least two factors, greatly reduced transcript levels and 'meffective
translation, resulting
from mutation of the structural gene encoding the'enzyme. In summary, the
results
indicate that the Ep gene regulatory elements can drive high level expression
in a
tightly coordinated, tissue and developmentally specific manner.

CA 02211018 2002-08-28
-36-
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 from the scope
of the
invention as described in the following claims.

CA 02211018 1997-09-19
-37-
References
Abrahams, S.L., Hayes, C.M., and Watson, J.M. (1994) Organ-specific expression
of three peroxidase-encoding cDNAs from lucerne (Medicago sativa).
GenBank Accession # L36156.
Baga, M. Chibbar, R.N., and Kartha, K.K., (1995) Molecular cloning and
expression analysis of peroxidase genes from wheat. Plant Molec. Biol. 29,
647-662
Baker, D.M., Minor, H.C., and Cumbie, B.G. (1987) Scanning electron microscopy
examination of soybean testa development. Can. J. Bot. 65, 2420-2424.
Bowles, D.J. (1990) Defense-related proteins in higher plants. Annu. Rev.
Biochem.
59, 873-907.
Buttery, B.R., and Buzzell, R.I. (1968) Peroxidase activity in the seeds of
soybean
varieties. Crop Sci. 8, 722-725.
Buzzell, R.I., and Buttery, B.R. (1969) Inheritance of peroxidase activity in
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Campa, A. (1991) Biological roles of plant peroxidases: known and potential
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In Peroxidases in Chemistry and Biology, Volume II (J. Everse, K.E. Everse
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Diaz-De-Leon, f., Klotz, K.L., and Lagrimini, M. (1993) Nucleotide Sequence of
the Tobacco (Nicotiana tabacum) anionic peroxidase gene. Plant Physiol. 101,
1117-1118.

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el-Turk, J., Asemota, 0., Leymarie, J., Sallaud, C., Mesnage, S., Breda, C.,
Buffard, D., Kondorosi, A., and Esnault, R. (1996) Nucleotide sequence of
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Seed Crops Indust. March, 4-9
Fujiyama, K., Takemura, H., Shibayama, S., Kobayashi, K., Choi, J.-K.,
Shinmyo, A., Takano, M., Yamada, Y,. and Okada, H. (1988) Structure fo
the Horseradish Peroxidase isozyme c genes. Eur. J. Biochem. 173, 681-687.
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Gillikin, J.W., and Graham, J.S. (1991) Purification and developmental
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CA 02211018 1997-09-19
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CA 02211018 2002-08-28
-41-
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Her Majesty in Right of Canada as represented by the
Minister of Agriculture and Agri-Food Canada
(B) STREET: Central Experimental Farm
(C) CITY: Ottawa
(D) PROVINCE: Ontario
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): K1A 0C6
(ii) TITLE OF INVENTION: Seed Coat DNA Regulatory Region And Peroxidase
(iii) NUMBER OF SEQUENCES: 19
(iv) CORRESPONDENCE ADDRESS
(A) NAME: GOWLING LAFLEUR HENDERSON LLP
(B) STREET: 160 ELGIN STREET, SUITE 2600
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 1C3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,211,018
(B) FILING DATE: 19-SEP-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,186,833
(B) FILING DATE: 30-SEP-1996
(A) APPLICATION NUMBER: US 08/723,414
(B) FILING DATE: 30-SEP-1996
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME: GOWLING LAFLEUR HENDERSON LLP
(B) REFERENCE NUMBER: 08-873726CA1
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1244 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear

CA 02211018 2002-08-28
-42-
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Glycine max
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..1056
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION:1..78
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
atg ggt tcc atg cgt cta tta gta gtg gca ttg ttg tgt gca ttt gct 48
Met Gly Ser Met Arg Leu Leu Val Val Ala Leu Leu Cys Ala Phe Ala
1 5 10 15
atg cat gca ggt ttt tca gtc tct tat gct cag ctt act cct acg ttc 96
Met His Ala Gly Phe Ser Val Ser Tyr Ala Gln Leu Thr Pro Thr Phe
20 25 30
tac aga gaa aca tgt cca aat ctg ttc cct att gtg ttt gga gta atc 144
Tyr Arg Glu Thr Cys Pro Asn Leu Phe Pro Ile Val Phe Gly Val Ile
35 40 45
ttc gat gct tct ttc acc gat ccc cga atc ggg gcc agt ctc atg agg 192
Phe Asp Ala Ser Phe Thr Asp Pro Arg Ile Gly Ala Ser Leu Met Arg
50 55 60
ctt cat ttt cat gat tgc ttt gtt caa ggt tgt gat gga tca gtt ttg 240
Leu His Phe His Asp Cys Phe Val G1n Gly Cys Asp Gly Ser Val Leu
65 70 75 80
ctg aac aac act gat aca ata gaa agc gag caa gat gca ctt cca aat 288
Leu Asn Asn Thr Asp Thr Ile Glu Ser Glu Gln Asp Ala Leu Pro Asn
85 90 95
atc aac tca ata aga gga ttg gac gtt gtc aat gac atc aag aca gcg 336
Ile Asn Ser Ile Arg Gly Leu Asp Val Val Asn Asp Ile Lys Thr Ala
100 105 110
gtg gaa aat agt tgt cca gac aca gtt tct tgt gct gat att ctt gct 384
Val Glu Asn Ser Cys Pro Asp Thr Val Ser Cys Ala Asp Ile Leu Ala
115 120 125
att gca gct gaa ata gct tct gtt ctg gga gga ggt cca gga tgg cca 432
Ile Ala Ala Glu Ile Ala Ser Val Leu Gly Gly Gly Pro Gly Trp Pro
130 135 140
gtt cca tta gga aga agg gac agc tta aca gca aac cga acc ctt gca 480
Val Pro Leu Gly Arg Arg Asp Ser Leu Thr Ala Asn Arg Thr Leu Ala
145 150 155 160

CA 02211018 2002-08-28
-43-
aat caa aac ctt cca gca cct ttc ttc aac ctc act caa ctt aaa gct 528
Asn Gln Asn Leu Pro Ala Pro Phe Phe Asn Leu Thr Gln Leu Lys Ala
165 170 175
tcc ttt gct gtt caa ggt ctc aac acc ctt gat tta gtt aca ctc tca 576
Ser Phe Ala Val Gln Gly Leu Asn Thr Leu Asp Leu Val Thr Leu Ser
180 185 190
ggt ggt cat acg ttt gga aga gct cgg tgc agt aca ttc ata aac cga 624
Gly Gly His Thr Phe Gly Arg Ala Arg Cys Ser Thr Phe Ile Asn Arg
195 200 205
tta tac aac ttc agc aac act gga aac cct gat cca act ctg aac aca 672
Leu Tyr Asn Phe Ser Asn Thr Gly Asn Pro Asp Pro Thr Leu Asn Thr
210 215 220
aca tac tta gaa gta ttg cgt gca aga tgc ccc cag aat gca act ggg 720
Thr Tyr Leu Glu Val Leu Arg Ala Arg Cys Pro Gln Asn Ala Thr Gly
225 230 235 240
gat aac ctc acc aat ttg gac ctg agc aca cct gat caa ttt gac aac 768
Asp Asn Leu Thr Asn Leu Asp Leu Ser Thr Pro Asp Gln Phe Asp Asn
245 250 255
aga tac tac tcc aat ctt ctg cag ctc aat ggc tta ctt cag agt gac 816
Arg Tyr Tyr Ser Asn Leu Leu Gln Leu Asn Gly Leu Leu Gln Ser Asp
260 265 270
caa gaa ctt ttc tcc act cct ggt gct gat acc att ccc att gtc aat 864
Gln Glu Leu Phe Ser Thr Pro Gly Ala Asp Thr Ile Pro Ile Val Asn
275 280 285
agc ttc agc agt aac cag aat act ttc ttt tcc aac ttt aga gtt tca 912
Ser Phe Ser Ser Asn Gln Asn Thr Phe Phe Ser Asn Phe Arg Val Ser
290 295 300
atg ata aaa atg ggt aat att gga gtg ctg act ggg gat gaa gga gaa 960
Met Ile Lys Met Gly Asn Ile Gly Val Leu Thr Gly Asp Glu Gly Glu
305 310 315 320
att cgc ttg caa tgt aat ttt gtg aat gga gac tcg ttt gga tta gct 1008
Ile Arg Leu Gln Cys Asn Phe Val Asn Gly Asp Ser Phe Gly Leu Ala
325 330 335
agt gtg gcg tcc aaa gat gct aaa caa aag ctt gtt gct caa tct aaa 1056
Ser Val Ala Ser Lys Asp Ala Lys Gln Lys Leu Val Ala Gln Ser Lys
340 345 350
taaaccaata attaatgggg atgtgcatgc tagctagcat gtaaaggcaa attaggttgt 1116
aaacctcttt gctagctata ttgaaataaa ccaaaggagt agtgtgcatg tcaattcgat 1176
tttgccatgt acctcttgga atattatgta ataattattt gaatctcttt aaggtactta 1236
attaatca 1244

CA 02211018 2002-08-28
-44-
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4700 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Glycine max
(ix) FEATURE:
(A) NAME/KEY: promoter
(B) LOCATION:1..1532
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION:1533..1610
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION:1533..1751
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION:2383..2574
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION:3605..3769
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION:4033..4515
(ix) FEATURE:
(A) NAME/KEY: Intron
(B) LOCATION:1752..2382
(ix) FEATURE:
(A) NAME/KEY: Intron
(B) LOCATION:2575..3604
(ix) FEATURE:
(A) NAME/KEY: Intron
(B) LOCATION:3770..4032
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1533..1751
(ix) FEATURE:
(A) NAME/KEY: CDS

CA 02211018 2002-08-28
-45-
(B) LOCATION:2383..2574
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:3605..3769
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:4033..4512
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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

CA 02211018 2002-08-28
-46-
gtcgtcgtca taggcaattg ggcatcatat cataaacaat acgtacgtga tattatctag 1380
tgtctctcag tttactttat gagaaattat ttttctttaa aaaaagttaa ttaataaaaa 1440
catttgcgat accgtgagtt acaagaaatc cgccgaattc atctctataa ataaaaggat 1500
ctatatgaga ggtaaaatca tattaactca aa atg ggt tcc atg cgt cta tta 1553
Met Gly Ser Met Arg Leu Leu
1 5
gta gtg gca ttg ttg tgt gca ttt gct atg cat gca ggt ttt tca gtc 1601
Val Val Ala Leu Leu Cys Ala Phe Ala Met His Ala Gly Phe Ser Val
15 20
tct tat gct cag ctt act cct acg ttc tac aga gaa aca tgt cca aat 1649
Ser Tyr Ala Gln Leu Thr Pro Thr Phe Tyr Arg Glu Thr Cys Pro Asn
25 30 35
ctg ttc cct att gtg ttt gga gta atc ttc gat gct tct ttc acc gat 1697
Leu Phe Pro Ile Val Phe Gly Val Ile Phe Asp Ala Ser Phe Thr Asp
40 45 50 55
ccc cga atc ggg gcc agt ctc atg agg ctt cat ttt cat gat tgc ttt 1745
Pro Arg Ile Gly Ala Ser Leu Met Arg Leu His Phe His Asp Cys Phe
60 65 70
gtt caa gtacgtactt ttttttttcc ttccaaaatg ccctgcatat ttaacaagat 1801
Val Gln
tgctttgttc acctagaaaa atgtgttttt ttcaacgatc ttacgtacgt ttgtttggtt 1861
tgaaaaataa atcagaaaga gatcaagaaa atagctagaa agaaagcaac gtttttttaa 1921
aaggtattta gtgtgagaaa aatattaaaa ctgaagagaa agaaattaaa taagcttttc 1981
ttgaatgata tttacatgtc ttattaactt aaagtcacct tttttcttta agttgtgctt 2041
gaagaaaaaa gatgtctttc agtttagttt tgattaatgc taattatatt tttaattaat 2101
taattaatac tatatatcta tttaccatat taattattac tatatttcat gatgacaaca 2161
gacaagtatt ctaaagaggt atcggtagat gattaatttt tttataaaaa aatcttttgc 2221
gtgtatagat attcttttat aattggtgca gaaacttgta atgctaattg caattaatct 2281
tacattgatt aactaatagc tataatcaat atttaggtta ggtataggag acaaatcaag 2341
tgatctgaac aaattaagtt gttatatttg cattgtgaca g ggt tgt gat gga tca 2397
Gly Cys Asp Gly Ser
gtt ttg ctg aac aac act gat aca ata gaa agc gag caa gat gca ctt 2445
Val Leu Leu Asn Asn Thr Asp Thr Ile Glu Ser Glu Gln Asp Ala Leu
85 90

CA 02211018 2002-08-28
-47-
cca aat atc aac tca ata aga gga ttg gac gtt gtc aat gac atc aag 2493
Pro Asn Ile Asn Ser Ile Arg Gly Leu Asp Val Val Asn Asp Ile Lys
95 100 105 110
aca gcg gtg gaa aat agt tgt cca gac aca gtt tct tgt gct gat att 2541
Thr Ala Val Glu Asn Ser Cys Pro Asp Thr Val Ser Cys Ala Asp Ile
115 120 125
ctt gct att gca gct gaa ata gct tct gtt ctg gtaattaata actcctaatt 2594
Leu Ala Ile Ala Ala Glu Ile Ala Ser Val Leu
130 135
aattcccaac cattaaaaag ttgcatgatt ggattcaaaa ttctatggta ttggggttct 2654
gatataaatt tgtaattaaa ttgcactaaa aaaaattatc atatactttt aataaaaaaa 2714
atttatctaa tttaatttat tattaaaact atttttaaaa ttcaatccta actctttttt 2774
aatcggagca tgtaagctgg cacccaccgt atatcgttgg aagatgctat aaaaccattt 2834
aattaatgga tggaatcagt caaaacattt aattcaaaat actcttaatt gtgattagta 2894
atcatgttcg ggcaagttac gttgtgtata attaatttga cttaatcaga taaaaaaaca 2954
aatggacgca agccggttgg tatagatatc actggcctgt agaatatgtg gtttttcacg 3014
tttaaataaa agctagctac tatattatat ttagtctttt tttttcttaa acccatttaa 3074
cgtgatttat tgactgtgaa acatgtttcc acacacaggc ttagaaactc ctcgcaacta 3134
acatctccaa aatttgacta tttatttatg aagataattc atctatgatg ttcaactcta 3194
ttatatatat gtatcatcgc agtattaaga attataatag tcaaatatag aagtatatcg 3254
ggtaaatgta gttgcatgtg cgacctgttt cgtgtaaaat gcttattcta tatagctttt 3314
tttattggaa aataacgatg aactaaaaac gaaagggtat catatagttt gacttttatg 3374
ttagagagag acatcttaat ttggtcatat gttaaataat taattacaat gcatacacaa 3434
atatttatgc catatctaaa aaatgataaa atatcatagg tatactcaac tatatgatat 3494
ccccataaca gaaattgtac ttttcttcag gcaatgaact taacatttct gtttgctaaa 3554
aacaaacatc cacttaaagt ggttcaacat atttatgtaa taatttacag gga gga 3610
Gly Gly
ggt cca gga tgg cca gtt cca tta gga aga agg gac agc tta aca gca 3658
Gly Pro Gly Trp Pro Val Pro Leu Gly Arg Arg Asp Ser Leu Thr Ala
140 145 150 155
aac cga acc ctt gca aat caa aac ctt cca gca cct ttc ttc aac ctc 3706
Asn Arg Thr Leu Ala Asn Gln Asn Leu Pro Ala Pro Phe Phe Asn Leu
160 165 170

CA 02211018 2002-08-28
-48-
act caa ctt aaa gct tcc ttt gct gtt caa ggt ctc aac acc ctt gat 3754
Thr Gln Leu Lys Ala Ser Phe Ala Val Gln Gly Leu Asn Thr Leu Asp
175 180 185
tta gtt aca ctc tca ggtatacata atcaattttt tatttgctat tagctagcaa 3809
Leu Val Thr Leu Ser
190
taaaaagtct ctgatacaga catatttaga taaattaatt tctccataaa catttataat 3869
aaaattatca atttatgtac ttaaaaatta tggattgaag ctcttttcat ccaactttta 3929
ctaaagttaa ggtgcatata atataaaata aactatctct tgtttcttat aaaaagattg 3989
aagataagtt aaagtctact tataaatcat taatatatgt ata ggt ggt cat acg 4044
Gly Gly His Thr
195
ttt gga aga gct cgg tgc agt aca ttc ata aac cga tta tac aac ttc 4092
Phe Gly Arg Ala Arg Cys Ser Thr Phe Ile Asn Arg Leu Tyr Asn Phe
200 205 210
agc aac act gga aac cct gat cca act ctg aac aca aca tac tta gaa 4140
Ser Asn Thr Gly Asn Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Glu
215 220 225
gta ttg cgt gca aga tgc ccc cag aat gca act ggg gat aac ctc acc 4188
Val Leu Arg Ala Arg Cys Pro Gln Asn Ala Thr Gly Asp Asn Leu Thr
230 235 240
aat ttg gac ctg agc aca cct gat caa ttt gac aac aga tac tac tcc 4236
Asn Leu Asp Leu Ser Thr Pro Asp Gln Phe Asp Asn Arg Tyr Tyr Ser
245 250 255 260
aat ctt ctg cag ctc aat ggc tta ctt cag agt gac caa gaa ctt ttc 4284
Asn Leu Leu Gln Leu Asn Gly Leu Leu Gln Ser Asp Gln Glu Leu Phe
265 270 275
tcc act cct ggt gct gat acc att ccc att gtc aat agc ttc agc agt 4332
Ser Thr Pro Gly Ala Asp Thr Ile Pro Ile Val Asn Ser Phe Ser Ser
280 285 290
aac cag aat act ttc ttt tcc aac ttt aga gtt tca atg ata aaa atg 4380
Asn Gln Asn Thr Phe Phe Ser Asn Phe Arg Val Ser Met Ile Lys Met
295 300 305
ggt aat att gga gtg ctg act ggg gat gaa gga gaa att cgc ttg caa 4428
Gly Asn Ile Gly Val Leu Thr Gly Asp Glu Gly Glu Ile Arg Leu Gln
310 315 320
tgt aat ttt gtg aat gga gac tcg ttt gga tta gct agt gtg gcg tcc 4476
Cys Asn Phe Val Asn Gly Asp Ser Phe Gly Leu Ala Ser Val Ala Ser
325 330 335 340

CA 02211018 2002-08-28
-49-
aaa gat gct aaa caa aag ctt gtt gct caa tct aaa taa accaataatt 4525
Lys Asp Ala Lys Gln Lys Leu Val Ala Gln Ser Lys
345 350
aatggggatg tgcatgctag ctagcatgta aaggcaaatt aggttgtaaa cctctttgct 4585
agctatattg aaataaacca aaggagtagt gtgcatgtca attcgatttt gccatgtacc 4645
tcttggaata ttatgtaata attatttgaa tctctttaag gtacttaatt aatca 4700
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: degenerate probe
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ttycaygayt gyttygt 17
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: primer prx2+
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
cttccaaata tcaactcaat 20
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA

CA 02211018 2002-08-28
-50-
(ix) FEATURE:
(D) OTHER INFORMATION: primer prx6-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
taaagttgga aaagaaagta 20
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: primer prx9
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
atgcatgcag gtttttcagt 20
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: primer prx10-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ttgctcgctt tctattgtat 20
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:

CA 02211018 2002-08-28
-51-
(D) OTHER INFORMATION: primer prx12+
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
tcttcgatgc ttctttcacc 20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: primer prx29+
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
cataaacaat acgtacgtga t 21
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1031 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Glycine max
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
tttcatgatt gctttgttca aggttgtgat ggatcagttt tactgaacaa cactgataca 60
atagaaagcg agcaagatgc acttccaaat atcaactcaa taagaggatt ggacgttgtc 120
aatgacatca agacagcggt ggaaaatagt tgtccagaca cagtttcttg tgctgatatt 180
cttgctattg cagctgaaat agcttctgtt gctgggagga ggtcaggatg gccagttcca 240
ttaggaagaa gggacagctt aacagcaaac cgaacccttg caaatcaaaa ccttccagca 300
cctttcttca acctcactca acttaaagct tcctttgctg ttcaaggtct caacaccctt 360
gatttagtta cactctcagg tggtcatacg tctggaagag ctcggtgcag tacattcata 420
aaccgattat acaacttcag caacactgga ctgatccact tggacacaac atacttagaa 480

CA 02211018 2002-08-28
-52-
gtattgcgtg caagatgccc ccagaatgca actggggata acctcaccaa tttggacctg 540
agcacacctg atcaatttga caacagatac tactccaatc ttctgcagct caatggctta 600
cttcagagtg accaagaacg tttctccact cctggtgctg ataccattcc attgtcaata 660
gcttcagcga accagaatac tttcttttcc aactttagag tttcaatgat aaaaatgggt 720
aatattggag tgctgactgg ggatgaagga gaaattcgct tgcaatgtaa ttttgtgaat 780
ggagactcgt ttggattagc tagtgtggcg tccaaagatg ctaaacaaaa gcttgttgct 840
caatctaaat aaaccaataa ttaatgggga tgtcgatgct agctacgatg taaaggcaaa 900
ttaggttgaa acctctttgc tagctatatt gaaataaacc aaaggagtag tgtcgatgtc 960
aattcgattt tgccatgtac ctcttggaat attatgtaat aattatttga atctcaaaaa 1020
aaaaaaaaaa a 1031
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ggcaaacaat gaactccctt cgtgctgtag caatagcttt gtgctgtatt gtggttgtgc 60
ttggagggtt acccttctct tcaaatgcgc aacttgatcc atccttttac aggaacactt 120
gtccaaatgt tagttccatt gttcgtgaag tcataaggag tgtttctaag aaagatcctc 180
gtatgcttgc tagtcttgtc aggcttcact ttcatgactg ttttgttcaa ggttgtgatg 240
catcagtttt actaaacaaa actgataccg ttgtgagtga acaagatgct tttccaaaca 300
gaaactcatt aagaggtttg gatgttgtga atcaaatcaa aacagctgtg gaaaaggctt 360
gtcctaacac agtttcttgt gctgatattc ttgctctttc tgctgaatta tcatctacac 420
tggcagatgg tcctgactgg aaggttcctt taggaagaag agatggttta acggcaaacc 480
agttacttgc taatcaaaat cttccagctc ctttcaatac tactgatcaa cttaaagctg 540
catttgctgc tcaaggtctc gatactactg atctggttgc actctccggt gctcatacat 600

CA 02211018 2002-08-28
-53-
ttggaagagc tcattgctct ttatttgtta gccgattgta caacttcagc ggtacgggaa 660
gtcccgatcc aactcttaac acaacttact tacaacaatt gcgcacaata tgtcccaatg 720
gtggacctgg cacgaacctt accaatttcg atccaacgac tcctgataaa tttgacaaga 780
actattactc taatcttcaa gtgaaaaaag gtttgcttca aagtgatcaa gagttgttct 840
caacatctgg ttcagatacc attagcattg tcaacaaatt cgcaaccgat caaaaagctt 900
tttttgagag ctttagggct gctatgatca aaatgggaaa tattggtgtg ttaaccggga 960
accaaggaga gattagaaaa caatgcaact ttgttaattc aaaatcagca gaacttggtc 1020
ttatcaatgt tgcctcagca gattcatctg aggagggtat ggttagctca atgtaaatgt 1080
agtgattgga agcaactaat aaattaagaa gctataacta tgcacattca tggtatgtgt 1140
gagatagtta ttagatgctt tgtgagcaaa aatcttttgg atttcatttg aagtgtttct 1200
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
gctcttcaaa acaatgaact ccttagcaac ttctatgtgg tgtgttgtgc ttttagttgt 60
gcttggagga ctaccctttt cctcagatgc acaacttagt cccacttttt acagcaaaac 120
gtgtccaact gttagttcca ttgttagcaa tgtcttaaca aacgtttcta agacagatcc 180
tcgcatgctt gctagtctcg tcaggcttca ctttcatgac tgttttgttc tgggatgtga 240
tgcctcagtt ttgctgaaca atactgctac aatcgtaagc gaacaacaag cttttccaaa 300
taacaactct ctaagaggtt tggatgttgt gaatcagatc aaactggctg tagaagtgcc 360
ttgtcctaac acagtttctt gtgctgatat tcttgcactt gctgctcaag catcctctgt 420
tctggcacaa ggtcctagtt ggacggttcc tttaggaaga agggatggtt taaccgcaaa 480
ccgaacactt gcaaatcaaa atcttccggc tccattcaat tccttggatc aacttaaagc 540
tgcatttact gctcaaggcc tcaatactac tgatctagtt gcactctcgg gtgctcatac 600

CA 02211018 2002-08-28
-54-
atttggaaga gctcattgcg cacaatttgt tagtcgattg tacaacttca gcagtactgg 660
aagtcccgat ccaactctta acacaactta cttacaacaa ctgcgcacaa tatgtcccaa 720
tggtggacct ggcacaaacc ttaccaattt cgatccaacg actcctgata aatttgacaa 780
gaactattac tccaatcttc aagtgaaaaa gggtttgctc caaagtgatc aagagttgtt 840
ctcaacttct ggtgcagata ccattagcat tgtcaacaaa ttcagcaccg atcaaaatgc 900
tttctttgag agctttaagg ctgcaatgat taaaatgggc aatattggtg tgctaacagg 960
gacaaaagga gagattagaa aacaatgcaa ctttgtgaac tttgtgaact caaattctgc 1020
agaactagat ttagccacca tagcatccat agtagaatca ttagaggatg gtattgctag 1080
tgtaatataa ataaattagc gtaaatgcac ttattgaaat cttgtgacta gatgccacta 1140
ataaataagt tataactagg cacatttcat gtcacttgaa atttcatgcc tgtatatgag 1200
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
ctccttagca acttctatgt ggtgtgttgt gcttttagtt gtgcttggag gactaccctt 60
ttcctcagat gcacaactta gtcccacttt ttacagcaaa acgtgtccaa ctgttagttc 120
cattgttagc aatgtcttaa caaacgtttc taagacagat cctcgcatgc ttgctagtct 180
cgtcaggctt cactttcatg actgttttgt tctgggatgt gatgcctcag ttttgctgaa 240
caatactgct acaatcgtaa gcgaacaaca agcttttcca aataacaact ctctaagggg 300
tttggatgtt gtgaatcaga tcaaaactgc tgtagaaagt gcttgtccta acacagtttc 360
ttgtgctgat attcttgcac ttgctcaagc atcctctgtt ctggcacaag gtcctagttg 420
gacggttcct ttaggaagaa gggatggttt aaccgcaaac cgaacacttg caaatcaaaa 480
tcttccggct ccattcaatt ccttggatca ccttaaactg catttgactg ctcaaggcct 540
cattactcct gttctagttg ccctctcggg tgctcataca tttggaagag ctcattgcgc 600

CA 02211018 2002-08-28
-54- 1
acaatttgtt agtcgattgt acaacttcag cagtactgga agtcccgatc caactcttaa 660
cacaacttac ttacaacaac tgcgcacaat atgtcccaat ggtggacctg gcacaaacct 720
taccaatttc gatccaacga ctcctgataa atttgacaag aactattact ccaatcttca 780
agtgaaaaag ggtttgctcc aaagtgatca agagttgttc tcaacttctg gtgcagatac 840
cattagcatt gtcgacaaat tcagcaccga tcaaaatgct ttctttgaga gctttaaggc 900
tgcaatgatt aaaatgggca atattggtgt gctaacaggg acaaaaggag agattagaaa 960
acaatgcaac tttgtgaact caaattctgc agaactagat ttagccacca tagcatccat 1020
agtagaatca ttagaggatg gaattgctag tgtaatataa ataaattagc gaaaatgcac 1080
ttattgaaat cttgtgacta gatcccacta ataaataagt tataactagg cacatttcat 1140
gtcacttgaa atcctatgcc ttgtatatta gaggacgtgt tcttcttggt attatactat 1200
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
aatgcttggt ctaagtgcaa cagctttttg ctgtatggtg tttgtgctaa ttggaggagt 60
acccttttca aatgcacaac tagatccttc attttacaac agtacatgtt ctaatcttga 120
ttcaatcgta cgtggtgtgc tcacaaatgt ttcacaatct gatcccagaa tgcttggtag 180
tctcatcagg ctacattttc atgactgttt tgttcaaggt tgcgatgcct cgattttgct 240
gaacgatacg gctacaatag tgagcgagca aagtgcacca ccaaataaca actccataag 300
aggtttggat gtgataaacc agatcaaaac agcggtggaa aatgcttgtc ctaacacagt 360
ttcttgtgct gatattcttg ctctttctgc tgaaatatca tctgatctgg caaatggtcc 420
tacttggcaa gttccattag gaagaaggga tagtttgaca gcaaataatt cccttgcagc 480
tcaaaatctt cctgccccca ctttcaacct tactcgacta aaatctaact ttgataatca 540
aaacctcagt actactgatc tagttgcact ctcaggtggc catacaattg gaagaggtca 600

CA 02211018 2002-08-28
-54-2
atgcagattt ttcgttgatc gattatacaa tttcagcaac actggaaacc ccgattcaac 660
tcttaacacg acctatttac aaacattgca agcaatatgt cccaatggtg gacctggtac 720
aaacctaacc gatttggacc caaccacacc agatacattt gactccaact actactccaa 780
tctccaagtt ggaaagggct tgtttcagag tgaccaagag cttttttcca gaaatggttc 840
tgacactatt tctattgtca atagtttcgc caataatcaa actctcttct ttgaaaattt 900
tgtagcctca atgataaaaa tgggtaatat tggagtttta actggatctc aaggtgaaat 960
tagaacacag tgtaatgctg tgaatgggaa ttcttctgga ttggctactg tagtcaccaa 1020
agaatcatca gaagatggaa tggctagctc attctaaata taagcttgga aaatattgaa 1080
gaggttctat aattttgtgc atacatatat ggtatgtgca tgtggtgtat tatgtttttg 1140
ttatgttctt caagttgatc agggactgta gaagctccct aataatattt gtgtcaaagt 1200
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 283
(B) TYPE: a1 t1(hO acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(vi) ORIGINAL SOURCE: Glycine max
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Phe His Asp Cys Phe Val Gln Gly Cys Asp Gly Ser Val Leu Leu Asn
1 5 10 15
Asn Thr Asp Thr Ile Glu Ser Glu Gln Asp Ala Leu Pro Asn Ile Asn
20 25 30
Ser Ile Arg Gly Leu Asp Val Val Asn Asp Ile Lys Thr Ala Val Glu
35 40 45
Asn Ser Cys Pro Asp Thr Val Ser Cys Ala Asp Ile Leu Ala Ile Ala
50 55 60
Ala Glu Ile Ala Ser Val Ala Gly Arg Arg Ser Gly Trp Pro Val Pro
65 70 75 80
Leu Gly Arg Arg Asp Ser Leu Thr Ala Asn Arg Thr Leu Ala Asn Gln
85 90 95
Asn Leu Pro Ala Pro Phe Phe Asn Leu Thr Gln Leu Lys Ala Ser Phe
100 105 110

CA 02211018 2002-08-28
-54- 3
Ala Val Gln Gly Leu Asn Thr Leu Asp Leu Val Thr Leu Ser Gly Gly
115 120 125
His Thr Ser Gly Arg Ala Arg Cys Ser Thr Phe Ile Asn Arg Leu Tyr
130 135 140
Asn Phe Ser Asn Thr Gly Leu Ile His Leu Asp Thr Thr Tyr Leu Glu
145 150 155 160
Val Leu Arg Ala Arg Cys Pro Gln Asn Ala Thr Gly Asp Asn Leu Thr
165 170 175
Asn Leu Asp Leu Ser Thr Pro Asp Gln Phe Asp Asn Arg Tyr Tyr Ser
180 185 190
Asn Leu Leu Gln Leu Asn Gly Leu Leu Gln Ser Asp Gln Glu Arg Phe
195 200 205
Ser Thr Pro Gly Ala Asp Thr Ile Pro Leu Ser Ile Ala Ser Ala Asn
210 215 220
Gln Asn Thr Phe Phe Ser Asn Phe Arg Val Ser Met Ile Lys Met Gly
225 230 235 240
Asn Ile Gly Val Leu Thr Gly Asp Glu Gly Glu Ile Arg Leu Gln Cys
245 250 255
Asn Phe Val Asn Gly Asp Ser Phe Gly Leu Ala Ser Val Ala Ser Lys
260 265 270
Asp Ala Lys Gln Lys Leu Val Ala Gln Ser Lys
275 280
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 355
(B) TYPE: cTmif)o acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Asn Ser Leu Arg Ala Val Ala Ile Ala Leu Cys Cys Ile Val Val
1 5 10 15
Val Leu Gly Gly Leu Pro Phe Ser Ser Asn Ala Gln Leu Asp Pro Ser
20 25 30

CA 02211018 2002-08-28
-54-4
Phe Tyr Arg Asn Thr Cys Pro Asn Val Ser Ser Ile Val Arg Glu Val
35 40 45
Ile Arg Ser Val Ser Lys Lys Asp Pro Arg Met Leu Ala Ser Leu Val
50 55 60
Arg Leu His Phe His Asp Cys Phe Val Gln Gly Cys Asp Ala Ser Val
65 70 75 80
Leu Leu Asn Lys Thr Asp Thr Val Val Ser Glu Gln Asp Ala Phe Pro
85 90 95
Asn Arg Asn Ser Leu Arg Gly Leu Asp Val Val Asn Gln Ile Lys Thr
100 105 110
Ala Val Glu Lys Ala Cys Pro Asn Thr Val Ser Cys Ala Asp Ile Leu
115 120 125
Ala Leu Ser Ala Glu Leu Ser Ser Thr Leu Ala Asp Gly Pro Asp Trp
130 135 140
Lys Val Pro Leu Gly Arg Arg Asp Gly Leu Thr Ala Asn Gin Leu Leu
145 150 155 160
Ala Asn Gln Asn Leu Pro Ala Pro Phe Asn Thr Thr Asp Gln Leu Lys
165 170 175
Ala Ala Phe Ala Ala Gln Gly Leu Asp Thr Thr Asp Leu Val Ala Leu
180 185 190
Ser Gly Ala His Thr Phe Gly Arg Ala His Cys Ser Leu Phe Val Ser
195 200 205
Arg Leu Tyr Asn Phe Ser Gly Thr Gly Ser Pro Asp Pro Thr Leu Asn
210 215 220
Thr Thr Tyr Leu Gln Gln Leu Arg Thr Ile Cys Pro Asn Gly Gly Pro
225 230 235 240
Gly Thr Asn Leu Thr Asn Phe Asp Pro Thr Thr Pro Asp Lys Phe Asp
245 250 255
Lys Asn Tyr Tyr Ser Asn Leu Gln Val Lys Lys Gly Leu Leu Gln Ser
260 265 270
Asp Gln Glu Leu Phe Ser Thr Ser Gly Ser Asp Thr Ile Ser Ile Val
275 280 285
Asn Lys Phe Ala Thr Asp Gln Lys Ala Phe Phe Glu Ser Phe Arg Ala
290 295 300
Ala Met Ile Lys Met Gly Asn Ile Gly Val Leu Thr Gly Asn Gln Gly
305 310 315 320

CA 02211018 2002-08-28
-54- 5
Glu Ile Arg Lys Gln Cys Asn Phe Val Asn Ser Lys Ser Ala Glu Leu
325 330 335
Gly Leu Ile Asn Val Ala Ser Ala Asp Ser Ser Glu Glu Gly Met Val
340 345 350
Ser Ser Met
355
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 358
(B) TYPE: aminv acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Asn Ser Leu Ala Thr Ser Met Trp Cys Val Val Leu Leu Val Val
1 5 10 15
Leu Gly Gly Leu Pro Phe Ser Ser Asp Ala Gln Leu Ser Pro Thr Phe
20 25 30
Tyr Ser Lys Thr Cys Pro Thr Val Ser Ser Ile Val Ser Asn Val Leu
35 40 45
Thr Asn Val Ser Lys Thr Asp Pro Arg Met Leu Ala Ser Leu Val Arg
50 55 60
Leu His Phe His Asp Cys Phe Val Leu Gly Cys Asp Ala Ser Val Leu
65 70 75 80
Leu Asn Asn Thr Ala Thr Ile Val Ser Glu Gln Gln Ala Phe Pro Asn
85 90 95
Asn Asn Ser Leu Arg Gly Leu Asp Val Val Asn Gln Ile Lys Leu Ala
100 105 110
Val Glu Val Pro Cys Pro Asn Thr Val Ser Cys Ala Asp Ile Leu Ala
115 120 125
Leu Ala Ala Gln Ala Ser Ser Val Leu Ala Gln Gly Pro Ser Trp Thr
130 135 140
Val Pro Leu Gly Arg Arg Asp Gly Leu Thr Ala Asn Arg Thr Leu Ala
145 150 155 160

CA 02211018 2002-08-28
-54- 6
Asn Gln Asn Leu Pro Ala Pro Phe Asn Ser Leu Asp Gln Leu Lys Ala
165 170 175
Ala Phe Thr Ala Gln Gly Leu Asn Thr Thr Asp Leu Val Ala Leu Ser
180 185 190
Gly Ala His Thr Phe Gly Arg Ala His Cys Ala Gln Phe Val Ser Arg
195 200 205
Leu Tyr Asn Phe Ser Ser Thr Gly Ser Pro Asp Pro Thr Leu Asn Thr
210 215 220
Thr Tyr Leu Gln Gln Leu Arg Thr Ile Cys Pro Asn Gly Gly Pro Gly
225 230 235 240
Thr Asn Leu Thr Asn Phe Asp Pro Thr Thr Pro Asp Lys Phe Asp Lys
245 250 255
Asn Tyr Tyr Ser Asn Leu Gln Val Lys Lys Gly Leu Leu Gln Ser Asp
260 265 270
Gln Glu Leu Phe Ser Thr Ser Gly Ala Asp Thr Ile Ser Ile Val Asn
275 280 285
Lys Phe Ser Thr Asp Gln Asn Ala Phe Phe Glu Ser Phe Lys Ala Ala
290 295 300
Met Ile Lys Met Gly Asn Ile Gly Val Leu Thr Gly Thr Lys Gly Glu
305 310 315 320
Ile Arg Lys Gln Cys Asn Phe Val Asn Phe Val Asn Ser Asn Ser Ala
325 330 335
Glu Leu Asp Leu Ala Thr Ile Ala Ser Ile Val Glu Ser Leu Glu Asp
340 345 350
Gly Ile Ala Ser Val Ile
355
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 347
(B) TYPE: ciminO acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

CA 02211018 2002-08-28
-54- 7
Met Trp Cys Val Val Leu Leu Val Val Leu Gly Gly Leu Pro Phe Ser
1 5 10 15
Ser Asp Ala Gln Leu Ser Pro Thr Phe Tyr Ser Lys Thr Cys Pro Thr
20 25 30
Val Ser Ser Ile Val Ser Asn Val Leu Thr Asn Val Ser Lys Thr Asp
35 40 45
Pro Arg Met Leu Ala Ser Leu Val Arg Leu His Phe His Asp Cys Phe
50 55 60
Val Leu Gly Cys Asp Ala Ser Val Leu Leu Asn Asn Thr Ala Thr Ile
65 70 75 80
Val Ser Glu Gln Gln Ala Phe Pro Asn Asn Asn Ser Leu Arg Gly Leu
85 90 95
Asp Val Val Asn Gln Ile Lys Thr Ala Val Glu Ser Ala Cys Pro Asn
100 105 110
Thr Val Ser Cys Ala Asp Ile Leu Ala Leu Ala Gln Ala Ser Ser Val
115 120 125
Leu Ala Gln Gly Pro Ser Trp Thr Val Pro Leu Gly Arg Arg Asp Gly
130 135 140
Leu Thr Ala Asn Arg Thr Leu Ala Asn Gln Asn Leu Pro Ala Pro Phe
145 150 155 160
Asn Ser Leu Asp His Leu Lys Leu His Leu Thr Ala Gln Gly Leu Ile
165 170 175
Thr Pro Val Leu Val Ala Leu Ser Gly Ala His Thr Phe Gly Arg Ala
180 185 190
His Cys Ala Gln Phe Val Ser Arg Leu Tyr Asn Phe Ser Ser Thr Gly
195 200 205
Ser Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Gln Leu Arg Thr
210 215 220
Ile Cys Pro Asn Gly Gly Pro Gly Thr Asn Leu Thr Asn Phe Asp Pro
225 230 235 240
Thr Thr Pro Asp Lys Phe Asp Lys Asn Tyr Tyr Ser Asn Leu Gln Val
245 250 255
Lys Lys Gly Leu Leu Gln Ser Asp Gln Glu Leu Phe Ser Thr Ser Gly
260 265 270
Ala Asp Thr Ile Ser Ile Val Asp Lys Phe Ser Thr Asp Gln Asn Ala
275 280 285

CA 02211018 2002-08-28
-54- 8
Phe Phe Glu Ser Phe Lys Ala Ala Met Ile Lys Met Gly Asn Ile Gly
290 295 300
Val Leu Thr Gly Thr Lys Gly Glu Ile Arg Lys Gln Cys Asn Phe Val
305 310 315 320
Asn Ser Asn Ser Ala Glu Leu Asp Leu Ala Thr Ile Ala Ser Ile Val
325 330 335
Glu Ser Leu Glu Asp Gly Ile Ala Ser Val Ile
340 345
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351
(B) TYPE: aml'np acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: PRT
(vi) ORIGINAL SOURCE: Medicago sativa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Leu Gly Leu Ser Ala Thr Ala Phe Cys Cys Met Val Phe Val Leu
1 5 10 15
Ile Gly Gly Val Pro Phe Ser Asn Ala Gln Leu Asp Pro Ser Phe Tyr
20 25 30
Asn Ser Thr Cys Ser Asn Leu Asp Ser Ile Val Arg Gly Val Leu Thr
35 40 45
Asn Val Ser Gin Ser Asp Pro Arg Met Leu Gly Ser Leu Ile Arg Leu
50 55 60
His Phe His Asp Cys Phe Val Gln Gly Cys Asp Ala Ser Ile Leu Leu
65 70 75 80
Asn Asp Thr Ala Thr Ile Val Ser Glu Gln Ser Ala Pro Pro Asn Asn
85 90 95
Asn Ser Ile Arg Gly Leu Asp Val Ile Asn Gln Ile Lys Thr Ala Val
100 105 110
Glu Asn Ala Cys Pro Asn Thr Val Ser Cys Ala Asp Ile Leu Ala Leu
115 120 125
Ser Ala Glu Ile Ser Ser Asp Leu Ala Asn Gly Pro Thr Trp Gln Val
130 135 140

CA 02211018 2002-08-28
-54- 9
Pro Leu Gly Arg Arg Asp Ser Leu Thr Ala Asn Asn Ser Leu Ala Ala
145 150 155 160
Gln Asn Leu Pro Ala Pro Thr Phe Asn Leu Thr Arg Leu Lys Ser Asn
165 170 175
Phe Asp Asn Gln Asn Leu Ser Thr Thr Asp Leu Val Ala Leu Ser Gly
180 185 190
Gly His Thr Ile Gly Arg Gly Gln Cys Arg Phe Phe Val Asp Arg Leu
195 200 205
Tyr Asn Phe Ser Asn Thr Gly Asn Pro Asp Ser Thr Leu Asn Thr Thr
210 215 220
Tyr Leu Gln Thr Leu Gln Ala Ile Cys Pro Asn Gly Gly Pro Gly Thr
225 230 235 240
Asn Leu Thr Asp Leu Asp Pro Thr Thr Pro Asp Thr Phe Asp Ser Asn
245 250 255
Tyr Tyr Ser Asn Leu Gln Val Gly Lys Gly Leu Phe Gln Ser Asp Gln
260 265 270
Glu Leu Phe Ser Arg Asn Gly Ser Asp Thr Ile Ser Ile Val Asn Ser
275 280 285
Phe Ala Asn Asn Gin Thr Leu Phe Phe Glu Asn Phe Vai Ala Ser Met
290 295 300
Ile Lys Met Gly Asn Ile Gly Val Leu Thr Gly Ser Gln Gly Glu Ile
305 310 315 320
Arg Thr Gln Cys Asn Ala Val Asn Gly Asn Ser Ser Gly Leu Ala Thr
325 330 335
Val Val Thr Lys Glu Ser Ser Glu Asp Gly Met Ala Ser Ser Phe
340 345 350

Representative Drawing