U.S. patent application number 11/478024 was filed with the patent office on 2006-10-26 for nucleic acid fragments encoding isoflavone synthase.
Invention is credited to Gary M. Fader, Woosuk Jung, Brian McGonigle, Joan T. Odell, Xiaodan Yu.
Application Number | 20060242735 11/478024 |
Document ID | / |
Family ID | 36915508 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060242735 |
Kind Code |
A1 |
Fader; Gary M. ; et
al. |
October 26, 2006 |
Nucleic acid fragments encoding isoflavone synthase
Abstract
This invention relates to an isolated nucleic acid sequence
encoding isoflavone synthase. The invention also relates to the
construction of chimeric sequences encoding all or a substantial
portion of the enzymes, in sense or antisense orientation, wherein
expression of the chimeric sequence results in production of
altered levels of the enzyme in a transformed host cell.
Inventors: |
Fader; Gary M.; (Newark,
DE) ; Jung; Woosuk; (Greenville, DE) ;
McGonigle; Brian; (Wilmington, DE) ; Odell; Joan
T.; (Unionville, PA) ; Yu; Xiaodan;
(Chesterfield, MO) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
36915508 |
Appl. No.: |
11/478024 |
Filed: |
June 29, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09857581 |
Jun 5, 2001 |
7098011 |
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PCT/US00/01772 |
Jan 26, 2000 |
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11478024 |
Jun 29, 2006 |
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60117769 |
Jan 27, 1999 |
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60144783 |
Jul 20, 1999 |
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60156094 |
Sep 24, 1999 |
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Current U.S.
Class: |
800/284 ;
435/193; 435/254.2; 435/412; 435/415; 435/468; 800/312;
800/320.1 |
Current CPC
Class: |
C12N 9/0077 20130101;
C12N 15/8243 20130101; C12P 17/06 20130101; C12N 15/825
20130101 |
Class at
Publication: |
800/284 ;
800/320.1; 435/193; 435/468; 435/412; 435/415; 800/312;
435/254.2 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 9/10 20060101 C12N009/10; C12N 1/00 20060101
C12N001/00; C12N 5/04 20060101 C12N005/04; A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82 |
Claims
1-19. (canceled)
20. A plant comprising in its genome a chimeric polynucleotide
comprising an isolated nucleic acid sequence encoding a polypeptide
with isoflavone synthase activity having the amino acid sequence
set forth in SEQ ID NO:66 wherein said chimeric polynucleotide is
operably linked to at least one regulatory sequence.
21. The plant of claim 20 further comprising in its genome a second
chimeric sequence comprising a nucleic acid sequence encoding a
polypeptide that regulates expression of at least one enzyme of the
phenylpropanoid pathway.
22. The plant of claim 20 wherein the plant is a soybean plant.
23. The plant of claim 20 wherein the plant is a corn plant.
24. A seed from the plant of claim 20.
25. A seed from the plant of claim 21.
26-33. (canceled)
34. A method of producing a plant with increased isoflavonoid
content comprising (a) transforming a plant cell with a first
chimeric polynucleotide comprising an isolated nucleic acid
sequence encoding a polypeptide with isoflavone synthase activity
having the amino acid sequence set forth in SEQ ID NO:66; (b)
optionally transforming the plant cell with a second chimeric
sequence comprising a nucleic acid sequence encoding a polypeptide
that regulates expression of at least one enzyme of the
phenylpropanoid pathway; and (c) growing the transformed plant cell
under conditions that promote the regeneration of a whole plant
from the transformed cell wherein the transformed plant regenerated
from the transformed cell produces an amount of an isoflavonoid
that is greater than the amount of the isoflavonoid that is
produced in a plant that is regenerated from a plant cell that is
not transformed with the chimeric polynucleotide of part (a).
35. The method of claim 34 wherein the plant is a soybean
plant.
36. The method of claim 34 wherein the plant is a corn plant.
37. The transgenic plant produced by the method of claim 34.
38. The transgenic plant of claim 37 wherein the plant is a soybean
plant.
39. The transgenic plant of claim 37 wherein the plant is a corn
plant.
40. A seed from the plant of claim 37.
41-50. (canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/117,769, filed Jan. 27, 1999, U.S. Provisional
Application No. 60/144,783, filed Jul. 20, 1999, and U.S.
Provisional Application No. 60/156,094, filed Sep. 24, 1999.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
sequences encoding isoflavone synthase and their use in producing
isoflavones.
BACKGROUND OF THE INVENTION
[0003] Isoflavonoids represent a class of secondary metabolites
produced in legumes by a branch of the phenylpropanoid pathway and
include such compounds as isoflavones, isoflavanones, rotenoids,
pterocarpans, isoflavans, quinone derivatives,
3-aryl-4-hydroxy-coumarins, 3-arylcoumarins, isoflav-3-enes,
coumestans, alpha-methyldeoxybenzoins, 2-arylbenzofurans,
isoflavanol, coumaronochromone and the like. In plants, these
compounds are known to be involved in interactions with other
organisms and to participate in the defense responses of legumes
against phytopathogenic microorganisms (Dewick, P. M. (1993) in The
Flavonoids, Advances in Research Since 1986, Harborne, J. B. Ed.,
pp. 117-238, Chapman and Hall, London). Isoflavonoid-derived
compounds also are involved in symbiotic relationships between
roots and rhizobial bacteria which eventually result in nodulation
and nitrogen-fixation (Phillips, D. A. (1992) in Recent Advances in
Phytochemistry. Vol. 26, pp 201-231, Stafford, H. A. and Ibrahim,
R. K., Eds, Pleneum Press, New York), and overall they have been
shown to act as antibiotics, repellents, attractants, and signal
compounds (Barz, W. and Welle, R. (1992) Phenolic Metabolism in
Plants, pg 139-164, Ed by H. A. Stafford and R. K. Ibrahim, Plenum
Press, New York).
[0004] Isoflavonoids have also been reported to have physiological
activity in animal and human studies. For example, it has been
reported that the isoflavones found in soybean seeds possess
antihemolytic (Naim, M., et al. (1976) J. Agric. Food Chem.
24:1174-1177), antifungal (Naim, M., et al. (1974) J. Agr. Food
Chem. 22:806-810), estrogenic (Price, K. R. and Fenwick, G. R.
(1985) Food Addit. Contam. 2:73-106), tumor-suppressing (Messina,
M. and Barnes, S. (1991) J. Natl. Cancer Inst. 83:541-546;
Peterson, G., et al. (1991) Biochem. Biophys. Res. Commun.
179:661-667), hypolipidemic (Mathur, K., et al. (1964) J. Nutr.
84:201-204), and serum cholesterol-lowering (Sharma, R. D. (1979)
Lipids 14:535-540) effects. These epidemiological studies indicate
that isoflavones in soybean protein products, when taken as a
dietary supplement, may produce many significant health
benefits.
[0005] Free isoflavones rarely accumulate to high levels in
soybeans. Instead they are usually conjugated to carbohydrates or
organic acids. Soybean seeds contain three types of isoflavones in
four different forms: the aglycones, daidzein, genistein and
glycitein; the glucosides, daidzin, genistin and glycitin; the
acetylgucosides, 6''-O-acetyldaidzin, 6''-O-acetylgenistin and
6''-O-acetylglycitin; and the malonylglucosides,
6''-O-malonyldaidzin, 6''-O-malonylgenistin and
6''-O-malonylglycitin. In accordance with the present invention,
all of these compounds are included in the term isoflavonoids. The
content of isoflavonoids in soybean seeds is quite variable and is
affected by both genetics and environmental conditions such as
growing location and temperature during seed fill (Tsukamoto, C.,
et al. (1995) J. Agric. Food Chem. 43:1184-1192; Wang, H. and
Murphy, P. A. (1994) J. Agric. Food Chem. 42:1674-1677). In
addition, isoflavonoid content in legumes can be stress-induced by
pathogenic attack, wounding, high UV light exposure and pollution
(Dixon, R. A. and Paiva, N. L. (1995) Plant Cell 7:1085-1097).
[0006] The biosynthetic pathway for isoflavonoids in soybean and
their relationship with several other classes of phenylpropanoids
is presented in FIG. 1. Many of the enzymes involved in the
synthesis of isoflavonoids in legumes have been identified and many
of the genes in the pathway have been cloned. These include three
P450-dependent monooxygenases, cinnamate 4-hydoxylase (Potts, J. R.
M., et al. (1974) J. Biol. Chem. 249:5019-5026), isoflavone
2'-hydroxylase (Akashi, T. et al. (1998) Biochem. Biophys. Res.
Commun. 251:67-70), and dihydroxypterocarpan 6a-hydroxylase
(Schopfer, C. R., et. al. (1998) FEBS Lett. 432:182-186). However,
to date the gene encoding isoflavone synthase, the first step in
the phenylpropanoid branch that commits metabolic intermediates to
the synthesis of isoflavonoids, has been neither identified nor
cloned from any species. In this central reaction, 2S-flavanone is
converted into an isoflavonoid such as genistein and daidzein. The
enzymatic reaction for this oxidative aryl migration step was first
reported by Hagmann, M. L. and Grisebach, H. ((1984) FEBS Lett.
175:199-202). The reaction involves a P450 monoxygenase-mediated
conversion of the 2S-flavanone to a 2-hydroxyisoflavanone, followed
by conversion to the isoflavonoid. This last step is possibly
mediated by a soluble dehydratase (Kochs, G. and Grisenbach, H.
(1985) Eur. J. Biochem. 155:311-318). However, the
2-hydroxyisoflavanone intermediate was described as unstable and
could convert directly to genistein.
[0007] Cytochrome P450-dependant monooxygenases comprise a large
group of heme-containing enzymes, most of which catalyze NADPH- and
O.sub.2-dependant hydroxylation reactions. Most of these enzymes do
not use NADPH directly, but rely upon an interaction with a
flavoprotein known as a P450 reductase that transfers electrons
from the cofactor to the P450. Cloning of plant P450s by
traditional protein purification strategies has been difficult, as
these membrane-bound proteins are often very unstable and are
typically present in low abundance. PCR-based cloning strategies
using sequence homologies between P450s has increased dramatically
the number of P450 genes cloned. However, the in vivo activity of
many of these cloned genes remains unknown and they are classified
simply as P450s, and are grouped into families based solely on
sequence homology (Chapple, C. (1998) Annu. Rev. Plant Physiol.
Plant Mol. Bio. 49:311-343). Proteins that are greater than 55%
identical are designated as members of the same subfamily, while
P450s that are 97% identical, or greater, are assumed to be allelic
variants of the same gene (Chapple, C. (1998) Annu. Rev. Plant
Physiol. Plant Mol. Bio. 49:311-343).
[0008] Efforts to determine in vivo activities of existing P450
clones are increasing. Most efforts involve expressing genes or
cDNAs for P450s in yeast or insect cell systems, and then screening
for a particular activity. For example, isoflavone 2'-hydroxylase
(Akashi, T., et al. (1998) Biochem. Biophys. Res. Commun.
251:67-70) and dihydroxypterocarpan 6a-hydroxylase (Schopfer, C.
R., et al. (1998) FEBS Letters 432:182-186) were identified in this
manner.
[0009] The physiological activities associated with isoflavonoids
in both plants and humans makes the manipulation of their contents
in crop plants highly desirable. For example, increasing levels of
isoflavonoid in soybean seeds would increase the efficiency of
extraction and lower the cost of isoflavone-related products sold
today for use in either reduction of serum cholesterol or in
estrogen replacement therapy. Decreasing levels of isoflavonoid in
soybean seeds would be beneficial for production of soy-based
infant formulas where the estrogenic effects of isoflavonoid are
undesirable. Raising levels of isoflavonoid phytoalexins in
vegetative plant tissue could increase plant defenses to pathogen
attack, thereby improving plant disease resistance and lowering
pesticide use rates. Manipulation of isoflavonoid levels in roots
could lead to improved nodulation and increased efficiencies of
nitrogen fixation. To date, however, it has proven difficult to
develop soybean or other plant lines with consistently high levels
of isoflavonoid. Because isoflavone synthase is the central
reaction in pathways producing isoflavonoids, identification of
this functional gene is extremely important, and its manipulation
via molecular techniques is expected to allow production of
soybeans and other plants with high, stable levels of isoflavonoid.
Introduction of the isoflavone synthase gene in non-legume crop
species including, but not limited to, corn, wheat, rice,
sunflower, and canola could lead to synthesis of isoflavonoids. The
expression of isoflavonoids would confer to these species disease
resistance and/or properties which produce human/livestock health
benefits.
[0010] Substrates for isoflavone synthase may be limiting for
synthesizing very high levels of isoflavonoids in soybean, or for
synthesizing isoflavonoids in non-legumes. It is desirable to
increase the flux of metabolites through the phenylpropanoid
pathway to provide additional amounts of substrate to those
occurring naturally. Different stress conditions such as UV
irradiation, phosphate starvation, prolonged exposure to cold, and
chemical (such as herbicide) treatment can cause activation of the
phenylpropanoid pathway. While these treatments may produce the
desired substrate availability, it is more desirable to have a
genetic means of activating the phenylpropanoid pathway. It is
known that expression of genes encoding certain transcription
factors can regulate the expression of various genes that encode
enzymes of the phenylpropanoid pathway. These include, but are not
limited to, the C1 myb-type transcription factor of maize and the
AmMyb305 of Antirrhinum majus. The C1 myb-type transcription factor
of maize, in conjunction with the myc-type transcription factor R,
activates chalcone synthase and chalcone isomerase genes
(Grotewold, E., et al. (1998) Plant Cell 10:721-740). The
Antirrhinum majus AmMyb305 activates the phenylalanine ammonia
lyase promoter (Sablowski, R. W., et al. (1994) EMBO J.
13:128-137). Transcription factors such as these may be expressed
in host plant cells to activate expression of genes in the
phenylpropanoid pathway thereby increasing the encoded enzyme
activities and the flux of compounds through the pathway. Increases
in the precursors to substrates of isoflavone synthase would
enhance the production of isoflavonoids.
SUMMARY OF THE INVENTION
[0011] The instant invention relates to isolated nucleic acid
sequences encoding isoflavone synthase. In addition, this invention
relates to nucleic acid sequences that are complementary to nucleic
acid sequences encoding isoflavone synthase. The nucleic acid
sequences may be of genomic or cDNA origin and may contain
introns.
[0012] In another embodiment, the instant invention relates to
chimeric genes encoding isoflavone synthase or to chimeric genes
that comprise nucleic acid sequences that are complementary to the
nucleic acid sequences encoding the enzyme, operably linked to
suitable regulatory sequences, wherein expression of the chimeric
genes results in production of levels of isoflavone synthase in
transformed host cells that are altered (i.e., increased or
decreased) from the levels produced in untransformed host
cells.
[0013] In a further embodiment, the instant invention concerns a
transformed host cell comprising in its genome a chimeric gene
encoding an isoflavone synthase that is operably linked to suitable
regulatory sequences. Expression of the chimeric gene results in
production of altered levels of the enzyme in the transformed host
cell. The transformed host cell can be of eukaryotic or prokaryotic
origin, and includes cells derived from higher plants and
microorganisms. The invention also includes transformed plants that
arise from transformed host cells of higher plants, and seeds
derived from such transformed plants.
[0014] An additional embodiment of the instant invention concerns a
method of altering the level of expression of a plant isoflavone
synthase in a transformed host cell comprising transforming a host
cell with a chimeric gene comprising a nucleic acid sequence (cDNA
or genomic DNA) encoding an isoflavone synthase operably linked to
suitable regulatory sequences and growing the transformed host cell
under conditions that are suitable for expression of the chimeric
gene wherein expression of the chimeric gene results in production
of altered levels of isoflavone synthase in the transformed host
cell. The altered levels of isoflavone synthase may be higher due
to overexpression, or may be lower due to cosuppression or anti
sense suppression.
[0015] A further embodiment of the instant invention is a method
for increasing the amount of one or more isoflavonoids in a host
cell. The method comprising the steps of transforming a host cell
with a chimeric gene comprising a nucleic acid sequence encoding an
isoflavone synthase operably linked to suitable regulatory
sequences and growing the transformed host cell under conditions
that are suitable for expression of the chimeric gene wherein
expression of the chimeric gene results in production of an amount
of isoflavonoids in the transformed host cell that is greater than
the amount of isoflavonoids that are produced in a cell that is not
transformed with the chimeric gene.
[0016] A further embodiment of the instant invention is a method
for decreasing the amount of one or more isoflavonoids in a host
cell. The method comprising the steps of transforming a host cell
with a chimeric gene comprising a nucleic acid sequence encoding
all or a substantial portion of an isoflavone synthase operably
linked to suitable regulatory sequences and growing the transformed
host cell under conditions that are suitable for expression of the
chimeric gene wherein expression of the chimeric gene results in
production of an amount of isoflavonoids in the transformed host
cell that is less than the amount of isoflavonoids that are
produced in a cell that is not transformed with the chimeric gene.
The invention also includes transformed plants that arise from
transformed host cells of higher plants, and seeds derived from
such transformed plants.
[0017] An additional embodiment of the instant invention concerns a
method for obtaining a nucleic acid sequence encoding all or
substantially all of an amino acid sequence encoding isoflavone
synthase.
[0018] A still further embodiment of the instant invention concerns
a transformed host cell comprising a chimeric gene encoding
isoflavone synthase and at least one chimeric gene encoding a
transcription factor that can regulate expression of one or more
genes in the phenylpropanoid pathway. The invention also includes
transformed plants that arise from transformed host cells of higher
plants, and seeds derived from such transformed plants.
[0019] A further embodiment is a method of increasing the amount of
one or more isoflavonoids in a host cell comprising transforming a
host cell with a chimeric gene having a nucleic acid sequence
encoding an isoflavone synthase operably linked to suitable
regulatory sequences and with at least one chimeric gene having a
nucleic acid sequence encoding a transcription factor that
regulates expression of genes in the phenylpropanoid pathway, and
growing the transformed host cell under conditions that are
suitable for expression of the chimeric genes wherein expression of
the chimeric genes result in production of an amount of one or more
isoflavonoids in the transformed host cell that is greater than the
amount of the isoflavonoids that are produced in a cell that is not
transformed with the chimeric genes. The invention also includes
transformed plants that arise from transformed host cells of higher
plants, and seeds derived from such transformed plants.
[0020] Yet a further embodiment of the present invention is a
method of altering the level of isoflavonoids in a plant cell that
is transformed with a chimeric isoflavone synthase gene comprising
exposing said cell to a phenylpropanoid pathway-altering agent. The
phenylpropanoid pathway-altering agent may be a transcription
factor or stress, for example. Stress includes and is not limited
to ultraviolet light, temperature, pressure, phosphate level, and
herbicide treatment. The transcription factors may be a C1 myb-type
transcription factor of maize and a myc-type transcription factor
R, or a chimera containing the maize R region between the C1 DNA
binding domain and the C1 activation domain.
BIOLOGICAL DEPOSIT
[0021] The following transformed yeast strain and vector plasmid
have been deposited with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, and
bears the following designation, accession number and date of
deposit. TABLE-US-00001 Yeast Strain Accession Number Date of
Deposit Isoflavone Synthase GM1 ATCC 203606 Jan. 27, 1999 Plasmid
DP7951 ATCC PTA-371 Jul. 20, 1999
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0022] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application.
[0023] FIG. 1 depicts the phenylpropanoid metabolic pathway, and
illustrates particularly the biosynthesis of isoflavonoids.
[0024] FIGS. 2A and B presents the results of HPLC analyses of
naringenin standards. FIG. 2A presents the absorption spectra
recorded at 260 nm and FIG. 2B presents the absorption spectra
recorded at 280 nm.
[0025] FIGS. 3A and B presents the results of HPLC analyses of
genistein standards. FIG. 3A presents the absorption spectra
recorded at 260 nm and FIG. 3B presents the absorption spectra
recorded at 280 nm.
[0026] FIGS. 4A and B presents the results of HPLC analyses of
genistein and naringenin from microsomes derived from
elicitor-treated soybean hypocotyls. Absorption spectra was
recorded at 260 nm (FIG. 4A) and 280 nm (FIG. 4B). Naringenin and
genistein peaks are indicated.
[0027] FIGS. 5A and B presents the results of HPLC analyses of
genistein and naringenin from microsomes derived from non-treated
soybean hypocotyls. Absorption spectra was recorded at 260 nm (FIG.
5A) and 280 nm (FIG. 5B). Naringenin and genistein peaks are
indicated.
[0028] FIGS. 6A and B presents the results of HPLC analyses of
genistein and naringenin from microsomes derived from
elicitor-treated soybean cell suspension cultures. Absorption
spectra was recorded at 260 nm (FIG. 6A) and 280 nm (FIG. 6B).
Naringenin and genistein peaks are indicated.
[0029] FIGS. 7A and B presents the results of HPLC analyses of
genistein and naringenin from microsomes derived from non-treated
soybean cell suspension cultures. Absorption spectra was recorded
at 260 nm (FIG. 7A) and 280 nm (FIG. 7B). Naringenin peak is
indicated.
[0030] FIGS. 8A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
prior to incubation in the presence of NADPH cofactor (negative
control). Absorption spectra was recorded at 260 nm (FIG. 8A) and
280 nm (FIG. 8B).
[0031] FIGS. 9A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 1 h incubation in the presence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 9A) and 280 nm (FIG. 9B).
[0032] FIGS. 10A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 2 h incubation in the presence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 10A) and 280 nm (FIG.
10B).
[0033] FIGS. 11A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 3 h incubation in the presence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 11A) and 280 nm (FIG.
11B).
[0034] FIG. 12 A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 4 h incubation in the presence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 12A) and 280 nm (FIG.
12B).
[0035] FIGS. 13A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 14 h incubation in the presence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 13A) and 280 nm (FIG.
13B).
[0036] FIGS. 14A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 40 minutes incubation in the presence of NADPH cofactor.
Absorption spectra was recorded at 260 nm (FIG. 14A) and 280 nm
(FIG. 14B).
[0037] FIGS. 15A and B presents the results of HPLC analyses of
genistein and naringenin in 150 .mu.g of yeast microsomal proteins
after 40 minutes incubation in the presence of NADPH cofactor.
Absorption spectra was recorded at 260 nm (FIG. 15A) and 280 nm
(FIG. 15B).
[0038] FIGS. 16A and B presents the results of HPLC analyses of
genistein and naringenin in 75 .mu.g of yeast microsomal proteins
after 4 h incubation in the absence of NADPH cofactor. Absorption
spectra was recorded at 260 nm (FIG. 16A) and 280 nm (FIG.
16B).
[0039] FIGS. 17A and B presents a comparison of the absorption
spectra recorded by a diode array detector of a genistein standard
(FIG. 17A; with an HPLC retention time of 3.128), and a reference
spectrum (FIG. 17B).
[0040] FIGS. 18A and B presents a comparison of the absorption
spectra recorded by a diode array detector of the newly synthesized
peak located at the retention time of 3.131 in the HPLC analysis of
yeast microsomes incubated for 14 h in the presence of NADPH on
FIG. 18A and the reference spectrum on FIG. 18B.
[0041] FIGS. 19A, B, C, D and E presents the electropositive mass
spectrum obtained for the peaks observed by HPLC analysis of yeast
microsome samples incubated with liquiritigenin. FIG. 19A
corresponds to the peak at 273.2 m/z, FIG. 19B corresponds to the
peak at 271 m/z, FIG. 19C corresponds to "peak 2", FIG. 19D
corresponds to liquiritigenin standard (the substrate), and FIG.
19E corresponds to daidzein standard (the product).
[0042] FIG. 20 depicts the plasmid map of pOY160.
[0043] FIG. 21 depicts the plasmid map of pOY206.
[0044] FIG. 22 depicts the plasmid map of pDP7951, having an ATCC
accession No. PTA-371.
[0045] FIG. 23 depicts the plasmid map of pOY162.
[0046] FIG. 24 depicts the plasmid map of pKS93s.
[0047] FIG. 25 depicts the distribution of the isoflavonoid content
of 25 transgenic lines transformed with the isoflavone synthase
sequence from clone sgs1c.pk006.o20 and a control line. Bars
represent the mean of three analyses for each line. The result of
single factor ANOVA is presented along with the least significant
difference (LSD) at P.ltoreq.0.01. The asterisk above the bars
represents those lines with mean isoflavonoid concentrations
significantly lower than control (bars 1 through 6), or those lines
with mean isoflavonoid concentrations significantly greater than
control (bars 15 through 25) based on the LSD test at
P.ltoreq.0.01.
[0048] FIG. 26 depicts the comparison of the rates of genistein and
daidzein synthesis by microsomes of the yeast transformant GM 1.
Samples representing incubation periods of 2, 4, 6, 8 and 10 h were
analyzed by HPLC and the peak areas for genistein and daidzein were
quantitated by calibration with authentic genistein and daidzein
standards. Assays were repeated three times and the average amount
of isoflavonoid synthesized at each time point was plotted, with
vertical lines representing error bars.
[0049] FIG. 27 presents the results of HPLC analyses of daidzein
and liquiritigenin in extracts from BMS cells before incubation in
the presence of NADPH cofactor (Panels A and B) and after 10 h
incubation in the presence of NADPH cofactor (Panels C and D).
Absorption spectra was recorded at 260 nm (Panels A and C) and 280
nm (Panels B and D).
[0050] FIG. 28 depicts the plasmid map of pCW109-IFS.
[0051] The following sequence descriptions and Sequences Listing
attached hereto comply with the rules governing nucleotide and/or
amino acid sequence disclosures in patent applications as set forth
in 37 C.F.R. .sctn.1.821-1.825. The Sequence Listing contains the
one letter code for nucleotide sequence characters and the three
letter codes for amino acids as defined in conformity with the
IUPAC-IUB standards described in Nucleic Acids Research
13:3021-3030 (1985) and in the Biochemical Journal 219 (No.
2):345-373 (1984) which are herein incorporated by reference. The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37 C.F.R. .sctn. 1.822.
[0052] SEQ ID NO:1 is the nucleotide sequence comprising the
soybean cDNA insert in clone sgs1c.pk006.o20 encoding an
enzymatically active isoflavone synthase.
[0053] SEQ ID NO:2 is the deduced amino acid sequence of an
enzymatically active soybean isoflavone synthase derived from the
nucleotide sequence of SEQ ID NO:1.
[0054] SEQ ID NO:3 is the nucleotide sequence of an oligonucleotide
primer used in the construction of yeast strain WHT1.
[0055] SEQ ID NO:4 is the nucleotide sequence of an oligonucleotide
primer used in the construction of the yeast strain WHT1.
[0056] SEQ ID NO:5 is the nucleotide sequence of an oligonucleotide
primer used to amplify the cDNA insert from clone
sgs1c.pk006.o20.
[0057] SEQ ID NO:6 is the nucleotide sequence of an oligonucleotide
primer used to amplify the cDNA insert from clone
sgs1c.pk006.o20.
[0058] SEQ ID NO:7 is the nucleotide sequence of an oligonucleotide
primer used for PCR amplification of the soybean clone with
sequence corresponding to the one found in NCBI General Identifier
No. 2739005. This oligonucleotide sequence corresponds to
nucleotides 3 to 26 of the NCBI sequence.
[0059] SEQ ID NO:8 is the nucleotide sequence of an oligonucleotide
primer used for PCR amplification of the soybean clone with
sequence corresponding to the one found in NCBI General Identifier
No. 2739005. This oligonucleotide sequence corresponds to the
complement of nucleotides 1798 to 1824 of the NCBI sequence.
[0060] SEQ ID NO:9 is the nucleotide sequence of an enzymatically
active soybean isoflavone synthase having an NCBI General
Identifier No. 2739005.
[0061] SEQ ID NO:10 is the deduced amino acid sequence of an
enzymatically active soybean isoflavone synthase derived from of
SEQ ID NO:9 and having an NCBI General Identifier No. 2739006.
[0062] SEQ ID NO:11 is the nucleotide sequence of an
oligonucleotide primer used for PCR amplification of the isoflavone
synthase genes from mung bean, red clover, white clover, lentil,
hairy vetch, alfalfa, lupine and snow pea.
[0063] SEQ ID NO:12 is the nucleotide sequence of an
oligonucleotide primer used for PCR amplification of the isoflavone
synthase genes from mung bean, red clover, white clover, lentil,
hairy vetch, alfalfa, lupine and snow pea.
[0064] SEQ ID NO:13 is the nucleotide sequence of an
oligonucleotide primer used in the second round of PCR
amplification of the white clover, lentil, hairy vetch, alfalfa and
lupine isoflavone synthase genes.
[0065] SEQ ID NO:14 is the nucleotide sequence of an
oligonucleotide primer used in the second round of PCR
amplification of the white clover, lentil, hairy vetch, alfalfa and
lupine isoflavone synthase genes.
[0066] SEQ ID NO:15 is the nucleotide sequence comprising the
alfalfa cDNA insert in clone alfalfa1 encoding an almost entire
alfalfa isoflavone synthase.
[0067] SEQ ID NO:16 is the deduced amino acid sequence of an almost
entire alfalfa isoflavone synthase derived from the nucleotide
sequence of SEQ ID NO:15.
[0068] SEQ ID NO:17 is the nucleotide sequence comprising the hairy
vetch cDNA insert in clone hairy vetch1 encoding an almost entire
hairy vetch isoflavone synthase.
[0069] SEQ ID NO:18 is the deduced amino acid sequence of an almost
entire hairy vetch isoflavone synthase derived from the nucleotide
sequence of SEQ ID NO:17.
[0070] SEQ ID NO:19 is the nucleotide sequence comprising the
lentil cDNA insert in clone lentil1 encoding an almost entire
lentil isoflavone synthase.
[0071] SEQ ID NO:20 is the deduced amino acid sequence of an almost
entire lentil isoflavone synthase derived from the nucleotide
sequence of SEQ ID NO:19.
[0072] SEQ ID NO:21 is the nucleotide sequence comprising the
lentil cDNA insert in clone lentil2 encoding an almost entire
lentil isoflavone synthase.
[0073] SEQ ID NO:22 is the deduced amino acid sequence of an almost
entire lentil isoflavone synthase derived from the nucleotide
sequence of SEQ ID NO:21.
[0074] SEQ ID NO:23 is the nucleotide sequence comprising the mung
bean cDNA insert in clone mung bean1 encoding an entire mung bean
isoflavone synthase.
[0075] SEQ ID NO:24 is the deduced amino acid sequence of an entire
mung bean isoflavone synthase derived from SEQ ID NO:23.
[0076] SEQ ID NO:25 is the nucleotide sequence comprising the mung
bean cDNA insert in clone mung bean2 encoding an entire mung bean
isoflavone synthase.
[0077] SEQ ID NO:26 is the deduced amino acid sequence of an entire
mung bean isoflavone synthase derived from SEQ ID NO:25.
[0078] SEQ ID NO:27 is the nucleotide sequence comprising the mung
bean cDNA insert in clone mung bean3 encoding an entire mung bean
isoflavone synthase.
[0079] SEQ ID NO:28 is the deduced amino acid sequence of an entire
mung bean isoflavone synthase derived from SEQ ID NO:27.
[0080] SEQ ID NO:29 is the nucleotide sequence comprising the mung
bean cDNA insert in clone mung bean4 encoding an entire mung bean
isoflavone synthase.
[0081] SEQ ID NO:30 is the deduced amino acid sequence of an entire
mung bean isoflavone synthase derived from SEQ ID NO:30.
[0082] SEQ ID NO:31 is the nucleotide sequence comprising the red
clover cDNA insert in clone red clover1 encoding an entire red
clover isoflavone synthase.
[0083] SEQ ID NO:32 is the deduced amino acid sequence of an entire
red clover isoflavone synthase derived from SEQ ID NO:31.
[0084] SEQ ID NO:33 is the nucleotide sequence comprising the red
clover cDNA insert in clone red clover2 encoding an entire red
clover isoflavone synthase.
[0085] SEQ ID NO:34 is the deduced amino acid sequence of an entire
red clover isoflavone synthase derived from SEQ ID NO:33.
[0086] SEQ ID NO:35 is the nucleotide sequence comprising the snow
pea cDNA insert in clone snow peal encoding an entire snow pea
isoflavone synthase.
[0087] SEQ ID NO:36 is the deduced amino acid sequence of an entire
snow pea isoflavone synthase derived from SEQ ID NO:37.
[0088] SEQ ID NO:37 is the nucleotide sequence comprising the white
clover cDNA insert in clone white clover1 encoding an almost entire
white clover isoflavone synthase.
[0089] SEQ ID NO:38 is the deduced amino acid sequence of an almost
entire white clover isoflavone synthase derived from SEQ ID
NO:37.
[0090] SEQ ID NO:39 is the nucleotide sequence comprising the white
clover cDNA insert in clone white clover2 encoding an almost entire
white clover isoflavone synthase.
[0091] SEQ ID NO:40 is the deduced amino acid sequence of an almost
entire white clover isoflavone synthase derived from SEQ ID
NO:39.
[0092] SEQ ID NO:41 is the nucleotide sequence of an
oligonucleotide primer used for PCR amplification of the isoflavone
synthase coding region in clone sgs1c.pk006.o20. SEQ ID NO:42 is
the nucleotide sequence of an oligonucleotide primer used for PCR
amplification of the isoflavone synthase coding region in clone
sgs1c.pk006.o20.
[0093] SEQ ID NO:43 is the nucleotide sequence of an
oligonucleotide primer used to determine the transcription of the
soybean isoflavone synthase in transgenic tobacco.
[0094] SEQ ID NO:44 is the nucleotide sequence of an
oligonucleotide primer used to determine the transcription of the
soybean isoflavone synthase in transgenic tobacco.
[0095] SEQ ID NO:45 is the nucleotide sequence of an
oligonucleotide primer to the maize R coding region used to amplify
genomic DNA to determine the presence of a chimera containing the
maize R region between the region encoding the C1 DNA binding
domain and the C1 activation domain (CRC) in transgenic corn
cells.
[0096] SEQ ID NO:46 is the nucleotide sequence of an
oligonucleotide primer to the 3' untranslated region from potato
protease inhibitor II gene used to amplify genomic DNA to determine
the presence of CRC in transgenic corn cells.
[0097] SEQ ID NO:47 is the nucleotide sequence comprising the
sugarbeet cDNA insert in clone sugarbeet1, encoding an almost
entire sugarbeet isoflavone synthase.
[0098] SEQ ID NO:48 is the deduced amino acid sequence of an almost
entire sugarbeet isoflavone synthase derived from SEQ ID NO:47.
[0099] SEQ ID NO:49 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean isoflavone synthase coding region in clone
sgs1c.pk006.o20.
[0100] SEQ ID NO:50 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean isoflavone synthase coding region in clone
sgs1c.pk006.o20.
[0101] SEQ ID NO:51 is the nucleotide sequence of an
oligonucleotide primer used to amplify the genomic sequence
comprising the isoflavone synthase in clone sgs1c.pk006.o20.
[0102] SEQ ID NO:52 is the nucleotide sequence of a genomic
fragment encoding the isoflavone synthase in clone
sgs1c.pk006.o20.
[0103] SEQ ID NO:53 is the nucleotide sequence of a genomic
fragment encoding the CYP93C1 isoflavone synthase.
[0104] SEQ ID NO:54 is the nucleotide sequence comprising the
lupine cDNA insert in clone lupine1 encoding an entire lupine
isoflavone synthase.
[0105] SEQ ID NO:55 is the deduced amino acid sequence of an entire
lupine isoflavone synthase derived from SEQ ID NO:54.
[0106] SEQ ID NO:56 is the nucleotide sequence comprising the
alfalfa cDNA insert in clone alfalfa2 encoding an almost entire
alfalfa isoflavone synthase.
[0107] SEQ ID NO:57 is the amino acid sequence of an almost entire
alfalfa isoflavone synthase derived from SEQ ID NO:56.
[0108] SEQ ID NO:58 is the nucleotide sequence comprising the
alfalfa cDNA insert in clone alfalfa3 encoding an almost entire
alfalfa isoflavone synthase.
[0109] SEQ ID NO:59 is the amino acid sequence of an almost entire
alfalfa isoflavone synthase derived from SEQ ID NO:58.
[0110] SEQ ID NO:60 is the amino acid sequence comprising the
sugarbeet cDNA insert in clone sugarbeet2, encoding an almost
entire sugarbeet isoflavone synthase.
[0111] SEQ ID NO:61 is the deduced amino acid sequence of an almost
entire sugarbeet isoflavone synthase derived from SEQ ID NO:60.
[0112] SEQ ID NO:62 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean chalcone reductase coding region in clone
src3c.pk009.e4.
[0113] SEQ ID NO:63 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean chalcone reductase coding region in clone
src3c.pk009.e4.
[0114] SEQ ID NO:64 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean chalcone reductase present in monocot cells.
[0115] SEQ ID NO:65 is the nucleotide sequence of an
oligonucleotide primer used for the PCR amplification of the
soybean chalcone reductase present in monocot cells.
[0116] SEQ ID NO:66 is the amino acid sequence of the consensus
sequence produced by the Megalign Program using the Clustal method
and the amino acid sequences depicted in SEQ ID NOs:2, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 48, 55, 57, 59, and 61.
DETAILED DESCRIPTION OF THE INVENTION
[0117] The instant invention discloses nucleotide and amino acid
sequences for isoflavone synthases from legumes such as soybean,
alfalfa, lupine, hairy vetch, lentil, mung bean, red clover, snow
pea, and white clover and non-legumes such as sugarbeet. As the
enzyme that catalyzes the first step of the isoflavonoid branch of
the phenylpropanoid pathway (see FIG. 1), altering the level of
this enzyme may be useful for changing isoflavonoid content.
[0118] Plant P450 enzymes catalyze a diverse range of reactions,
including molecular transformations in primary metabolism, and in
the metabolism and detoxification of xenobiotics. Although
tentative identification of any given gene or conceptual
translation product as a P450 is relatively simple based on its
similarity to other known P450s, the assignment of actual catalytic
function cannot necessarily be inferred from nucleic acid or
protein sequence information alone. The instant disclosure
demonstrates and teaches the identification of a cDNA from soybean
that encodes isoflavone synthase based on the ability of the
encoded polypeptide to convert the normal substrate for the
reaction, 2S-flavanone, to genistein. Demonstration of activity has
been accomplished in subcellular fractions of a yeast strain, WHT1,
which has been specifically altered to also express a P450
reductase from Helianthus tuberosum. In this manner, and using the
materials identified and described herein, other nucleic acid
sequences from soybean and from other plants that are predicted to
encode P450s may be tested to determine whether any of those P450's
possess isoflavone synthase activity.
[0119] "The isoflavonoids are biogeneticaly related to the
flavonoids but constitute a distinctly separate class in that they
contain a rearranged C15 skeleton and may be regarded as
derivatives of 3-phenylchroman." Isoflavonoids. Dewick, P. M.
(1982) in The Flavonoids: Advances in Research, Harborne, J. B. and
Mabry, T. J., Ed., pp 535-640, Chapman and Hall Ltd, New York.
Oxidative rearrangement of a flavanone precursor with a 2,3-aryl
shift yields an isoflavonoid. Isoflavones are the most abundant of
the natural isoflavonoid derivatives, with over 160 isoflavone
aglycones being recognized.
[0120] In the context of this disclosure, a number of terms shall
be utilized. As used herein, a "nucleic acid sequence" is a polymer
of RNA or DNA that is single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases. A
nucleic acid sequence in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA or synthetic
DNA.
[0121] As used herein, "substantially similar" refers to nucleic
acid sequences wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the polypeptide encoded by the
nucleotide sequence. "Substantially similar" also refers to nucleic
acid sequences wherein changes in one or more nucleotide bases does
not affect the ability of the nucleic acid sequence to mediate
alteration of gene expression by gene silencing through for example
antisense or co-suppression technology. "Substantially similar"
also refers to modifications of the nucleic acid fragments of the
instant invention such as deletion or insertion of one or more
nucleotides that do not substantially affect the functional
properties of the resulting transcript vis-a-vis the ability to
mediate gene silencing or alteration of the functional properties
of the resulting protein molecule. It is therefore understood that
the invention encompasses more than the specific exemplary
nucleotide or amino acid sequences and includes functional
equivalents thereof.
[0122] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a nucleic acid sequence which
result in the production of a chemically equivalent amino acid at a
given site, but do not effect the functional properties of the
encoded polypeptide, are well known in the art. Thus, a codon for
the amino acid alanine, a hydrophobic amino acid, may be
substituted by a codon encoding another less hydrophobic residue,
such as glycine, or a more hydrophobic residue, such as valine,
leucine, or isoleucine. Similarly, changes which result in
substitution of one negatively charged residue for another, such as
aspartic acid for glutamic acid, or one positively charged residue
for another, such as lysine for arginine, can also be expected to
produce a functionally equivalent product. Nucleotide changes which
result in alteration of the N-terminal and C-terminal portions of
the polypeptide molecule would also not be expected to alter the
activity of the polypeptide. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
[0123] Moreover, substantially similar nucleic acid sequences may
also be characterized by their ability to hybridize. Estimates of
such homology are provided by either DNA-DNA or DNA-RNA
hybridization under conditions of stringency as is well understood
by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic
Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions
can be adjusted to screen for moderately similar sequences, such as
homologous sequences from distantly related organisms, to highly
similar sequences, such as genes that duplicate functional enzymes
from closely related organisms. Post-hybridization washes determine
stringency conditions. One set of preferred conditions uses a
series of washes starting with 6.times.SSC, 0.5% SDS at room
temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at
45.degree. C. for 30 min, and then repeated twice with
0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more
preferred set of stringent conditions uses higher temperatures in
which the washes are identical to those above except for the
temperature of the final two 30 min washes in 0.2.times.SSC, 0.5%
SDS was increased to 60.degree. C. Another preferred set of highly
stringent conditions uses two final washes in 0.1.times.SSC, 0.1%
SDS at 65.degree. C.
[0124] Substantially similar nucleic acid sequences of the instant
invention may also be characterized by their percent identity to
the nucleic acid sequences disclosed herein, as determined by
algorithms commonly employed by those skilled in this art.
Preferred are those nucleic acid sequences whose sequences are at
least about 85% identical and more preferably at least about 90%
identical to the nucleotide sequences reported herein. More
preferred are nucleic acid sequences that are at least about 90%
identical and more preferably at least about 95% identical to the
nucleotide sequences reported herein. More preferred are nucleic
acid sequences that are 95% identical to the nucleotide sequences
reported herein. Sequence alignments and percent identity
calculations were performed using the Megalign program of the
LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences was performed using the
Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 2, GAP PENALTY=5, WINDOW=4 and DIAGONALS
SAVED=4.
[0125] Substantially similar nucleic acid sequences of the instant
invention may also be characterized by the percent identity of the
amino acid sequences that they encode to the amino acid sequences
disclosed herein, as determined by algorithms commonly employed by
those skilled in this art. Preferred are those nucleic acid
sequences whose nucleotide sequences encode amino acid sequences
that are at least about 95% identical and even more preferably at
least about 98% identical to the amino acid sequences reported
herein. Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASARGENE
bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).
Multiple alignment of the sequences was performed using the Clustal
method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)
with the default parameters (GAP PENALTY-=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5.
[0126] A "substantial portion" of an amino acid or nucleotide
sequence comprises an amino acid or a nucleotide sequence that is
sufficient to afford putative identification of the protein or gene
that the amino acid or nucleotide sequence comprises. Amino acid
and nucleotide sequences can be evaluated either manually by one
skilled in the art, or by using computer-based sequence comparison
and identification tools that employ algorithms such as BLAST
(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol.
Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In
general, a sequence of ten or more contiguous amino acids or thirty
or more contiguous nucleotides is necessary in order to putatively
identify a polypeptide or nucleic acid sequence as homologous to a
known protein or gene. Moreover, with respect to nucleotide
sequences, gene-specific oligonucleotide probes comprising 30 or
more contiguous nucleotides may be used in sequence-dependent
methods of gene identification (e.g., Southern hybridization) and
isolation (e.g., in situ hybridization of bacterial colonies or
bacteriophage plaques). In addition, short oligonucleotides of 12
or more nucleotides may be used as amplification primers in PCR in
order to obtain a particular nucleic acid sequence comprising the
primers. Accordingly, a "substantial portion" of a nucleotide
sequence comprises a nucleotide sequence that will afford specific
identification and/or isolation of a nucleic acid sequence
comprising the sequence. The instant specification teaches amino
acid and nucleotide sequences encoding polypeptides that comprise
one or more particular plant proteins. The skilled artisan, having
the benefit of the sequences as reported herein, may now use all or
a substantial portion of the disclosed sequences for purposes known
to those skilled in this art. Accordingly, the instant invention
comprises the complete sequences as reported in the accompanying
Sequence Listing, as well as substantial portions of those
sequences as defined above.
[0127] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid sequence comprising a
nucleotide sequence that encodes all or a substantial portion of
the amino acid sequences set forth herein. The skilled artisan is
well aware of the "codon-bias" exhibited by a specific host cell in
usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a nucleic acid sequence for improved
expression in a host cell, it is desirable to design the nucleic
acid fragment such that its frequency of codon usage approaches the
frequency of preferred codon usage of the host cell.
[0128] "Synthetic nucleic acid fragments" can be assembled from
oligonucleotide building blocks that are chemically synthesized
using procedures known to those skilled in the art. These building
blocks are ligated and annealed to form larger nucleic acid
sequences which may then be enzymatically assembled to construct
the entire desired nucleic acid sequence. "Chemically synthesized",
as related to nucleic acid sequence, means that the component
nucleotides were assembled in vitro. Manual chemical synthesis of
nucleic acid sequences may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the nucleic acid sequences can be tailored for optimal gene
expression based on optimization of nucleotide sequence to reflect
the codon bias of the host cell. The skilled artisan appreciates
the likelihood of successful gene expression if codon usage is
biased towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0129] "Gene" refers to a nucleic acid sequence that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers any gene
that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0130] "Coding sequence" refers to a nucleotide sequence that codes
for a specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0131] "Promoter" refers to a nucleotide sequence capable of
controlling the expression of a coding sequence or functional RNA.
In general, a coding sequence is located 3' to a promoter sequence.
The promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a nucleotide sequence which can
stimulate promoter activity. It may be an innate element of the
promoter or a heterologous element inserted to enhance the level
and/or tissue-specificity of a promoter. Promoters may be derived
in their entirety from a native gene, or be composed of different
elements derived from different promoters found in nature, or even
comprise synthetic nucleotide segments. It is understood by those
skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental conditions. Promoters which cause a nucleic acid
sequence to be expressed in most cell types at most times are
commonly referred to as "constitutive promoters". "Organ-specific"
or "development-specific" promoters are those that direct gene
expression almost exclusively in specific organs, such as leaves or
seeds, or at specific development stages in an organ, such as in
early or late embryogenesis, respectively. New promoters of various
types useful in plant cells are constantly being discovered;
numerous examples may be found in the compilation by Okamuro and
Goldberg (1989) Biochemistry of Plants 15:1-82. It is further
recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, nucleic acid
sequences of different lengths may have identical promoter
activity.
[0132] The expression of foreign genes in plants is well
established (De Blaere et al. (1987) Meth. Enzymol. 143:277-291).
Proper level of expression of mRNAs may require the use of
different chimeric genes utilizing different promoters. Such
chimeric genes can be transferred into host plants either together
in a single expression vector or sequentially using more than one
vector. Expression in plants will use regulatory sequences
functional in such plants.
[0133] The origin of the promoter chosen to drive the expression of
the coding sequence is not critical as long as it has sufficient
transcriptional activity to accomplish the invention by expressing
translatable mRNA for the desired protein genes in the desired host
tissue.
[0134] The "translation leader sequence" refers to a nucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Molecular Biotechnology
3:225-236).
[0135] The "3' non-coding sequences" refer to nucleotide sequences
located downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.
[0136] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into polypeptide by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to an RNA transcript that includes the mRNA and
so can be translated into a polypeptide by the cell. "Antisense
RNA" refers to an RNA transcript that is complementary to all or
part of a target primary transcript or mRNA and that blocks the
expression of a target gene (see U.S. Pat. No. 5,107,065,
incorporated herein by reference). The complementarity of an
antisense RNA may be with any part of the specific nucleotide
sequence, i.e., at the 5' non-coding sequence, 3' non-coding
sequence, introns, or the coding sequence. "Functional RNA" refers
to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may
not be translated but yet has an effect on cellular processes.
[0137] The term "operably linked" refers to the association of two
or more nucleic acid sequences on a single nucleic acid sequence so
that the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0138] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid sequence of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0139] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0140] "Transformation" refers to the transfer of a nucleic acid
sequence into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0141] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989
(hereinafter "Sambrook").
[0142] A nucleic acid sequence encoding a soybean isoflavone
synthase was isolated and identified from a cDNA library. Nucleic
acid sequences encoding three alfalfa, one hairy vetch, one snow
pea, one lupine, two lentil, two red clover, two white clover, two
sugarbeet, and four mung bean isoflavone synthases have been
isolated-using RT-PCR. Nucleic acid sequences encoding two soybean
isoflavone synthases have been isolated from genomic DNA. The
nucleic acid sequences of the instant invention may be used to
isolate cDNAs and genes encoding homologous enzymes from the same
or other plant species. Isolation of homologous genes using
sequence-dependent protocols is well known in the art. Examples of
sequence-dependent protocols include, but are not limited to,
methods of nucleic acid hybridization, and methods of DNA and RNA
amplification as exemplified by various uses of nucleic acid
amplification technologies (e.g., polymerase chain reaction, ligase
chain reaction).
[0143] For example, genes encoding other isoflavone synthase
proteins, either as cDNAs or genomic DNAs, could be isolated
directly by using all or a portion of the instant nucleic acid
sequence as aDNA hybridization probe to screen libraries from any
desired plant employing methodology well known to those skilled in
the art. Specific oligonucleotide probes based upon the instant
nucleic acid sequence can be designed and synthesized by methods
known in the art (Sambrook). Moreover, the entire sequence can be
used directly to synthesize DNA probes by methods known to the
skilled artisan such as random primers DNA labeling, nick
translation, or end-labeling techniques, or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part of or
full-length of the instant sequences. The resulting amplification
products can be labeled directly during amplification reactions or
labeled after amplification reactions, and used as probes to
isolate full-length cDNA or genomic fragments under conditions of
appropriate stringency.
[0144] In addition, two short segments of the instant nucleic acid
sequences may be used in polymerase chain reaction protocols to
amplify longer nucleic acid sequences encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid sequences wherein the
sequence of one primer is derived from the instant nucleic acid
sequences, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA
85:8998-9002) to generate cDNAs by using PCR to amplify copies of
the region between a single point in the transcript and the 3' or
5' end. Primers oriented in the 3' and 5' directions can be
designed from the instant sequences. Using commercially available
3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA sequences
can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA
86:5673-5677; Loh et al. (1989) Science 243:217-220). Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin (1989) Techniques
1:165).
[0145] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner (1984) Adv. Immunol. 36:1; Sambrook).
[0146] The nucleic acid sequence of the instant invention may be
used to create transgenic plants and transgenic seeds in which
expression of nucleic acid sequences (or their complements)
encoding the disclosed enzyme result in levels of the corresponding
endogenous enzyme that are higher or lower than normal.
Alternatively, expression of the instant nucleic acid sequence may
result in the production of the encoded enzyme in cell types or
developmental stages in which they are not normally found. Either
strategy would have the effect of altering the level of
isoflavonoids.
[0147] For example, overexpression of isoflavone synthase may
result in an increase in isoflavonoid content in legumes. Increased
isoflavonoid content in legumes has been shown to be associated
with beneficial health effects in humans. In contrast, certain soy
food products would benefit from lower levels of isoflavonoid due
to adverse effects on flavor.
[0148] Overexpression of the proteins of the instant invention may
be accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. The chimeric gene may comprise promoter sequences and
translation leader sequences derived from the same genes. 3'
Non-coding sequences encoding transcription termination signals may
also be provided. The instant chimeric gene may also comprise one
or more introns in order to facilitate gene expression.
[0149] Plasmid vectors comprising the isolated polynucleotide (or
chimeric gene) may be constructed. The choice of plasmid vector is
dependent upon the method that will be used to transform host
plants. The skilled artisan is well aware of the genetic elements
that must be present on the plasmid vector in order to successfully
transform, select and propagate host cells containing the chimeric
gene. The skilled artisan will also recognize that different
independent transformation events will result in different levels
and patterns of expression (Jones et al. (1985) EMBO J.
4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics
218:78-86), and thus that multiple events must be screened in order
to obtain lines displaying the desired expression level and
pattern. Such screening may be accomplished by Southern analysis of
DNA, Northern analysis of mRNA expression, Western analysis of
protein expression, or phenotypic analysis.
[0150] The nucleic acid sequence of the instant invention may be
used to create transgenic plants that have increased expression of
the disclosed enzyme and that are additionally transformed with a
chimeric gene encoding a transcription factor that regulates
expression of one or more genes in the phenylpropanoid pathway. The
chimeric transcription factor gene has regulatory sequences such
that its expression is coordinated with that of the isoflavone
synthase gene developmentally and preferably within the same cell
type. This combination of expression of isoflavone synthase and
transcription factor regulating phenylpropanoid pathway genes has
the effect of enhancing the level of isoflavonoid synthesis due to
increased levels of substrates for isoflavone synthase. The
chimeric transcription factor gene regulates expression of at least
one gene in the phenylpropanoid pathway. While not intending to be
bound by any theory or theories of operation it is believed to
regulate as many as two, three or four genes in the phenylpropanoid
pathway.
[0151] For example, a plant cell that does not naturally produce
isoflavonoids and does not have an active phenylpropanoid pathway
would not produce the substrates for isoflavone synthase to convert
to isoflavonoids. Activation of the phenylpropanoid pathway in the
desired cells or at the desired developmental stage would provide
these substrates allowing the synthesis of isoflavonoids.
[0152] The present invention is also directed to a method of
altering the level of isoflavonoids in a cell comprising exposing
said cell to a phenylpropanoid pathway altering agent. The cell may
be a plant cell such as a monocot, including and not limited to
corn, or a dicot, such as soybean, for example. A phenylpropanoid
pathway altering agent may be any agent that results in an increase
or decrease in the level of expression of an enzyme in the
phenylpropanoid pathway, such as isoflavone synthase, phenylalanine
ammonia lyase, chalcone synthase, among others. Such
phenylpropanoid pathway altering agents include and are not limited
to a transcription factor and stress. Transcription factors include
and are not limited to chimeric transcription factors, a chimera
containing the maize R region between the region encoding the C1
DNA binding domain and the C1 activation domain (CRC) for example.
Stresses to a plant cell include ultraviolet light, temperature,
pressure, chemicals including and not limited to herbicides, and
phosphate level. Phosphate levels may be increased or decreased
such that decreasing phosphate levels may result in phosphate
starvation.
[0153] It may also be desirable to reduce or eliminate expression
of genes encoding the instant polypeptides in plants for some
applications. In order to accomplish this, a chimeric gene designed
for co-suppression of the instant polypeptide can be constructed by
linking a gene or gene sequence encoding that polypeptide to plant
promoter sequences. Alternatively, a chimeric gene designed to
express antisense RNA for all or part of the instant nucleic acid
sequence can be constructed by linking the gene or gene sequence in
reverse orientation to plant promoter sequences. Either the
co-suppression or antisense chimeric genes could be introduced into
plants via transformation wherein expression of the corresponding
endogenous genes are reduced or eliminated.
[0154] Molecular genetic solutions to the generation of plants with
altered gene expression have a decided advantage over more
traditional plant breeding approaches. Changes in plant phenotypes
can be produced by specifically inhibiting expression of one or
more genes by antisense inhibition or cosuppression (U.S. Pat. Nos.
5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression
construct would act as a dominant negative regulator of gene
activity. While conventional mutations can yield negative
regulation of gene activity these effects are most likely
recessive. The dominant negative regulation available with a
transgenic approach may be advantageous from a breeding
perspective. In addition, the ability to restrict the expression of
specific phenotype to the reproductive tissues of the plant by the
use of tissue specific promoters may confer agronomic advantages
relative to conventional mutations which may have an effect in all
tissues in which a mutant gene is ordinarily expressed.
[0155] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppresion technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
sense or antisense genes may require the use of different chimeric
genes utilizing different regulatory elements known to the skilled
artisan. Once transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenics for those that most effectively display the desired
phenotype. Accordingly, the skilled artisan will develop methods
for screening large numbers of transformants. The nature of these
screens will generally be chosen on practical grounds. For example,
one can screen by looking for changes in gene expression by using
antibodies specific for the protein encoded by the gene being
suppressed, or one could establish assays that specifically measure
enzyme activity. A preferred method will be one which allows large
numbers of samples to be processed rapidly, since it will be
expected that a large number of transformants will be negative for
the desired phenotype.
[0156] The instant isoflavone synthases (or portions of the
enzymes) may be produced in heterologous host cells, particularly
in the cells of microbial hosts, and can be used to prepare
antibodies to the enzymes by methods well known to those skilled in
the art. The antibodies are useful for detecting the enzymes in
situ in cells or in vitro in cell extracts. Preferred heterologous
host cells for production of isoflavone synthase are yeast hosts.
Yeast expression systems and expression vectors containing
regulatory sequences that direct high level expression of foreign
proteins are well known to those skilled in the art. Any of these
could be used to construct chimeric genes for production of the
instant isoflavone synthase. These chimeric genes could then be
introduced into appropriate hosts via transformation to provide
high level expression of the enzymes. An example of a vector for
high level expression of the instant isoflavone synthase in a yeast
host is provided (Example 5).
[0157] All or a substantial portion of the nucleic acid sequences
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid sequences may be
used as restriction sequence length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid sequences of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al. (1987) Genomics 1: 174-181) in order to construct a genetic
map. In addition, the nucleic acid sequences of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein et
al. (1980) Am. J. Hum. Genet. 32:314-331).
[0158] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bematzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications
describe genetic mapping of specific cDNA clones using the
methodology outlined above or variations thereof. For example, F2
intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
[0159] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel et al. In: Nonmammalian
Genomic Analysis: A Practical Guide, Academic press 1996, pp.
319-346, and references cited therein).
[0160] In another embodiment, nucleic acid probes derived from the
instant nucleic acid sequences may be used in direct fluorescence
in situ hybridization (FISH) mapping (Trask (1991) Trends Genet.
7:149-154). Although current methods of FISH mapping favor use of
large clones (several to several hundred KB; see Laan et al. (1995)
Genome Research 5:13-20), improvements in sensitivity may allow
performance of FISH mapping using shorter probes.
[0161] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian (1989) J Lab. Clin. Med 114(2):95-96),
polymorphism of PCR-amplified fragments (CAPS; Sheffield et al.
(1993) Genomics 16:325-332), allele-specific ligation (Landegren et
al. (1988) Science 241:1077-1080), nucleotide extension reactions
(Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid
Mapping (Walter et al. (1997) Nature Genetics 7:22-28) and Happy
Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For
these methods, the sequence of a nucleic acid fragment is used to
design and produce primer pairs for use in the amplification
reaction or in primer extension reactions. The design of such
primers is well known to those skilled in the art. In methods
employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0162] The physiological activities associated with isoflavonoids
in both plants and humans makes the manipulation of their contents
in crop plants highly desirable. For example, increasing levels of
isoflavonoids in soybean seeds would increase the efficiency of
extraction and lower the cost of isoflavonoid-related products
sold. Decreasing levels of isoflavonoids in soybean seeds would be
beneficial for production of soy-based infant formulas where the
estrogenic effects of isoflavonoids are undesirable. Decreasing
levels of isoflavonoids may also increase palatability of soy
foods. Raising levels of isoflavonoid phytoalexins in vegetative
plant tissue could increase plant defenses to pathogen attack,
thereby improving resistance and lowering the need for pesticide
use. Manipulation of isoflavonoid levels in roots could lead to
improved nodulation and increased efficiencies of nitrogen
fixation. To date, however, it has proven difficult to develop
soybean or other plant lines with consistently high levels of
isoflavonoids.
[0163] Identification of the functional isoflavone synthase gene is
extremely important because isoflavone synthase catalyzes the
central reaction in pathways producing isoflavonoids. Manipulation
of the isoflavone synthase gene via molecular techniques is
expected to allow production of soybeans and other plants with
high, stable levels of isoflavonoids. Introduction of the
isoflavone synthase gene in non-legume crop species including, but
not limited to, corn, wheat, rice, sunflower, and canola could lead
to synthesis of isoflavonoids in these species. Synthesis of
isoflavonoids would 1) confer disease resistance to the crops
and/or 2) produce crops which would benefit human and/or livestock
health.
EXAMPLES
[0164] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
Example 1
Microsome Preparation from Elicitor-Treated Soybean Hypocotyls and
Elicitor-Treated Cell Suspension Culture
Elicitor Treatment of Soybean Seeds
[0165] Soybean seeds were placed on a bed of vermiculite (5 to 6 cm
thick) and covered with a layer of vermiculite about 2 cm thick.
Seeds were germinated for five days in a growth chamber until the
average length of hypocotyls reached to about 3 to 4 cm. The growth
chamber was kept at a cycle that consisted of a 14 h light period
at 25.degree. C. and a 10 h dark period at 21.degree. C.
Illumination was supplied from cool white fluorescent and
incandescent lamps that provide a photon flux density of 450
.mu.Em.sup.-2s.sup.-1. Soybean hypocotyls were pulled out from the
vermiculite bed and were placed on wet paper towels. The soybean
hypocotyls were divided into two groups: one of the groups was
treated with elicitor and the other was not treated.
[0166] Elicitor treatment was conducted as follows. The epidermal
surfaces of the hypocotyls were opened using a razor blade. The
incisions were approximately 2 cm long and 1 to 2 mm deep; one was
made on each hypocotyl. Fungal-derived elictors were prepared by
the method of Sharp et al. (Sharp, J. K. et al. (1984) J. Biol.
Chem. 259:11312-11320). Twenty micrograms of acidified fungal
elicitors were dissolved in 20 .mu.L of 10 mM KH.sub.2PO.sub.4, and
were then applied to the wound of a hypocotyl The treated
hypocotyls were incubated for 15 h in the dark at room temperature
and 100% humidity. At the end of the incubation period, the
hypocotyls were sectioned closely below the cotyledonal node and
were immediately frozen in liquid nitrogen and stored at
-76.degree. C. until used. Non-elicitor-treated hypocotyls were
handled in the same manner as were elicitor-treated hypocotyls,
except for wounding and elicitor application. The non-treated
hypocotyls were used as a negative control of isoflavone synthase
induction.
Elicitor Treatment of Soybean Cell Suspension Culture
[0167] Soybean suspension cell cultures were grown at 25.degree. C.
in 250 mL flasks that were tightly covered with two layers of
aluminum foil to prevent illumination. Cells were grown in 35 mL of
Murashige and Skoog medium (Gibco BRL) supplemented with 0.75 mg/L
2,4-dichlorophenoxyacetic acid and 0.55 mg/mL 6-benzyl aminopurine.
Cells were diluted (1:3 ratio) into fresh medium every 7 days and
elicitor treatment was conducted 3 days after cell dilution. One
hundred fifty milligrams of the same fungal elicitor used to treat
the hypocotyls was dissolved in 15 mL of 10 mM KH.sub.2PO.sub.4 and
was filter sterilized. Five milligrams of sterile fungal elicitor
dissolved in 333 .mu.L 10 mM KH.sub.2PO.sub.4 was added per flask.
Cells were harvested 15 h after addition of elicitor. The same
suspension culture conditions were used before and after elicitor
treatment. Cells were recovered using a Nalgene PES filter unit
(0.2 .mu.m) followed by 3 minutes of air flow. Filtered cells were
immediately frozen in liquid nitrogen and kept at -76.degree. C.
until used. Non-elicitor-treated cells were handled in the same
manner, except for the addition of elicitor.
Microsome Preparation from Soybean Hypocotyls and
Suspension-Cultured Cells
[0168] For preparation of the crude extracts, 3 to 5 g of
previously frozen, elicitor-treated and non-treated soybean
hypocotyls and elicitor-treated and non-treated suspension cultured
cells were ground in liquid nitrogen using a pre-chilled pestle and
mortar. The powder was added to 25 mL of extraction buffer (buffer
A: 0.1M Tris-HCl, pH 7.5, 14 mM .beta.-mercaptoethanol, 20% (w/v)
sucrose and 0.8 g of Dowex 1X2 resin (mesh 200-400)), and the
slurry was stirred for 20 to 30 minutes in an ice-water bath. The
slurry was transferred to Nalgene Oak Ridge tubes and centrifuged
at 8000 g for 10 minutes at 4.degree. C. The supernate was
carefully transferred into 13 mL polyallomer tubes which fit into a
Sorvall TH641 rotor and centrifuged at 160,000 g for 40 minutes to
2 h at 4.degree. C. The precipitated microsomes were washed twice
with the storage buffer (buffer B: 80 mM KH.sub.2PO.sub.4, pH 8.5,
14 mM .beta.-mercaptoethanol, 30% (v/v) glycerol) and resuspended
with storage buffer. The microsomal pellet was gently homogenized
by hand using a disposable plastic pestle, and the suspension was
divided into several aliquots which were frozen on dry-ice.
Bradford protein micro assays were used to quantify the protein
content of the microsomal preparations (Bio-Rad, Richmond, Calif.).
Two microliters of a microsome preparation were diluted with 198
.mu.L of distilled water. Forty microliters of this dilution was
mixed with 10 .mu.L of Bio-Rad protein assay solution in a
microtiter plate, and the total protein concentration was
determined by reading the sample in a kinetic microplate reader
(Molecular Devices Inc.), according to the manufacturer's
instructions (Bio-Rad). Microsomes were stored at -76.degree. C.
until used.
Example 2
Development of Isoflavone Synthase Assay
[0169] An assay to measure isoflavone synthase activity was
developed using either of the two substrates of isoflavone
synthase, (.+-.) naringenin (4',5,7-trihydroxyflavanone; Sigma,
N-5893) or liquiritigenin monohydrate (4',7-dihydroxyflavanone;
Indofine, 02-1150S), dissolved in 80% ethanol. The reaction mixture
was prepared at room temperature and consisted of 100 .mu.M
naringenin or liquiritigenin, 80 mM K.sub.2HPO.sub.4, 0.5 mM
glutathione (Sigma, G-4251), 20% w/v sucrose, and 30 to 150 .mu.g
of microsome preparation. The reaction mixtures were preincubated
for 5 minutes without NADPH (synthesis of genistein and daidzein
requires NADPH as a co-factor). The volume of microsomes and
substrate added to any one reaction did not exceed 5% and 1%,
respectively, of the total reaction volume. A typical reaction
volume was 250 .mu.L. The reaction was started by the addition of
40 nmol of NADPH per each 100 .mu.L of final reaction volume. The
pH of the reaction mixture was 8.0 before the addition of the
substrate, NADPH and microsomes.
[0170] Microsomes were thawed, an aliquot removed and the remaining
sample was immediately frozen on dry ice and stored in the freezer.
The reactions using microsomes prepared from soybean
elicitor-treated hypocotyls were run for incubation periods of up
to 24 h, while the reactions using the yeast microsomes were
allowed to run for incubation periods of up to 14 h. Following
incubation, 200 .mu.L of ethyl acetate was added directly to the
mixture and the mixture was shaken for 1 minute using a vortex
mixer. Separation of the organic phase was accelerated by
centrifugation for 2 minutes at 4.degree. C. The organic phase was
removed and analyzed.
[0171] Qualitative and quantitative analyses were performed using a
Hewlett Packard 1100 series HPLC and a Hewlett-Packard/Micromass
LC/MS. Samples were assayed on a Hewlett Packard 1100 series HPLC
system using either a Li-Chrospher 100 RP-18 column (5 .mu.m) or a
Phenomenex Luna 3u C18 (2) column (150.times.4.6 mm). Using either
column, samples from in vitro microsome assays in ethyl acetate,
were isocratically separated for 5 minutes employing 65% methanol
as the mobile phase. The second column was used for plant samples
where the ethyl acetate was evaporated and the samples resuspended
in 80% methanol. In these cases separation used a 10 minutes linear
gradient from 20% methanol/80% 10 mM ammonium acetate, pH 8.3 to
100% methanol using a flow rate of 0.8 ml per minute. Genistein and
daidzein were monitored by the absorbance at 260 mm and naringenin
and liquiritigenin were monitored by the absorbance at 280 nm. Peak
areas were converted to nanograms using, as standards for
calibration, authentic naringenin, liquiritigenin, genistein, and
daidzein (Indofine Chemical Company, Inc., Somerville, N.J.)
dissolved in ethanol.
[0172] Analyses using LC/MS employed 10 .mu.L of the ethyl acetate
phase that had been first evaporated with nitrogen gas and
resuspended in 100 .mu.L of 25% acetonitrile in water. These
samples were analyzed by a Hewlett-Packard/Micromass LC/MS
instrument. A twenty-five microliter sample was run on a Zorbax
Eclipse XDB-C8 reverse-phase column (3.times.150 mm, 3.5 micron)
isocratically with 25% of solvent B in solvent A. Solvent A was
0.1% formic acid in water, and solvent B was 0.1% formic acid in
acetonitrile. Mass spectrometry was carried out by electro-spray
scanning from 200-400 m/e, using +60 volt cone voltage. The diode
array signals were monitored between 200-400 nm in both
instruments.
[0173] The genistein and liquiritigenin signals observed in the in
vitro assay samples were verified by comparisons of retention time,
diode array detected absorption spectra and mass spectrometry data
to the standards. FIG. 2 presents the results of HPLC analyses of
naringenin standards and FIG. 3 presents the results of HPLC
analyses of genistein standards.
[0174] Incubations in the absence of an essential component
required for isoflavone synthase-catalyzed synthesis of
isoflavonoid (e.g., NADPH, naringenin, liquiritigenin, or
microsomes) were performed as negative controls.
[0175] Positive control samples consisting of soybean microsomes
which were prepared from elicitor-treated hypocotyls and suspension
culture cells were used to establish the in vitro assay system.
Optimization of this in vitro assay system was critical for
validation of the yeast expression system for functional cloning.
We observed positive results (i.e., the synthesis of genistein) in
assays that used either the microsomes of elicitor-treated soybean
hypocotyls (FIG. 4) or those obtained from elicitor-treated cell
suspension cultures (FIG. 6). We observed about six times higher
specific enzyme activities of isoflavone synthase in the microsomes
of elicitor-treated hypocotyls and cell cultures (FIG. 4 and FIG.
6, respectively) than in the microsomes obtained from non-treated
hypocotyls and cell cultures (FIG. 5 and FIG. 7, respectively).
Example 3
Composition of Soybean cDNA Library, Isolation and Sequencing of
cDNA Clone
[0176] A cDNA library was prepared using mRNAs from soybean seeds
that had been allowed to germinate for 4 hours. The library was
prepared in Uni-ZAP.TM. XR vector according to the manufacturer's
protocol (Stratagene Cloning Systems, La Jolla, Calif.). Conversion
of the Uni-ZAP.TM. XR library into a plasmid library was
accomplished according to the protocol provided by Stratagene. Upon
conversion, cDNA inserts were contained in the plasmid vector
pBluescript. cDNA inserts from randomly picked bacterial colonies
containing recombinant pBluescript plasmids were amplified via
polymerase chain reaction using primers specific for vector
sequences flanking the inserted cDNA sequences or plasmid DNA was
prepared from cultured bacterial cells. Amplified insert DNAs or
plasmid DNAs were sequenced in dye-primer sequencing reactions to
generate partial cDNA sequences (expressed sequence tags or "ESTs";
see Adams, M. D. et al. (1991) Science 252:1651-1656). The
resulting ESTs were analyzed using a Perkin Elmer Model 377
fluorescent sequencer.
Example 4
Identification and Characterization of a cDNA Clone for Isoflavone
Synthase
[0177] ESTs encoding candidate isoflavone synthases were identified
by conducting BLAST (Basic Local Alignment Search Tool; Altschul,
S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences obtained in Example 3 were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm provided by the
National Center for Biotechnology Information (NCBI). The DNA
sequences were translated in all reading frames and compared for
similarity to all publicly available protein sequences contained in
the "nr" database using the BLASTX algorithm (Gish, W. and States,
D. J. (1993) Nature Genetics 3:266-272) provided by the NCBI.
[0178] The insert in cDNA clone sgs1c.pk006.o20 was identified as a
candidate isoflavone synthase gene by a BLAST search against the
NCBI database. The 5' sequence of this insert was determined to be
related to Glycine max cytochrome P450 monooxygenase CYP93C1p
(CYP93C1) mRNA, the complete coding sequence of which may be found
as NCBI General Identifier No. 2739005. The CYP93C1p cDNA sequence
was obtained using random isolation and screening to identify
soybean P450s involved in herbicide metabolism (Siminszky B., et
al. (1999) Proc. Natl. Acad. Sci. USA. 96:1750-1755). Isoflavone
synthase catalyzes in soybeans the oxidation of
7,4'dihyroxyflavanone (liquiritigenein) or
5,7,4'trihydroxyflananone (naringenin) to daidzein or genistein
respectively. Earlier published work (Kochs and Griesbach (1986)
Eur. J. Biochem 155:311-318; Hashim et al. (1990) FEBS 271:219-222)
suggested that the enzyme that catalyzes this reaction is a
cytochrome P450. Accordingly, in order to confirm the identity of
the polypeptide encoded by the insert in cDNA clone sgs1c.pk006.o20
as an isoflavone synthase, the polypeptide encoded by this insert
was evaluated for its ability to catalyze the formation of
genistein from naringenin.
[0179] The ability of the cDNA insert in clone sgs1c.pk006.o20 to
encode an isoflavone synthase was evaluated by expression of the
encoded polypeptide in an engineered yeast (Saccharomyces
cerivisae) strain. Microsomes prepared from the engineered yeast
strain transformed with a plasmid encoding the putative isoflavone
synthase were assayed for their ability to mediate the synthesis of
genistein in the presence of substrate (naringenin).
[0180] Yeast strain W303-1B was used as the starting material and
modified by homologous recombination. The coding sequence of the
P450 reductase HT1 isolated from Helianthus tuberosus (NCBI General
Identifier No. 1359894) was inserted into the integrative plasmid
pYeDP110 (Pompon, D. et al. (1996) Meth. Enz. 272:51-64). Insertion
was achieved after PCR amplification for addition of Bam HI and Eco
RI restriction sites 5' and 3' of the coding region, respectively,
using the primers listed as SEQ ID NO:3 and SEQ ID NO:4.
TABLE-US-00002 [SEQ ID NO:3] 5'-CGGGATCCATGCAACCGGAAACGGTCG-3' [SEQ
ID NO:4] 5'-CCGGAATTCTCACCAAACATCACGGAGGTATG-3'
[0181] Transformation of W303-1B with the linearized plasmid led to
homologous recombination with the promoter and terminator sequences
of the endogenous yeast reductase (CPR1) resulting in the
disruption of the CPR1 gene and replacement with the URA3 gene and
HT1 under the control of the galactose-inducible promoter
GAL10-CYC1. The resulting strain is designated WHT1.
[0182] Plasmid DNA (200 ng) from cDNA clone sgs1c.pk006.o20 was
used as template for PCR with primers that are homologous to the
vector sequences flanking the cDNA cloning site (SEQ ID NO:5 and
SEQ ID NO:6). TABLE-US-00003 [SEQ ID NO:5]
5'-TCAAGGAGAAAAAACCCCGGATCCATGTTGCTGGAACTTGCAC TTGG-3' [SEQ ID
NO:6] 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGCG-3'
[0183] Amplification was performed using the GC melt kit (Clontech)
with a 1 M final concentration of GC melt reagent. Amplification
took place in a Perkin Elmer 9700 thermocycler for 30 cycles as
follows: 94.degree. C. for 30 seconds, 60.degree. C. for 30
seconds, and 72.degree. C. for 1 minute. The amplified insert was
then incubated with a modified pRS315 plasmid (NCBI General
Identifier No. 984798; Sikorski, R. S. and Hieter, P. (1989)
Genetics 122:19-27) that had been digested with Not I and Spe I.
Plasmid pRS315 had been previously modified by the insertion of a
bidirectional gal 1/10 promoter between the Xho I and Hind III
sites. The plasmid was then transformed into the WHT1 yeast strain
using standard procedures. The insert recombines though gap repair
to form the desired plasmid (Hua, S. B., et al. (1997) Plasmid
38:91-96.). The resulting transformed yeast strain is named
Isoflavone Synthase GM1 (hereinafter referred to as "GM1"), and
bears ATCC Accession No. 203606.
[0184] Yeast microsomes were prepared according to the methods of
Pompon et al. (Pompon, D., et al. (1996) Meth. Enz. 272:51-64).
Briefly, a yeast colony was grown overnight (to saturation) in SG
(-Leucine) medium at 30.degree. C. with good aeration. A 1:50
dilution of this culture was made into 500 mL of YPGE medium with
adenine supplementation and allowed to grow at 30.degree. C. with
good aeration to an OD.sub.600 of 1.6 (24-30 h). Fifty mL of 20%
galactose was added, and the culture was allowed to grow overnight
at 30.degree. C. The cells were recovered by centrifugation at
5,500 rpm for five minutes in a Sorvall GS-3 rotor. The cell pellet
was resuspended in 80 mL of TEK buffer (0.1M KCl in TE) and left at
room temperature for five minutes. The cells were recovered by
centrifugation as described above. The cell pellet was resuspended
in 5 mL of TES-B (0.6M sorbitol in TE), and glass beads (0.5 mm
diameter) were gently added until they reached the surface of the
suspension. The cells were disrupted by shaking up and down for
five minutes, with an agitation frequency of at least once every
0.5 second. Five mL of TES-B were added to the crude extract, and
the beads were washed with some agitation. The supernatant was
withdrawn and saved. The wash was repeated twice and the liquid
fractions were pooled. The combined fractions were clarified by
spinning at 11,000 rpm in a Sorvall SS34 rotor. The pellet was
discarded and the microsomes were precipitated by the addition of
NaCl to a final concentration of 0.15 M. PEG 4000 was added to a
final concentration of 0.1 g/mL. The mixture was incubated on ice
for at least 15 minutes, and the microsomal fraction was recovered
by at 8,500 rpm for 10 minutes in an SS34 rotor. The pellets were
resuspended in TEG (glycerol, 20% by volume, in TE) at a
concentration of 20-40 mgs of protein per mL at which point they
may be stored at -70.degree. C. for months without any detectable
loss of activity.
Example 5
Demonstration of Functional Expression of Isoflavone Synthase in
Yeast
[0185] The synthesis of genistein or daidzein from either
naringenin or liquiritigenin was observed in an in vitro assay that
was mediated by yeast microsomes prepared from the yeast
transformant GM1 expressing the polypeptide encoded by the insert
in soybean cDNA clone sgs1c.pk006.o20. Samples were prepared and
run on a LiChrospher 100 RP-18 column (5 .mu.m) or a Phenomenex
Luna 3u C18 (2) column (150.times.4.6 mm) as described in Example
2. Peaks in the yeast microsome assay samples were identified as
being genistein or daidzein by their HPLC retention time and
absorption spectrum. The retention time and the absorption spectrum
of the peak found in the expected location of genistein was
identical to the retention time and spectrum of authentic genistein
(compare FIGS. 3 and 4, FIGS. 17 and 18). The daidzein peak also
had identical retention time and absorption spectrum to the
standard. More direct evidence was obtained using LC/MS. Data for
daidzein is shown in FIG. 19. The molecular weights of the
materials corresponding to the expected genistein and daidzein
peaks from the yeast microsome assay samples were 270.32 and 255.2,
respectively. The molecular weights of authentic genistein and
daidzein are 270.23 and 255.2, respectively.
[0186] The synthesis of genistein in yeast microsomes obtained from
the yeast strain Isoflavone Synthase GM1 was monitored over the
course of incubation with the substrate naringenin. Samples
representing incubation periods of 0 minutes and 1, 2, 3, 4 and 14
h were analyzed. Results are presented in FIGS. 8 through 13. A
simultaneous increase of genistein, the product, and decrease of
naringenin, the substrate of isoflavone synthase, was observed. A
detectable amount of genistein was synthesized as early as 40
minutes (FIG. 14). Incubation of microsomes with either naringenin
or liquiritigenin as substrate shows an increase in accumulation of
genistein and daidzein (the product) over ten hours as seen in FIG.
26.
[0187] Genistein synthesis corresponds quantitatively with the
amount of input GM1 microsomes (FIG. 14 and FIG. 15). The genistein
peak in the assay using GM1 as a source was about 10 times higher
than the peak observed from soybean microsome prepared from
elicitor-treated hypocotyls (compare FIG. 4 and FIG. 13). Genistein
synthesis by yeast microsomes using GM1 also demonstrated an
absolute requirement for NADPH. Without the cofactor, the reaction
mixture did not synthesize any detectable genistein over a 4-h
incubation (FIG. 16).
[0188] An unidentified peak, designated "peak 2," with a retention
time of 1.59, was also detected during monitoring of reactions
catalyzed by yeast microsomes at 280 nm (see FIG. 9 to FIG. 15).
This peak was not significant in negative controls (FIG. 8 and FIG.
16). Koch and Grisebach proposed a hypothesis for the synthesis of
an intermediate during the conversion of naringenin to genistein
(Kochs, G. and Grisenbach, H. (1985) Eur. J. Biochem. 155:311-318).
This proposal stated that the oxidative aryl migration required to
convert naringenin to genistein proceeds via a cytochrome P450
monooxygenase-mediated conversion of the 2S-flavanone to a
2-hydroxyisoflavone, followed by dehydration to the isoflavonoid,
possibly mediated by a soluble dehydratase. The 2-hydroxyisoflavone
intermediate was described as unstable and could spontaneously
convert to genistein. In electrospray LC/MS the most prominent peak
in the spectrum of "peak 2" is at m/z=289, consistent with it being
the [MH].sup.+ form of the proposed hydroxylated intermediate. The
height of "peak 2" detected in the 4 h incubation sample was bigger
than that for "peak 2" in the 14 h incubation sample. That sample
showed the largest genistein peak among the microsome assays that
were performed. It is suspected that "peak 2" may represent this
proposed intermediate that may be formed transiently during the
synthesis of genistein by isoflavone synthase. A similar
intermediate (at m/z=273) was also detected in the conversion of
liquiritigenin to daidzein (FIG. 19).
[0189] To compare the rates of genistein and daidzein synthesis by
microsomes of the yeast transformant GM1, samples representing
incubation periods of 2, 4, 6, 8 and 10 h were analyzed. The peak
areas for genistein and daidzein were quantitated by calibration
with authentic genistein and daidzein standards. Assays were
repeated three times and the average amount of isoflavonoid
synthesized at each time point was plotted, with vertical lines
representing error bars (FIG. 26).
Example 6
Identification of CYP93C1 as a Soybean Isoflavone Synthase
[0190] The sequence of the mRNA encoding CYP93C1, a cytochrome P450
monooxygenase, is found in the NCBI database having General
Identifier No. 2739005. The function of the protein encoded by this
mRNA has yet to be identified. The cDNA insert in clone
sgs1c.pk006.o20 encodes an isoflavone synthase and has sequence
similarities with CYP93C1. To determine whether CYP93C1 encodes a
functional isoflavone synthase, cDNA was prepared and cloned into
the yeast vector pRS315-gal and transformed into yeast strain WHT1
to assay for its ability to produce genistein. The CYP93C1 mRNA was
amplified from RNA isolated from soybean tissue (cv. S1990)
infected with the fungal pathogen Sclerotinia slerotiorum using
RT-PCR. Fungal infection causes an increase in the amount of
isoflavonoid produced and thus the amount of isoflavone synthase
transcript was increased in the infected tissue. Soybean plants
were infected 45 days after planting seeds and were harvested two
days later. Total RNA was prepared using the TRIzol Reagent
following the manufacturer's instructions (Gibco BRL) and 1 .mu.g
of the resulting total RNA was converted into a first strand cDNA
using the Superscript.TM. Preamplification system and using oligodT
as the reverse transcription primer. One microliter of first strand
cDNA was amplified by PCR using the primers listed as SEQ ID NO:7
and SEQ ID NO:8: TABLE-US-00004 5'-AAAATTAGCCTCACAAAAGCAAAG-3' [SEQ
ID NO:7] 5'-ATATAAGGATTGATAGTTTATAGTAGG-3' [SEQ ID NO:8]
[0191] The nucleotide sequence in SEQ ID NO:7 corresponds to
nucleotides 3 to 26 of the sequence found in NCBI General
Identifier No. 2739005. The nucleotide sequence in SEQ ID NO:8
corresponds to the complement of nucleotides 1798 to 1824 of the
sequence found in NCBI General Identifier No. 2739005.
Amplification was performed on a Perkin Elmer Applied Biosystems
GeneAmp PCR System using the Advantage-GC cDNA polymerase mix
(Clontech), following the manufacturer's instructions, with a 1 M
final concentration of GC melt reagent. Previous to amplification,
the mixture was incubated at 94.degree. C. for 5 minutes.
Amplification was performed using 30 cycles of: 94.degree. C. for
30 seconds, 53.degree. C. for 30 seconds and 72.degree. C. for 2
minutes. Following amplification, the mixture was incubated at
72.degree. C. for 7 minutes. The amplified product was then cloned
into pCR2.1 using "The Original TA Cloning Kit" (Invitrogen).
Plasmid DNA was purified using QIAFilter cartridges (Qiagen Inc)
according to the manufacturer's instructions. Sequence was
generated on an ABI Automatic sequencer using dye terminator
technology and using a combination of vector and insert-specific
primers. Sequence editing was performed using DNAStar (DNASTAR,
Inc.). The sequence generated represents coverage at least two
times in each direction. The sequence of the resulting clone,
presented in SEQ ID NO:9, was identical with that of CYP93C1 (NCBI
General Identifier No. 2739005); the deduced amino acid sequence of
this cDNA is shown in SEQ ID NO:10.
[0192] The above plasmid was then cloned into the yeast vector
pRS315-gal using gap repair as described in Example 4. Standard
procedures were used to transform the resulting plasmid into the
WHT1 yeast strain. Microsomes were prepared from the WHT1 yeast
strain containing the soybean CYP93C1 sequence and assayed for the
production of genistein and daidzein as described in Example 5. The
resulting microsomes exhibited isoflavone synthase activities. To
compare the rates of genistein and daidzein synthesis by microsomes
of the yeast transformant containing the soybean CYP93C1 sequence,
samples representing incubation periods of 2, 4, 6, 8 and 10 h were
analyzed. The peak areas for genistein and daidzein were
quantitated by calibration with authentic genistein and daidzein
standards as prepared in Example 2. Daidzein and genistein
accumulated linearly over the time course.
Example 7
Amplification and Identification of Isoflavone Synthase From Other
Legume Species
[0193] Nucleic acid sequences encoding isoflavone synthases from
lupine, mung bean, snow pea, alfalfa, red clover, white clover,
hairy vetch and lentil were derived from total RNA prepared from
young seedlings. Mung bean sprouts and snow pea sprouts were
obtained from the local grocery store. Seeds for alfalfa, red
clover, white clover, hairy vetch, and lentil were obtained from
Pinetree Garden Seeds while seeds for lupine (cv Russell Mix) were
obtained from Botanical Interests, Inc. Seedlings were germinated
in a controlled temperature growth chamber (14 h light at
25.degree. C. and 10 h dark at 21.degree. C.) and harvested after
approximately two weeks except for lupine, which was harvested
after approximately three weeks. Total RNA was prepared using
TRizol Reagent (Gibco BRL) according to the manufacturer's
instructions. For each plant, a first strand cDNA was prepared from
1 .mu.g total RNA using the Superscript.TM. Preamplification System
(Gibco BRL) following the manufacturer's instructions. OligodT was
used as the reverse transcription primer in all cases except white
clover where random hexamers were used.
[0194] Amplification was performed on a Perkin-Elmer Applied
Biosystems GeneAmp PCR System 9700PCR using Advantage-GC cDNA
polymerase mix (Clontech) according to the manufacturer's
instructions and with a final concentration of GC melt reagent
equal to 1 M. Amplification was preceded in all cases by incubation
at 94.degree. C. for 5 minutes and was followed by incubation at
72.degree. C. for 7 minutes. Two sets of primers were used for PCR
amplification. Primer set one is composed of SEQ ID NO:11 and SEQ
ID NO:12 and primer set two is composed of SEQ ID NO:13 and SEQ ID
NO:14: TABLE-US-00005 5'-ATGTTGCTGGAACTTGCACTT-3' [SEQ ID NO:11]
5'-TTAAGAAAGGAGTTTAGATGCAACG-3' [SEQ ID NO:12]
5'-TGTTTCTGCACTTGCGTCCCAC-3' [SEQ ID NO:13]
5'-CCGATCCTTGCAAGTGGAACAC-3' [SEQ ID NO:14]
[0195] The initial amplification of all samples was done using 1
.mu.L of first strand cDNA and primer set one (SEQ ID NO:11 and SEQ
ID NO:12). Amplification of mung bean was performed using 30 cycles
of 94.degree. C. for 30 seconds, 48.degree. C. for 30 seconds and
72.degree. C. for 2 minutes. Amplification of red clover was
performed using 30 cycles of 94.degree. C. for 30 seconds,
50.degree. C. for 30 seconds and 72.degree. C. for 1 minute.
Amplification of white clover, lentil, hairy vetch, alfalfa and
lupine was carried out in two steps. The first amplification
reaction was performed using 30 cycles of 94.degree. C. for 30
seconds, 50.degree. C. for 30 seconds and 72.degree. C. for one
minute. A second amplification reaction was done with 1 .mu.L of
the resulting product and primer set two (SEQ ID NO:13 and SEQ ID
NO:14) using 30 cycles of 94.degree. C. for 30 seconds,
50.5.degree. C. for 30 seconds and 72.degree. C. for one minute.
Amplification of snow pea was performed in three different PCR
reactions. The first reaction was performed using 30 cycles of
94.degree. C. 30 seconds, 50.5.degree. C. for 30 seconds and
72.degree. C. for one minute. One microliter from the resulting
product was used for a second amplification reaction using primer
set one and 30 cycles of 94.degree. C. for 30 seconds, 60.degree.
C. for 30 seconds and 72.degree. C. for one minute. The resulting
reaction was analyzed on a 1% agarose gel and the band at the
expected size was gel purified using the QIAquick Gel Extraction
Kit (Qiagen). The purified DNA was resuspended in 30 .mu.L of water
and 1 .mu.L was used as a template for a third PCR reaction using
primer set one with 30 cycles of 94.degree. C. for 30 seconds,
60.degree. C. for 30 seconds and 72.degree. C. for 90 seconds.
[0196] The resulting mung bean, red clover and snow pea PCR
sequences were cloned into pCR2.1 using "The Original TA Cloning
Kit" (Invitrogen). The resulting white clover, lentil, hairy vetch,
alfalfa and lupine PCR sequences were cloned into pCR2.1 using
TOPO.TM. TA Cloning Kit (Invitrogen). Plasmid DNA was purified
using QIAFilter cartridges (Qiagen Inc) or Wizard Plus Minipreps
DNA Purification System (Promega) following the manufacturer's
instructions. Sequence was generated on an ABI Automatic sequencer
using dye terminator technology and using a combination of vector
and insert-specific primers. Sequence editing was performed using
DNAStar (DNASTAR, Inc.). All sequences represent coverage at least
two times in both directions.
[0197] The nucleotide sequence of comprising the cDNA insert in
clone alfalfa 1 is shown in SEQ ID NO:15; the deduced amino acid
sequence of this DNA is shown in SEQ ID NO:16. The nucleotide
sequence comprising the cDNA insert in clone alfalfa 2 is shown in
SEQ ID NO:57; the deduced amino acid sequence of this DNA is shown
in SEQ ID NO:58. The nucleotide sequence comprising the cDNA insert
in clone alfalfa 3 is shown in SEQ ID NO:59; the deduced amino acid
sequence of this DNA is shown in SEQ ID NO:60. The nucleotide
sequence comprising the cDNA insert in clone hairy vetch 1 is shown
in SEQ ID NO:17; the deduced amino acid sequence of this DNA is
shown in SEQ ID NO:18. The nucleotide sequence comprising the cDNA
insert in clone lentil 1 is shown in SEQ ID NO:19; the deduced
amino acid sequence of this DNA is shown in SEQ ID NO:20. The
nucleotide sequence comprising the cDNA insert in clone lentil 2 is
shown in SEQ ID NO:21; the deduced amino acid sequence of this DNA
is shown in SEQ ID NO:22. The nucleotide sequence comprising the
cDNA insert in clone mung bean 1 is shown in SEQ ID NO:23; the
deduced amino acid sequence of this DNA is shown in SEQ ID NO:24.
The nucleotide sequence comprising the cDNA insert in clone mung
bean 2 is shown in SEQ ID NO:25; the deduced amino acid sequence of
this DNA is shown in SEQ ID NO:26. The nucleotide sequence
comprising the cDNA insert in clone mung bean 3 is shown in SEQ ID
NO:27; the deduced amino acid sequence of this DNA is shown in SEQ
ID NO:28. The nucleotide sequence comprising the cDNA insert in
clone mung bean 4 is shown in SEQ ID NO:29; the deduced amino acid
sequence of this DNA is shown in SEQ ID NO:30. The nucleotide
sequence comprising the cDNA insert in clone red clover 1 is shown
in SEQ ID NO:31; the deduced amino acid sequence of this DNA is
shown in SEQ ID NO:32. The nucleotide sequence comprising the cDNA
insert in clone red clover 2 is shown in SEQ ID NO:33; the deduced
amino acid sequence of this DNA is shown in SEQ ID NO:34. The
nucleotide sequence comprising the cDNA insert in clone snow pea 1
is shown in SEQ ID NO:35; the deduced amino acid sequence of this
DNA is shown in SEQ ID NO:36. The nucleotide sequence comprising
the cDNA insert in clone white clover 1 is shown in SEQ ID NO:37;
the deduced amino acid sequence of this DNA is shown in SEQ ID
NO:38. The nucleotide sequence comprising the cDNA insert in clone
white clover 2 is shown in SEQ ID NO:39; the deduced amino acid
sequence of this DNA is shown in SEQ ID NO:40. The nucleotide
sequence comprising the cDNA insert in clone lupine 1 is shown in
SEQ ID NO:54; the deduced amino acid sequence of this DNA is shown
in SEQ ID NO:55.
[0198] Plasmids corresponding to mung bean 2, red clover 2 and snow
pea 1 were amplified and the plant-specific DNA (corresponding to
SEQ ID NO:25, SEQ ID NO:33 and SEQ ID NO:35) were transferred to
the yeast vector pRS315-gal following the gap repair method
explained in Example 4 to produce the yeast expression strains
isoflavone synthase VR2, isoflavone synthase TP2, and isoflavone
synthase PS1, respectively. The eight amino acids at the amino- and
carboxy-terminus correspond to those translated from the primers
used in PCR amplification and not necessarily belong to the
endogenous genes. Microsomes were isolated from the resulting yeast
WHT1 strains containing the mung bean, red clover or snow pea
genes, and assayed for isoflavone synthase activity as described in
Example 5, with minor modifications. After incubation for 16 hours,
200 .mu.L of ethyl acetate was added to recover the isoflavonoids
from the assay solution, the ethyl acetate was evaporated under
nitrogen using a heating module evaporation system and the sample
resuspended in 200 .mu.L of 80% methanol. A 10 .mu.L sample of this
solution was injected into a Phenomenex Luna 3.mu. C18 (2) column
(size: 150.times.4.6 mm. The samples were eluted over 10 minutes
using an increasing methanol gradient (from 20% methanol/80% 100 mM
ammonium acetate buffer (pH 5.9) to 100% methanol (v/v)) at a flow
rate of 1 mL per minute. The levels of genistein and naringenin in
the eluted samples were monitored through the absorption spectrum
at 260 and 290 nm. The genistein signal was verified by comparisons
of retention time, diode array detected absorption spectra. As seen
in Table 1, microsomes from all three strains produced genistein
and therefore exhibited isoflavone synthase activity.
TABLE-US-00006 TABLE 1 Genistein Synthesis Using in vitro Yeast
Assay System Yeast expression strain Genistein Synthesized
Isoflavone Synthase VR2 1298 ng Isoflavone Synthase TP2 59 ng
Isoflavone Synthase PS1 19 ng pRS315-gal Not detectable
Example 8
Amplification and Identification of Isoflavone Synthase From
Non-Legume Species
[0199] Isoflavonoids are most often found in the legumes, although
there are occasional examples of isoflavonoids in non-legume plants
(Dewick, P. M., Isoflavonoids in The Flavonoids: Advances in
Research edited by J. B. Harborne and T. J. Mabry pp. 535-640). To
obtain isoflavone synthases with greater molecular diversity,
isoflavone synthase genes from Beta vulgaris (sugarbeet) were
cloned and their activity tested. Sugarbeet, a member of the family
Chenopodiaceae, is one of the few non-legume species to have been
shown to have isoflavonoids present (Geigert, et al. (1973)
Tetrahedron. 29:2703-2706).
[0200] Sugarbeet seeds were germinated in a growth chamber as
described in Example 7 (14 h light at 25.degree. C. and 10 h dark
at 21.degree. C.) and harvested after two weeks. Total RNA was
prepared using TRIzol Reagent (Gibco BRL) according to the
manufacturer's instructions. First strand cDNA was prepared from 1
.mu.g total RNA using the Superscript.TM. Preamplification System
(Gibco BRL) following the manufacturer's instructions with OligodT
as the reverse transcription primer.
[0201] Amplification was performed on a Perkin-Elmer Applied
Biosystems GeneAmp PCR System 9700PCR using Advantage-GC cDNA
polymerase mix (Clontech) according to the manufacturer's
instructions and with a final concentration of GC melt reagent
equal to 1 M. Amplification was preceded in all cases by incubation
at 94.degree. C. for 5 minutes and was followed by incubation at
72.degree. C. for 7 minutes.
[0202] Amplification was carried out in two steps. The first
amplification reaction was performed using 1 .mu.L of first strand
cDNA and primer set one (SEQ ID NO:11 and SEQ ID NO:12) with 30
cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds and 72.degree. C. for one minute. A second amplification
reaction was done with 1 .mu.L of the resulting product with primer
set two (SEQ ID NO:13 and SEQ ID NO:14) and using 30 cycles of
94.degree. C. for 30 seconds, 50.5.degree. C. for 30 seconds and
72.degree. C. for one minute. The resulting PCR sequence was cloned
into pCR2.1 using TOPO.TM. TA Cloning Kit (Invitrogen). Plasmid DNA
was purified using QIAFilter cartridges (Qiagen Inc) or Wizard Plus
Minipreps DNA Purification System (Promega) following the
manufacturer's instructions. Sequence was generated on an ABI
Automatic sequencer using dye terminator technology and using a
combination of vector and insert-specific primers. Sequence editing
was performed using DNAStar (DNASTAR, Inc.). All sequences
represent coverage at least two times in both directions. The
nucleotide sequence comprising the cDNA insert in clone sugarbeet 1
is shown in SEQ ID NO:47; the deduced amino acid sequence of this
DNA is shown in SEQ ID NO:48. The nucleotide sequence comprising
the cDNA insert in clone sugarbeet 2 is shown in SEQ ID NO:61; the
deduced amino acid sequence of this DNA is shown in SEQ ID
NO:61.
[0203] The data in Table 2 summarizes the relationship of the
isoflavone synthase nucleotide and amino acid sequences disclosed
herein. Reported are the percent identity of the nucleotide
sequences set forth in SEQ ID NOs:9, 15, 17, 19, 21, 23, 25, 27,
29, 31, 33, 35, 37, 39, 47 and 54 to instant soybean isoflavone
synthase sequence set forth in SEQ ID NO:1. In addition, the
percent identity of the amino acid sequences deduced from the
instant nucleotide sequences as set forth in SEQ ID NOs: 10, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 48 and 55 are
compared to the amino acid sequence set forth in SEQ ID NO:2.
TABLE-US-00007 TABLE 2 Percent Identity of Nucleotide Coding
Sequences and Amino Acid Sequences of Polypeptides Homologous to
Isoflavone Synthase SEQ ID Percent Identity NO. length to SEQ ID
NO: 1/2 nt aa Crop (nts)* nucleotides (nt) amino acids (aa) 9 10
Soybean 1824 85.9 96.7 15 16 Alfalfa1 1501 99.5 99.0** 56 57
Alfalfa2 1501 92.2 96.2** 58 59 Alfalfa3 1501 92.3 96.6** 17 18
Hairy vetch 1501 92.3 96.2** 19 20 Lentil1 1501 97.9 98.8** 21 22
Lentil2 1501 92.3 96.4** 23 24 Mung bean1 1566 92.5 96.7 25 26 Mung
bean2 1566 92.5 96.7 27 28 Mung bean3 1566 92.6 96.7 29 30 Mung
bean4 1566 92.7 96.7 31 32 Red clover 1566 92.5 96.4 33 34 Red
clover 1566 92.6 96.7 35 36 Snow pea 1563 99.3 99.0 37 38 White
clover1 1496 99.3 98.4** 39 40 White clover2 1501 98.3 99.0** 60 61
Sugarbeet1 1497 91.9 95.6** 47 48 Sugarbeet2 1501 92.3 96.6** 54 55
Lupine 1501 92.2 96.2** *SEQ ID NO: 1 contains 1756 nucleotides.
**These sequences are 22 amino acids shorter because the primers
used for PCR were derived from the soybean sequence.
[0204] The data presented in Table 2 indicates that the nucleotide
and amino acid sequences encoding the various isoflavone synthases
are highly conserved among divergent species. Sequence alignments
and percent identity calculations were performed using the Megalign
program of the LASARGENE bioinformatics computing suite (DNASTAR
Inc., Madison, Wis.). Multiple alignment of the sequences was
performed using the Clustal method of alignment (Higgins and Sharp
(1989) CABIOS. 5:151-153) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10).
[0205] A consensus sequence was determined by aligning the amino
acid sequences of the present invention using the Clustal method of
alignment and this sequence is shown in SEQ ID NO:66. Amino acids
not conserved are indicated by Xaa. These are: TABLE-US-00008
Xaa.sub.10 Phe or Leu Xaa.sub.16 Ser or Leu Xaa.sub.23 Ser or Thr
Xaa.sub.25 Ile or Lys Xaa.sub.39 Lys or Arg Xaa.sub.48 Pro or Leu
Xaa.sub.60 Pro or Leu Xaa.sub.73 Leu or His Xaa.sub.74 Ser or Tyr
Xaa.sub.95 Ala or Thr Xaa.sub.96 Asn or His Xaa.sub.102 Asn or Ser
Xaa.sub.110 Ile, Val, or Thr Xaa.sub.112 Arg or His Xaa.sub.117 Asn
or Ser Xaa.sub.118 Ser or Leu Xaa.sub.121 Met or Arg Xaa.sub.122
Ala or Val Xaa.sub.124 Phe or Ile Xaa.sub.129 Lys or Arg
Xaa.sub.147 Lys or Glu Xaa.sub.159 Leu or Phe Xaa.sub.162 Ala or
Val Xaa.sub.166 Ser or Gly Xaa.sub.170 Gln or Arg Xaa.sub.175 Val
or Leu Xaa.sub.183 Ala or Thr Xaa.sub.187 Thr or Ile Xaa.sub.191
Met or Val Xaa.sub.209 Phe or Tyr Xaa.sub.219 Arg or Trp
Xaa.sub.223 Tyr or His Xaa.sub.253 Gly or Glu Xaa.sub.259 Lys or
Glu Xaa.sub.263 Val or Asp Xaa.sub.264 Val, Asp, or Ile Xaa.sub.268
Ala or Val Xaa.sub.272 Phe or Leu Xaa.sub.285 Thr or Met
Xaa.sub.293 Glu or Asp Xaa.sub.294 Thr, or Ile Xaa.sub.301 Phe or
Leu Xaa.sub.306 Thr or Ile Xaa.sub.311 Val or Glu Xaa.sub.312 Val
or Ala Xaa.sub.325 Arg or Lys Xaa.sub.328 Gln or Glu Xaa.sub.334
Val or Ala Xaa.sub.342 Arg or Ile Xaa.sub.377 Thr or Ile
Xaa.sub.381 Glu or Gly Xaa.sub.385 Tyr, His, or Cys Xaa.sub.387 Ile
or Thr Xaa.sub.393 Val or Ile Xaa.sub.394 Leu or Pro Xaa.sub.402
Arg or Lys Xaa.sub.404 Ser or Pro Xaa.sub.413 Ser or Phe
Xaa.sub.422 Glu or Gly Xaa.sub.428 Gly or Arg Xaa.sub.429 Pro or
Leu Xaa.sub.435 Gln or Arg Xaa.sub.447 Arg or Gly Xaa.sub.453 Asn,
Ser, or Ile Xaa.sub.459 Met or Thr, and Xaa.sub.485 Asp or Gly
[0206] To verify that the similarity between the isoflavone
synthase nucleotide sequences from soybean and from sugarbeet were
not due to artifacts of PCR, a nucleic acid sequence containing the
soybean isoflavone synthase set forth in SEQ ID NO:1 was used as a
probe for Southern blot analysis against sugarbeet genomic DNA.
Hybridization was done overnight at 65.degree. C. in 6.times.SSC,
5.times. Denhardts. Filters were washed 2 times in 2.times.SSC, 1%
SDS at room temperature and 2 times in 0.2.times.SSC, 0.5% SDS at
65.degree. C. Hybridizing bands were detected indicating that
sugarbeet does contain genes with high homology to the soybean
isoflavone synthase sequence.
Example 9
Preparation of Transgenic Tobacco with Chimeric Isoflavone Synthase
Gene
[0207] The ability to obtain isoflavone synthase activity by
expressing the gene from soybean clone sgs1c.pk006.o20 in other
plants was tested by preparing transgenic tobacco plants expressing
the isoflavone synthase gene and assaying for genistein production.
The 1.6 Kb isoflavone synthase coding region from clone
sgs1c.pk006.o20 (SEQ ID NO:1) was amplified using a standard PCR
reaction in a GeneAmp PCR System with the primers shown in SEQ ID
NO:41 and SEQ ID NO:42: TABLE-US-00009 [SEQ ID NO:41]
5'-TTGCTGGAACTTGCACTTGGT-3' [SEQ ID NO:42]
5'-GTATATGATGGGTACCTTAATTAAGAAAGGAG-3'
[0208] The resulting DNA sequence (IFS) contains from the second
codon to the stop codon of the soybean isoflavone synthase gene
sequence followed by a Kpn I site. The following three sequences
(in 5' to 3' order) were assembled in pUC18 vector (New England
Biolabs) to yield plasmid pOY160 (depicted in FIG. 20): [0209]
35S/cabL, a promoter sequence comprising 1.3 Kb from the
cauliflower mosaic virus (CaMV) 35S promoter extending to 8 bp
downstream from the transcription start site followed by a 60 bp
leader sequence derived from the chlorophyll a/b binding protein
gene 22L (Harpster M. H. et al. (1988) Mol. Gen. Genet.
212:182-190); [0210] IFS, the isoflavone synthase gene fragment
generated by PCR amplification using the primers from SEQ ID NO:41
and SEQ ID NO:42. [0211] Nos3'; an 800 bp fragment which contains
the polyadenylation signal sequence from the nopaline synthase gene
(Depicker A. et al. (19820 J. Mol. Appl. Genet. 1:561-573).
[0212] The 5' end of IFS was ligated to Nco I-digested, filled-in,
35S/cabL. The 3' end of IFS was digested with Kpn I and ligated to
Kpn I-digested Nos3'.
[0213] The following three fragments were ligated to create plasmid
pOY204: [0214] 1) The Hind III/Pst I fragment comprising the
35S/cabL-5'IFS from pOY160, [0215] 2) The Pst I/Sal I fragment
comprising the 3'IFS-Nos3' from pOY160, [0216] 3) The Hind III/Sal
I fragment from vector pPZP211.
[0217] The vector pPZP211 contains an npt II gene fragment under
the control of the 35S CaMV promoter conferring kanamycin
resistance as the plant selectable marker (Hajdukiewicz P. et al.
(1994) Plant Mol. Biol. 25:989-994).
[0218] The plasmid pOY204 was transformed into the Agrobacterium
tumefaciens strain LBA4404 and was subsequently introduced into
Nicotiana tobaccum by leaf disc co-cultivation following standard
procedures (De Blaere et al. 1987 Meth. Enzymol. 143:277). The leaf
discs were incubated for three weeks on selection medium (MS salts
with vitamins (Gibco BRL), 1 mg/L 6-benzylaminopurine (BA), 100
mg/L kanamycin, and 500 mg/L Claforan). The regenerating plants
were transferred to rooting medium (selection medium without BA)
for another two weeks. Transformed plants were identified by the
appearance of roots in this selection media. Following standard
protocols, DNA samples were prepared from six randomly-selected
shoots and used as templates for PCR using the primers from SEQ ID
NO:41 and SEQ ID NO:42. Verification of the presence of the
isoflavone synthase coding region in the genome of the tested
tobacco shoots was done by separating the reaction product using a
1% agarose gel and staining with ethidium bromide. The expected 1.6
Kb fragment was obtained as the reaction product in all the
transgenic tobacco shoots and not in the untransformed tobacco
controls.
Transcription of Soybean Isoflavone Synthase in Transgenic Tobacco
Shoots
[0219] Transcription of the isoflavone synthase gene in the
transgenic tobacco shoots was confirmed using RT-PCR. Total
steady-state plant RNA was extracted from four randomly-selected
tobacco shoots resulting from transformation with pOY204 using the
RNeasy Plant Mini Kit (Qiagen) following standard protocols. RT-PCR
amplification was performed using "The SuperScript One Step RT-PCR
Kit" (Gibco BRL) with the primers: TABLE-US-00010
5'-GACGCCTCACTTACGACAACTCTGTG-3' [SEQ ID NO:43]
5'-CCTCTCGGGACGGAATTCTGATGGT-3' [SEQ ID NO:44]
[0220] After incubation at 50.degree. C. for 45 minutes,
amplification was carried out using 37 cycles of 93.degree. C. for
30 seconds, 64.degree. C. for 30 seconds and 72.degree. C. for 1
minute. The resulting DNA was separated on a 1% agarose gel.
Samples from the putative isoflavone synthase-containing tobacco
showed an 840 bp band not seen in the sample from the untransformed
tobacco control.
Example 10
Expression of Soybean Isoflavone Synthase in Transgenic Tobacco
Activity of Soybean Isoflavone Synthase in Tobacco Shoots
[0221] The activity of the soybean isoflavone synthase in the
transgenic tobacco was determined by analyzing shoots for the
presence of genistein. Approximately one gram of tissue from shoots
of five-week-old rooting transformants and from untransformed
tobacco plants were ground in liquid nitrogen and extracted for 20
minutes at room temperature using 10 mL of 80% ethanol. After
filtration through Acrodisc CR-PTFE syringe filters (Gelman
Sciences), 3 mL from each extraction solution were concentrated to
1 mL by evaporation under nitrogen gas flow using a 50.degree. C.
heating block. To hydrolyze any malonyl or glucosyl-derivatized
compounds present, 3 mL of 1 N HCl were added and the samples
incubated at 95.degree. C. for 2 h followed by extraction using 1
mL ethyl acetate. Five hundred .mu.L of the ethyl acetate phase
were dried under nitrogen and resuspended in 20 .mu.L chloroform.
The presence of genistein in the samples was determined by gas
chromatography/mass spectroscopy (GC/MS) analysis.
[0222] Before injection into a Hewlett Packard 6890 gas
chromatograph, the hydroxyl groups in the samples were derivatized
to trimethylsilylate by the addition of 100 .mu.L of BSTFA (N,
O-bis(trimethylsilyl)-trifluoroacetamide; Supelco) and incubation
at 37.degree. C. for 1 h. The samples were dried under nitrogen gas
and re-dissolved in 20 .mu.L chloroform immediately before manual
injection into the gas chromatograph. Two .mu.L of sample were
manually injected onto a 15 meter dry bed GC capillary column
(J&W, Jones Chromatography, Mid Glamorgan, UK) through an
injector port operated in the split mode (5:1). The initial oven
temperature was set at 200.degree. C. and the column was set at a
linear temperature gradient from 200.degree. C. to 300.degree. C.
in 20 minutes with a helium gas flow rate of 1.5 mL/minute. The
mass spectrum was monitored using a Hewlett Packard 5973
mass-selective detector at an ionization potential of 70 eV. The
mass ions identified from the cracking pattern of pure genistein
treated as mentioned above are 414 and 399 m/z. These peaks
represent the products of partially derivatized genistein, the form
obtained following the above procedure. Twenty nine of thirty three
tobacco transformants analyzed by gas chromatography had an
identifiable genistein peak at 8.7 minutes. The presence of
genistein in these peaks was confirmed by the detection of peaks at
414 and 399 m/z in the mass spectra. These results confirmed that
the soybean isoflavone synthase coding region is expressed in
tobacco plants under control of the 35S CaMV promoter and causes
novel production of genistein in tobacco shoot tissue.
Presence of Genistein in Tobacco Flowers
[0223] Flowers from the tobacco transformants were assayed for the
presence of genistein. Extracts were prepared as described above,
except that after hydrolysis, the dried ethyl acetate extracts were
resuspended in 1 mL of 80% methanol. The HPLC protocol was the same
as in Example 2 using a Phenomenex Luna 3u C18 (2) column
(150.times.4.6 mm). As compared to extracts from wild type plants,
the transformant flowers contained two additional large peaks in
the HPLC profile. One of these peaks was identified as genistein
while the other is unknown. Detection of the large genistein peak
in the HPLC profile of the tobacco flower extracts indicated that
there was a much higher amount of genistein present in the tobacco
flowers than in the tobacco shoots, since the genistein in the
shoot samples was only detectable by GC/MS. The prevalence of
genistein in the flowers relates to the expression of the
anthocyanin biosynthetic pathway, which is active in the flowers as
indicated by the pink flower color. An active anthocyanin pathway
produces the naringenin substrate for isoflavone synthase.
Example 11
Expression of Soybean Isoflavone Synthase in Transgenic
Arabidopsis
[0224] Arabidopsis thaliana was transformed with the plasmid pOY204
via in planta vacuum infiltration following standard protocols
(Bechtold et al. (1993) CR Life Sciences 316:1194-1199). Briefly,
three-week-old Arabidopsis thaliana ectotype WS plants were
submerged in 500 mL of Agrobacterium, strain GV3101 harboring
pOY204, suspended in basic MS media (Gibco BRL) and vacuum was
applied repeatedly for 10 minutes. The infiltrated plants were
allowed to set seeds for another three weeks. The harvested seeds
were surface-sterilized, then germinated and grown for three weeks
on plates containing 75 mg/L kanamycin. Approximately 120 green
healthy plants were recovered in the first round of screening and
were transferred to soil for two more weeks. The plants at this
stage had green immature pods and few leaves. Extracts were
prepared and analyzed by HPLC and GC/MS as described in Example 2,
except that after hydrolysis, the dried ethyl acetate extracts were
resuspended in 1 mL of 80% methanol. Five of twelve
randomly-selected Arabidopsis transformants analyzed by HPLC had an
identifiable genistein peak at 8.7 minutes. GC MS analysis
confirmed the presence of genistein in these peaks by detection of
the characteristic peaks at 414 and 399 m/z in the mass spectra.
These results show that the soybean isoflavone synthase gene is
functional in the Arabidopsis plants and genistein is produced.
Example 12
Enhancing Isoflavonoid Levels in Transgenic Arabidopsis
[0225] To determine whether activation of the phenylpropanoid
pathway results in increased accumulation of isoflavonoids in
IFS-transformed Arabidopsis, the pathway was activated by UV light
treatments. Homozygous Arabidopsis transformants of line A109-4,
which synthesize genistein, were identified through germination on
kanamycin-containing medium by first selecting a transformant that
segregated kanamycin resistance in a 3:1 ratio. A resistant progeny
from this generation that then produced 100% resistant progeny was
identified as a homozygote. Plants from this population and wild
type Arabidopsis plants were transferred to 2-inch pots 10 days
after germination and grown for 10 more days. Plants were placed
directly under 366 nm UV light for 16 h (46 mWatt/cm.sup.2, using
an UVL-56 BLAK-Ray Lamp from UV Products, Inc., San Gabriel,
Calif.). Control plants were placed under the same described
environment except for the UV illumination. The above ground parts
of Arabidopsis plants were pulverized in liquid nitrogen to fine
powder immediately after UV treatment. The tissues were extracted
with 10 mL 80% methanol per 1 gram of fresh weight. The genistein
content from tissue extracts of UV-treated and untreated plants was
determined by HPLC using a Phenomenex Luna 3u (2) column
(150.times.4.6 mm) and a mobil phase linear gradient which goes in
15 minutes from 20% methanol, 80% 10 mM ammonium acetate, pH 8.3 to
100% methanol followed by 100% methanol for 5 minutes as described
in Example 2. Aliquots from the same extracts were also assayed for
anthocyanin accumulation using photospectrometry as described by
Bariola, P. A., et. al. ((1999) Plant Physiol. 119:331-342).
Briefly, one mL of extract was mixed with one mL of 0.5% (v/v) HCl
followed by the addition of two mL of chloroform and vortexing for
ten seconds. The mixture was allowed to separate to two phases at
room temperature. The absorbance of the aqueous phase was assayed
at 530 nm and 657 nm. The anthocyanin content was calculated by
subtracting the absorbance value at 657 from the absorbance value
at 530 and normalizing to fresh weight. As seen in Table 3, the
anthocyanin content and genistein level in IFS-transformed
Arabidopsis varies with UV treatment (The average and standard
deviations of four independent plants from each group are shown).
TABLE-US-00011 TABLE 3 Anthocyanin Content and Genistein Levels in
Transgenic Arabidopsis Plants Anthocyanin Genistein (by HPLC)
(A530-A657) (mAu/25 uL) Sample Control UV Control UV Control 0.0463
.+-. 0.0148 0.0591 .+-. 0.0202 0 0 Plants (no IFS gene) A109-4
0.0339 .+-. 0.0100 0.0368 .+-. 0.0116 121 .+-. 41 303 .+-. 58
(35S-IFS)
[0226] Anthocyanins are products of one branch of the
phenylpropanoid pathway, and the level of their accumulation is an
indication of the activity of this pathway. As seen in the table
above, genistein was not detectable and the anthocyanin levels
increased by about 28% after UV treatment in the control plants. In
plants expressing IFS the anthocyanin levels were not significantly
increased while the genistein levels more than doubled. A
duplication of this experiment also showed an increase in genistein
level (anthocyanin levels without UV treatment: 0.1426+/-0.0245;
and with UV treatment: 0.1463+/-0.0145 (units as described above);
genistein without UV treatment: 602+/-94; and with UV treatment:
857+/-46 (units as described above)). In this case the level of
anthocyanins in non-treated plants was much higher, probably due to
insect infestation. The level of genistein was higher in
non-treated plants and the increase with UV treatment was not as
large as in the first experiment. These results demonstrate that
activation of the phenylpropanoid pathway, in this case by stress
treatment (UV or insect infestation), results in an increased level
of genistein accumulation in transformants expressing isoflavone
synthase.
Example 13
Expression of Soybean Isoflavone Synthase in Monocot Cells
[0227] The ability to obtain isoflavone synthase activity in
monocot cells was tested by transforming the soybean gene from
clone sgs1c.pk006.o20 into corn suspension cells and assaying for
genistein production. The soybean isoflavone synthase gene was
cloned in a vector for expression in monocot cells and its activity
determined by the expression of genistein in corn. A chimeric
isoflavone synthase gene plasmid was prepared (pOY206) using the
pGEM9Zf cloning vector (Promega) for expression of the instant
isoflavone synthase in monocots. The following fragments were
inserted between two copies of the 3 Kb SAR fragment (the A
element, originally located between 8.7 and 11.7 kb upstream of the
chicken lysozyme gene coding region (Loc P. V. and Stratling W. H.
(1988) EMBO J. 7:655-664): [0228] 1. the 35S/cabL promoter fragment
from Example 9, [0229] 2. a 490 bp fragment containing the sixth
intron from the maize Adh1 gene (Mascarenhas, D. et al. (1990)
Plant Mol. Biol. 15:913-920) and ending with an Nco I site, [0230]
3. IFS, the isoflavone synthase fragment from Example 9, [0231] 4.
a 285 bp fragment containing the polyadenylation signal sequence
from the nopaline synthase gene (Depicker A. et al. (1982) J. Mol.
Appl. Genet. 1:561-573). Gene Combinations used for Corn Cell
Transformation
[0232] The plasmid pOY206 (FIG. 21) containing the chimeric
isoflavone synthase gene for expression in monocots was transformed
into corn cells in conjunction with plasmid pDETRIC. Plasmid
pDETRIC contains the bar gene from Streptomyces hygroscopicus that
confers resistance to the herbicide glufosinate (Thompson et al.
(1987) EMBO J. 6:2519). In the pDETRIC plasmid the bar gene is
under the control of the CaMV 35S promoter, its
translation-initiation codon has been changed from GTG to ATG for
proper translation initiation in plants (De Block et al. (1987)
EMBO J. 6:2513), and uses the Agrobacterium tumefaciens octopine
synthase polyadenylation signal.
[0233] Since the phenylpropanoid pathway is not active in corn
suspension cells a third plasmid containing a gene encoding a
transcription factor that activates the phenylpropanoid pathway
was, in some cases, bombarded into the corn cells in conjunction
with isoflavone synthase gene. This plasmid, pDP7951 (depicted in
FIG. 22 and bearing ATCC accession number PTA-371), contains in the
5'-3' orientation: [0234] the Agrobacterium nopaline synthase gene
promoter region, [0235] a tobacco mosaic virus (TMV) omega enhancer
sequence, [0236] the fifth intron from the maize adh1 gene, [0237]
CRC (a chimera containing the maize R region between the region
encoding the C1 DNA binding domain and the C1 activation domain),
[0238] the potato protease inhibitor II polyadenylation signal
sequence.
[0239] Additionally, a chimeric gene consisting of the CRC coding
region expressed from the CaMV 35S promoter was prepared and used
in corn cell transformations. The Sma I fragment of DP7951
containing CRC was ligated to Nco I and Kpn I ends that had been
blunt ended with Mung bean nuclease (New England Biolabs) to create
the chimeric gene: 35S/cabL-IFS-Nos3'. This plasmid is called
pOY162, and its restriction enzyme map is shown in FIG. 23.
Transformation of Monocot Cells
[0240] Black Mexican Sweet (BMS) suspension culture is a commonly
used, corn-derived, monocot cell line. Cultures were maintained in
MS2D medium (MS salts with vitamins (Gibco BRL), 20 g/L sucrose, 2
mg/L 2,4-dichlorophenoxyacetic acid, pH 5.8), incubated with
shaking (125 rpm) at 26.degree. C. in the dark, and subcultured
with fresh medium every five days.
[0241] Transformations were performed by microprojectile
bombardment using a DuPont Biolistic PDS 1000/He system (Klein T.
M. et al. (1987) Nature 327:70-73). Gold particles (0.6 microns)
were coated with mixtures of plasmid DNAs as indicated in Table 4:
TABLE-US-00012 TABLE 4 Plasmid Groups used in Maize Transformations
Group Plasmids 1 3 .mu.g pDETRIC + 6 .mu.g pOY206 2 3 .mu.g pDETRIC
+ 6 .mu.g pOY206 + 6 .mu.g pDP7951 3 3 .mu.g pDETRIC + 6 .mu.g
pDP7951 4 3 .mu.g pDETRIC + 6 .mu.g pOY206 + 6 .mu.g pOY162
[0242] Two days after subculture, BMS suspension culture aliquots
(6 mL each), were evenly distributed over Whatman#1 filter disks,
transferred onto solid MS2D medium (MS2D, 7 g/L agar) and incubated
at 26.degree. C. overnight. Filter disks containing the BMS cells
were positioned approximately 3.5 inches away from the retaining
screen and bombarded twice. Membrane rupture pressure was set at
1,100 psi and the chamber was evacuated to -28 inches of mercury.
Bombarded tissues were incubated for four days at 26.degree. C. in
the dark and then transferred to MS2D selection medium (solid MS2D
medium containing 3 mg/L Bialaphos). Resistant tissue was
transferred to fresh MS2D selection medium after seven weeks and
tissue was harvested for analysis two weeks later.
Analysis of Transformed Corn Cells for Synthesis of Anthocyanins
and Genistein
[0243] All control tissue and BMS lines transformed with group 1
were white in color. Approximately half of the Bialaphos-selected
resistant tissue that grew in plates bombarded with groups
containing CRC (groups 2 and 3) showed the wild type white color,
while the other half showed various degrees of red coloration, a
visual indication of anthocyanin accumulation. The red phenotype
indicates that expression of CRC in these lines is sufficient to
transcriptionally activate the expression of genes in the
phenylpropanoid pathway leading to anthocyanin synthesis and
accumulation (Grotewold E. et al. (1998) Plant Cell 10:721-740).
Presence of the isoflavone synthase gene in these tissues was
confirmed by the appearance of the appropriate sized fragments when
performing PCR on genomic DNA using primers from SEQ ID NO:43 and
SEQ ID NO:44. The presence of the CRC coding region in these
tissues was verified by the production of an appropriate fragment
when performing PCR on genomic DNA using the primers from SEQ ID
NO:45 (to the R region) and SEQ ID NO:46 (to the 3' untranslated
region from potato protease inhibitor II gene). TABLE-US-00013
5'-GCGGTGCACGGGCGGACTCTTCTTC-3' [SEQ ID NO:45]
5'-CGCCCAATACGCAAACCGCCTCTCC-3' [SEQ ID NO:46]
[0244] Tissue from 25 lines transformed with Group 1, 5 white lines
resulting from transformation with Group 2, 7 red lines transformed
with Group 2, 6 white lines transformed with Group 3, and 6 red
lines transformed with Group 3 was harvested and analyzed for the
presence of genistein using HPLC and GC-MS. Extracts were prepared
and analyzed as described in Example 2. The genistein HPLC peak and
the identifying 414 and 399 m/z MS peaks were detected in the
extracts from all seven red lines transformed with Group 2 while no
genistein was detected in any of the white lines transformed with
the same plasmids. Lines transformed with Group 3 did not have
genistein whether they were red or white. Sixteen lines transformed
with Group 4 also produced genistein. A summary of these results is
shown in Table 5. TABLE-US-00014 TABLE 5 Genistein Synthesis in
Transformed BMS Tissue Tissue Naringenin Genistein Group No. Color
Produced Produced 1 25 White NO NO 2 5 White NO NO 2 7 Red YES YES
3 6 White NO NO 3 6 Red YES NO 4 16 Red YES YES
[0245] The synthesis of genistein in BMS lines transformed with a
soybean isoflavone synthase-containing construct indicated that the
soybean protein was expressed and was functional in monocot cells.
Genistein was only produced in cell lines producing naringenin
indicating that the soybean isoflavone synthase gene was only
effective in the presence of an activated phenylpropanoid pathway.
The intermediate naringenin in the phenylpropanoid pathway provided
the substrate for isoflavone synthase to produce genistein.
Example 14
Synthesis of Daidzein in Monocot Cells
[0246] The activity of chalcone reductase determines the relative
levels of substrates available for isoflavone synthase to produce
genistein or daidzein (see FIG. 1). Chalcone reductase reduces
4,2',4',6'-tetrahydroxychalcone to 4,2',4'-trihydroxychalcone, thus
producing liquiritigenin as the substrate for isoflavone synthase
to produce daidzein. Chalcone reductases are present in legumes,
but have not been found in most non-legume plants including
Arabidopsis, tobacco, and corn. To produce daidzein in non-legume
plants, a plasmid DNA containing a soybean chalcone reductase gene
was introduced into corn suspension cells by microprojectile
bombardment, together with a selection marker, CRC, and IFS
constructs as described in Example 13.
[0247] A soybean cDNA clone encoding chalcone reductase was
identified by homology to known chalcone reductase genes of alfalfa
(Ballance and Dixon (1995) Plant Phys. 107:1027-1028). The cDNA
library was prepared using mRNAs from eight-day-old soybean roots
inoculated with cyst Nematode for four days, and sequenced as
described in Example 3. BLAST analysis was performed as described
in Example 4. The DNA containing the entire coding region from the
identified clone, src3c.pk009.e4, was amplified using PCR with the
primers shown in SEQ ID NO:62 and SEQ ID NO:63 TABLE-US-00015
5'-GTTACCATGGCTGCTGCTATTG-3' [SEQ ID NO:62]
5'-TTAAACGTAAAATGAAACAAGAGG-3' [SEQ ID NO:63]
[0248] The 5' primer had an Nco I site at the start of the coding
region. The 1.3 kb PCR product was subcloned into the pTOPO2.1
vector (Invitrogen Inc., Carlsbad, Calif.). The 1.3 kb coding
region fragment was excised as a Nco I/Kpn I fragment, using the
Nco I site and the Kpn I site from the vector. This fragment was
isolated and ligated between the 35S/CabL promoter and Nos
3'polyadenylation signal sequence in the pUC18 vector as described
in Example 9, to produce plasmid pCHR40, which was used in the BMS
transformation experiments.
[0249] Transformation of corn suspension cells was done as
described in Example 13, using pDETRIC, pCHR40, pOY206 and pOY162.
Selection and culturing were as described in Example 13. Each
selected line was assayed for the presence of the IFS and CRC genes
using PCR as in Example 13. The presence of the CHR gene was
determined by the appearance of a 0.6 kb fragment when performing
PCR on the tissues using the primers shown in SEQ ID NO:64 and SEQ
ID NO:65: TABLE-US-00016 5'-GACACTTCGACACTGCTGCTGCTTAT-3' [SEQ ID
NO:64] 5'-TCTCAAACTCACCTGGGCTATGGAT-3' [SEQ ID NO:65]
[0250] Of 32 lines screened, five carried all three transgenes.
Extracts were prepared, as described in Example 13, from these 32
lines and a control line that carries the CRC and IFS genes, but
not the CHR gene. All of the extracts were treated with 1 N HCl to
hydrolyze all possible oligosaccharide derivatives as described in
Example 10. HPLC and GC-MS were performed as described in Examples
2 and 10. One out of the five lines was shown to produce daidzein.
In the HPLC assay, in addition to the peaks of naringenin and
genistein, a small peak occurred at the same retention time as the
daidzein standard (9.6 min) (FIGS. 27C and D). This peak was not
present in the control samples (FIGS. 27A and B). In the GC-MS
assay, the daidzein-specific cracking pattern was found at the same
retention time as the standard (8.0 min). All of the major ions of
the daidzein spectrum were present (m/z: 398, 383, 218, 97). This
example shows that introduction of the soybean chalcone reductase
gene into corn cells together with the isoflavone synthase and CRC
genes results in the production of both daidzein and genistein.
Example 15
Alteration of Isoflavonoid Levels-in Soybean Somatic Embryos
[0251] The ability to change the levels of isoflavonoids by
overexpressing the gene from soybean clone sgs1c.pk006.o20 in
soybean somatic embryos was tested by preparing transgenic soybean
somatic embryos and assaying the isoflavonoid levels. The entire
insert from clone sgs1c.pk006.o20 (SEQ ID NO:1) was amplified in a
standard PCR reaction on a Perkin Elmer Applied Biosystems GeneAmp
PCR System using Pfu polymerase (Stratagene) with the primers shown
in SEQ ID NO:49 and SEQ ID NO:50: TABLE-US-00017
5'-GAATTCGCGGCCGCTCTAGAACTAGTGGAT-3' [SEQ ID NO:49]
5'-GAATTCGCGGCCGCGAATTGGGTACCGGGC-3' [SEQ ID NO:50]
[0252] The resulting fragment is bound by Not I sites in the primer
sequences and contains a 5' leader sequence, the coding region for
isoflavone synthase, the untranslated 3' region from SEQ ID NO:1,
and a stretch of 18 A residues at the 3' end. This fragment was
digested with Not I and ligated to Not 1-digested and
phosphatase-treated pKS67. The plasmid pKS67 was prepared by
replacing in pRB20 (described in U.S. Pat. No. 5,846,784) the 800
bp Nos 3' fragment, described in Example 9, with the 285 bp Nos 3'
fragment, described in Example 12. Clones were screened for the
sense orientation of the isoflavone synthase insert fragment by
digestion with Bam HI. The resulting plasmid pKS93s, shown in FIG.
24, has the beta-conglycinin promoter operably linked to the
fragment encoding isoflavone synthase followed by the Nos 3'end.
Plasmid pKS93s contains a T7 promoter/HPT/T7 terminator cassette
for expression of the HPT enzyme in certain strains of E. coli,
such as NovaBlue (DE3) (from Novagen), that are lysogenic for
lambda DE3 (which carries the T7 RNA Polymerase gene under lacV5
control). Plasmid pK93s also contains the 35S/HPT/NOS 3' cassette
for constitutive expression of the HPT enzyme in plants. These two
expression systems allow selection for growth in the presence of
hygromycin to be used as a means of identifying cells that contain
plasmid DNA sequences in both bacterial and plant systems.
Transformation of Soybean Somatic Embryo Cultures
[0253] The following stock solutions and media were used for
transformation and propagation of soybean somatic embryos:
TABLE-US-00018 Stock Solutions (g/L) MS Sulfate 100x stock
MgSO.sub.4.7H.sub.2O 37.0 MnSO.sub.4.H.sub.2O 1.69
ZnSO.sub.4.7H.sub.2O 0.86 CuSO.sub.4.5H.sub.2O 0.0025 MS Halides
100x stock CaCl.sub.2.2H.sub.2O 44.0 KI 0.083 CoCl.sub.2.6H.sub.2O
0.00125 KH.sub.2PO.sub.4 17.0 H.sub.3BO.sub.3 0.62
Na.sub.2MoO.sub.4.2H.sub.2O 0.025 Na.sub.2EDTA 3.724
FeSO.sub.4.7H.sub.2O 2.784 B5 Vitamin stock myo-inositol 100.0
nicotinic acid 1.0 pyridoxine HCl 1.0 thiamine 10.0 Media SB55 (per
Liter) 10 mL of each MS stock 1 mL of B5 Vitamin stock 0.8 g
NH.sub.4NO.sub.3 3.033 g KNO.sub.3 1 mL 2,4-D (10 mg/mL stock)
0.667 g asparagine pH 5.7 SB103 (per Liter) 1 pk. Murashige &
Skoog salt mixture* 60 g maltose 2 g gelrite pH 5.7 SB148 (per
Liter) 1 pk. Murashige & Skoog salt mixture* 60 g maltose 1 mL
B5 vitamin stock 7 g agarose pH 5.7 *(Gibco BRL)
[0254] Soybean embryonic suspension cultures were maintained in 35
mL liquid media (SB55) on a rotary shaker (150 rpm) at 28.degree.
C. with a mix of fluorescent and incandescent lights providing a 16
h day 8 h night cycle. Cultures were subcultured every 2 to 3 weeks
by inoculating approximately 35 mg of tissue into 35 mL of fresh
liquid media.
[0255] Soybean embryonic suspension cultures were transformed with
pKS93s by the method of particle gun bombardment (see Klein et al.
(1987) Nature 327:70-73) using a DuPont Biolistic PDS 1000/He
instrument. Five .mu.L of pKS93s plasmid DNA (1 g/L), 50 .mu.L
CaCl.sub.2 (2.5 M), and 20 .mu.L spermidine (0.1 M) were added to
50 .mu.L of a 60 mg/mL 1 mm gold particle suspension. The particle
preparation was agitated for 3 minutes, spun in a microfuge for 10
seconds and the supernate removed. The DNA-coated particles were
then washed once with 400 .mu.L of 70% ethanol and resuspended in
40 mL of anhydrous ethanol. The DNA/particle suspension was
sonicated three times for 1 second each. Five .mu.L of the
DNA-coated gold particles were then loaded on each macro carrier
disk.
[0256] Approximately 300 to 400 mg of two-week-old suspension
culture was placed in an empty 60 mm.times.15 mm petri dish and the
residual liquid removed from the tissue using a pipette. The tissue
was placed about 3.5 inches away from the retaining screen and
bombarded twice. Membrane rupture pressure was set at 1100 psi and
the chamber was evacuated to -28 inches of Hg. Two plates were
bombarded, and following bombardment, the tissue was divided in
half, placed back into liquid media, and cultured as described
above.
[0257] Fifteen days after bombardment, the liquid media was
exchanged with fresh SB55 containing 50 mg/mL hygromycin. The
selective media was refreshed weekly. Six weeks after bombardment,
green, transformed tissue was isolated and inoculated into flasks
to generate new transformed embryonic suspension cultures.
[0258] Transformed embryonic clusters were removed from liquid
culture media and placed on a solid agar media, SB103, containing
0.5% charcoal to begin maturation. After 1 week, embryos were
transferred to SB103 media minus charcoal. After 5 weeks on SB103
media, maturing embryos were separated and placed onto SB148 media.
During maturation embryos were kept at 26.degree. C. with a mix of
fluorescent and incandescent lights providing a 16 h day 8 h night
cycle. After 3 weeks on SB148 media, embryos were analyzed for the
expression of the isoflavonoids. Each embryonic cluster gave rise
to 5 to 20 somatic embryos.
[0259] Non-transformed somatic embryos were cultured by the same
method as used for the transformed somatic embryos.
Analysis of Transformed Somatic Embryos
[0260] At the end of the 8.sup.th week on SB103 medium somatic
embryos were harvested from 12 independently transformed lines.
Somatic embryos were collected individually and stored in 96-well
plates at -80.degree. until lyophilized. Somatic embryos were
lyophilized for 24 hours. Three to five lyophilized somatic embryos
were pooled in a micro centrifuge tube and the dry weight was
measured three times. Three samples of dried embryos were assayed
for each transformed line. An 80% methanol solution was added to
the lyophilized somatic embryos and the samples incubated for 24 h
in the dark at room temperature to extract isoflavonoids. The 80%
methanol solution was filtered through a Costar nylon membrane
microcentrifuge filter with 0.22 .mu.m pore size (Sigma).
[0261] For HPLC analysis of the extracts, twenty .mu.l of the 80%
methanol sample was applied to a Phenomenex Luna 3.mu. C18 (2)
column (size: 150.times.4.6 mm). Separation occurred during the
gradient elution of 10 mM ammonium buffer, pH 8.35 (solvent A) and
methanol (solvent B) as the mobile phase. Continuous increasing of
solvent B in solvent A, from 20 to 100% for 10 min was employed.
Standards for the isoflavonoids daidzin, daidzein, glycitin,
glycitein, genistin, genistein, liquiritigenin and naringenin were
prepared by the gradual addition of 80% methanol to each powder.
The peaks and spectra corresponding to daidzein, glycitin and
genistein conjugated with malonylated glucosides were determined by
LC/MS. Isoflaovonoids were monitored through the absorption spectra
at 260 and 280 nm. The isoflavonoid signals observed in the soybean
somatic embryo samples were verified by comparisons of the
retention times and diode array detected absorption spectra with
those of the standards. The areas of all peaks corresponding to the
isoflaovones in a sample were added and divided by the dry weight
of that sample. These dry weight based normalized area sums were
used for statistical analysis.
[0262] An analysis of variance test (ANOVA; Steel, R. G. D. and
Torrie, J. H. (1996) Principles and Procedures of Statistics: A
Biometrical Approach (McGraw-Hill Series in Probability and
Statistics, New York) was conducted using Microsoft Excel 97
(Microsoft). Data were analyzed as a single factor design with
single gene transformation as the main effect. Experimental units
were the sum of peak areas of identified isoflavonoids normalized
to dry weight. The mean square from the ANOVA was used to calculate
the least significant difference (LSD) for each comparison. The sum
of isoflavonoid peak areas of samples from a non-transformed
control line were compared with those of 25 independent
pKS93s-transformed, hygromycin resistant lines. FIG. 25 shows a
graph depicting the distribution of the sum of isoflavone area per
mg of dry weight of soybean somatic embryos transgenic for the
isoflavone synthase gene and a control line. The results are
depicted in the graph in ascending order of the amount of total
isoflavones produced. Some lines, such as the ones represented in
bars 7 through 14, contained approximately the same levels of
isoflavones as the control line. While most of the lines showed
intermediate increases or decreases in the amounts of isoflavones
produced, there are clear examples of lines having markedly
increased or decreased amounts of isoflavones. For example, bar 25
represents a line which expresses 208% as much isoflavones as the
control line, bar 24 represents a line which expresses 184% as much
isoflavones as the control line, and bar 1 represents a line which
produces only 25% of the isoflavones as the control line. These
differences in the amounts of isoflavones produced may be caused by
the position of the transgene in the chromosome, the number of
copies of the gene that are integrated in the chromosome, DNA
methylation, gene silencing, etc. These results indicate that
transgenic expression of isoflavone synthase affords the ability to
manipulate isoflavonoid levels as desired for a particular
application; i.e., transformants may be chosen for advancement that
have large changes in isoflavonoid levels (i.e., very high as in
IS19 or very low as in IS6) or more subtle changes in the content
of isoflavonoids.
Example 16
Amplification and Analysis of Soybean Genomic Isoflavone Synthase
DNA
[0263] Genomic sequences encoding isoflavone synthase may be used
to express isoflavone synthase as well as the cDNA sequences.
Therefore the genomic sequences containing the coding regions for
the soybean isoflavone synthase genes were isolated.
[0264] Soybean genomic DNA was prepared from Glycine max cv. Wye
following standard protocols (DNeasy Plant Maxi Kit, Qiagen,
Valencia, Calif.). Using this DNA as template, a genomic DNA
fragment including the sequence corresponding to the soybean insert
in sgs1c.pk006.o20 was produced by PCR with the primers listed as
SEQ ID NO:41 and SEQ ID NO:42. A genomic DNA fragment including the
sequence of CYP93C1 was produced with the primers listed as SEQ ID
NO:7 and SEQ ID NO:51: TABLE-US-00019
5'-AAAATTAGCCTCACAAAAGCAAAG-3' [SEQ ID NO:7]
5'-GCAAACGAAGACAAATGGGAGATGATA3' [SEQ ID NO:51]
[0265] Amplification was performed on a Perkin Elmer Applied
Biosystems GeneAmp PCR System using the Expand.TM. Hi fidelity PCR
system from Boehringer Mannheim (Indianapolis, Ind.). These PCR
fragments were cloned into the pCR2.1 vector (Invitrogen) and
sequenced as described in Example 6. The nucleotide sequence of the
genomic fragment comprising the isoflavone synthase sequence from
clone sgs1c.pk006.o20 is given in SEQ ID NO:52. The nucleotide
sequence of the genomic fragment comprising the isoflavone synthase
sequence of CYP93C1 is given in SEQ ID NO:53. Both genes were found
to contain one intron. The splice junction for both introns is
within the codon for amino acid 300. The intron sequence in SEQ ID
NO:52 corresponds to nucleotides 895 to 1112 (217 nucleotides),
while the intron sequence in SEQ ID NO:53 corresponds to
nucleotides 947 to 1082 (135 nucleotides) in SEQ ID NO:53.
Alignment of the intron nucleotide sequences using the Clustal
method of alignment and the default parameters (KTUPLE 2, GAP
PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4) shows that the intron
sequences are 46.3% identical.
Example 17
Alteration of Isoflavonoid Levels in Soybean Plants
[0266] The ability to alter the isoflavonoid levels in transgenic
soybean plants expressing the gene from soybean clone
sgs1c.pk006.o20 was tested by transforming somatic embryo cultures
with a vector containing the gene, allowing the plant to
regenerate, and meassuring the levels of isoflavonoids produced. In
addition, the soybean IFS gene was transformed in conjunction with
the CRC gene.
Construction of Vectors for Transformation of Glycine max
[0267] A vector containing a chimeric isoflavone synthase gene was
constructed as follows. The 1.6 Kb isoflavone synthase coding
region from clone sgs1c.pk006.o20 (SEQ ID NO:1) was amplified using
a standard PCR reaction in a GeneAmp PCR System using Pfu
polymerase (Stratagene) with the primers shown in SEQ ID NO:41 and
SEQ ID NO:42 as in Example 9. The plasmid pCW109 (World Patent
Publication No. WO94/11516) was digested with Nco I. The resulting
DNA fragments were treated with T4 DNA polymerase in the presence
of dATP; dCTP, dGTP and dTTP to obtain blunt ends followed by
digestion with Kpn I. The ligation of these two DNA fragments
created the plasmid pCW109--IFS, shown in FIG. 28, which has
operably linked: [0268] the beta-conglycinin promoter [0269] the
isoflavone synthase coding region [0270] the phaseolin 3' end
[0271] The 3.2 Kb fragment containing the
beta-conglycinin/P-IFS-phaseolin 3' chimeric gene was purified from
pCW109-IFS as a Hind III fragment and ligated with Hind
III-digested and phosphatase-treated pZBL102. pZBL102 is derived
from pKS18HH (described in U.S. Pat. No. 5,846,784) by replacing
the long Nos 3' fragment in pKS18HH with the short Nos 3' fragment
described in Example 13. The Sal I site between the two hygromycin
phosphotransferase coding regions was deleted, and a Not I site was
added between the Hind III and Sal I sites 5' to the 35S promoter
of the 35S-HPT gene.
[0272] The resulting plasmid, named pWSJ001, has a T7
promoter/HPT/T7 terminator cassette for expression of the HPT
enzyme in certain strains of E. coli that are lysogenic for lambda
DE3. The lambda DE3 carries the T7 RNA Polymerase gene under lacV5
control and is found in commercially available E. coli strains such
as NovaBlue (DE3) (from Novagen). Plasmid pWSJ001 also contains the
35S/HPT/NOS 3' cassette for constitutive expression of the HPT
enzyme in plants. These two expression systems allow selection for
growth in the presence of hygromycin to be used as a means of
identifying cells that contain plasmid DNA sequences in both
bacterial and plant systems.
[0273] A vector containing a chimeric CRC gene was constructed as
follows. The plasmid pDP7951 of Example 13, FIG. 22, was digested
with SmaI and the fragment containing the CRC coding region was
purified. This CRC fragment was ligated to a modified vector
containing the sequences of pCW109 (World Patent Publication No.
WO94/11516) with the substitution of a phaseolin promoter fragment
extending to -410 and including leader sequences to +77 (Slightom
et al., 1991 Plant Mol Biol Man B16:1) instead of the
beta-conglycinin promoter. Modification included digestion with
NcoI and S1 nuclease treatment followed by religation to remove the
ATG sequence of the NcoI site that follows the promoter fragment.
The vector was then digested with KpnI and the ends filled in so
that the SmaI CRC fragment was inserted in a blunt-end ligation.
From the resulting plasmid, the HindIII fragment containing the
phaseolin promoter-CRC-phaseolin 3' chimeric gene was isolated and
ligated with HindIII digested pZBL 102 (described above). The
resulting plasmid was called pOY203.
Transformation of Somatic Soybean Embryo Cultures and Regeneration
of Soybean Plants
[0274] Soybean embryogenic suspension cultures were transformed
with pWSJ001 or pWSJ001 in conjunction with pOY203 by the method of
particle gun bombardment as in Example 15. Besides the media used
for the soybean somatic embryo cultures described in Example 15,
the following media were used: TABLE-US-00020 Media SBP6 SB55 with
only 0.5 mL 2,4-D SB71-1 (per liter) B5 salts 1 ml B5 vitamin stock
30 g sucrose 750 mg MgCl2 2 g gelrite pH 5.7
[0275] Eleven days post bombardment, the liquid media was exchanged
with fresh SB55 containing 50 mg/mL hygromycin. The selective media
was refreshed weekly. Seven weeks post bombardment, green,
transformed tissue was observed growing from untransformed,
necrotic embryogenic clusters. Isolated green tissue was removed
and inoculated into individual flasks to generate new, clonally
propagated, transformed embryogenic suspension cultures. Thus each
new line was treated as independent transformation event. These
suspensions can then be maintained as suspensions of embryos
clustered in an immature developmental stage through subculture or
regenerated into whole plants by maturation and germination of
individual somatic embryos.
[0276] Transformed embryogenic clusters were removed from liquid
culture and placed on a solid agar media (SB103) containing no
hormones or antibiotics. Embryos were cultured for eight weeks at
26.degree. C. with mixed florescent and incandescent lights on a
16:8 h day/night schedule. During this period, individual embryos
were removed from the clusters and analyzed at various stages of
embryo development. Selected lines were assayed by PCR for the
presence of the an additional IFS gene using the primers shown in
SEQ ID NO:43 and SEQ ID NO:44. Separation of the PCR products on an
agarose gel yielded a 1062 bp fragment indicative of the endogenous
IFS gene (i.e., containing introns) and an 845 bp fragment in the
embryos containing the transgene IFS. Somatic embryos become
suitable for germination after eight weeks and were then removed
from the maturation medium and dried in empty petri dishes for 1 to
5 days. The dried embryos were then planted in SB71-1 medium where
they were allowed to germinate under the same lighting and
germination conditions described above. Germinated embryos were
transferred to sterile soil and grown to maturity. Seed were
harvested.
[0277] Seed from IFS-transformed and IFS+CRC-transformed soybean
plants are analyzed for isoflavonoid levels. Extracts are prepared
and analyzed by HPLC as described in Example 15 except that a 150
to 200 mg chip of soybean seed is used for the analysis. Seeds with
statistically significant variation in the level of isoflavonoid
concentration are further analyzed.
[0278] Various modifications of the invention in addition to those
shown and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims.
[0279] The disclosure of each reference set forth above is
incorporated herein by reference in its entirety.
Sequence CWU 1
1
66 1 1756 DNA Glycine max 1 gtaattaacc tcactcaaac tcgggatcac
agaaaccaac aacagttctt gcactgaggt 60 ttcacgatgt tgctggaact
tgcacttggt ttgtttgtgt tagctttgtt tctgcacttg 120 cgtcccacac
caagtgcaaa atcaaaagca cttcgccacc tcccaaaccc tccaagccca 180
aagcctcgtc ttcccttcat tggccacctt cacctcttaa aagataaact tctccactat
240 gcactcatcg atctctccaa aaagcatggc cccttattct ctctctcctt
cggctccatg 300 ccaaccgtcg ttgcctccac ccctgagttg ttcaagctct
tcctccaaac ccacgaggca 360 acttccttca acacaaggtt ccaaacctct
gccataagac gcctcactta cgacaactct 420 gtggccatgg ttccattcgg
accttactgg aagttcgtga ggaagctcat catgaacgac 480 cttctcaacg
ccaccaccgt caacaagctc aggcctttga ggacccaaca gatccgcaag 540
ttccttaggg ttatggccca aagcgcagag gcccagaagc cccttgacgt caccgaggag
600 cttctcaaat ggaccaacag caccatctcc atgatgatgc tcggcgaggc
tgaggagatc 660 agagacatcg ctcgcgaggt tcttaagatc ttcggcgaat
acagcctcac tgacttcatc 720 tggcctttga agtatctcaa ggttggaaag
tatgagaaga ggattgatga catcttgaac 780 aagttcgacc ctgtcgttga
aagggtcatc aagaagcgcc gtgagatcgt cagaaggaga 840 aagaacggag
aagttgttga gggcgaggcc agcggcgtct tcctcgacac tttgcttgaa 900
ttcgctgagg acgagaccat ggagatcaaa attaccaagg agcaaatcaa gggccttgtt
960 gtcgactttt tctctgcagg gacagattcc acagcggtgg caacagagtg
ggcattggca 1020 gagctcatca acaatcccag ggtgttgcaa aaggctcgtg
aggaggtcta cagtgttgtg 1080 ggcaaagata gactcgttga cgaagttgac
actcaaaacc ttccttacat tagggccatt 1140 gtgaaggaga cattccgaat
gcacccacca ctcccagtgg tcaaaagaaa gtgcacagaa 1200 gagtgtgaga
ttaatgggta tgtgatccca gagggagcat tggttctttt caatgtttgg 1260
caagtaggaa gggaccccaa atactgggac agaccatcag aattccgtcc cgagaggttc
1320 ttagaaactg gtgctgaagg ggaagcaggg cctcttgatc ttaggggcca
gcatttccaa 1380 ctcctcccat ttgggtctgg gaggagaatg tgccctggtg
tcaatttggc tacttcagga 1440 atggcaacac ttcttgcatc tcttatccaa
tgctttgacc tgcaagtgct gggccctcaa 1500 ggacaaatat tgaaaggtga
tgatgccaaa gttagcatgg aagagagagc tggcctcaca 1560 gttccaaggg
cacatagtct cgtttgtgtt ccacttgcaa ggatcggcgt tgcatctaaa 1620
ctcctttctt aattaagata atcatcatat acaatagtag tgtcttgcca tcgcagttgc
1680 tttttatgta ttcataatca tcatttcaat aaggtgtgac tggtacttaa
tcaagtaatt 1740 aaggttacat acatgc 1756 2 521 PRT Glycine max 2 Met
Leu Leu Glu Leu Ala Leu Gly Leu Phe Val Leu Ala Leu Phe Leu 1 5 10
15 His Leu Arg Pro Thr Pro Ser Ala Lys Ser Lys Ala Leu Arg His Leu
20 25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly
His Leu 35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu
Ile Asp Leu Ser 50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Ser
Phe Gly Ser Met Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu
Phe Lys Leu Phe Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn
Thr Arg Phe Gln Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp
Asn Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe
Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140
Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145
150 155 160 Arg Val Met Ala Gln Ser Ala Glu Ala Gln Lys Pro Leu Asp
Val Thr 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser
Met Met Met Leu 180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala
Arg Glu Val Leu Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp
Phe Ile Trp Pro Leu Lys Tyr Leu 210 215 220 Lys Val Gly Lys Tyr Glu
Lys Arg Ile Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val
Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg
Arg Lys Asn Gly Glu Val Val Glu Gly Glu Ala Ser Gly Val Phe 260 265
270 Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys
275 280 285 Ile Thr Lys Glu Gln Ile Lys Gly Leu Val Val Asp Phe Phe
Ser Ala 290 295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala
Leu Ala Glu Leu 305 310 315 320 Ile Asn Asn Pro Arg Val Leu Gln Lys
Ala Arg Glu Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu
Val Asp Glu Val Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala
Ile Val Lys Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val
Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr
Val Ile Pro Glu Gly Ala Leu Val Leu Phe Asn Val Trp Gln Val 385 390
395 400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro
Glu 405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro
Leu Asp Leu 420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly
Ser Gly Arg Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser
Gly Met Ala Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp
Leu Gln Val Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly
Asp Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr
Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510
Ile Gly Val Ala Ser Lys Leu Leu Ser 515 520 3 27 DNA Artificial
Sequence Oligonucleotide primer used in construction of WHT1 3
cgggatccat gcaaccggaa accgtcg 27 4 32 DNA Artificial Sequence
Oligonucleotide primer used in construction of yeast strain WHT1 4
ccggaattct caccaaacat cacggaggta tc 32 5 47 DNA Artificial Sequence
Oligonucleotide primer 5 tcaaggagaa aaaaccccgg atccatgttg
ctggaacttg cacttgg 47 6 35 DNA Artificial Sequence Oligonucleotide
primer 6 ggccagtgaa ttgtaatacg actcactata gggcg 35 7 24 DNA
Artificial Sequence Oligonucleotide primer 7 aaaattagcc tcacaaaagc
aaag 24 8 27 DNA Artificial Sequence Oligonucleotide primer 8
atataaggat tgatagttta tagtagg 27 9 1824 DNA Glycine max 9
ggaaaattag cctcacaaaa gcaaagatca aacaaaccaa ggacgagaac acgatgttgc
60 ttgaacttgc acttggttta ttggttttgg ctctgtttct gcacttgcgt
cccacaccca 120 ctgcaaaatc aaaagcactt cgccatctcc caaacccacc
aagcccaaag cctcgtcttc 180 ccttcatagg acaccttcat ctcttaaaag
acaaacttct ccactacgca ctcatcgacc 240 tctccaaaaa acatggtccc
ttattctctc tctactttgg ctccatgcca accgttgttg 300 cctccacacc
agaattgttc aagctcttcc tccaaacgca cgaggcaact tccttcaaca 360
caaggttcca aacctcagcc ataagacgcc tcacctatga tagctcagtg gccatggttc
420 ccttcggacc ttactggaag ttcgtgagga agctcatcat gaacgacctt
cccaacgcca 480 ccactgtaaa caagttgagg cctttgagga cccaacagac
ccgcaagttc cttagggtta 540 tggcccaagg cgcagaggca cagaagcccc
ttgacttgac cgaggagctt ctgaaatgga 600 ccaacagcac catctccatg
atgatgctcg gcgaggctga ggagatcaga gacatcgctc 660 gcgaggttct
taagatcttt ggcgaataca gcctcactga cttcatctgg ccattgaagc 720
atctcaaggt tggaaagtat gagaagagga tcgacgacat cttgaacaag ttcgaccctg
780 tcgttgaaag ggtcatcaag aagcgccgtg agatcgtgag gaggagaaag
aacggagagg 840 ttgttgaggg tgaggtcagc ggggttttcc ttgacacttt
gcttgaattc gctgaggatg 900 agaccatgga gatcaaaatc accaaggacc
acatcgaggg tcttgttgtc gactttttct 960 cggcaggaac agactccaca
gcggtggcaa cagagtgggc attggcagaa ctcatcaaca 1020 atcctaaggt
gttggaaaag gctcgtgagg aggtctacag tgttgtggga aaggacagac 1080
ttgtggacga agttgacact caaaaccttc cttacattag agcaatcgtg aaggagacat
1140 tccgcatgca cccgccactc ccagtggtca aaagaaagtg cacagaagag
tgtgagatta 1200 atggatatgt gatcccagag ggagcattga ttctcttcaa
tgtatggcaa gtaggaagag 1260 accccaaata ctgggacaga ccatcggagt
tccgtcctga gaggttccta gagacagggg 1320 ctgaagggga agcagggcct
cttgatctta ggggacaaca ttttcaactt ctcccatttg 1380 ggtctgggag
gagaatgtgc cctggagtca atctggctac ttcgggaatg gcaacacttc 1440
ttgcatctct tattcagtgc ttcgacttgc aagtgctggg tccacaagga cagatattga
1500 agggtggtga cgccaaagtt agcatggaag agagagccgg cctcactgtt
ccaagggcac 1560 atagtcttgt ctgtgttcca cttgcaagga tcggcgttgc
atctaaactc ctttcttaat 1620 taagatcatc atcatatata atatttactt
tttgtgtgtt gataatcatc atttcaataa 1680 ggtctcgttc atctactttt
tatgaagtat ataagccctt ccatgcacat tgtatcatct 1740 cccatttgtc
ttcgtttgct acctaaggca atcttttttt ttttagaatc acatcatcct 1800
actataaact atcaatcctt atat 1824 10 521 PRT Glycine max 10 Met Leu
Leu Glu Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15
His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20
25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His
Leu 35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile
Asp Leu Ser 50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe
Gly Ser Met Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe
Lys Leu Phe Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr
Arg Phe Gln Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser
Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val
Arg Lys Leu Ile Met Asn Asp Leu Pro Asn Ala Thr Thr 130 135 140 Val
Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Thr Arg Lys Phe Leu 145 150
155 160 Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu
Thr 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met
Met Met Leu 180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg
Glu Val Leu Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe
Ile Trp Pro Leu Lys His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys
Arg Ile Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val
Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg
Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270
Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275
280 285 Ile Thr Lys Asp His Ile Glu Gly Leu Val Val Asp Phe Phe Ser
Ala 290 295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu
Ala Glu Leu 305 310 315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala
Arg Glu Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val
Asp Glu Val Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile
Val Lys Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val
Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val
Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395
400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu
405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu
Asp Leu 420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser
Gly Arg Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly
Met Ala Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu
Gln Val Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly
Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val
Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile
Gly Val Ala Ser Lys Leu Leu Ser 515 520 11 21 DNA Artificial
Sequence Oligonucleotide primer 11 atgttgctgg aacttgcact t 21 12 25
DNA Artificial Sequence Oligonucleotide primer 12 ttaagaaagg
agtttagatg caacg 25 13 22 DNA Artificial Sequence Oligonucleotide
primer 13 tgtttctgca cttgcgtccc ac 22 14 22 DNA Artificial Sequence
Oligonucleotide primer 14 ccgatccttg caagtggaac ac 22 15 1501 DNA
Medicago sativa 15 tgtttctgca cttgcgtccc acaccaagtg caaaatcaaa
agcacttcgc cacctcccaa 60 accccccaag cccaaagcct cgtcttccct
tcattggcca ccttcacctc ttaaaagata 120 aacttctcca ctatgcactc
atcgatctct ccaaaaagca tggcccctta ttctctctct 180 ccttcggctc
catgccaacc gtcgttgcct ccacccctga gttgttcaag ctcttcctcc 240
aaacccacga ggcaacttcc ttcaacacaa ggttccaaac ctctgccaca agacgcctca
300 cttacgacaa ctctgtggcc atggttccat tcggacctta ctggaggttc
gtgaggaagc 360 tcatcatgaa cgaccttctc aacgccacca ccgtcaacaa
gctcaggcct ttgaggaccc 420 aacagatccg caagttcctt agggttatgg
cccaaagcgc agaggcccag aagccccttg 480 acgtcaccga ggagcttctc
aaatggacca acagcaccat ctccatgatg atgctcggcg 540 aggctgagga
gatcagagac atcgctcgcg aggttcttaa gatcttcggc gaatacagcc 600
tcactgactt catctggcct ttgaagtatc tcaaggttgg aaagtatgag aagaggattg
660 atgacatctt gaacaagttc gaccctgtcg ttgaaagggt catcaagaag
cgccgtggga 720 tcgtcagaag gagagagaac ggagaagttg ttgagggcga
ggccagcggc gtcttcctcg 780 acactttgct tgaattcgct gaggacgaga
ccatggagat caaaattacc aaggagcaaa 840 tcaagggcct tgttgtcgac
cttttctctg cagggacaga ttccacagcg gtggcaacag 900 agtgggcatt
ggcagagctc atcaacaatc ccagggtgtt gcaaaaggct cgtgaggagg 960
tctacagtgt tgtgggcaaa gatagactcg ttgacgaagt tgacactcaa aaccttcctt
1020 acattagggc cattgtgaag gagacattcc gaatgcaccc accactccca
gtggtcaaaa 1080 gaaagtgcac agaagagtgt gagattaatg ggtatgtgat
cccagaggga gcattggttc 1140 ttttcaatgt ttggcaagta ggaagggacc
ccaaatactg ggacagacca tccgaattcc 1200 gtcccgagag gttcttagaa
actggtgctg aaggggaagc agggcctctt gatcttaggg 1260 gccagcattt
ccaactcctc ccatttgggt ctgggaggag aatgtgccct ggtgtcaatt 1320
tggctacttc aggaatggca acacttcttg catctcttat ccaatgcttt gacctgcaag
1380 tgctgggccc tcaaggacaa atattgaaag gtgatgatgc caaagttagc
atggaagaga 1440 gagctggcct cacagttcca agggcacata gtctcgtttg
tgttccactt gcaaggatcg 1500 g 1501 16 499 PRT Medicago sativa 16 Phe
Leu His Leu Arg Pro Thr Pro Ser Ala Lys Ser Lys Ala Leu Arg 1 5 10
15 His Leu Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly
20 25 30 His Leu His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu
Ile Asp 35 40 45 Leu Ser Lys Lys His Gly Pro Leu Phe Ser Leu Ser
Phe Gly Ser Met 50 55 60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu
Phe Lys Leu Phe Leu Gln 65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn
Thr Arg Phe Gln Thr Ser Ala Thr 85 90 95 Arg Arg Leu Thr Tyr Asp
Asn Ser Val Ala Met Val Pro Phe Gly Pro 100 105 110 Tyr Trp Arg Phe
Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr
Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140
Phe Leu Arg Val Met Ala Gln Ser Ala Glu Ala Gln Lys Pro Leu Asp 145
150 155 160 Val Thr Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser
Met Met 165 170 175 Met Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala
Arg Glu Val Leu 180 185 190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp
Phe Ile Trp Pro Leu Lys 195 200 205 Tyr Leu Lys Val Gly Lys Tyr Glu
Lys Arg Ile Asp Asp Ile Leu Asn 210 215 220 Lys Phe Asp Pro Val Val
Glu Arg Val Ile Lys Lys Arg Arg Gly Ile 225 230 235 240 Val Arg Arg
Arg Glu Asn Gly Glu Val Val Glu Gly Glu Ala Ser Gly 245 250 255 Val
Phe Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu 260 265
270 Ile Lys Ile Thr Lys Glu Gln Ile Lys Gly Leu Val Val Asp Leu Phe
275 280 285 Ser Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala
Leu Ala 290 295 300 Glu Leu Ile Asn Asn Pro Arg Val Leu Gln Lys Ala
Arg Glu Glu Val 305 310 315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu
Val Asp Glu Val Asp Thr Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala
Ile Val Lys Glu Thr Phe Arg Met His
340 345 350 Pro Pro Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu Cys
Glu Ile 355 360 365 Asn Gly Tyr Val Ile Pro Glu Gly Ala Leu Val Leu
Phe Asn Val Trp 370 375 380 Gln Val Gly Arg Asp Pro Lys Tyr Trp Asp
Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro Glu Arg Phe Leu Glu Thr
Gly Ala Glu Gly Glu Ala Gly Pro Leu 405 410 415 Asp Leu Arg Gly Gln
His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg 420 425 430 Arg Met Cys
Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu 435 440 445 Leu
Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln 450 455
460 Gly Gln Ile Leu Lys Gly Asp Asp Ala Lys Val Ser Met Glu Glu Arg
465 470 475 480 Ala Gly Leu Thr Val Pro Arg Ala His Ser Leu Val Cys
Val Pro Leu 485 490 495 Ala Arg Ile 17 1501 DNA Vicia villosa 17
tgtttctgca cttgcgtccc acacccactg caaaatcaaa agcacttcgc catctcccaa
60 acccaccaag cccaaagcct cgtcttccct tcataggaca ccttcatctc
ttaaaagaca 120 aacttctcca ctacgcactc atcgacctct ccaaaaaaca
tggtccctta ttctctctct 180 actttggctc catgccaacc gttgttgcct
ccacaccaga attgttcaag ctcttcctcc 240 aaacgcacga ggcaacttcc
ttcaacacaa ggttccaaac ctcagccata agacgcctca 300 cctatgatag
cttagtggcc atggttccct tcggacctta ctggaagttc gtgaggaagc 360
tcatcatgaa cgaccttctc aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc
420 aacagatccg caagttcctt agggttatgg cccaaggcgc agaggcacag
aagccccttg 480 acttgaccga ggagcttctg aaatggacca acagcaccat
ctctatgatg atgctcggcg 540 aggctgagga gatcagagac atcgctcgcg
aggttcttaa gatctatggc gaatacagcc 600 tcactgactt catctggcca
ttgaagcatc tcaaggttgg aaagtatgag aagaggatcg 660 acgacatctt
gaacaagttc gaccctgtcg ttgaaagagt catcaagaag cgccgtgaga 720
tcgtgaggag gagaaagaac ggagaggttg ttgagggtga ggtcagcggg gttttccttg
780 acactttgct tgaattcgct gaggatgaga ccacggagat caaaatcacc
aaggaccaca 840 tcaagggtct tgttgtcgac tttttctcgg caggaataga
ctccacagcg gtggcaacag 900 agtgggcatt ggcagaactc atcaacaatc
ctaaggtgtt ggaaaaggct cgtgaggagg 960 tctacagtgt tgtgggaaag
gacagacttg tggacgaagt tgacactcaa aaccttcctt 1020 acattagagc
aatcgtgaag gagacattcc gcatgcaccc gccactccca gtggtcaaaa 1080
gaaagtgcac agaagagtgt gagattaatg gatatgtgat cccagaggga gcattgattc
1140 tcttcaatgt atggcaagta ggaagggacc ccaaatactg ggacagacca
tcggagttcc 1200 gtcctgagag gttcctagag acaggggctg aaggggaagc
aaggcctctt gatcttaggg 1260 gacaacattt tcaacttctc ccatttgggt
ctgggagggg aatgtgccct ggagtcaatc 1320 tggctacttc gggaatggca
acacttcttg catctcttat tcagtgcttt gacttgcaag 1380 tgctgggtcc
acaaggacag atattgaagg gtggtgacgc caaagttagc atggaagaga 1440
gggccggcct cactgttcca agggcacata gtcttgtctg tgttccactt gcaaggatcg
1500 g 1501 18 499 PRT Vicia villosa 18 Phe Leu His Leu Arg Pro Thr
Pro Thr Ala Lys Ser Lys Ala Leu Arg 1 5 10 15 His Leu Pro Asn Pro
Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly 20 25 30 His Leu His
Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp 35 40 45 Leu
Ser Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met 50 55
60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln
65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser
Ala Ile 85 90 95 Arg Arg Leu Thr Tyr Asp Ser Leu Val Ala Met Val
Pro Phe Gly Pro 100 105 110 Tyr Trp Lys Phe Val Arg Lys Leu Ile Met
Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr Val Asn Lys Leu Arg Pro
Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140 Phe Leu Arg Val Met Ala
Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp 145 150 155 160 Leu Thr Glu
Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met 165 170 175 Met
Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu 180 185
190 Lys Ile Tyr Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys
195 200 205 His Leu Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile
Leu Asn 210 215 220 Lys Phe Asp Pro Val Val Glu Arg Val Ile Lys Lys
Arg Arg Glu Ile 225 230 235 240 Val Arg Arg Arg Lys Asn Gly Glu Val
Val Glu Gly Glu Val Ser Gly 245 250 255 Val Phe Leu Asp Thr Leu Leu
Glu Phe Ala Glu Asp Glu Thr Thr Glu 260 265 270 Ile Lys Ile Thr Lys
Asp His Ile Lys Gly Leu Val Val Asp Phe Phe 275 280 285 Ser Ala Gly
Ile Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala 290 295 300 Glu
Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Val 305 310
315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr
Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe
Arg Met His 340 345 350 Pro Pro Leu Pro Val Val Lys Arg Lys Cys Thr
Glu Glu Cys Glu Ile 355 360 365 Asn Gly Tyr Val Ile Pro Glu Gly Ala
Leu Ile Leu Phe Asn Val Trp 370 375 380 Gln Val Gly Arg Asp Pro Lys
Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro Glu Arg Phe
Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu 405 410 415 Asp Leu
Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg 420 425 430
Gly Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu 435
440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro
Gln 450 455 460 Gly Gln Ile Leu Lys Gly Gly Asp Ala Lys Val Ser Met
Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr Val Pro Arg Ala His Ser
Leu Val Cys Val Pro Leu 485 490 495 Ala Arg Ile 19 1501 DNA Lens
culinaris 19 tgtttctgca cttgcgtccc acacccactg caaaatcaaa agcacttcgc
catctcccaa 60 acccaccaag cccaaagcct cgtcttccct tcataggaca
ccctcatctc ttaaaagaca 120 aacttctcca ctacgcactc atcgacctct
ccaaaaaaca tggtccctta ttctccctct 180 actttggctc catgccaacc
gttgttgcct ccacaccaga attgttcaag ctcttcctcc 240 aaacgcacga
ggcaacttcc ttcaacacaa ggttccaaac ctcagccata agacgcctca 300
cctatgatag ctcagtggcc atggttccat tcggacctta ctggaagttc gtgaggaagc
360 tcatcatgaa cgaccttctc aacgccacca ccgtcaacaa gctcaggcct
ttgaggaccc 420 aacagatccg caagttcctt agggttatgg cccaaagcgc
agaggcccag aagccccttg 480 acgtcaccga ggagcttctc aaatggacca
acagcaccat ctccatgatg atgctcggcg 540 aggctgagga gatcagagac
atcgctcgcg aggttcttaa gatcttcggc gaatacagcc 600 tcactgactt
catctggcct ttgaagtatc tcaaggttgg aaagtatgag aagaggattg 660
atgacatctt gaacaagttc gaccctgtcg ttgaaagggt catcaagaag cgccgtgaga
720 tcgtcagaag gagaaagaac ggagaagttg ttgagggcga ggccagcggc
gtcttcctcg 780 acactttgct tgaattcgct gaggacgaga ccatggagat
caaaattacc aaggagcaaa 840 tcaagggcct tgttgtcgac tttttctctg
cagggacaga ttccacagcg gtggcaacag 900 agtgggcatt ggcagagctc
atcaacaatc ccagggtgtt gcaaaaggct cgtgaggagg 960 tctacagtgt
tgtgggcaaa gatatactcg ttgacgaagt tgacactcaa aaccttcctt 1020
acattagggc cattgtgaag gagacattcc gaatgcaccc accactccca gtggtcaaaa
1080 gaaagtgcac agaagagtgt gagattaatg ggcatgtgat cccagaggga
gcattggttc 1140 ttttcaatgt ttggcaagta ggaagggacc ccaaatactg
ggacagacca tcagaattcc 1200 gtcccgagag gttcttagaa actggtgctg
aaggggaagc agggcctctt gatcttaggg 1260 gccagcattt ccaactcctc
ccatttgggt ctgggaggag aatgtgccct ggtgtcaatt 1320 tggctacttc
aggaatggca acacttcttg catctcttat ccaatgcttt gacctgcaag 1380
tgctgggccc tcaaggacaa atattgaaag gtgatgatgc caaagttagc atggaagaga
1440 gagctggcct cacagttcca agggcacata gtctcgtttg tgttccactt
gcaaggatcg 1500 g 1501 20 499 PRT Lens culinaris 20 Phe Leu His Leu
Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg 1 5 10 15 His Leu
Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly 20 25 30
His Pro His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp 35
40 45 Leu Ser Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser
Met 50 55 60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu
Phe Leu Gln 65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn Thr Arg Phe
Gln Thr Ser Ala Ile 85 90 95 Arg Arg Leu Thr Tyr Asp Ser Ser Val
Ala Met Val Pro Phe Gly Pro 100 105 110 Tyr Trp Lys Phe Val Arg Lys
Leu Ile Met Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr Val Asn Lys
Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140 Phe Leu Arg
Val Met Ala Gln Ser Ala Glu Ala Gln Lys Pro Leu Asp 145 150 155 160
Val Thr Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met 165
170 175 Met Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val
Leu 180 185 190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp
Pro Leu Lys 195 200 205 Tyr Leu Lys Val Gly Lys Tyr Glu Lys Arg Ile
Asp Asp Ile Leu Asn 210 215 220 Lys Phe Asp Pro Val Val Glu Arg Val
Ile Lys Lys Arg Arg Glu Ile 225 230 235 240 Val Arg Arg Arg Lys Asn
Gly Glu Val Val Glu Gly Glu Ala Ser Gly 245 250 255 Val Phe Leu Asp
Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu 260 265 270 Ile Lys
Ile Thr Lys Glu Gln Ile Lys Gly Leu Val Val Asp Phe Phe 275 280 285
Ser Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala 290
295 300 Glu Leu Ile Asn Asn Pro Arg Val Leu Gln Lys Ala Arg Glu Glu
Val 305 310 315 320 Tyr Ser Val Val Gly Lys Asp Ile Leu Val Asp Glu
Val Asp Thr Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys
Glu Thr Phe Arg Met His 340 345 350 Pro Pro Leu Pro Val Val Lys Arg
Lys Cys Thr Glu Glu Cys Glu Ile 355 360 365 Asn Gly His Val Ile Pro
Glu Gly Ala Leu Val Leu Phe Asn Val Trp 370 375 380 Gln Val Gly Arg
Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro
Glu Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu 405 410
415 Asp Leu Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg
420 425 430 Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala
Thr Leu 435 440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val
Leu Gly Pro Gln 450 455 460 Gly Gln Ile Leu Lys Gly Asp Asp Ala Lys
Val Ser Met Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr Val Pro Arg
Ala His Ser Leu Val Cys Val Pro Leu 485 490 495 Ala Arg Ile 21 1501
DNA Lens culinaris 21 tgtttctgca cttgcgtccc acacccactg caaaatcaaa
agcacttcgc catctcccaa 60 acccaccaag cccaaagcct cgtcttccct
tcataggaca ccttcatctc ttaaaagaca 120 aacttctcca ctacgcactc
atcgacctct ccaaaaaaca tggtccctta ttctctctct 180 actttggctc
catgccaacc gttgttgcct ccacaccaga attgttcaag ctcttcctcc 240
aaacgcacga ggcaacttcc ttcaacacaa ggttccaaac ctcagccata agacgcctca
300 cctatgatag ctcagtggcc atggttccct tcggacctta ctggaagttc
gtgaggaagc 360 tcatcatgaa cgaccttctc aacgccacca ctgtaaacaa
gttgaggcct ttgaggaccc 420 aacagatccg caagttcctt agggttatgg
cccaaggcgc agaggcacag aagccccttg 480 acttgaccga ggagcttctg
aaatggacca acagcaccat ctccatgatg gtgctcggcg 540 aggctgagga
gatcagagac atcgctcgcg aggttcttaa gatctttggc gaatacagcc 600
tcactgactt catctggcca ttgaagcatc tcaaggttgg aaagtatgag aagaggatcg
660 acgacatctt gaacaagttc gaccctgtcg ttgaaagagt catcaagaag
cgccgtgaga 720 tcgtgaggag gagaaagaac ggagaggttg ttgagggtga
ggtcagcggg gttttccttg 780 acactttgct tgaattcgct gaggatgaga
ccatggagat caaaatcacc aaggaccaca 840 tcaagggtct tgttgtcgac
tttttctcgg caggaacaga ctccacagcg gtggcaacag 900 agtgggcatt
ggcagaactc atcaacaatc ctaaggtgtt ggaaaaggct cgtgaggagg 960
tctacagtgt tgtgggaaag gacagacttg tggacgaagt tgacactcaa aaccttcctt
1020 acattagagc aatcgtgaag gagacattcc gcatgcaccc gccactccca
gtggtcaaaa 1080 gaaagtgcac agaagagtgt gagattaatg gatgtgtgac
cccagaggga gcattgattc 1140 tcttcaatgt atggcaagta ggaagagacc
ccaaatactg ggacagacca tcggagttcc 1200 gtcctgagag gttcctagag
acaggggctg aaggggaagc aaggcctctt gatcttaggg 1260 gacgacattt
tcaacttctc ccatttgggt ctgggaggag aatgtgccct ggagtcaatc 1320
tggctacttc gggaatggca acacttcttg catctcttat tcagtgcttt gacttgcagg
1380 tgctgggtcc acaaggacag atattgaagg gtggtgacgc caaagttagc
atggaagaga 1440 gagccggcct cactgttcca agggcacata gtcttgtctg
tgttccactt gcaaggatcg 1500 g 1501 22 499 PRT Lens culinaris 22 Phe
Leu His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg 1 5 10
15 His Leu Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly
20 25 30 His Leu His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu
Ile Asp 35 40 45 Leu Ser Lys Lys His Gly Pro Leu Phe Ser Leu Tyr
Phe Gly Ser Met 50 55 60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu
Phe Lys Leu Phe Leu Gln 65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn
Thr Arg Phe Gln Thr Ser Ala Ile 85 90 95 Arg Arg Leu Thr Tyr Asp
Ser Ser Val Ala Met Val Pro Phe Gly Pro 100 105 110 Tyr Trp Lys Phe
Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr
Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140
Phe Leu Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp 145
150 155 160 Leu Thr Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser
Met Met 165 170 175 Val Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala
Arg Glu Val Leu 180 185 190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp
Phe Ile Trp Pro Leu Lys 195 200 205 His Leu Lys Val Gly Lys Tyr Glu
Lys Arg Ile Asp Asp Ile Leu Asn 210 215 220 Lys Phe Asp Pro Val Val
Glu Arg Val Ile Lys Lys Arg Arg Glu Ile 225 230 235 240 Val Arg Arg
Arg Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly 245 250 255 Val
Phe Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu 260 265
270 Ile Lys Ile Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe
275 280 285 Ser Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala
Leu Ala 290 295 300 Glu Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala
Arg Glu Glu Val 305 310 315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu
Val Asp Glu Val Asp Thr Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala
Ile Val Lys Glu Thr Phe Arg Met His 340 345 350 Pro Pro Leu Pro Val
Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile 355 360 365 Asn Gly Cys
Val Thr Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp 370 375 380 Gln
Val Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390
395 400 Pro Glu Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro
Leu 405 410 415 Asp Leu Arg Gly Arg His Phe Gln Leu Leu Pro Phe Gly
Ser Gly Arg 420 425 430 Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser
Gly Met Ala Thr Leu 435 440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp
Leu Gln Val Leu Gly Pro Gln 450 455 460 Gly Gln Ile Leu Lys Gly Gly
Asp Ala Lys Val Ser Met Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr
Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu 485 490 495 Ala Arg
Ile 23 1566 DNA Phaseolus aureus 23 atgttgctgg aacttgcact
tggtttattg gttttggctc tgtttctgca cttgcgtccc 60 actcccactg
caaaatcaaa agcacttcgc catctcccaa acccaccaag cccaaagcct 120
cgtcttccct tcataggaca ccttcatctc ttaaaagaca aacttctcca ctacgcactc
180 atcgacctct ccaaaaaaca tggtccctta ttctctctct actttggctc
catgccaacc 240 gttgttgcct ccacaccaga attgttcaag ctcttcctcc
aaacgcacga ggcaacttcc 300 ttcaacacaa ggttccaaac ctcagccata
agacgcctca cctatgatag ctcagtggcc 360 atggttccct tcggacctta
ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc 420 aacgccacca
ctgtaaacaa gttgaggcct ttgaggaccc aacagatccg caagttcctt 480
agggttatgg cccaaggcgc agaggcacag aagccccttg acttgaccga ggagcttctg
540 aaatggacca acagcaccat ctccatgatg atgctcggcg aggctgagga
gatcagagac 600 atcgctcgcg aggttcttaa gatctttggc gaatacagcc
tcactgactt catctggcca 660 ttgaagcatc tcaaggttgg aaagtatgag
aagaggatcg acgacatctt gaacaagttc 720 gaccctgtcg ttgaaagagt
catcaagaag cgccgtgaga tcgtgaggag gagaaagaac 780 ggagaggttg
ttgagggtga ggtcagcggg gttttccttg acactttgct tgaattcgct 840
gaggatgaga ccatggagat caaaatcacc aaggaccaca tcaagggtct tgttgtcgac
900 tttttctcgg caggaacaga ctccacagcg gtggcaacag agtgggcatt
ggcagaactc 960 atcaacaatc ctaaggtgtt ggaaaaggct cgtgaggagg
cctacagtgt tgtgggaaag 1020 gacagacttg tggacgaagt tgacactcaa
aaccttcctt acattagagc aatcgtgaag 1080 gagacattcc gcatgcaccc
gccactccca gtggtcaaaa gaaagtgcac agaagagtgt 1140 gagattaatg
gatatgtgat cccagaggga gcattgattc tcttcaatgt atggcaagta 1200
ggaagagacc ccaaatactg ggacagacca tcggagttcc gtcctgagag gttcctagag
1260 acaggggctg aaggggaagc aaggcctctt gatcttaggg gacaacattt
tcaacttctc 1320 ccatttgggt ctgggaggag aatgtgccct ggagtcaatc
tggctacttc gggaatggca 1380 acacttctcg catctcttat tcagtgcttt
gacttgcaag tgctgggtcc acaaggacag 1440 atattgaagg gtggtgacgc
caaagttagc atggaagaga gagccggcct cactgttcca 1500 agggcacata
gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc taaactcctt 1560 tctaaa
1566 24 522 PRT Phaseolus aureus 24 Met Leu Leu Glu Leu Ala Leu Gly
Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15 His Leu Arg Pro Thr Pro
Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20 25 30 Pro Asn Pro Pro
Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 35 40 45 His Leu
Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser 50 55 60
Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met Pro Thr 65
70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln
Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser
Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser Ser Val Ala Met Val
Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val Arg Lys Leu Ile Met
Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Lys Leu Arg Pro
Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg Val Met
Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu Thr 165 170 175 Glu
Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu 180 185
190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile
195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys
His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile
Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val Ile Lys
Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg Lys Asn Gly Glu Val
Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270 Leu Asp Thr Leu Leu
Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275 280 285 Ile Thr Lys
Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 290 295 300 Gly
Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu Leu 305 310
315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Ala Tyr
Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr
Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe
Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys Cys Thr
Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro Glu Gly Ala
Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly Arg Asp Pro
Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410 415 Arg Phe
Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu Asp Leu 420 425 430
Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Met 435
440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu Leu
Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro
Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly Asp Ala Lys Val Ser
Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala His Ser
Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala Ser Lys
Leu Leu Ser Lys 515 520 25 1566 DNA Phaseolus aureus 25 atgttgctgg
aacttgcact tggtttattg gttttggctc tgtttctgca cttgcgtccc 60
acacccactg caaaatcaaa agcacttcgc catctcccaa acccaccaag cccaaagcct
120 cgtcttccct tcataggaca ccttcatctc ttaaaagaca aacttctcca
ctacgcgctc 180 atcgacctct ccaaaaaaca tggtccctta ttctctctct
actttggctc catgccaacc 240 gttgttgcct ccacaccaga attgttcaag
ctcttcctcc aaacgcacga ggcaacttcc 300 ttcaacacaa ggttccaaac
ctcagccata agacgcctca cctatgatag ctcagtggcc 360 atggttccct
tcggacctta ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc 420
aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc aacagatccg caagttcctt
480 agggctatgg cccaaggcgc agaggcacag aagccccttg acttgaccga
ggagcttctg 540 aaatggacca acagcaccat ctccatgatg atgctcggcg
aggctgagga gatcagagac 600 atcgctcgcg aggttcttaa gatctttggc
gaatacagcc tcactgactt catctggcca 660 ttgaagcatc tcaaggttgg
aaagtatgag aagaggatcg acgacatctt gaacaagttc 720 gaccctgtcg
ttgaaagagt catcaagaag cgccgtgaga tcgtgaggag gagaaagaac 780
ggagaggttg ttgagggtga ggtcagcggg gttttccttg acactttgct tgaattcgct
840 gaggatgaga ccatggagat caaaatcacc aaggaccaca tcaagggtct
tgttgtcgac 900 tttttctcgg caggaacaga ctccacagcg gtggcaacag
agtgggcatt ggcagaactc 960 atcaacaatc ctaaggtgtt ggaaaaggct
cgtgaggagg tctacagtgt tgtgggaaag 1020 gacagacttg tggacgaagt
tgacactcaa aaccttcctt acattagagc aatcgtgaag 1080 gagacattcc
gcatgcaccc gccactccca gtggtcaaaa gaaagtgcac ggaagagtgt 1140
gagattaatg gatatgtgat cccagaggga gcattgattc tcttcaatgt atggcaagta
1200 ggaagagacc ccaaatactg ggacagacca tcggagttcc gtcctgagag
gttcctagag 1260 acaggggctg aaggggaagc aaggcctctt gatcttaggg
gacaacattt tcaacttctc 1320 ccatttgggt ctgggaggag aatgtgccct
ggagtcaatc tggctacttc gggaatggca 1380 acacttcttg catctcttat
tcagtgcttt gacttgcaag tgctgggtcc acaaggacag 1440 atattgaagg
gtggtgacgc caaagttagc atggaagaga gagccggcct cactgttcca 1500
agggcacata gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc taaactcctt
1560 tcttaa 1566 26 521 PRT Phaseolus aureus 26 Met Leu Leu Glu Leu
Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15 His Leu Arg
Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20 25 30 Pro
Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 35 40
45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser
50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met
Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe
Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln
Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser Ser Val Ala
Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val Arg Lys Leu
Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Lys Leu
Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg
Ala Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu Thr 165 170
175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu
180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu
Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro
Leu Lys His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp
Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val
Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg Lys Asn Gly
Glu Val Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270 Leu Asp Thr
Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275 280 285 Ile
Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 290 295
300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu Leu
305 310 315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu
Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val
Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu
Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys
Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro Glu
Gly Ala Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly Arg
Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410 415
Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu Asp Leu 420
425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg
Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr
Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu
Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly Asp Ala Lys
Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala
His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala
Ser Lys Leu Leu Ser 515 520 27 1566 DNA Phaseolus aureus 27
atgttgctgg aacttgcact tggtttattg gttttggctc tgtttctgca cttgcgtccc
60 acacccactg caaaatcaaa agcacttcgc catctcccaa acccaccaag
cccaaagcct 120 cgtcttccct tcataggaca ccttcatctc ttaaaagaca
aacttctcca ctacgcactc 180 atcgacctct ccaaaaaaca tggtccctta
ttctctctct actttggctc catgccaacc 240 gttgttgcct ccacaccaga
attgttcaag ctcttcctcc aaacgcacga ggcaacttcc 300 ttcaacacaa
ggttccaaac ctcagccata agacgcctca cctatgatag ctcagtggcc 360
atggttccct tcggacctta ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc
420 aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc aacagatccg
caagttcctt 480 agggttatgg cccaaggcgc agaggcacag aagccccttg
acttgaccga ggagcttctg 540 aaatggacca acagcaccat ctccatgatg
atgctcggcg aggctgagga gatcagagac 600 atcgctcgcg aggttcttaa
gatctttggc gaatacagcc tcactgactt catctggcca 660 ttgaagcatc
tcaaggttgg aaagtatgag aagaggatcg acgacatctt gaacaagttc 720
gaccctgtcg ttgaaagagt catcaagaag cgccgtgaga tcgtgaggag gagaaagaac
780 ggagaggttg ttgagggtga ggtcagcggg gttttccttg acactttgct
tgaattcgct 840 gaggatgaga ccacggagat caaaatcacc aaggaccaca
tcaagggtct tgttgtcgac 900 tttttctcgg caggaacaga ctccacagcg
gtggcaacag agtgggcatt ggcagaactc 960 atcaacaatc ctaaggtgtt
ggaaaaggct cgtgaggagg tctacagtgt tgtgggaaag 1020 gacagacttg
tggacgaagt tgacactcaa aaccttcctt acattagagc aatcgtgaag 1080
gagacattcc gcatgcaccc gccactccca gtggtcaaaa gaaagtgcac agaagagtgt
1140 gagattaatg gatatgtgat cccagaggga gcattgattc tcttcaatgt
atggcaagta 1200 ggaagagacc ccaaatactg ggacagacca tcggagttcc
gtcctgagag gttcctagag 1260 acaggggctg aaggggaagc aaggcctctt
gatcttaggg gacaacattt tcaacttctc 1320 ccatttgggt ctgggaggag
aatgtgccct ggagtcaatc tggctacttc gggaatggca 1380 acacttcttg
catctcttat tcagtgcttt gacttgcaag tgctgggtcc acaaggacag 1440
atattgaagg gtggtgacgc caaagttagc atggaagaga gggccggcct cactgttcca
1500 agggcacata gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc
taaactcctt 1560 tcttaa 1566 28 521 PRT Phaseolus aureus 28 Met Leu
Leu Glu Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15
His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20
25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His
Leu 35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile
Asp Leu Ser 50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe
Gly Ser Met Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe
Lys Leu Phe Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr
Arg Phe Gln Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser
Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val
Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val
Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150
155 160 Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu
Thr 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met
Met Met Leu 180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg
Glu Val Leu Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe
Ile Trp Pro Leu Lys His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys
Arg Ile Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val
Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg
Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270
Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Thr Glu Ile Lys 275
280 285 Ile Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser
Ala 290 295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu
Ala Glu Leu 305 310 315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala
Arg Glu Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val
Asp Glu Val Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile
Val Lys Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val
Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val
Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395
400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu
405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu
Asp Leu 420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser
Gly Arg Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly
Met Ala Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu
Gln Val Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly
Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val
Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile
Gly Val Ala Ser Lys Leu Leu Ser 515 520 29 1566 DNA Phaseolus
aureus 29 atgttgctgg aacttgcact tggtttattg gttttggctc tgtttctgca
cttgcgtccc 60 acacccactg caaaatcaaa agcacttcgc catctcccaa
acccaccaag cccaaagcct 120 cgtcttccct tcataggaca ccttcatctc
ttaaaagaca aacttctcca ctacgcactc 180 atcgacctct ccaaaaaaca
tggtccctta ttctctctct actttggctc catgccaacc 240 gttgttgcct
ccacaccaga attgttcaag ctcttcctcc aaacgcacga ggcaacttcc 300
ttcaacacaa ggttccaaac ctcagccata agacgcctca cctatgatag ctcagtggcc
360 atggttccct tcggacctta ctggaagttc gtgaggaagc tcatcatgaa
cgaccttctc 420 aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc
aacagatccg caagttcctt 480
agggttatgg cccaaggcgc agaggcacag aagccccttg acttgaccga ggagcttctg
540 aaatggacca acagcaccat ctccatgatg atgctcggcg aggctgagga
gatcagagac 600 atcgctcgcg aggttcttaa gatctttggc gaatacagcc
tcactgactt catctggcca 660 ttgaagcatc tcaaggttgg aaagtatgag
aagaggatcg acgacatctt gaacaagttc 720 gaccctgtcg ttgaaagagt
catcaagaag cgccgtgaga tcgtgaggag gagaaagaac 780 ggagaggttg
ttgagggtga ggtcagcggg gttttccttg acactttgct tgaattcgct 840
gaggatgaga ccatggagat caaaatcacc aaggaccaca tcaagggtct tgttgtcgac
900 tttttctcgg caggaacaga ctccacagcg gaggcaacag agtgggcatt
ggcagaactc 960 atcaacaatc ctaaggtgtt ggaaaaggct cgtgaggagg
tctacagtgt tgtgggaaag 1020 gacagacttg tggacgaagt tgacactcaa
aaccttcctt acattagagc aatcgtgaag 1080 gagacattcc gcatgcaccc
gccactccca gtggtcaaaa gaaagtgcac agaagagtgt 1140 gagattaatg
gatatgtgat cccagaggga gcattgattc tcttcaatgt atggcaagta 1200
ggaagagacc ccaaatactg ggacagacca tcggagttcc gtcctgagag gttcctagag
1260 acaggggctg aaggggaagc aaggcctctt gatcttaggg gacaacattt
tcaacttctc 1320 ccatttgggt ctgggaggag aatgtgccct ggagtcaatc
tggctacttc gggaatggca 1380 acacttcttg catctcttat tcagtgcttt
gacttgcaag tgctgggtcc acaaggacag 1440 atattgaagg gtggtgacgc
caaagttagc atggaagaga gagccggcct cactgttcca 1500 agggcacata
gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc taaactcctt 1560 tcttaa
1566 30 521 PRT Phaseolus aureus 30 Met Leu Leu Glu Leu Ala Leu Gly
Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15 His Leu Arg Pro Thr Pro
Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20 25 30 Pro Asn Pro Pro
Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 35 40 45 His Leu
Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser 50 55 60
Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met Pro Thr 65
70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln
Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser
Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser Ser Val Ala Met Val
Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val Arg Lys Leu Ile Met
Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Lys Leu Arg Pro
Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg Val Met
Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu Thr 165 170 175 Glu
Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu 180 185
190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile
195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys
His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile
Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val Ile Lys
Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg Lys Asn Gly Glu Val
Val Glu Gly Glu Val Ser Gly Val Phe 260 265 270 Leu Asp Thr Leu Leu
Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275 280 285 Ile Thr Lys
Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 290 295 300 Gly
Thr Asp Ser Thr Ala Glu Ala Thr Glu Trp Ala Leu Ala Glu Leu 305 310
315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Val Tyr
Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr
Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe
Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys Cys Thr
Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro Glu Gly Ala
Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly Arg Asp Pro
Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410 415 Arg Phe
Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu Asp Leu 420 425 430
Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Met 435
440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu Leu
Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro
Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly Asp Ala Lys Val Ser
Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala His Ser
Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala Ser Lys
Leu Leu Ser 515 520 31 1566 DNA Trifolium pratense 31 atgttgctgg
aacttgcact tggtttattg gttttggctc tgtttctgca cttgcgtccc 60
acacccactg caaaatcaaa agcacttcgc catctcccaa acccaccaag cccaaagcct
120 cgtcttccct tcataggaca ccttcatctc ttaaaagaca aacttctcca
ctacgcactc 180 atcgacctct ccaaaaaaca tggtccctta ttctctctct
actttggctc catgccaacc 240 gttgttgcct ccacaccaga attgttcaag
ctcttcctcc aaacgcacga ggcaacttcc 300 ttcaacacaa ggttccaaac
ctcagccata agacgcctca cctatgatag ctcagtggcc 360 atggttccca
tcggacctta ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc 420
aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc aacagatccg caagttcctt
480 agggttatgg cccaaggcgc agaggcacag aagccccttg acttgaccga
ggagcttctg 540 aaatggacca acagcaccat ctccatgatg atgctcggcg
aggctgagga gatcagagac 600 atcgctcgcg aggttcttaa gatctttggc
gaatacagcc tcactgactt catctggcca 660 ttgaagcatc tcaaggttgg
aaagtatgag aagaggatcg acgacatctt gaacaagttc 720 gaccctgtcg
ttgaaagagt catcaagaag cgccgtgaga tcgtgaggag gagaaagaac 780
ggagaggttg atgagggtga ggtcagcggg gttttccttg acactttgct tgaattcgct
840 gaggatgaga ccacggagat caaaatcacc aaggaccaca tcaagggtct
tgttgtcgac 900 tttttctcgg cagggacaga ctccacagcg gtggcaacag
agtgggcatt ggcagaactc 960 atcaacaatc ctaaggtgtt ggaaaaggct
cgtgaggagg tctacagtgt tgtgggaaag 1020 gacagacttg tggacgaagt
tgacactcaa aaccttcctt acattagagc aatcgtgaag 1080 gagacattcc
gcatgcaccc gccactccca gtggtcaaaa gaaagtgcac agaagagtgt 1140
gagattaatg gatatgtgat cccagaggga gcattgattc tcttcaatgt atggcaagta
1200 ggaagagacc ccaaatactg ggacagacca tcggagttcc gtcctgagag
gttcctagag 1260 acaggggctg aaggggaagc aaggcctctt gatcttaggg
gacaacattt tcaacttctc 1320 ccatttgggt ctgggaggag aatgtgccct
ggagtcaatc tggctacttc gggaatggca 1380 acacttcttg catctcttat
tcagtgcttt gacttgcaag tgctgggtcc acaaggacag 1440 atattgaagg
gtggtgacgc caaagttagc atggaagaga gggccggcct cactgttcca 1500
agggcacata gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc taaactcctt
1560 tcttaa 1566 32 521 PRT Trifolium pratense 32 Met Leu Leu Glu
Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 1 5 10 15 His Leu
Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu 20 25 30
Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 35
40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu
Ser 50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser
Met Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu
Phe Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn Thr Arg Phe
Gln Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp Ser Ser Val
Ala Met Val Pro Ile Gly Pro Tyr Trp 115 120 125 Lys Phe Val Arg Lys
Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Lys
Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160
Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp Leu Thr 165
170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met
Leu 180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val
Leu Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp
Pro Leu Lys His Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile
Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg
Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg Lys Asn
Gly Glu Val Asp Glu Gly Glu Val Ser Gly Val Phe 260 265 270 Leu Asp
Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Thr Glu Ile Lys 275 280 285
Ile Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 290
295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu
Leu 305 310 315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu
Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu
Val Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys
Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg
Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro
Glu Gly Ala Leu Ile Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly
Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410
415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu Asp Leu
420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg
Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala
Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val
Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Gly Asp Ala
Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg
Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val
Ala Ser Lys Leu Leu Ser 515 520 33 1566 DNA Trifolium pratense 33
atgttgctgg aacttgcact tggtttattg gttttggctc tgtttctgca cttgcgtccc
60 acacccactg caaaatcaaa agcacttcgc catctcccaa acccaccaag
cccaaagcct 120 cgtcttccct tcataggaca ccttcatctc ttaaaagaca
aacttctcca ctacgcactc 180 atcgacctct ccaaaaaaca tggtccctta
ttctctctct actttggctc catgccaacc 240 gttgttgcct ccacaccaga
attgttcaag ctcttcctcc aaacgcacga ggcaacttcc 300 ttcaacacaa
ggttccaaac ctcagccata agacgcctca cctatgatag ctcagtggcc 360
atggttccct tcggacctta ctggaagttc gtgaggaagc tcatcatgaa cgaccttctc
420 aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc aacagatccg
caagttcctt 480 agggttatgg cccaaggcgc agaggcacag aagccccttg
acttgaccga ggagcttctg 540 aaatggacca acagcaccat ctccatgatg
atgctcggcg aggctgagga gatcagagac 600 atcgctcgcg aggttcttaa
gatctttggc gaatacagcc tcactgactt catctggcca 660 ttgaagcatc
tcaaggttgg aaagtatgag aagaggatcg acgacatctt gaacaagttc 720
gaccctgtcg ttgaaagagt catcaagaag cgccgtgaga tcgtgaggag gagaaagaac
780 ggagaggttg ttgagggtga ggtcagcggg gttttccttg acactttgct
tgaattcgct 840 gaggatgaga ccacggagat caaaatcacc aaggaccaca
tcaagggtct tgttgtcgac 900 tttttctcgg caggaacaga ctccacagcg
gtggcaacag agtgggcatt ggcagaactc 960 atcaacaatc ctaaggtgtt
ggaaaaggct cgtgaggagg tctacagtgt tgtgggaaag 1020 gacagacttg
tggacgaagt tgacactcaa aaccttcctt acattagagc aatcgtgaag 1080
gagacattcc gcatgcaccc gccactccca gtggtcaaaa gaaagtgcac agaagagtgt
1140 gagattaatg gatatgtgat cccagaggga gcattgattc tcttcaatgt
atggcaagta 1200 ggaagagacc ccaaatactg ggacagacca tcggagttcc
gtcctgagag gttcctagag 1260 acaggggctg aaggggaagc aaggcctctt
gatcttaggg gacaacattt tcaacttctc 1320 ccatttgggt ctgggaggag
aatgtgccct ggagtcaatc tggctacttc gggaatggca 1380 acacttcttg
catctcttat tcagtgcttt gacttgcaag tgctgggtcc acaaggacag 1440
atattgaagg gtggtgacgc caaagttagc atggaagaga gggccggcct cactgttcca
1500 agggcacata gtcttgtctg tgttccactt gcaaggatcg gcgttgcatc
taaactcctt 1560 tcttaa 1566 34 521 PRT Trifolium pratense 34 Met
Leu Leu Glu Leu Ala Leu Gly Leu Leu Val Leu Ala Leu Phe Leu 1 5 10
15 His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His Leu
20 25 30 Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly
His Leu 35 40 45 His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu
Ile Asp Leu Ser 50 55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Tyr
Phe Gly Ser Met Pro Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu
Phe Lys Leu Phe Leu Gln Thr His 85 90 95 Glu Ala Thr Ser Phe Asn
Thr Arg Phe Gln Thr Ser Ala Ile Arg Arg 100 105 110 Leu Thr Tyr Asp
Ser Ser Val Ala Met Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe
Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140
Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145
150 155 160 Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp
Leu Thr 165 170 175 Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser
Met Met Met Leu 180 185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala
Arg Glu Val Leu Lys Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp
Phe Ile Trp Pro Leu Lys His Leu 210 215 220 Lys Val Gly Lys Tyr Glu
Lys Arg Ile Asp Asp Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val
Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg
Arg Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly Val Phe 260 265
270 Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Thr Glu Ile Lys
275 280 285 Ile Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe
Ser Ala 290 295 300 Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala
Leu Ala Glu Leu 305 310 315 320 Ile Asn Asn Pro Lys Val Leu Glu Lys
Ala Arg Glu Glu Val Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu
Val Asp Glu Val Asp Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala
Ile Val Lys Glu Thr Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val
Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr
Val Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp Gln Val 385 390
395 400 Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro
Glu 405 410 415 Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro
Leu Asp Leu 420 425 430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly
Ser Gly Arg Arg Met 435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser
Gly Met Ala Thr Leu Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp
Leu Gln Val Leu Gly Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly
Gly Asp Ala Lys Val Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr
Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510
Ile Gly Val Ala Ser Lys Leu Leu Ser 515 520 35 1563 DNA Pisum
sativum 35 atgttgctgg aacttgcact tggtttgttt gtgttagctt tgtttctgca
cttgcgtccc 60 acaccaagcg caaaatcaaa agcacttcgc cacctcccaa
accctccaag cccaaagcct 120 cgtcttccct tcattggcca ccttcacctc
ttaaaagata aacttctcca ctatgcactc 180 atcgatctct ccaaaaagca
tggcccctta ttctctctct ccttcggctc catgccaacc 240 gtcgttgcct
ccacccctga gttgttcaag ctcttcctcc aagcccacga ggcaacttcc 300
ttcagcacaa ggttccaaac ctctgccgta agacgcctca cttacgacaa ctctgtggcc
360 atggttccat tcggacctta ctggaagttc gtgaggaagc tcatcatgaa
cgaccttctc 420 aacgccacca ccgtcaacga gctcaggcct ttgaggaccc
aacagatccg caagttcctt 480 agggttatgg cccaaagcgc agaggcccag
aagccccttg acgtcaccga ggagcttctc 540 aaatggacca acagcaccat
ctccatgatg atgctcggcg aggctgagga gatcagagac 600 atcgctcgcg
aggtccttaa gatcttcggc gaatacagcc tcactgactt catctggcct 660
ttgaagtatc tcaaggttgg aaagtatgag aagaggattg atgacatctt gaacaagttc
720 gaccctgtcg ttgaaagggt catcaagaag cgccgtgaga tcgtcagaag
gagaaagaac 780 ggagaagttg ttgagggcga ggccagcggc gtcttcctcg
acactttgct tgaattcgct 840 gaggacgaga ccatggagat caaaattacc
aaggagcaaa tcaagggcct
tgttgtcgac 900 tttttctctg cagggacaga ttccacagcg gtggcaacag
agtgggcatt ggcagagctc 960 atcaacaatc ccagggtgtt gcaaaaggct
cgtgaggagg tctacagtgt tgtgggcaaa 1020 gatagactcg ttgacgaagt
cgacactcaa aaccttcctt acattagggc cattgtgaag 1080 gagacattcc
gaatgcaccc accactccca gtggtcaaaa gaaagtgcac agaagagtgt 1140
gagattaatg ggtatgtgat cccagaggga gcattggttc ttttcaatgt ttggcaagta
1200 ggaaaggacc ccaaatactg ggacagacca tcagaattcc gtcccgagag
gttcttagaa 1260 actggcgctg aaggggaagc agggcctctt gatcttaggg
gccagcattt ccaactcctc 1320 ccatttgggt ctgggaggag aatgtgccct
ggtgtcaatt tggctacttc aggaatggca 1380 acacttcttg catctcttat
ccaatgcttt gacctgcaag tgctgggccc tcaaggacaa 1440 atattgaaag
gtgacgatgc caaagttagc atggaagaga gagctggcct caccgttcca 1500
agggcacata gtctcgtttg tgttccactt gcaaggatcg gcgttgcatc taaactcctt
1560 tct 1563 36 521 PRT Pisum sativum 36 Met Leu Leu Glu Leu Ala
Leu Gly Leu Phe Val Leu Ala Leu Phe Leu 1 5 10 15 His Leu Arg Pro
Thr Pro Ser Ala Lys Ser Lys Ala Leu Arg His Leu 20 25 30 Pro Asn
Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His Leu 35 40 45
His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp Leu Ser 50
55 60 Lys Lys His Gly Pro Leu Phe Ser Leu Ser Phe Gly Ser Met Pro
Thr 65 70 75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu
Gln Ala His 85 90 95 Glu Ala Thr Ser Phe Ser Thr Arg Phe Gln Thr
Ser Ala Val Arg Arg 100 105 110 Leu Thr Tyr Asp Asn Ser Val Ala Met
Val Pro Phe Gly Pro Tyr Trp 115 120 125 Lys Phe Val Arg Lys Leu Ile
Met Asn Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Glu Leu Arg
Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe Leu 145 150 155 160 Arg Val
Met Ala Gln Ser Ala Glu Ala Gln Lys Pro Leu Asp Val Thr 165 170 175
Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met Met Leu 180
185 190 Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys
Ile 195 200 205 Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu
Lys Tyr Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp
Ile Leu Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val Ile
Lys Lys Arg Arg Glu Ile Val Arg 245 250 255 Arg Arg Lys Asn Gly Glu
Val Val Glu Gly Glu Ala Ser Gly Val Phe 260 265 270 Leu Asp Thr Leu
Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile Lys 275 280 285 Ile Thr
Lys Glu Gln Ile Lys Gly Leu Val Val Asp Phe Phe Ser Ala 290 295 300
Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala Glu Leu 305
310 315 320 Ile Asn Asn Pro Arg Val Leu Gln Lys Ala Arg Glu Glu Val
Tyr Ser 325 330 335 Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp
Thr Gln Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr
Phe Arg Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys Cys
Thr Glu Glu Cys Glu Ile Asn Gly 370 375 380 Tyr Val Ile Pro Glu Gly
Ala Leu Val Leu Phe Asn Val Trp Gln Val 385 390 395 400 Gly Lys Asp
Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro Glu 405 410 415 Arg
Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu Asp Leu 420 425
430 Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Arg Met
435 440 445 Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu
Leu Ala 450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly
Pro Gln Gly Gln 465 470 475 480 Ile Leu Lys Gly Asp Asp Ala Lys Val
Ser Met Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala His
Ser Leu Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala Ser
Lys Leu Leu Ser 515 520 37 1496 DNA Trifolium repens 37 tctcacttgc
gtcccacacc aagtgcaata tcaaaagcac ttcgccacct cccaaaccct 60
ccaagcccaa ggcctcgtct tcccttcatt ggccaccttc acctcttaaa agataaactt
120 ctccactatg cacccatcga tctctccaaa aagcatggcc ccttattctc
tctctccttc 180 ggctccatgc caaccgtcgt tgcctccacc cctgagttgt
tcaagctctt cctccaaacc 240 cacgaggcaa cttccttcaa cacaaggttc
caaacctctg ccataagaca cctcacttac 300 gacaactctg tggccatggt
tccattcgga ccttactgga agttcgtgag gaagctcatc 360 atgaacgacc
ttctcaacgc caccaccgtc aacaagctca ggcctttgag gacccaacag 420
atccgcaagt tccttagggt tatggcccaa agcgcagagg cccagaagcc ccttgacgtc
480 accgaggagc ttctcaaatg gaccaacagc accatctcca tgatgatgct
cggcgaggct 540 gaggagatca gagacatcgc tcgcgaggtt cttaagatct
tcggcgaata cagcctcact 600 gacttcatct ggcctttgaa gtacctcaag
gttggaaagt atgagaagag gattgatgac 660 atcttgaaca agttcgaccc
tgtcgttgaa agggtcatca agaagcgccg tgagatcgtc 720 agaaggagaa
agaacggaga agttgttgag ggcgaggcca gcggcgtctt cctcgacact 780
ttgcttgaat tcgctgagga cgagaccatg gagatcaaaa ttaccaagga gcaaatcaag
840 ggccttgttg tcgacttttt ctctgcaggg acagattcca cagcggtggt
aacagagtgg 900 gcattggcag agctcatcaa caatcccagg gtgttgcaaa
aggctcgtga ggaggtctac 960 agtgttgtgg gcaaagatag actcgttgac
gaagttgaca ctcaaaacct tccttacatt 1020 agggccattg tgaaggagac
attccgaatg cacccaccac tcccagtggt caaaagaaag 1080 tgcacagaag
agtgtgagat taatgggtat gtgatcccag agggagcatt ggttcttttc 1140
aatgtttggc aagtaggaag ggaccccaaa tactgggaca gaccatcaga atcccgtccc
1200 gagaggttct tagaaactgg tgctgaaggg gaagcagggc ctcttgatct
taggggccag 1260 catttccaac tcctcccatt tgggtctggg aggagaatgt
gccctggtgt cagtttggct 1320 acttcaggaa tggcaacact tcttgcatct
cttatccaat gctttgacct gcaagtgctg 1380 ggccctcaag gacaaatatt
gaaaggtgat gatgccaaag ttagcatgga agagagagct 1440 ggcctcacag
ttccaagggc acatagtctc gtttgtgttc cacttgcaag gatcgg 1496 38 498 PRT
Trifolium repens 38 Ser His Leu Arg Pro Thr Pro Ser Ala Ile Ser Lys
Ala Leu Arg His 1 5 10 15 Leu Pro Asn Pro Pro Ser Pro Arg Pro Arg
Leu Pro Phe Ile Gly His 20 25 30 Leu His Leu Leu Lys Asp Lys Leu
Leu His Tyr Ala Pro Ile Asp Leu 35 40 45 Ser Lys Lys His Gly Pro
Leu Phe Ser Leu Ser Phe Gly Ser Met Pro 50 55 60 Thr Val Val Ala
Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln Thr 65 70 75 80 His Glu
Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser Ala Ile Arg 85 90 95
His Leu Thr Tyr Asp Asn Ser Val Ala Met Val Pro Phe Gly Pro Tyr 100
105 110 Trp Lys Phe Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala
Thr 115 120 125 Thr Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile
Arg Lys Phe 130 135 140 Leu Arg Val Met Ala Gln Ser Ala Glu Ala Gln
Lys Pro Leu Asp Val 145 150 155 160 Thr Glu Glu Leu Leu Lys Trp Thr
Asn Ser Thr Ile Ser Met Met Met 165 170 175 Leu Gly Glu Ala Glu Glu
Ile Arg Asp Ile Ala Arg Glu Val Leu Lys 180 185 190 Ile Phe Gly Glu
Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys Tyr 195 200 205 Leu Lys
Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu Asn Lys 210 215 220
Phe Asp Pro Val Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile Val 225
230 235 240 Arg Arg Arg Lys Asn Gly Glu Val Val Glu Gly Glu Ala Ser
Gly Val 245 250 255 Phe Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu
Thr Met Glu Ile 260 265 270 Lys Ile Thr Lys Glu Gln Ile Lys Gly Leu
Val Val Asp Phe Phe Ser 275 280 285 Ala Gly Thr Asp Ser Thr Ala Val
Val Thr Glu Trp Ala Leu Ala Glu 290 295 300 Leu Ile Asn Asn Pro Arg
Val Leu Gln Lys Ala Arg Glu Glu Val Tyr 305 310 315 320 Ser Val Val
Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr Gln Asn 325 330 335 Leu
Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg Met His Pro 340 345
350 Pro Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn
355 360 365 Gly Tyr Val Ile Pro Glu Gly Ala Leu Val Leu Phe Asn Val
Trp Gln 370 375 380 Val Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser
Glu Ser Arg Pro 385 390 395 400 Glu Arg Phe Leu Glu Thr Gly Ala Glu
Gly Glu Ala Gly Pro Leu Asp 405 410 415 Leu Arg Gly Gln His Phe Gln
Leu Leu Pro Phe Gly Ser Gly Arg Arg 420 425 430 Met Cys Pro Gly Val
Ser Leu Ala Thr Ser Gly Met Ala Thr Leu Leu 435 440 445 Ala Ser Leu
Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln Gly 450 455 460 Gln
Ile Leu Lys Gly Asp Asp Ala Lys Val Ser Met Glu Glu Arg Ala 465 470
475 480 Gly Leu Thr Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu
Ala 485 490 495 Arg Ile 39 1501 DNA Trifolium repens 39 tgtttctgca
cttgcgtccc acacccactg caaaatcaaa agcacttcgc catctcccaa 60
acccaccaag cccaaagcct cgtcttccct tcataggaca ccttcatctc ttaaaagaca
120 aacttctcca ctacgcactc atcgacctct ccaaaaaaca tggtccctta
ttctctctct 180 actttggctc catgccaacc gttgttgcct ccacaccaga
attgttcaag ctcttcctcc 240 aaacgcacga ggcaacttcc ttcaacacaa
ggttccaaac ctcagccata agacgcctca 300 cctacgacaa ctctgtggcc
atggttccat tcggacctta ctggaagttc gtgaggaagc 360 tcatcatgaa
cgaccttctc aacgccacca ccgtcaacaa gctcaggcct ttgaggaccc 420
aacagatccg caagttcctt agggttatgg cccaaagcgc agaggcccag aagccccttg
480 acgtcaccga ggagcttctc aaatggacca acagcaccat ctccatgatg
atgctcggcg 540 aggctgagga gatcagagac atcgctcgcg aggttcttaa
gatcttcggc gaatacagcc 600 tcactgactt catctggcct ttgaagtatc
tcaaggttgg aaagtatgag aagaggattg 660 atgacatctt gaacaagttc
gaccctgtcg ttgaaagagt catcaagaag cgccgtgaga 720 tcgtcagaag
gagaaagaac ggagaagttg ttgagggcga ggccagcggc gtcttcctcg 780
acactttgct tgaattcgct gaggacgaga ccatggagat caaaattacc aaggagcaaa
840 tcaagggcct tgttgtcgac tttttctctg cagggacaga ttccacagcg
gtggcaacag 900 agtgggcatt ggcagagctc atcaacaatc ccaaggtgtt
gcaaaaggct cgtgaggagg 960 cctacagtgt tgtgggcaaa gatagactcg
ttgacgaagt tgacactcaa aaccttcctt 1020 acattagggc cattgtgaag
gagacattcc gaatgcaccc accactccca gtggtcaaaa 1080 gaaagtgcac
agaagagtgt gggattaatg ggtatgtgat cccagaggga gcattggttc 1140
ttttcaatgt ttggcaagta ggaagggacc ccaaatactg ggacagacca tcagaattcc
1200 gtcccgagag gttcttagaa actggtgctg aaggggaagc agggcctctt
gatcttaggg 1260 gccagcattt ccaactcctc ccatttgggt ctgggaggag
aatgtgccct ggtgtcaatt 1320 tggctacttc aggaatggca acacttcttg
catctcttat ccaatgcttt gacctgcaag 1380 tgctgggccc tcaaggacaa
atattgaaag gtgatgatgc caaagttagc atggaagaga 1440 gagctggcct
cacagttcca agggcacata gtctcgtttg tgttccactt gcaaggatcg 1500 g 1501
40 499 PRT Trifolium repens 40 Phe Leu His Leu Arg Pro Thr Pro Thr
Ala Lys Ser Lys Ala Leu Arg 1 5 10 15 His Leu Pro Asn Pro Pro Ser
Pro Lys Pro Arg Leu Pro Phe Ile Gly 20 25 30 His Leu His Leu Leu
Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp 35 40 45 Leu Ser Lys
Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met 50 55 60 Pro
Thr Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln 65 70
75 80 Thr His Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser Ala
Ile 85 90 95 Arg Arg Leu Thr Tyr Asp Asn Ser Val Ala Met Val Pro
Phe Gly Pro 100 105 110 Tyr Trp Lys Phe Val Arg Lys Leu Ile Met Asn
Asp Leu Leu Asn Ala 115 120 125 Thr Thr Val Asn Lys Leu Arg Pro Leu
Arg Thr Gln Gln Ile Arg Lys 130 135 140 Phe Leu Arg Val Met Ala Gln
Ser Ala Glu Ala Gln Lys Pro Leu Asp 145 150 155 160 Val Thr Glu Glu
Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met 165 170 175 Met Leu
Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu 180 185 190
Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys 195
200 205 Tyr Leu Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu
Asn 210 215 220 Lys Phe Asp Pro Val Val Glu Arg Val Ile Lys Lys Arg
Arg Glu Ile 225 230 235 240 Val Arg Arg Arg Lys Asn Gly Glu Val Val
Glu Gly Glu Ala Ser Gly 245 250 255 Val Phe Leu Asp Thr Leu Leu Glu
Phe Ala Glu Asp Glu Thr Met Glu 260 265 270 Ile Lys Ile Thr Lys Glu
Gln Ile Lys Gly Leu Val Val Asp Phe Phe 275 280 285 Ser Ala Gly Thr
Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala 290 295 300 Glu Leu
Ile Asn Asn Pro Lys Val Leu Gln Lys Ala Arg Glu Glu Ala 305 310 315
320 Tyr Ser Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr Gln
325 330 335 Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg
Met His 340 345 350 Pro Pro Leu Pro Val Val Lys Arg Lys Cys Thr Glu
Glu Cys Gly Ile 355 360 365 Asn Gly Tyr Val Ile Pro Glu Gly Ala Leu
Val Leu Phe Asn Val Trp 370 375 380 Gln Val Gly Arg Asp Pro Lys Tyr
Trp Asp Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro Glu Arg Phe Leu
Glu Thr Gly Ala Glu Gly Glu Ala Gly Pro Leu 405 410 415 Asp Leu Arg
Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg 420 425 430 Arg
Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu 435 440
445 Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln
450 455 460 Gly Gln Ile Leu Lys Gly Asp Asp Ala Lys Val Ser Met Glu
Glu Arg 465 470 475 480 Ala Gly Leu Thr Val Pro Arg Ala His Ser Leu
Val Cys Val Pro Leu 485 490 495 Ala Arg Ile 41 21 DNA Artificial
Sequence PCR primer 41 ttgctggaac ttgcacttgg t 21 42 32 DNA
Artificial Sequence PCR primer 42 gtatatgatg ggtaccttaa ttaagaaagg
ag 32 43 26 DNA Artificial Sequence PCR primer 43 gacgcctcac
ttacgacaac tctgtg 26 44 25 DNA Artificial Sequence PCR primer 44
cctctcggga cggaattctg atggt 25 45 25 DNA Artificial Sequence PCR
primer 45 gcggtgcacg ggcggactct tcttc 25 46 25 DNA Artificial
Sequence PCR primer 46 cgcccaatac gcaaaccgcc tctcc 25 47 1501 DNA
Beta vulgaris 47 tgtttctgca cttgcgtccc acacccactg caaaatcaaa
agcacttcgc catctcccaa 60 acccaccaag cccaaagcct cgtcttccct
tcataggaca ccttcatctc ttaaaagaca 120 aacttctcca ctacgcactc
atcgacctct ccaaaaaaca tggtccctta ttctctctct 180 actttggctc
catgccaacc gttgttgcct ccacaccaga attgttcaag ctcttcctcc 240
aaacgcacga ggcaacttcc ttcaacacaa ggttccaaac ctcagccata agacgcctca
300 cctatgatag ctcagtggcc atggttccct tcggacctta ctggaagttc
gtgaggaagc 360 tcatcatgaa cgaccttctc aacgccacca ctgtaaacaa
gttgaggcct ttgaggaccc 420 aacagatccg caagttcctt agggttatgg
cccaaggcgc agaggcacag aagccccttg 480 acttgaccga ggagcttctg
aaatggacca acagcaccat ctccatgatg atgctcggcg 540 aggctgagga
gatcagagac atcgctcgcg aggttcttaa gatctttggc gaatacagcc 600
tcactgactt catctggcca ttgaagcatc tcaaggttgg aaagtatgag aagaggatcg
660 acgacatctt gaacaagttc gaccctgtcg ttgaaagagt catcaagaag
cgccgtgaga 720 tcgtgaggag gagaaagaac ggagaggatg ttgagggtga
ggtcagcggg gttttccttg 780 acactttgct tgaattcgct gaggatgaga
ccatggagat caaaatcacc aaggaccaca 840 tcaagggtct tgttgtcgac
tttttctcgg caggaacaga ctccacagcg gtggcaacag 900 agtgggcatt
ggcagaactc atcaacaatc ctaaggtgtt ggaaaaggct cgtgaggagg 960
tctacagtgt tgtgggaaag gacagacttg tggacgaagt agacactcaa aaccttcctt
1020 acattagagc aatcgtgaag gagacattcc gcatgcaccc gccactccca
gtggtcaaaa 1080 gaaagtgcat agaagagtgt gagattaatg gatatgtgat
cccagaggga gcattgattc 1140 tcttcaatgt atggcaagta ggaagagacc
ctaaatactg ggacagacca
tcggagttcc 1200 gtcctgagag gttcctagag acaggggctg aaggggaagc
aaggcttctt gatcttaggg 1260 gacaacattt tcaacttctc ccatttgggt
ctgggaggag aatgtgccct ggagtcaatc 1320 tggctacttc gggaatggca
acacttcttg catctcttat tcagtgcttt gacttgcaag 1380 tgctgggtcc
acaaggacag atattgaagg gtggtgacgc caaagttagc atggaagaga 1440
gagccggcct cactgttcca agggcacata gtcttgtctg tgttccactt gcaaggatcg
1500 g 1501 48 499 PRT Beta vulgaris 48 Phe Leu His Leu Arg Pro Thr
Pro Thr Ala Lys Ser Lys Ala Leu Arg 1 5 10 15 His Leu Pro Asn Pro
Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly 20 25 30 His Leu His
Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp 35 40 45 Leu
Ser Lys Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met 50 55
60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln
65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser
Ala Ile 85 90 95 Arg Arg Leu Thr Tyr Asp Ser Ser Val Ala Met Val
Pro Phe Gly Pro 100 105 110 Tyr Trp Lys Phe Val Arg Lys Leu Ile Met
Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr Val Asn Lys Leu Arg Pro
Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140 Phe Leu Arg Val Met Ala
Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp 145 150 155 160 Leu Thr Glu
Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser Met Met 165 170 175 Met
Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu 180 185
190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys
195 200 205 His Leu Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile
Leu Asn 210 215 220 Lys Phe Asp Pro Val Val Glu Arg Val Ile Lys Lys
Arg Arg Glu Ile 225 230 235 240 Val Arg Arg Arg Lys Asn Gly Glu Asp
Val Glu Gly Glu Val Ser Gly 245 250 255 Val Phe Leu Asp Thr Leu Leu
Glu Phe Ala Glu Asp Glu Thr Met Glu 260 265 270 Ile Lys Ile Thr Lys
Asp His Ile Lys Gly Leu Val Val Asp Phe Phe 275 280 285 Ser Ala Gly
Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala 290 295 300 Glu
Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Val 305 310
315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp Thr
Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe
Arg Met His 340 345 350 Pro Pro Leu Pro Val Val Lys Arg Lys Cys Ile
Glu Glu Cys Glu Ile 355 360 365 Asn Gly Tyr Val Ile Pro Glu Gly Ala
Leu Ile Leu Phe Asn Val Trp 370 375 380 Gln Val Gly Arg Asp Pro Lys
Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro Glu Arg Phe
Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Leu Leu 405 410 415 Asp Leu
Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg 420 425 430
Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu 435
440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro
Gln 450 455 460 Gly Gln Ile Leu Lys Gly Gly Asp Ala Lys Val Ser Met
Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr Val Pro Arg Ala His Ser
Leu Val Cys Val Pro Leu 485 490 495 Ala Arg Ile 49 30 DNA
Artificial Sequence PCR primer 49 gaattcgcgg ccgctctaga actagtggat
30 50 30 DNA Artificial Sequence PCR primer 50 gaattcgcgg
ccgcgaattg ggtaccgggc 30 51 27 DNA Artificial Sequence PCR primer
51 gcaaacgaag acaaatggga gatgata 27 52 1801 DNA Glycine max Intron
(895)..(1112) 52 ttgctggaac ttgcacttgg tttgtttgtg ttagctttgt
ttctgcactt gcgtcccaca 60 ccaagtgcaa aatcaaaagc acttcgccac
ctcccaaacc ctccaagccc aaagcctcgt 120 cttcccttca ttggccacct
tcacctctta aaagataaac ttctccacta tgcactcatc 180 gatctctcca
aaaagcatgg ccccttattc tctctctcct tcggctccat gccaaccgtc 240
gttgcctcca cccctgagtt gttcaagctc ttcctccaaa cccacgaggc aacttccttc
300 aacacaaggt tccaaacctc tgccataaga cgcctcactt acgacaactc
tgtggccatg 360 gttccattcg gaccttactg gaagttcgtg aggaagctca
tcatgaacga ccttctcaac 420 gccaccaccg tcaacaagct caggcctttg
aggacccaac agatccgcaa gttccttagg 480 gttatggccc aaagcgcaga
ggcccagaag ccccttgacg tcaccgagga gcttctcaaa 540 tggaccaaca
gcaccatctc catgatgatg ctcggcgagg ctgaggagat cagagacatc 600
gctcgcgagg ttcttaagat cttcggcgaa tacagcctca ctgacttcat ctggcctttg
660 aagtatctca aggttggaaa gtatgagaag aggattgatg acatcttgaa
caagttcgac 720 cctgtcgttg aaagggtcat caagaagcgc cgtgagatcg
tcagaaggag aaagaacgga 780 gaagttgttg agggcgaggc cagcggcgtc
ttcctcgaca ctttgcttga attcgctgag 840 gacgagacca tggagatcaa
aattaccaag gagcaaatca agggccttgt tgtcgtaagt 900 ttccttcttc
tctcctactt tattactttc tttcattcat catatgtatt ggcattaaat 960
agtatactat atgagaaaat atgttacgca ctcacggtgt aaagatatgt ggtgtttttt
1020 taaaaagaga tacagaagtt gcttttatgc atgtatgtta acgtatattt
actcaagtgg 1080 aaactaatta attctcaatt ttgggtatgt aggacttttt
ctctgcaggg acagattcca 1140 cagcggtggc aacagagtgg gcattggcag
agctcatcaa caatcccagg gtgttgcaaa 1200 aggctcgtga ggaggtctac
agtgttgtgg gcaaagatag actcgttgac gaagttgaca 1260 ctcaaaacct
tccttacatt agggccattg tgaaggagac attccgaatg cacccaccac 1320
tcccagtggt caaaagaaag tgcacagaag agtgtgagat taatgggtat gtgatcccag
1380 agggagcatt ggttcttttc aatgtttggc aagtaggaag ggaccccaaa
tactgggaca 1440 gaccatcaga attccgtccc gagaggttct tagaaactgg
tgctgaaggg gaagcagggc 1500 ctcttgatct taggggccag catttccaac
tcctcccatt tgggtctggg aggagaatgt 1560 gccctggtgt caatttggct
acttcaggaa tggcaacact tcttgcatct cttatccaat 1620 gctttgacct
gcaagtgctg ggccctcaag gacaaatatt gaaaggtgat gatgccaaag 1680
ttagcatgga agagagagct ggcctcacag ttccaagggc acatagtctc gtttgtgttc
1740 cacttgcaag gatcggcgtt gcatctaaac tcctttctta attaagggat
ccatcatata 1800 c 1801 53 1900 DNA Glycine max Intron (947)..(1082)
53 aattagcctc acaaaagcaa agatcaaaca aaccaaggac gagaacacga
tgttgcttga 60 acttgcactt ggtttattgg ttttggctct gtttctgcac
ttgcgtccca cacccactgc 120 aaaatcaaaa gcacttcgcc atctcccaaa
cccaccaagc ccaaagcctc gtcttccctt 180 cataggacac cttcatctct
taaaagacaa acttctccac tacgcactca tcgacctctc 240 caaaaaacat
ggtcccttat tctctctcta ctttggctcc atgccaaccg ttgttgcctc 300
cacaccagaa ttgttcaagc tcttcctcca aacgcacgag gcaacttcct tcaacacaag
360 gttccaaacc tcagccataa gacgcctcac ctatgatagc tcagtggcca
tggttccctt 420 cggaccttac tggaagttcg tgaggaagct catcatgaac
gaccttccca acgccaccac 480 tgtaaacaag ttgaggcctt tgaggaccca
acagacccgc aagttcctta gggttatggc 540 ccaaggcgca gaggcacaga
agccccttga cttgaccgag gagcttctga aatggaccaa 600 cagcaccatc
tccatgatga tgctcggcga ggctgaggag atcagagaca tcgctcgcga 660
ggttcttaag atctttggcg aatacagcct cactgacttc atctggccat tgaagcatct
720 caaggttgga aagtatgaga agaggatcga cgacatcttg aacaagttcg
accctgtcgt 780 tgaaagggtc atcaagaagc gccgtgagat cgtgaggagg
agaaagaacg gagaggttgt 840 tgagggtgag gtcagcgggg ttttccttga
cactttgctt gaattcgctg aggatgagac 900 catggagatc aaaatcacca
aggaccacat cgagggtctt gttgtcgtga gtttcctgct 960 tcattcattg
atcgaaatat gcagtatttt gttaacaaga gatcgagaat tgacatttat 1020
atattcatgt ggtggcaatt aattaacggt acgcattctt aatcgatatt gtgtatgtgc
1080 aggacttttt ctcggcagga acagactcca cagcggtggc aacagagtgg
gcattggcag 1140 aactcatcaa caatcctaag gtgttggaaa aggctcgtga
ggaggtctac agtgttgtgg 1200 gaaaggacag acttgtggac gaagttgaca
ctcaaaacct tccttacatt agagcaatcg 1260 tgaaggagac attccgcatg
cacccgccac tcccagtggt caaaagaaag tgcacagaag 1320 agtgtgagat
taatggatat gtgatcccag agggagcatt gattctcttc aatgtatggc 1380
aagtaggaag agaccccaaa tactgggaca gaccatcgga gttccgtcct gagaggttcc
1440 tagagacagg ggctgaaggg gaagcagggc ctcttgatct taggggacaa
cattttcaac 1500 ttctcccatt tgggtctggg aggagaatgt gccctggagt
caatctggct acttcgggaa 1560 tggcaacact tcttgcatct cttattcagt
gcttcgactt gcaagtgctg ggtccacaag 1620 gacagatatt gaagggtggt
gacgccaaag ttagcatgga agagagagcc ggcctcactg 1680 ttccaagggc
acatagtctt gtctgtgttc cacttgcaag gatcggcgtt gcatctaaac 1740
tcctttctta attaagatca tcgtcatcat catcatatat aatatttact ttttgtgtgt
1800 tgataatcat catttcaata aggtctcgtt catctacttt ttatgaagta
tataagccct 1860 tccatgcaca ttgtatcatc tcccatttgt cttcgtttgc 1900 54
1501 DNA Lupinus albus 54 tgtttctgca cttgcgtccc acacccactg
caaaatcaaa agcacttcgc catctcccaa 60 acccaccaag cccaaagcct
cgtcttccct tcataggaca ccttcatctc ttaaaagaca 120 aacttctcca
ctacgcactc atcgacctct ccaaaaaaca tggtccctta ttctctctct 180
actttggctc catgccaacc gttgttgcct ccacaccaga attgttcaag ctcttcctcc
240 aaacgcacga ggcaacttcc ttcaacacaa ggttccaaac ctcagccata
agacgcctca 300 cctatgatag ctcagtggcc agggttccct tcggacctta
ctggaagttc gtgaggaagc 360 tcatcatgaa cgaccttctt aacgccacca
ctgtaaacaa gttgaggcct ttgaggaccc 420 aacagatccg caagttcctt
agggttatgg cccaaggcgc agaggcacag aagccccttg 480 acttgaccga
ggagcttctg aaatggacca acagcaccat ctccatgatg atgctcggcg 540
aggctgagga gatcagagac atcgctcgcg aggttcttaa gatctttggc gaatacagcc
600 tcactgactt catctggcca ttgaagcatc tcaaggttgg aaagtatgag
aagaggatcg 660 acgacatctt gaacaagttc gaccctgtcg ttgaaagagt
catcaagaag cgccgtgaga 720 tcgtgaggag gagaaagaac ggagaggttg
ttgagggtga ggtcagcggg gttctccttg 780 acactttgct tgaattcgct
gaggatgaga ccatggagat caaaatcacc aaggaccaca 840 tcaagggtct
tgttgtcgac tttttctcgg caggaacaga ctccacagcg gtggcaacag 900
agtgggcatt ggcagaactc atcaacaatc ctaaggtgtt ggaaagggct cgtgaggagg
960 tctacagtgt tgtgggaaag gacagacttg tggacgaagt tgacactcaa
aaccttcctt 1020 acattagagc aatcgtgaag gagacattcc gcatgcaccc
gccactccca gtggtcaaaa 1080 gaaagtgcac agaagagtgt gagattaatg
gatatgtgat cccagaggga gcattgattc 1140 tcttcaatgt atggcaagta
ggaagagacc ccaaatactg ggacagacca tcggagttcc 1200 gtcctgagag
gttcctagag acagaggctg aaggggaagc aaggcctctt gatcttaggg 1260
gacaacattt tcaacttctc ccatttgggt ctgggaggag aatgtgccct ggagtcattc
1320 tggctacttc gggaatggca acacttcttg catctcttat tcagtgcttt
gacttgcaag 1380 tgctgggtcc acaaggacag atattgaagg gtggtgacgc
caaagttagc atggaagaga 1440 gagccggcct cactgttcca agggcacata
gtcttgtctg tgttccactt gcaaggatcg 1500 g 1501 55 499 PRT Lupinus
albus 55 Phe Leu His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala
Leu Arg 1 5 10 15 His Leu Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu
Pro Phe Ile Gly 20 25 30 His Leu His Leu Leu Lys Asp Lys Leu Leu
His Tyr Ala Leu Ile Asp 35 40 45 Leu Ser Lys Lys His Gly Pro Leu
Phe Ser Leu Tyr Phe Gly Ser Met 50 55 60 Pro Thr Val Val Ala Ser
Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln 65 70 75 80 Thr His Glu Ala
Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser Ala Ile 85 90 95 Arg Arg
Leu Thr Tyr Asp Ser Ser Val Ala Arg Val Pro Phe Gly Pro 100 105 110
Tyr Trp Lys Phe Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala 115
120 125 Thr Thr Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg
Lys 130 135 140 Phe Leu Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys
Pro Leu Asp 145 150 155 160 Leu Thr Glu Glu Leu Leu Lys Trp Thr Asn
Ser Thr Ile Ser Met Met 165 170 175 Met Leu Gly Glu Ala Glu Glu Ile
Arg Asp Ile Ala Arg Glu Val Leu 180 185 190 Lys Ile Phe Gly Glu Tyr
Ser Leu Thr Asp Phe Ile Trp Pro Leu Lys 195 200 205 His Leu Lys Val
Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu Asn 210 215 220 Lys Phe
Asp Pro Val Val Glu Arg Val Ile Lys Lys Arg Arg Glu Ile 225 230 235
240 Val Arg Arg Arg Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly
245 250 255 Val Leu Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr
Met Glu 260 265 270 Ile Lys Ile Thr Lys Asp His Ile Lys Gly Leu Val
Val Asp Phe Phe 275 280 285 Ser Ala Gly Thr Asp Ser Thr Ala Val Ala
Thr Glu Trp Ala Leu Ala 290 295 300 Glu Leu Ile Asn Asn Pro Lys Val
Leu Glu Arg Ala Arg Glu Glu Val 305 310 315 320 Tyr Ser Val Val Gly
Lys Asp Arg Leu Val Asp Glu Val Asp Thr Gln 325 330 335 Asn Leu Pro
Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg Met His 340 345 350 Pro
Pro Leu Pro Val Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile 355 360
365 Asn Gly Tyr Val Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp
370 375 380 Gln Val Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu
Phe Arg 385 390 395 400 Pro Glu Arg Phe Leu Glu Thr Glu Ala Glu Gly
Glu Ala Arg Pro Leu 405 410 415 Asp Leu Arg Gly Gln His Phe Gln Leu
Leu Pro Phe Gly Ser Gly Arg 420 425 430 Arg Met Cys Pro Gly Val Ile
Leu Ala Thr Ser Gly Met Ala Thr Leu 435 440 445 Leu Ala Ser Leu Ile
Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln 450 455 460 Gly Gln Ile
Leu Lys Gly Gly Asp Ala Lys Val Ser Met Glu Glu Arg 465 470 475 480
Ala Gly Leu Thr Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu 485
490 495 Ala Arg Ile 56 1501 DNA Medicago sativa 56 tgtttctgca
cttgcgtccc acacccactg caaaatcaaa agcacttcgc catctcccaa 60
acccaccaag cccaaagcct cgtcttccct tcataggaca ccttcatctc ttaaaagaca
120 aacttctcca ctacgcactc atcgacctct ccaaaaaaca tggtccctta
ttctctctct 180 actttggctc catgccaacc gttgttgcct ccacaccaga
attgttcaag ctcttccttc 240 aaacgcacga ggcaacttcc ttcaacacaa
ggttccaaac ctcagccata agacgcctca 300 cctatgatag ctcagtggcc
atggctccct tcggacctta ctggaagttc gtgaggaagc 360 tcatcatgaa
cgaccttctc aacgccacca ctgtaaacaa gttgaggcct ttgaggaccc 420
aacagatccg caagttcctt agggttatgg cccaaggcgc agaggcacag aagccccttg
480 acttgaccga ggagcttctg aaatggacca acagcaccac ctccatgatg
atgctcggcg 540 aggctgagga gatcagagac atcgcccgcg aggttcttaa
gatctttggc gaatacagcc 600 tcactgactt catccggcca ttgaagcatc
tcaaggttgg aaagtatgag aagaggatcg 660 acgacatctt gaacaagttc
gaccctgtcg ttgaaagagt catcaagaag cgccgtgaga 720 tcgtgaggag
gagaaagaac ggagaggttg ttgagggtga ggtcagcggg gttttccttg 780
acactttgct tgaattcgct gaggatgaga ccacggagat caaaatcacc aaggaccaca
840 tcaagggtct tgttgtcgac tttttctcgg caggaacaga ctccacagcg
gtggcaacag 900 agtgggcatt ggcagaactc atcaacaatc ctaaggtgtt
ggaaaaggct cgtgaggagg 960 tctacagtgt tgtgggaaag gacagacttg
tggacgaagt tgacactcaa aaccttcctt 1020 acattagagc aatcgtgaag
gagacattcc gcatgcaccc gccactccca gtggtcaaaa 1080 gaaagtgcac
agaagagtgt gagattaatg gatatgtgat cccagaggga gcattgattc 1140
tcttcaatgt atggcaagta ggaagagact ccaaatactg ggacagacca tcggagttcc
1200 gtcctgagag gttcctagag acaggggctg aaggggaagc aaggcctctt
gatcttaggg 1260 gacaacattt tcaacttctc ccatttgggt ctgggaggag
aatgtgccct ggagtcaatc 1320 tggctacttc gggaatggca acacttcttg
catctcttat tcagtgcttt gacttgcaag 1380 tgctgggtcc acaaggacag
atattgaagg gtggtgacgc caaagttagc atggaagaga 1440 gggccggcct
cactgttcca agggcacata gtcttgtctg tgttccactt gcaaggatcg 1500 g 1501
57 499 PRT Medicago sativa 57 Phe Leu His Leu Arg Pro Thr Pro Thr
Ala Lys Ser Lys Ala Leu Arg 1 5 10 15 His Leu Pro Asn Pro Pro Ser
Pro Lys Pro Arg Leu Pro Phe Ile Gly 20 25 30 His Leu His Leu Leu
Lys Asp Lys Leu Leu His Tyr Ala Leu Ile Asp 35 40 45 Leu Ser Lys
Lys His Gly Pro Leu Phe Ser Leu Tyr Phe Gly Ser Met 50 55 60 Pro
Thr Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln 65 70
75 80 Thr His Glu Ala Thr Ser Phe Asn Thr Arg Phe Gln Thr Ser Ala
Ile 85 90 95 Arg Arg Leu Thr Tyr Asp Ser Ser Val Ala Met Ala Pro
Phe Gly Pro 100 105 110 Tyr Trp Lys Phe Val Arg Lys Leu Ile Met Asn
Asp Leu Leu Asn Ala 115 120 125 Thr Thr Val Asn Lys Leu Arg Pro Leu
Arg Thr Gln Gln Ile Arg Lys 130 135 140 Phe Leu Arg Val Met Ala Gln
Gly Ala Glu Ala Gln Lys Pro Leu Asp 145 150 155 160 Leu Thr Glu Glu
Leu Leu Lys Trp Thr Asn Ser Thr Thr Ser Met Met 165 170 175 Met Leu
Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu 180
185 190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp Phe Ile Arg Pro Leu
Lys 195 200 205 His Leu Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp
Ile Leu Asn 210 215 220 Lys Phe Asp Pro Val Val Glu Arg Val Ile Lys
Lys Arg Arg Glu Ile 225 230 235 240 Val Arg Arg Arg Lys Asn Gly Glu
Val Val Glu Gly Glu Val Ser Gly 245 250 255 Val Phe Leu Asp Thr Leu
Leu Glu Phe Ala Glu Asp Glu Thr Thr Glu 260 265 270 Ile Lys Ile Thr
Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe 275 280 285 Ser Ala
Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu Ala 290 295 300
Glu Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg Glu Glu Val 305
310 315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu Val Asp Glu Val Asp
Thr Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr
Phe Arg Met His 340 345 350 Pro Pro Leu Pro Val Val Lys Arg Lys Cys
Thr Glu Glu Cys Glu Ile 355 360 365 Asn Gly Tyr Val Ile Pro Glu Gly
Ala Leu Ile Leu Phe Asn Val Trp 370 375 380 Gln Val Gly Arg Asp Ser
Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390 395 400 Pro Glu Arg
Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu 405 410 415 Asp
Leu Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg 420 425
430 Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly Met Ala Thr Leu
435 440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly
Pro Gln 450 455 460 Gly Gln Ile Leu Lys Gly Gly Asp Ala Lys Val Ser
Met Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr Val Pro Arg Ala His
Ser Leu Val Cys Val Pro Leu 485 490 495 Ala Arg Ile 58 1501 DNA
Medicago sativa 58 tgtttctgca cttgcgtccc acacccactg caaaatcaaa
agcacttcgc catctcccaa 60 acccaccaag cccaaagcct cgtcttccct
tcataggaca ccttcatctc ttaaaagaca 120 aacttctcca ctacgcactc
atcgacctct ccaaaaaaca tggtccctta ttctctctct 180 actttggctc
catgccaacc gttgttgcct ccacaccaga attgttcaag ctcttcctcc 240
aaacgcacga ggcaacttcc ttcaacacaa ggttccaaac ctcagccata agacgcctca
300 cctatgatag ctcagtggcc atggttccct tcggacctta ctggaagttc
gtgaggaagc 360 tcatcatgaa cgaccttctc aacgccacca ctgtaaacaa
gttgaggcct ttgaggaccc 420 aacagatccg caagctcctt agggttatgg
cccaaggcgc agaggcacag aagccccttg 480 acttgaccga ggagcttctg
aaatggacca acagcaccat ctccatgatg atgctcggcg 540 aggctgagga
gatcagagac atcgctcgcg aggttcttaa gatctttggc gaatacagcc 600
tcactgactt catctggcca ttgaagcatc tcaaggttgg aaagtatgag aagaggatcg
660 acgacatctt gaacaagttc gaccctgtcg ttgaaagagt catcaagaag
cgccgtgaga 720 tcgtgaggag gagaaagaac ggagaggtta ttgagggtga
ggtcagcggg gttttccttg 780 acactttgct tgaattcgct gaggatgaga
ccacggagat caaaatcacc aaggaccaca 840 tcaagggtct tgttgtcgac
tttttctcgg caggaacaga ctccacagcg gtggcaacag 900 agtgggcatt
ggcagaactc atcaacaatc ctaaggtgtt ggagaaggct cgtgaggagg 960
tctacagtgt tgtgggaaag gacagacttg tggacgaagt tgacactcaa aaccttcctt
1020 acattagagc aatcgtgaag gagacattcc gcatgcaccc gccactccca
gtggtcaaaa 1080 gaaagtgcac agaagagtgt gagattaatg gatatgtgat
cccagaggga gcattgattc 1140 tcttcaatgt atggcaagta ggaagagacc
ccaaatactg ggacagacca tcggagttcc 1200 gtcctgagag gttcctagag
acaggggctg aaggggaagc aaggcctctt gatcttaggg 1260 gacaacattt
tcaacttctc ccatttgggt ctgggaggag aatgtgccct ggagtcaatc 1320
tggctacttc gggaatggca acacttcttg catctcttat tcagtgcttt gacttgcaag
1380 tgctgggtcc acaaggacag atattgaagg gtggtgacgc caaagttagc
atggaagaga 1440 gggccggcct cactgttcca agggcacata gtcttgtctg
tgttccactt gcaaggatcg 1500 g 1501 59 499 PRT Medicago sativa 59 Phe
Leu His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg 1 5 10
15 His Leu Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly
20 25 30 His Leu His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu
Ile Asp 35 40 45 Leu Ser Lys Lys His Gly Pro Leu Phe Ser Leu Tyr
Phe Gly Ser Met 50 55 60 Pro Thr Val Val Ala Ser Thr Pro Glu Leu
Phe Lys Leu Phe Leu Gln 65 70 75 80 Thr His Glu Ala Thr Ser Phe Asn
Thr Arg Phe Gln Thr Ser Ala Ile 85 90 95 Arg Arg Leu Thr Tyr Asp
Ser Ser Val Ala Met Val Pro Phe Gly Pro 100 105 110 Tyr Trp Lys Phe
Val Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala 115 120 125 Thr Thr
Val Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys 130 135 140
Leu Leu Arg Val Met Ala Gln Gly Ala Glu Ala Gln Lys Pro Leu Asp 145
150 155 160 Leu Thr Glu Glu Leu Leu Lys Trp Thr Asn Ser Thr Ile Ser
Met Met 165 170 175 Met Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala
Arg Glu Val Leu 180 185 190 Lys Ile Phe Gly Glu Tyr Ser Leu Thr Asp
Phe Ile Trp Pro Leu Lys 195 200 205 His Leu Lys Val Gly Lys Tyr Glu
Lys Arg Ile Asp Asp Ile Leu Asn 210 215 220 Lys Phe Asp Pro Val Val
Glu Arg Val Ile Lys Lys Arg Arg Glu Ile 225 230 235 240 Val Arg Arg
Arg Lys Asn Gly Glu Val Ile Glu Gly Glu Val Ser Gly 245 250 255 Val
Phe Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Thr Glu 260 265
270 Ile Lys Ile Thr Lys Asp His Ile Lys Gly Leu Val Val Asp Phe Phe
275 280 285 Ser Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala
Leu Ala 290 295 300 Glu Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala
Arg Glu Glu Val 305 310 315 320 Tyr Ser Val Val Gly Lys Asp Arg Leu
Val Asp Glu Val Asp Thr Gln 325 330 335 Asn Leu Pro Tyr Ile Arg Ala
Ile Val Lys Glu Thr Phe Arg Met His 340 345 350 Pro Pro Leu Pro Val
Val Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile 355 360 365 Asn Gly Tyr
Val Ile Pro Glu Gly Ala Leu Ile Leu Phe Asn Val Trp 370 375 380 Gln
Val Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg 385 390
395 400 Pro Glu Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro
Leu 405 410 415 Asp Leu Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly
Ser Gly Arg 420 425 430 Arg Met Cys Pro Gly Val Asn Leu Ala Thr Ser
Gly Met Ala Thr Leu 435 440 445 Leu Ala Ser Leu Ile Gln Cys Phe Asp
Leu Gln Val Leu Gly Pro Gln 450 455 460 Gly Gln Ile Leu Lys Gly Gly
Asp Ala Lys Val Ser Met Glu Glu Arg 465 470 475 480 Ala Gly Leu Thr
Val Pro Arg Ala His Ser Leu Val Cys Val Pro Leu 485 490 495 Ala Arg
Ile 60 1497 DNA Beta vulgaris 60 tctgcacttg cgtcccacac ccactgcaaa
atcaaaagca cttcgccatc tcccaaaccc 60 accaagccca aagcctcgtc
ttcccttcat aggacacctt catctcttaa aagacaaact 120 tctccactac
gcactcatcg acctctccaa aaaacatggt cccttattct ctcactactt 180
tggctccatg ccaaccgttg ttgcctccac accagaattg ttcaagctct tcctccaaac
240 gaacgaggca acttccttca acacaaggtt ccaaacctca gccataagac
gcctcaccta 300 tgatagctca gtggccatgg ttcccttcgg accttactgg
aagttcgtga ggaagctcat 360 catgaacgac cttctcaacg ccaccactgt
aaacaagttg aggcctttga ggacccaaca 420 gatccgcaag ttccttaggg
ctatggccca aggcgcagag gcacggaagc cccttgactt 480 gaccgaggag
cttctgaaat gggccaacag caccatctcc atgatgatgc tcggcgaggc 540
tgaggagatc agagacatcg ctcgcgaggt tcttaagatc tttggcgaat acagcctcac
600 tgacttcatc tggccattga agcatctcaa ggttggaaag tatgagaaga
ggatcgacga 660 catcttgaac aagttcgacc ctgtcgttga aagagtcatc
aagaagcgcc gtgagatcgt 720 gaggaggaga aagaacggag aggttgttga
gggtgaggtc agcggggttt tccttgacac 780 tttgcttgaa ttcgctgagg
atgagaccat ggagatcaaa atcaccaagg accacaccaa 840 gggtcttgtt
gtcgacttct tctcggcagg aacagactcc acagcggtgg caacagagtg 900
ggcattggca gaactcatca acaatcctaa ggtgttggaa aaggctcgtg aggaggtcta
960 cagtgttgtg ggaaaggaca gacttgtgga cgaagttgac actcaaaacc
ttccttacat 1020 tagagcaatc gtgaaggaga cattccgcat gcacccgcca
ctcccagtgg tcaaaagaaa 1080 gtgcacagaa gagtgtgaga ttaatggata
tgtgatccca gagggagcat tgattccctt 1140 caatgtatgg caagtaggaa
gagaccccaa atactgggac agaccatcgg agttccgtcc 1200 tgagaggttc
ctagagacag gggctgaagg ggaagcaagg cctcttgatc ttaggggaca 1260
acattttcaa cttctcccat ttgggtctgg gaggagaatg tgccctggag tcaatctggc
1320 tacttcggga acggcaacac ttcttgcatc tcttattcag tgctttgact
tgcaagtgct 1380 gggtccacag ggacagatat tgaagggtgg tgacgccaaa
gttagcatgg aagagagagc 1440 cggcctcact gttccaaggg cacatagtct
tgtctgtgtt ccacttgcaa ggatcgg 1497 61 498 PRT Beta vulgaris 61 Leu
His Leu Arg Pro Thr Pro Thr Ala Lys Ser Lys Ala Leu Arg His 1 5 10
15 Leu Pro Asn Pro Pro Ser Pro Lys Pro Arg Leu Pro Phe Ile Gly His
20 25 30 Leu His Leu Leu Lys Asp Lys Leu Leu His Tyr Ala Leu Ile
Asp Leu 35 40 45 Ser Lys Lys His Gly Pro Leu Phe Ser His Tyr Phe
Gly Ser Met Pro 50 55 60 Thr Val Val Ala Ser Thr Pro Glu Leu Phe
Lys Leu Phe Leu Gln Thr 65 70 75 80 Asn Glu Ala Thr Ser Phe Asn Thr
Arg Phe Gln Thr Ser Ala Ile Arg 85 90 95 Arg Leu Thr Tyr Asp Ser
Ser Val Ala Met Val Pro Phe Gly Pro Tyr 100 105 110 Trp Lys Phe Val
Arg Lys Leu Ile Met Asn Asp Leu Leu Asn Ala Thr 115 120 125 Thr Val
Asn Lys Leu Arg Pro Leu Arg Thr Gln Gln Ile Arg Lys Phe 130 135 140
Leu Arg Ala Met Ala Gln Gly Ala Glu Ala Arg Lys Pro Leu Asp Leu 145
150 155 160 Thr Glu Glu Leu Leu Lys Trp Ala Asn Ser Thr Ile Ser Met
Met Met 165 170 175 Leu Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg
Glu Val Leu Lys 180 185 190 Ile Phe Gly Glu Tyr Ser Leu Thr Asp Phe
Ile Trp Pro Leu Lys His 195 200 205 Leu Lys Val Gly Lys Tyr Glu Lys
Arg Ile Asp Asp Ile Leu Asn Lys 210 215 220 Phe Asp Pro Val Val Glu
Arg Val Ile Lys Lys Arg Arg Glu Ile Val 225 230 235 240 Arg Arg Arg
Lys Asn Gly Glu Val Val Glu Gly Glu Val Ser Gly Val 245 250 255 Phe
Leu Asp Thr Leu Leu Glu Phe Ala Glu Asp Glu Thr Met Glu Ile 260 265
270 Lys Ile Thr Lys Asp His Thr Lys Gly Leu Val Val Asp Phe Phe Ser
275 280 285 Ala Gly Thr Asp Ser Thr Ala Val Ala Thr Glu Trp Ala Leu
Ala Glu 290 295 300 Leu Ile Asn Asn Pro Lys Val Leu Glu Lys Ala Arg
Glu Glu Val Tyr 305 310 315 320 Ser Val Val Gly Lys Asp Arg Leu Val
Asp Glu Val Asp Thr Gln Asn 325 330 335 Leu Pro Tyr Ile Arg Ala Ile
Val Lys Glu Thr Phe Arg Met His Pro 340 345 350 Pro Leu Pro Val Val
Lys Arg Lys Cys Thr Glu Glu Cys Glu Ile Asn 355 360 365 Gly Tyr Val
Ile Pro Glu Gly Ala Leu Ile Pro Phe Asn Val Trp Gln 370 375 380 Val
Gly Arg Asp Pro Lys Tyr Trp Asp Arg Pro Ser Glu Phe Arg Pro 385 390
395 400 Glu Arg Phe Leu Glu Thr Gly Ala Glu Gly Glu Ala Arg Pro Leu
Asp 405 410 415 Leu Arg Gly Gln His Phe Gln Leu Leu Pro Phe Gly Ser
Gly Arg Arg 420 425 430 Met Cys Pro Gly Val Asn Leu Ala Thr Ser Gly
Thr Ala Thr Leu Leu 435 440 445 Ala Ser Leu Ile Gln Cys Phe Asp Leu
Gln Val Leu Gly Pro Gln Gly 450 455 460 Gln Ile Leu Lys Gly Gly Asp
Ala Lys Val Ser Met Glu Glu Arg Ala 465 470 475 480 Gly Leu Thr Val
Pro Arg Ala His Ser Leu Val Cys Val Pro Leu Ala 485 490 495 Arg Ile
62 22 DNA Artificial Sequence PCR primer 62 gttaccatgg ctgctgctat
tg 22 63 24 DNA Artificial Sequence PCR primer 63 ttaaacgtaa
aatgaaacaa gagg 24 64 26 DNA Artificial Sequence PCR primer 64
gacacttcga cactgctgct gcttat 26 65 25 DNA Artificial Sequence PCR
primer 65 tctcaaactc acctgggcta tggat 25 66 521 PRT Artificial
Sequence Consensus sequence 66 Met Leu Leu Glu Leu Ala Leu Gly Leu
Xaa Val Leu Ala Leu Phe Xaa 1 5 10 15 His Leu Arg Pro Thr Pro Xaa
Ala Xaa Ser Lys Ala Leu Arg His Leu 20 25 30 Pro Asn Pro Pro Ser
Pro Xaa Pro Arg Leu Pro Phe Ile Gly His Xaa 35 40 45 His Leu Leu
Lys Asp Lys Leu Leu His Tyr Ala Xaa Ile Asp Leu Ser 50 55 60 Lys
Lys His Gly Pro Leu Phe Ser Xaa Xaa Phe Gly Ser Met Pro Thr 65 70
75 80 Val Val Ala Ser Thr Pro Glu Leu Phe Lys Leu Phe Leu Gln Xaa
Xaa 85 90 95 Glu Ala Thr Ser Phe Xaa Thr Arg Phe Gln Thr Ser Ala
Xaa Arg Xaa 100 105 110 Leu Thr Tyr Asp Xaa Xaa Val Ala Xaa Xaa Pro
Xaa Gly Pro Tyr Trp 115 120 125 Xaa Phe Val Arg Lys Leu Ile Met Asn
Asp Leu Leu Asn Ala Thr Thr 130 135 140 Val Asn Xaa Leu Arg Pro Leu
Arg Thr Gln Gln Ile Arg Lys Xaa Leu 145 150 155 160 Arg Xaa Met Ala
Gln Xaa Ala Glu Ala Xaa Lys Pro Leu Asp Xaa Thr 165 170 175 Glu Glu
Leu Leu Lys Trp Xaa Asn Ser Thr Xaa Ser Met Met Xaa Leu 180 185 190
Gly Glu Ala Glu Glu Ile Arg Asp Ile Ala Arg Glu Val Leu Lys Ile 195
200 205 Xaa Gly Glu Tyr Ser Leu Thr Asp Phe Ile Xaa Pro Leu Lys Xaa
Leu 210 215 220 Lys Val Gly Lys Tyr Glu Lys Arg Ile Asp Asp Ile Leu
Asn Lys Phe 225 230 235 240 Asp Pro Val Val Glu Arg Val Ile Lys Lys
Arg Arg Xaa Ile Val Arg 245 250 255 Arg Arg Xaa Asn Gly Glu Xaa Xaa
Glu Gly Glu Xaa Ser Gly Val Xaa 260 265 270 Leu Asp Thr Leu Leu Glu
Phe Ala Glu Asp Glu Thr Xaa Glu Ile Lys 275 280 285 Ile Thr Lys Xaa
Xaa Xaa Lys Gly Leu Val Val Asp Xaa Phe Ser Ala 290 295 300 Gly Xaa
Asp Ser Thr Ala Xaa Xaa Thr Glu Trp Ala Leu Ala Glu Leu 305 310 315
320 Ile Asn Asn Pro Xaa Val Leu Xaa Xaa Ala Arg Glu Glu Xaa Tyr Ser
325 330 335 Val Val Gly Lys Asp Xaa Leu Val Asp Glu Val Asp Thr Gln
Asn Leu 340 345 350 Pro Tyr Ile Arg Ala Ile Val Lys Glu Thr Phe Arg
Met His Pro Pro 355 360 365 Leu Pro Val Val Lys Arg Lys Cys Xaa Glu
Glu Cys Xaa Ile Asn Gly 370 375 380 Xaa Val Xaa Pro Glu Gly Ala Leu
Xaa Xaa Phe Asn Val Trp Gln Val 385 390 395 400 Gly Xaa Asp Xaa Lys
Tyr Trp Asp Arg Pro Ser Glu Xaa Arg Pro Glu 405 410 415 Arg Phe Leu
Glu Thr Xaa Ala Glu Gly Glu Ala Xaa Xaa Leu Asp Leu 420 425 430 Arg
Gly Xaa His Phe Gln Leu Leu Pro Phe Gly Ser Gly Arg Xaa Met 435 440
445 Cys Pro Gly Val Xaa Leu Ala Thr Ser Gly Xaa Ala Thr Leu Leu Ala
450 455 460 Ser Leu Ile Gln Cys Phe Asp Leu Gln Val Leu Gly Pro Gln
Gly Gln 465 470 475 480 Ile Leu Lys Gly Xaa Asp Ala Lys Val Ser Met
Glu Glu Arg Ala Gly 485 490 495 Leu Thr Val Pro Arg Ala His Ser Leu
Val Cys Val Pro Leu Ala Arg 500 505 510 Ile Gly Val Ala Ser Lys Leu
Leu Ser 515 520
* * * * *
References