U.S. patent application number 10/922732 was filed with the patent office on 2005-01-20 for hypoallergenic transgenic soybeans.
Invention is credited to Jung, Rudolf, Kinney, Anthony J..
Application Number | 20050015826 10/922732 |
Document ID | / |
Family ID | 22698915 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050015826 |
Kind Code |
A1 |
Kinney, Anthony J. ; et
al. |
January 20, 2005 |
Hypoallergenic transgenic soybeans
Abstract
Hypoallergenic transgenic soybeans and recombinant expression
constructs to lower soybean vacuolar protein, commonly know as P34,
as well as other allergens are disclosed. Soybean protein products
made from these hypoallergenic soybeans should be substantially
free of the major soy allergen, P34, and, in addition, other minor
soy allergens, such as, Gly m Bd 28K, alpha-subunit of
beta-conglycinin, KSTI, Gly m2, Gly m IA, Gly m IB, rGLY m3 and
glycinin G1.
Inventors: |
Kinney, Anthony J.;
(Wilmington, DE) ; Jung, Rudolf; (Des Moines,
IA) |
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: |
22698915 |
Appl. No.: |
10/922732 |
Filed: |
August 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10922732 |
Aug 20, 2004 |
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09805694 |
Mar 14, 2001 |
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60189823 |
Mar 16, 2000 |
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Current U.S.
Class: |
800/260 ; 426/21;
426/601; 435/320.1; 435/468; 530/350; 536/23.6; 800/266; 800/295;
800/298; 800/312 |
Current CPC
Class: |
A23V 2002/00 20130101;
C07K 14/811 20130101; A23K 20/158 20160501; A23L 33/185 20160801;
A23V 2300/21 20130101; A23D 9/00 20130101; C07K 2319/00 20130101;
C11B 7/00 20130101; A23L 33/115 20160801; C11C 3/10 20130101; C11C
1/04 20130101; C07K 14/415 20130101; C12N 15/8242 20130101; C11B
3/00 20130101; C11B 1/04 20130101; C11C 3/12 20130101; A23L 11/03
20160801; A23L 11/07 20160801; A23K 10/30 20160501; C12N 15/8251
20130101; A23V 2002/00 20130101 |
Class at
Publication: |
800/260 ;
435/320.1; 435/468; 800/312; 800/298; 800/295; 426/601; 426/021;
800/266; 536/023.6; 530/350 |
International
Class: |
C07H 021/04; A01H
001/00; A01H 005/00; C12N 015/09; C12N 015/63; C07K 014/00; C12N
015/82; A01H 005/10 |
Claims
1. A recombinant expression construct to lower Gly m Bd 30K
(Soybean vacuolar protein P34) content of a soybean which comprises
a promoter operably linked to an isolated Gly m Bd 30K nucleic acid
fragment corresponding substantially to the nucleotide sequence set
forth in SEQ ID NO:1 or a functionally equivalent subfragment
thereof.
2-90. (canceled)
91. A method for making a hypoallergenic soybean plant which
comprises: (a) crossing a (i) first parent soybean which is a
soybean plant comprising in its genome a recombinant expression
construct to lower the Gly m Bd 30K (Soybean vacuolar protein P34)
content of a soybean which comprises a beta-conglycinin promoter
operably linked to an isolated Gly m Bd 30K nucleic acid fragment
corresponding to all or a part of the nucleotide sequence set forth
in SEQ ID NO: 1, with a (ii) second parent soybean which is a
hybrid, mutant or transgenic soybean parent wherein said second
parent is substantially free of one or more allergens selected from
the group consisting of Gly m Bd 28K, alpha-subunit of
beta-conglycinin, KSTI, Gly m2, Gly m IA, Gly m IB, rGLY m3 and
Glycinin G1; and (b) selecting progeny plants of the cross of step
(a) which are hypoallergenic.
92. A method for making a hypoallergenic soybean plant which
comprises: (a) crossing a (1) first parent soybean which is a
soybean plant comprising in its genome at least one expression
construct selected from the group consisting of: (i) a recombinant
expression construct to lower Gly m Bd 30K (Soybean vacuolar
protein P34) content of a soybean which comprises a promoter
operably linked to an isolated Gly m Bd 30K nucleic acid fragment
corresponding to all or a part of to the nucleotide sequence set
forth in SEQ ID NO:1; (ii) the recombinant expression construct of
part (a)(i) wherein the promoter is selected from the group
consisting of an alpha-subunit beta-conglycinin promoter, a Kunitz
Trypsin Inhibitor (KSTI) promoter, a Gly m Bd 28K promoter, T7
promoter, a .sup.35S promoter and a beta-phaseolin promoter; (iii)
the expression construct of claim 91; and (iv) a recombinant
expression construct for producing a hypoallergenic soybean which
comprises an isolated KSTI nucleic acid fragment corresponding to
all or part of the nucleotide sequence set forth in SEQ ID NO:2
operably linked to an isolated Gly m Bd 28K nucleic acid fragment
corresponding to all or part of the nucleotide sequence set forth
in SEQ ID NO:3 with a (2) second parent soybean which is a hybrid,
mutant or transgenic soybean wherein the second parent is selected
from the group consisting of a soybean plant which is substantially
free of the alpha subunit of beta-conglycinin; and (b) selecting
progeny plants of the cross of step (a) which are
hypoallergenic.
93. A method for making a hypoallergenic soybean plant which
comprises: (a) crossing a (i) first parent soybean which is a
soybean plant comprising in its genome at least one of the
expression constructs of claim 92 with a (ii) second parent soybean
which is a hybrid, mutant or transgenic soybean wherein the second
parent is substantially free of KSTI allergen; and (b) selecting
progeny plants of the cross of step (a) which are
hypoallergenic.
94. A method for making a hypoallergenic soybean plant which
comprises: (a) crossing a (i) first parent soybean which is a
soybean plant comprising in its genome at least one of the
expression constructs of claim 92 with a (ii) second parent soybean
which is a hybrid, mutant or transgenic parent soybean wherein said
second parent is substantially free of soybean allergens selected
from the group consisting of Gly m Bd 28K, alpha-subunit of
beta-conglycinin, KSTI, Gly m2, Gly m IA, Gly m IB, rGLY m3 and
Glycinin G1; and (b) selecting progeny plants of the cross to step
(a) which are hypoallergenic.
95. Seeds obtained from a plant made by the method of any of claims
91, 92, 93 or 94.
Description
FIELD OF THE INVENTION
[0001] This invention relates to hypoallergenic transgenic soybeans
and, in particular, to the preparation of recombinant expression
constructs to lower soybean vacuolar protein, commonly know as P34,
as well as other allergens such as Gly m IA, Gly m IB, rGLY m3 and
Glycinin G1 (A1aB1b). Such constructs can be used to produce
hypoallergenic transgenic soybean plants that in turn can be used
to make hypoallergenic soybean products which can be used in a
variety of food and feed applications.
BACKGROUND OF THE INVENTION
[0002] Food allergy is a serious nutritional problem in children
and adults. Basically, any food that contains protein has the
potential to elicit an allergic reaction in a percentage of the
human population. Most food-allergic reactions are attributable to
cows' milk, eggs, fish, crustaceans, peanuts, soybeans, tree nuts
and wheat. Sometimes referred to as "the Big Eight", it is
estimated that these foods or food groups account for more than 90%
of all food allergies in the United States. (Taylor et al., (1999)
Nutrition Today 34:15-22).
[0003] The allergens in foods are almost always naturally occurring
proteins. Although foods contain millions of individual proteins,
only a comparative few food proteins have been documented as being
allergens. Some foods are known to contain multiple allergenic
proteins, including soybeans, peanuts, cows' milk and eggs. (Burks
et al., (1988) J. Allergy Clin. Immunol. 81:1135-42; Thanh et al.,
(1976) J. Agr. Food Chem. 24:1117-21).
[0004] Improved isolation techniques resulting in better flavor and
increased functionality has resulted in widespread use of soy
protein isolates and concentrates in a variety of food products in
amounts that could trigger an allergic reaction in
soybean-sensitive individuals. Soybean protein allergies pose a
significant problem for large numbers of people because soybean
protein is now a common constituent of many processed foods. For
sensitive individuals, avoiding soybean products is difficult and
poses significant limitations in choosing processed and convenience
foods. Since the incidence of soybean-related food allergies is
increasing in many countries including the U.S. (Taylor et al.,
Chemistry of Food Allergens in Food Allergy, Chandra R. K. (ed.):
Food Allergy, Nutrition Research Education Foundation, 1987, pp
21-44), there is an ever-growing need to develop hypoallergenic
soybean products to address this issue.
[0005] The major human allergen of soybean seeds is a protein
designated Gly m Bd 30K also referred to as P34 because this
protein has been shown to have an N-terminal amino acid sequence
and amino acid composition identical to that of the soybean seed 34
kDa seed vacuolar protein, P34. Gly m Bd 30K was described by
(Ogawa et al., (1991) J. Nutr. Sci. Vitaminol. 37:555-565), as a
30- kDa mol wt protein, a minor constituent of the 7S globulin
fraction. Gly m Bd 30K is an outlying member of the
papain-superfamily of cysteine-proteases and is present in
processed food products that contain soybean protein. (Yaklich et
al., (1999) Crop Science 39:1444). Results have indicated that it
may not be possible to eliminate P34 from the food supply by
breeding with an improved germplasm base. (Yaklich et al., (1999)
Crop Science 39:1444). Thus, the elimination of P34 from soybean
seeds, as well as other allergens allergens such as Gly m IA, Gly m
IB, rGLY m3 and Glycinin G1 (A1aB1b), by using recombinant
technology not only would enhance food safety but it would make the
use of soybean products available to sensitive individuals.
SUMMARY OF THE INVENTION
[0006] This invention concerns a recombinant expression construct
to lower Gly m Bd 30K (soybean vacuolar protein P34) content of a
soybean which comprises a promoter operably linked to an isolated
Gly m Bd 30K nucleic acid fragment corresponding substantially to
the nucleotide sequence set forth in SEQ ID NO: 1 or a functionally
equivalent subfragment thereof.
[0007] In a second embodiment, this invention concerns a
recombinant expression construct for producing a hypoallergenic
soybean which comprises an isolated KSTI nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO:2 or a functionally equivalent subfragment thereof
operably linked to an isolated Gly m Bd 28K nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO:3 or a functionally equivalent subfragment thereof.
[0008] In a third embodiment, this invention concerns a
hypoallergenic soybean plant comprising in its genome at least one
of the claimed recombinant expression constructs. Also of interest
are seeds obtained from such plants, oil obtained from these seeds
and products made from the hydrogenation, fractionation,
interesterification or hydrolysis of oil obtained from the seeds of
such plants.
[0009] In a fourth embodiment, this invention concerns a
hypoallergenic soybean product, and any food or any feed
incorporating this soybean product or oil.
[0010] In a fifth embodiment, this invention concerns a method for
making a hypoallergenic soy products from hypoallergenic soybean
seeds which comprises:
[0011] (a) cracking the seeds obtained from a transgenic
hypoallergenic soybean plant of the invention to remove the meats
from the hulls; and
[0012] (b) flaking the meats obtained in step (a) to obtain the
desired flake thickness.
[0013] In a sixth embodiment, this invention concerns a method for
making a hypoallergenic soybean plant which comprises:
[0014] (a) crossing a first parent soybean which is a soybean plant
comprising in its genome recombinant expression construct to lower
the Gly m Bd 30K (soybean vacuolar protein P34) content of a
soybean which comprises a beta-conglycinin promoter operably linked
to an isolated Gly m Bd 30K nucleic acid fragment corresponding
substantially to the nucleotide sequence set forth in SEQ ID NO: 1
or a functionally equivalent subfragment thereof, with a second
soybean parent which is substantially free of one or more allergens
selected from the group consisting Gly m Bd 28K, alpha-subunit of
beta-conglycinin, KSTI, Gly m2, Gly m IA, Gly m IB, rGLY m3 and
Glycinin G1; and
[0015] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0016] In a seventh embodiment, this invention concerns a method
for making a hypoallergenic soybean plant which comprises:
[0017] (a) crossing a first parent soybean which is a soybean plant
comprising in its genome the recombinant expression construct to
lower the Gly m Bd 30K (Soybean vacuolar protein P34) content of a
soybean which comprises a beta-conglycinin promoter operably linked
to an isolated Gly m Bd 30K nucleic acid fragment corresponding
substantially to the nucleotide sequence set forth in SEQ ID NO: 1
or a functionally equivalent subfragment thereof, with a second
soybean parent which is naturally occurring soybean mutant which is
substantially free of Gly m Bd 28K and which is substantially free
of the alpha-subunit of beta-conglycinin; and
[0018] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0019] In an eighth embodiment, this invention concerns a method
for making a hypoallergenic soybean plant which comprises:
[0020] (a) crossing a first parent soybean which is a soybean plant
comprising in its genome any of the claimed recombinant constructs
with a second soybean parent wherein the second parent is selected
from the group consisting of a soybean plant comprising in its
genome a recombinant expression construct which produces a lower
level of the alpha subunit of beta-conglycinin or a naturally
occuring variant therof; and
[0021] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0022] In a ninth embodiment, this invention concerns a method for
making a hypoallergenic soybean plant which comprises:
[0023] (a) crossing a first parent soybean which is the soybean
plant comprising in its genome any of the claimed recombinant
constructs with a second soybean parent wherein the second parent
comprises naturally occurring mutant soybean plants which are
substantially free of the KSTI allergen; and
[0024] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0025] Also of interest are seeds obtained from plants made by
these methods, oil obtained from these seeds, products made from
the hydrogenation, fractionation, interesterification or hydrolysis
of oil obtained from the seeds of such plants, hypoallergenic
soybean products, and food, infant formula and animal feed
incorporating any of the hypoallergenic soybean products or
oils.
[0026] In a tenth embodiment, this invention concerns an isolated
nucleic acid fragment comprising a nucleic acid sequence encoding a
soybean Gly m Bd 28K protein. The protein which is encoded can have
an amino acid identity of 49% or greater to the polypeptide encoded
by SEQ ID NO:4 or a functionally equivalent subfragment thereof. In
another aspect, this isolated nucleic acid fragment can have a
nucleic acid identity of 48% or greater to the sequence set forth
in SEQ ID NO:3. Also of interest is any plant Gly m Bd 28K protein
having an amino acid identity of 49% or greater to the polypeptide
sequence set forth in SEQ ID NO:4. Chimeric genes comprising such
nucleic acid fragments or the reverse complement thereof operably
to regulatory sequences are also of interest as well as
hypoallergenic soybean plants comprising such chimeric genes, seeds
obtained from such plants, oil obtained from such seeds, and
products made from the hydrogenation, fractionation,
interesterification or hydrolysis of oil obtained from the seeds of
such plants. In still another aspect, this invention concerns a
hypoallergenic soybean product, and any food or any feed
incorporating this soybean product or oil.
[0027] In an eleventh embodiment, this invention concerns an
isolated nucleic acid fragment comprising a nucleic acid sequence
encoding a soybean Gly m 2 protein. The protein which, is encoded
can have an amino acid identity of 95% or greater to the
polypeptide sequence set forth in SEQ ID NO:6 or a functionally
equivalent subfragment thereof. Also of interest is any plant Gly m
2 protein having an amino acid identity of 95% or greater to the
polypeptide sequence set forth in SEQ ID NO:4. Chimeric genes
comprising such nucleic acid fragments or the reverse complement
thereof operably to regulatory sequences are also of interest as
well as hypoallergenic soybean plants comprising such chimeric
genes, seeds obtained from such plants, oil obtained from such
seeds, and products made from the hydrogenation, fractionation,
interesterification or hydrolysis of oil obtained from the seeds of
such plants. In still another aspect, this invention concerns a
hypoallergenic soybean product, and any food or any feed
incorporating this soybean product or oil.
[0028] In a twelfth embodiment, this invention concerns a
recombinant expression construct to lower Gly m IB content of a
soybean which comprises a promoter operably linked to an isolated
Gly m IB nucleic acid fragment corresponding substantially to the
nucleotide sequence set forth in SEQ ID NO:9 or a functionally
equivalent subfragment thereof.
[0029] In a thirteenth embodiment, this invention concerns a
recombinant expression construct to lower Gly m IB content of a
soybean which comprises a promoter operably linked to an isolated
Gly m IB nucleic acid fragment corresponding substantially to the
nucleotide sequence set forth in SEQ ID NO:9 or a functionally
equivalent subfragment thereof.
[0030] In a fourteenth embodiment, this invention concerns a
recombinant expression construct to lower rGLY m3 content of a
soybean wherein which comprises a promoter operably linked to an
isolated rGly m3 nucleic acid fragment corresponding substantially
to the nucleotide sequence set forth in SEQ ID NOs: 11 and 13 or a
functionally equivalent subfragment thereof.
[0031] In a fifteenth embodiment, this invention concerns a
recombinant expression construct to lower Glycinin G1 (A1aB1b)
content of a soybean wherein which comprises a promoter operably
linked to an isolated Glycinin G1 nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO: 15 or a functionally equivalent subfragment thereof.
[0032] Also of interest are a hypoallergenic soybean plant
comprising in its genome at least one of the claimed recombinant
expression constructs. Also of interest are seeds obtained from
such plants, oil obtained from these seeds and products made from
the hydrogenation, fractionation, interesterification or hydrolysis
of oil obtained from the seeds of such plants. In still another
aspect, this invention concerns a hypoallergenic soybean product,
and any food or any feed incorporating this soybean product or
oil.
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS
[0033] FIG. 1 depicts soy protein processing.
[0034] FIG. 2 shows a stained protein gel and the subsequent
antibody detection of the Gly I m protein on the nitrocellulose
filter blot. The proteins were obtained from transgenic somatic
embryos that did (7-1) or did not (3-1 and 6-1) exhibit
co-suppression of Gly I m. A control positive is included in the
last lane of the blot.
[0035] FIG. 3 shows an SDS acrylamide gel of seed proteins taken
from independent isolates of delta-12 desaturase (Fad2)
co-suppressed soybean plants transformed with pKS68 (see Example
8). Note that in each lane where the .alpha.'-subunit of
beta-conglycinin is reduced or missing the .alpha.-subunit is also
missing (lanes 1, 3, 4, 5, 6, 7, and 9). Lanes 1 and 2 are a
positive and negative control (respectively).
[0036] FIG. 4 is a map of plasmid pKS73 containing the Gly m I gene
in sense orientation to the promoter from the .alpha.'-subunit of
beta-conglycinin and followed by the phaseolin 3' untranslated
region. This plasmid was used in the co-suppression experiments
outlined in Example 1.
[0037] FIG. 5 is a map of plasmid pKS68 containing the Fad2 gene in
sense orientation to the promoter from the .alpha.'-subunit of
beta-conglycinin and followed by the phaseolin 3' untranslated
region. This plasmid was used in the co-suppression experiments
outlined in Example 8.
[0038] SEQ ID NO: 1 is the nucleotide sequence of the cDNA insert
in clone pKS73 encoding a soybean P34 protein. The sequence starts
and ends with the NotI sites that were part of the primer sequences
used in the construction of the insert (see Example 1). The
promoter that directs the synthesis of P34 in pKS73 is from the
beta-conglycinin gene, and the 3'-untranslated region is from the
phaseolin gene.
[0039] SEQ ID NO:2 is the nucleotide sequence of the Kunitz soybean
trypsin inhibitor (KSTI) introduced into plants to co-suppress the
endogenous protein.
[0040] SEQ ID NO:3 is the nucleotide sequence portion of the cDNA
insert in clone se6.pk0050.c3 encoding a substantial portion of a
soybean Gly m Bd 28K protein.
[0041] SEQ ID NO:4 is the deduced amino acid sequence of a
substantial portion of a soybean Gly m Bd 28K protein derived from
the nucleotide sequence of SEQ ID NO:3.
[0042] SEQ ID NO:5 is the nucleotide sequence portion of the cDNA
insert in clone s1s1c.pk027.a11 encoding a substantial portion of a
soybean Gly m2 protein.
[0043] SEQ ID NO:6 is the deduced amino acid sequence of a
substantial portion of a soybean Gly m2 protein derived from the
nucleotide sequence of SEQ ID NO:5.
[0044] SEQ ID NO:7 is the sequence of a synthetic oligonucleotide
used to amplify the P34 coding region incorporated into the
construct of SEQ ID NO: 1.
[0045] SEQ ID NO:8 is the sequence of a synthetic oligonucleotide
used to amplify the P34 coding region incorporated into the
construct of SEQ ID NO: 1.
[0046] SEQ ID NO:9 is the nucleotide sequence portion of the cDNA
insert from Genbank accession number AF100160 (Odani et al. (1987)
Eur J. Biochem 162:485-491), encoding a substantial portion of a
Glycine max (soybean) Gly m IA, a "hydrophobic protein from
soybean". Gly m IB is identical to Gly m IA but is missing 3 amino
acids from the amino terminus. Both proteins are believed to be
minor human allergens.
[0047] SEQ ID NO: 10 is the deduced amino acid sequence of a
substantial portion of a soybean Gly m IA protein derived from the
nucleotide sequence of SEQ ID NO:9.
[0048] SEQ ID NO: 11 is the nucleotide sequence portion of the cDNA
insert from Genbank accession number AJ223981 (Rihs et al. (1999)
J. Allergy Clin Immunol 104: 1293-1301), encoding a substantial
portion of a Glycine max (soybean) rGly m3, a "soybean profilin
homologue". This protein binds IgE antibodies, and is tentatively
identified as a soybean allergen.
[0049] SEQ ID NO: 12 is the deduced amino acid sequence of a
substantial portion of a soybean rGly m3 protein derived from the
nucleotide sequence of SEQ ID NO: 11.
[0050] SEQ ID NO: 13 is the nucleotide sequence portion of the cDNA
insert from Genbank accession number AJ223982 (Rihs et al. (1999)
J. Allergy Clin Immunol 104:1293-1301), encoding a substantial
portion of a Glycine max (soybean) rGly m3, a "soybean profilin
homologue". This protein binds IgE antibodies, and is tentatively
identified as a soybean allergen.
[0051] SEQ ID NO: 14 is the deduced amino acid sequence of a
substantial portion of a soybean rGly m3 protein derived from the
nucleotide sequence of SEQ ID NO: 13.
[0052] SEQ ID NO: 15 is the nucleotide sequence portion of the cDNA
insert from Genbank accession number X02985 (Zeece et al. (1999)
Food and Agric Immunol 11:83-90), encoding a substantial portion of
a Glycine max (soybean) glycinin G1 (or A1aB1b). Soybean glycinin
G1 binds IgE antibodies in its acidic domain, and is tentatively
identified as a soybean allergen.
[0053] SEQ ID NO: 16 is the deduced amino acid sequence of a
substantial portion of a soybean glycinin G1 (or A1aB1b) protein
derived from the nucleotide sequence of SEQ ID NO:15.
DETAILED DESCRIPTION OF THE INVENTION
[0054] In the context of this disclosure, a number of terms shall
be utilized.
[0055] The terms "P34 (soybean vacuolar protein)" and "Gly m BD
30K" and "Gly m 1" [SEQ ID NO:1] are used interchangeably herein.
They all refer to the major soybean seed allergen. Major allergens
are generally defined as proteins for which 50% or more of the
allergic patients studied have specific IgE.
[0056] The terms "KSTI" and "KTi3" [SEQ ID NO:2] are used
interchangeably herein. They refer to a Kunitz soybean trypsin
inhibitor or a Kunitz-type soybean trypsin inhibitor which is a
minor soybean seed allergen. S-II is another minor soybean seed
allergen along with 68-kDa which is the .alpha.-subunit of
.beta.-conglycinin.
[0057] The terms "Gly m Bd 28K" and "28K protein" [SEQ ID NOs:3 and
4] are used interchangeably herein. They refer to a 28 kilodalton
protein which is a minor soybean seed allergen.
[0058] The term "Gly m2" [SEQ ID NOs:5 and 6] refers to a small
75-amino acid protein that is aminor soybean seed allergen.
[0059] The term Gly m IA [SEQ ID NOs:9 and 10] and Gly m IB refer
to a hydrophobic soybean seed protein that has similarity to lipid
transfer proteins. Gly m IA is a 119 amino acid protein and Gly m
IB is identical except it is missing the first three amino acid
residues of the polypeptide. Both are considered minor soybean seed
allergens.
[0060] The term rGly M3 [SEQ ID NOs:11, 12, 13, and 14] refers to a
soybean profilin-like protein of 131 amino acids that binds human
IgE antibody. Plant profilins have been reported to be a
pan-allergen in pollen (Valenta et al. (1991) Science
253:557-560).
[0061] The term glycinin G1 [SEQ ID NOs:15 and 16] refers to a 495
amino acid soybean glycinin protein. This protein contains an acid
domain that binds to IgE antibody. Glycinin G2 which contains a
shorter (by 20 amino acids) acidic chain does not bind IgE [Zeece
et al. (1999) Food and Agric Immunol 11:83-90]. As noted above for
the rGly m3 profilin, IgE binding proteins are thought to be
potential allergens.
[0062] The term "hypoallergenic" means substantially free of any
allergens, i.e., an immunological response, such as an allergic
reaction, should not be triggered.
[0063] As used herein, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA. Nucleotides are referred to by their single
letter designation as follows: "A" for adenosine, "C" for cytidine,
"G" for guanosine, "T" for thymidine, "R" for purines (A or G), "Y"
for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I"
for inosine, and "N" for any nucleotide.
[0064] The terms "subfragment that is functionally equivalent" and
"functionally equivalent subfragment" are used interchangeably
herein. These terms refer to a portion or subsequence of an
isolated nucleic acid fragment in which the ability to alter gene
expression or produce a certain phenotype is retained whether or
not the fragment or subfragment encodes an active enzyme. For
example, the fragment or subfragment can be used in the design of
chimeric genes to produce the desired phenotype in a transformed
plant. Chimeric genes can be designed for use in co-suppression or
antisense by linking a nucleic acid fragment or subfragment
thereof, whether or not it encodes an active enzyme, in the
appropriate orientation relative to a plant promoter sequence.
[0065] The terms "substantially similar" and "corresponding
substantially" as used herein refer to nucleic acid fragments
wherein changes in one or more nucleotide bases does not affect the
ability of the nucleic acid fragment to mediate gene expression or
produce a certain phenotype. These terms also refer 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 alter the functional properties of the
resulting nucleic acid fragment relative to the initial, unmodified
fragment. It is therefore understood, as those skilled in the art
will appreciate, that the invention encompasses more than the
specific exemplary sequences.
[0066] Moreover, the skilled artisan recognizes that substantially
similar nucleic acid sequences encompassed by this invention are
also defined by their ability to hybridize, under moderately
stringent conditions (for example, 0.5.times.SSC, 0.1% SDS,
60.degree. C.) with the sequences exemplified herein, or to any
portion of the nucleotide sequences reported herein and which are
functionally equivalent to the promoter of the invention. Preferred
substantially similar nucleic acid sequences encompassed by this
invention are those sequences that are 45% identical to the nucleic
acid fragments reported herein or which are 45% identical to any
portion of the nucleotide sequences reported herein. More preferred
are nucleic acid fragments which are 50% identical to the nucleic
acid sequences reported herein, or which are 50% identical to any
portion of the nucleotide sequences reported herein. Most preferred
are nucleic acid fragments which are 60% identical to the nucleic
acid sequences reported herein, or which are 60% identical to any
portion of the nucleotide sequences reported herein. Sequence
alignments and percent similarity calculations may be determined
using a variety of comparison methods designed to detect homologous
sequences including, but not limited to, the Megalign program of
the LASARGENE bioinformatics computing suite (DNASTAR Inc.,
Madison, Wis.). Multiple alignment of the sequences are 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 and
calculation of percent identity of protein sequences using the
Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS
SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP
PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.
[0067] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to afford putative
identification of that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Altschul, S. F., et al., (1993) J. Mol.
Biol. 215:403-410) and Gapped Blast (Altschul, S. F. et al., (1997)
Nucleic Acids Res. 25:3389-3402); see also
www.ncbi.nlm.nih.gov/BLAST/).
[0068] "Gene" refers to a nucleic acid fragment 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.
[0069] "Coding sequence" refers to a DNA 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, but are not limited to,
promoters, translation leader sequences, introns, and
polyadenylation recognition sequences.
[0070] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. The promoter
sequence consists of proximal and more distal upstream elements,
the latter elements often referred to as enhancers. Accordingly, an
"enhancer" is a DNA sequence which can stimulate promoter activity
and may be an innate element of the promoter or a heterologous
element inserted to enhance the level 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 DNA 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 gene to be expressed in most cell types at most times are
commonly referred to as "constitutive promoters". 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, DNA
fragments of some variation may have identical promoter
activity.
[0071] An "intron" is an intervening sequence in a gene that does
not encode a portion of the protein sequence. Thus, such sequences
are transcribed into RNA but are then excised and are not
translated. The term is also used for the excised RNA sequences. An
"exon" is a portion of the sequence of a gene that is transcribed
and is found in the mature messenger RNA derived from the gene, but
is not necessarily a part of the sequence that encodes the final
gene product.
[0072] The "translation leader sequence" refers to a DNA 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, R. and Foster, G. D. (1995) Molecular Biotechnology
3:225).
[0073] The "3' non-coding sequences" refer to DNA 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.
[0074] "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 protein by the cell. "cDNA" refers to a DNA that is
complementary to and synthesized from a mRNA template using the
enzyme reverse transcriptase. The cDNA can be single-stranded or
converted into the double-stranded form using the klenow fragment
of DNA polymerase I. "Sense" RNA refers to RNA transcript that
includes the mRNA and so can be translated into protein within a
cell or in vitro. "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 (U.S. Pat. No.
5,107,065). The complementarity of an antisense RNA may be with any
part of the specific gene transcript, i.e., at the 5' non-coding
sequence, 3' non-coding sequence, introns, or the coding sequence.
"Functional RNA" refers to antisense RNA, ribozyme RNA, or other
RNA that may not be translated but yet has an effect on cellular
processes. The terms "complement" and "reverse complement" are used
interchangeably herein with respect to mRNA transcripts, and are
meant to define the antisense RNA of the message.
[0075] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment 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.
[0076] The term "expression", as used herein, refers to the
production of a functional end-product. Expression or
overexpression of a gene involves transcription of the gene and
translation of the mRNA into a precursor or mature protein.
"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.
[0077] "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).
[0078] 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 fragment 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
fragment can be constructed by linking the gene or gene fragment 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.
[0079] 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.
[0080] The person skilled in the art will know that special
considerations are associated with the use of antisense or
cosuppression 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, and is not
an inherent part of the invention. 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.
[0081] "Altered expression" refers to the production of gene
product(s) in transgenic organisms in amounts or proportions that
differ significantly from that activity in comparable tissue (organ
and of developmental type) from wild-type organisms.
[0082] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
[0083] A "chloroplast transit peptide" is an amino acid sequence
which is translated in conjunction with a protein and directs the
protein to the chloroplast or other plastid types present in the
cell in which the protein is made. "Chloroplast transit sequence"
refers to a nucleotide sequence that encodes a chloroplast transit
peptide. A "signal peptide" is an amino acid sequence that is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant
Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed
to a vacuole, a vacuolar targeting signal (supra) can further be
added, or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included (Raikhel
(1992) Plant Phys.100:1627-1632).
[0084] "Transformation" refers to the transfer of a nucleic acid
fragment 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. The preferred method of cell transformation of rice,
corn and other monocots is the use of particle-accelerated or "gene
gun" transformation technology (Klein et al., (1987) Nature
(London) 327:70-73; U.S. Pat. No. 4,945,050), or an
Agrobacterium-mediated method using an appropriate Ti plasmid
containing the transgene (Ishida Y. et al., 1996, Nature Biotech.
14:745-750).
[0085] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, 1989 (hereinafter "Sambrook").
[0086] The term "recombinant" refers to an artificial combination
of two otherwise separated segments of sequence, e.g., by chemical
synthesis or by the manipulation of isolated segments of nucleic
acids by genetic engineering techniques.
[0087] "PCR" or "Polymerase Chain Reaction" is a technique for the
synthesis of large quantities of specific DNA segments, consists of
a series of repetitive cycles (Perkin Elmer Cetus Instruments,
Norwalk, Conn.). Typically, the double stranded DNA is heat
denatured, the two primers complementary to the 3' boundaries of
the target segment are annealed at low temperature and then
extended at an intermediate temperature. One set of these three
consecutive steps is referred to as a cycle.
[0088] The terms "expression construct" and "recombinant expression
construct" are used interchangeably herein. These terms, as used
herein, comprise any of the isolated nucleic acid fragments of the
invention or subfragment thereof used either alone or in
combination with each other as discussed herein. They can be
incorporated into recombinant nucleic acid constructs, typically
DNA constructs, capable of introduction into and replication in a
host cell. Such construct may be itself or may be used in
conjunction with a vector. If a vector is used then the choice of
vector is dependent upon the method that will be used to transform
host plants as is well known to those skilled in the art. For
example, a plasmid vector can be used. The skilled artisan is well
aware of the genetic elements that must be present on the vector in
order to successfully transform, select and propagate host cells
comprising any of the isolated nucleic acid fragments of the
invention. 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.
[0089] As was noted above P34 constitutes the major allergen in
soybeans and is present in processed food products that contain
soybean protein. Assays of IgE binding using immunoglobulins from
soybean sensitive individuals indicates that 65% of the total
allergenic response can be attributed to P34. Detailed
immunological analysis of the allergenicity of P34 by epitope
mapping has shown that there are at least 12 distinct epitopes on
the protein.
[0090] P34 possesses most of the conserved characteristics of
cysteine proteases including a large precursor domain that is
posttranslationally processed. The primary sequence contains
aligned and conserved amino acids that are important in the
conserved tertiary conformation of the papain superfamily. P34
exhibits some unique features that separate it from other members
of the papain superfamily. Among these are replacement of the
conserved cysteine in the active site found in all other papain
family proteins with a glycine, suggesting that the protein is
enzymatically inactive. Cysteine proteases are typically
self-processed under acid-reducing conditions resulting in the
cleavage of the large precursor domain. However, P34 is processed
after an asparagine residue in a single step, most likely by the
same enzyme that processes the 11S storage proteins. Sequence
comparisons and alignments indicate that although P34 is a member
of the papain superfamily, it is also quite dissimilar from the
enzymatically active cysteine proteases including those identified
in soybean.
[0091] P34 may have a function in defense against Pseudomonas
infection by binding syringolide elicitors secreted the bacteria.
P34 is very abundant in seeds, but it is also found in vegetative
cells that are subject to bacterial infections.
[0092] It has been found that the P34 allergen can be substantially
removed from soybean embryos, without resulting lethality to the
embryo, by using recombinant techniques such as sense suppression
of an isolated nucleic acid fragment encoding P34 protein.
[0093] Thus, in one embodiment, the instant invention concerns a
recombinant expression construct to lower Gly m Bd 30K (Soybean
vacuolar protein P34) content of a soybean which comprises a
promoter operably linked to an isolated Gly m Bd 30K nucleic acid
fragment corresponding substantially to the nucleotide sequence set
forth in SEQ ID NO: 1 or a functionally equivalent subfragment
thereof. A transgenic soybean plant which comprises the foregoing
recombinant expression construct in its genome should be
hypoallergenic with respect to P34.
[0094] Any promoter can be used to practice the invention. There
can be mentioned a beta-conglycinin promoter, a Kunitz Trypsin
Inhibitor (KSTI) promoter, a Gly m Bd 28K promoter, T7 promoter, a
.sup.35S promoter and a beta-phaseolin promoter. The preferred
promoter is that of the .alpha.'-subunit of beta-conglycinin
(referred to herein as the beta-conglycinin promoter).
Co-suppressed plants that contain recombinant expression constructs
with the promoter of the .alpha.'-subunit of beta-conglycinin will
often exhibit suppression of both the .alpha. and .alpha.' subunits
of beta-congylcinin (as described in PCT Publication No.
WO97/47731, published on Dec. 18, 1997, the disclosure of which is
hereby incorporated by reference). Particularly preferred promoters
are those that allow seed-specific expression. This may be
especially useful since seeds are the primary source consumable
protein and oil, and also since seed-specific expression will avoid
any potential deleterious effect in non-seed tissues. This may be
particularly important for plants with reduced or undetectable
levels of p34, since no naturally occurring or induced mutations
have been recovered in this gene, implying a deleterious effect for
plants lacking this protein.
[0095] Examples of seed-specific promoters include, but are not
limited to, the promoters of seed storage proteins, which can
represent up to 90% of total seed protein in many plants. The seed
storage proteins are strictly regulated, being expressed almost
exclusively in seeds in a highly tissue-specific and stage-specific
manner (Higgins et al., (1984) Ann. Rev. Plant Physiol. 35:191-221;
Goldberg et al., (1989) Cell 56:149-160). Moreover, different seed
storage proteins may be expressed at different stages of seed
development.
[0096] Expression of seed-specific genes has been studied in great
detail (See reviews by Goldberg et al., (1989) Cell 56:149-160 and
Higgins et al., (1984) Ann. Rev. Plant Physiol. 35:191-221). There
are currently numerous examples of seed-specific expression of seed
storage protein genes in transgenic dicotyledonous plants. These
include genes from dicotyledonous plants for bean .beta.-phaseolin
(Sengupta-Gopalan et al., (1985) Proc. Natl. Acad. Sci. USA 82:
3320-3324; Hoffman et al., (1988) Plant Mol. Biol. 11: 717-729),
bean lectin (Voelker et al., (1987) EMBO J. 6: 3571-3577), soybean
lectin (Okamuro et al., (1986) Proc. Natl. Acad. Sci. USA 83:
8240-8244), soybean Kunitz trypsin inhibitor (Perez-Grau et al.,
(1989) Plant Cell 1: 095-1109), soybean b-conglycinin (Beachy et
al., (1985) EMBO J. 4: 3047-3053; pea vicilin (Higgins et al.,
(1988) Plant Mol. Biol. 11:683-695), pea convicilin (Newbigin et
al., (1990) Planta 180:461-470), pea legumin (Shirsat et al.,
(1989) Mol. Gen. Genetics 215:326-331); rapeseed napin (Radke et
al., (1988) Theor. Appl. Genet. 75:685-694) as well as genes from
monocotyledonous plants such as for maize 15 kD zein (Hoffman et
al., (1987) EMBO J. 6:3213-3221), maize 18 kD oleosin (Lee at al.,
(1991) Proc. Natl. Acad. Sci. USA 88:6181-6185), barley
.beta.-hordein (Marris et al., (1988) Plant Mol. Biol. 10:359-366)
and wheat glutenin (Colot et al., (1987) EMBO J. 6:3559-3564).
Moreover, promoters of seed-specific genes operably linked to
heterologous coding sequences in chimeric gene constructs also
maintain their temporal and spatial expression pattern in
transgenic plants. Such examples include use of Arabidopsis
thaliana 2S seed storage protein gene promoter to express
enkephalin peptides in Arabidopsis and Brassica napus seeds
(Vandekerckhove et al., (1989) Bio/Technology 7:929-932), bean
lectin and bean .beta.-phaseolin promoters to express luciferase
(Riggs et al., (1989) Plant Sci. 63:47-57), and wheat glutenin
promoters to express chloramphenicol acetyl transferase (Colot et
al., (1987) EMBO J. 6:3559-3564).
[0097] Of particular use in the expression of the nucleic acid
fragment of the invention will be the heterologous soybean seed
storage protein gene promoter from beta-conglycinin (Harada et al.,
(1989) Plant Cell 1:415-425). This promoter will be particularly
useful for co-suppression in the cotyledons at mid- to late-stages
of seed development (Beachy et al., (1985) EMBO J. 4: 3047-3053) in
transgenic plants. This is because there is very little position
effect on its expression in transgenic seeds. An added benefit of
this promoter is realized because its use as a transgenic promoter
is known to cause high frequency co-suppression of the endogenous
beta-conglycinin protein. This protein is known to be a minor
allergen in soybeans (Bush and Hefle (1996) Critical Rev in Food
Science and Nutrition 36:S 19-S 163).
[0098] In a second embodiment, this invention concerns a
recombinant expression construct for producing a hypoallergenic
soybean which comprises an isolated KSTI nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO:2 or a functionally equivalent subfragment thereof
operably linked to an isolated Gly m Bd 28K nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO:3 or a functionally equivalent subfragment thereof. A
transgenic soybean plant which comprises at least one of the
recombinant expression constructs, described herein, in its genome
should be hypoallergenic with respect to one or more of the
following allergens: P34, KSTI, and SII.
[0099] In a third embodiment, this invention concerns a
hypoallergenic soybean plant comprising in its genome at least one
of the expression constructs of this invention, seeds obtained from
such plants, oil obtained from the seeds of these plants, products
made from the hydrogenation, fractionation, interesterification or
hydrolysis of oil obtained from the seeds of such plants,
hypoallergenic soybean products, and any food or any feed
incorporating any of the hypoallergenic soybean products or oils as
described herein.
[0100] Such transgenic soybean plants can be made using
conventional techniques well known to those skilled in the art as
is discussed above. Introduction of transgenes into plants, i.e.,
transformation is well known to the skilled artisan. A preferred
method of plant cell transformation is the use of
particle-accelerated or "gene gun" transformation technology (Klein
et al. (1978) Nature (London) 327:70-73; U.S. Pat. No.
4,945,050).
[0101] In a fourth embodiment, this invention concerns
hypoallergenic soybean products obtained from transgenic soybean
plants comprising at least one of the recombinant expression
constructs of the invention in its genome.
[0102] "Soy protein products" are defined as those items produced
from soybean seed and are then used as ingredients in the
production of any feed and any food, for example, breakfast
cereals, in baking applications (e.g., breads, rolls, etc.), in
dairy or meat based food products such as infant formula,
nutritional beverage, milk replacer, soy extended bologna,
imitation processed cheese spread, brine injected ham, yogurt and
frozen desserts and the like. Table 1 lists a variety of soybean
protein products derived from soybean seeds. The terms "soy protein
products" and "soy products" are used interchangeably herein. Soy
protein processing is depicted in FIG. 1.
1TABLE 1 Soy Protein Products Derived from Soybean Seeds.sup.a
Whole Soybean Products Roasted Soybeans Baked Soybeans Soy Sprouts
Soy Milk Speciality Soy Foods/Ingredients Soy Milk Tofu Tempeh Miso
Soy Sauce Hydrolyzed Vegetable Protein Whipping Protein Processed
Soy Protein Products Soybean Meal Soy Grits Full Fat and Defatted
Flours Soy Protein Isolates Soy Protein Concentrates Textured Soy
Proteins Textured Flours and Concentrates Structured Concentrates
Structured Isolates .sup.aSee Soy Protein Products:
Characteristics, Nutritional Aspects and Utilization (1987). Soy
Protein Council
[0103] "Processing" refers to any physical and chemical methods
used to obtain the products listed in Table 1 and includes, but is
not limited to heat conditioning, flaking and grinding, extrusion,
solvent extraction, or aqueous soaking and extraction of whole or
partial seeds.
[0104] Furthermore, "processing" includes the methods used to
concentrate and isolate soy protein from whole or partial seeds, as
well as the various traditional Oriental methods in preparing
fermented soy food products. Trading Standards and Specifications
have been established for many of these products (see National
Oilseed Processors Association Yearbook and Trading Rules
1991-1992). Products referred to as being "high protein" or "low
protein" are those as decribed by these Standard Specifications.
"NSI" refers to the Nitrogen Solubility Index as defined by the
American Oil Chemists' Society Method Ac441. "KOH Nitrogen
Solubility" is an indicator of soybean meal quality and refers to
the amount of nitrogen soluble in 0.036 M KOH under the conditions
as described by Araba and Dale (1990) Poultry Science 69:76-83.
[0105] "White" flakes refer to flaked, dehulled cotyledons that
have been defatted and treated with controlled moist heat to have
an NSI of about 85 to 90. This term can also refer to a flour with
a similar NSI that has been ground to pass through a No. 100 U.S.
Standard Screen size. "Cooked" refers to a soy protein product,
typically a flour, with an NSI of about 20 to 60. "Toasted" refers
to a soy protein product, typically a flour, with an NSI below
20.
[0106] "Grits" refer to defatted, dehulled cotyledons having a U.S.
Standard screen size of between No. 10 and 80. Soy flours and grits
are made by grinding and screening soybean flakes either before or
after removal of the oil. Their protein content is in the range of
40% to 54%. Soy flours and grits are the least refined forms of soy
protein products used for human consumption and may vary in fat
content, particle size, and degree of heat treatment. They are also
produced in lecithinated or refatted forms. The degree of heat
treatment creates varying levels of water dispersibility, a quality
that can be useful in tailoring functionality in many food
applications.
[0107] "Soy Protein Concentrates" refer to those products produced
from dehulled, defatted soybeans by three basic processes: acid
leaching (at about pH 4.5), extraction with alcohol (about 55-80%),
and denaturing the protein with moist heat prior to extraction with
water. Conditions typically used to prepare soy protein
concentrates have been described by Pass (1975) U.S. Pat. No.
3,897,574; Campbell et al., (1985) in New Protein Foods, ed. by
Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10, Seed
Storage Proteins, pp 302-338. Thus, term "soy protein concentrates"
as used herein refers to those products which are prepared from
high quality sound, clean dehulled soybean seeds by removing most
of the oil and water soluble non-protein constituents and must
contain not less than 65% protein on a moisture free basis as set
forth in [(1966) Official Publication of the Association of
American Feed Control Officials, Inc.]. Neutralized concentrates
prepared by acid leaching have a higher water-soluble protein
content than those prepared by either alcohol leaching or heat
denaturation techniques. In another process, low water-soluble soy
protein concentrate (aqueous alcohol extraction) is subjected to
heat treatment by steam injection or jet cooking to increase
solubility and functionality. Functionality may be improved further
by additional treatments. Concentrates function as emulsifiers and
emulsion stabilizers, they bind fat and water, and they offer
special adhesive properties similar to those of isolates.
[0108] The term "soy protein isolates" as used herein refers to
those products which are the major proteinaceous fraction of
soybeans prepared from dehulled soybeans by removing the majority
of non-protein compounds and must contain not less than 90% protein
on a moisture free basis as set forth in [(1996) Official
Publication of the Association of American Feed Control Officials,
Inc.]. Isolates may also be lecithinated to improve disperisibility
and to reduce dusting. Both gelling and non-gelling varieties are
available, as well as varying grades of viscosity.
[0109] "Extrusion" refers to processes whereby material (grits,
flour or concentrate) is passed through a jacketed auger using high
pressures and temperatures as a means of altering the texture of
the material. "Texturing" and "structuring" refer to extrusion
processes used to modify the physical characteristics of the
material. The characteristics of these processes, including
thermoplastic extrusion, have been described previously [Atkinson,
(1970) U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods,
ed. by Altschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp
367-414]. Moreover, conditions used during extrusion processing of
complex foodstuff mixtures that include soy protein products have
been described previously [Rokey (1983) Feed Manufacturing
Technology III, 222-237; McCulloch, U.S. Pat. No. 4,454,804].
[0110] Oil made from seeds obtained from the hypoallergenic soybean
plants of the invention can be used in a variety of applications.
These oils can be used in the preparation of foods. Examples
include, but are not limited to, uses as ingredients, as coatings,
as salad oils, as spraying oils, as roasting oils, and as frying
oils. Foods in which the oil may be used include, but are not
limited to, crackers and snack foods, confectionery products,
syrups and toppings, sauces and gravies, soups, batter and breading
mixes, baking mixes and doughs.
[0111] These oils can also be used as a blending source to make a
blended oil product. By a blending source, it is meant that the oil
of this invention can be mixed with other vegetables oils to
improve the characteristics, such as fatty acid composition,
flavor, and oxidative stability, of the other oils. The amount of
oil of this invention which can be used will depend upon the
desired properties sought to be achieved in the resulting final
blended oil product. Examples of blended oil products include, but
are not limited to, margarines, shortenings, frying oils, salad
oils, etc.
[0112] In another aspect, the oils of this invention can be
subjected to further processing such as hydrogenation,
fractionation, interesterification or fat splitting
(hydrolysis).
[0113] In still another aspect, this invention concerns by-products
made during the production of the oils of this invention.
[0114] Methods for the extraction and processing of soybean seeds
to produce soybean oil and meal are well known throughout the
soybean processing industry. In general, soybean oil is produced
using a series of steps that accomplish the extraction and
purification of an edible oil product from the oil bearing seed.
Soybean oils and soybean byproducts are produced using the
generalized steps shown in the diagram below.
[0115] Soybean seeds are cleaned, tempered, dehulled, and flaked
which increases the efficiency of oil extraction. Oil extraction is
usually accomplished by solvent (hexane) extraction but can also be
achieved by a combination of physical pressure and/or solvent
extraction. The resulting oil is called crude oil. The crude oil
may be degummed by hydrating phospholipids and other polar and
neutral lipid complexes which facilitate their separation from the
nonhydrating, triglyceride fraction (soybean oil). The resulting
lecithin gums may be further processed to make commercially
important lecithin products used in a variety of food and
industrial products as emulsification and release (antisticking)
agents.
[0116] Lecithin constitutes a member of a class of complex lipids
called phospholipids, phosphoglycerides or glycerol phosphatides.
They are characteristic major components of cell membranes. The
most abundant phospholipids in higher plants and animals are
phosphatidylcholine and phosphatidylethanolamine which contain as
head groups the amino alcohols ethanolamine and choline,
respectively. (The new names recommended for these
phosphoglycerides are phosphatidylcholine and
phosphatidylethanolamine. The old trivial names are lecithin and
cephalin, respectively). These two phosphoglycerides are major
components of most animal cell membranes. The so-called lecithin
products described above are actually a mixture of phospholipids,
predominantly phosphatidylcholine and phosphatidylethanolamine.
[0117] Degummed oil may be further refined for the removal of
impurities; primarily free fatty acids, pigments, and residual
gums. Refining is accomplished by the addition of caustic which
reacts with free fatty acid to form soap and hydrates phosphatides
and proteins in the crude oil. Water is used to wash out traces of
soap formed during refining. The soapstock byproduct may be used
directly in animal feeds or acidulated to recover the free fatty
acids. Color is removed through adsorption with a bleaching earth
which removes most of the chlorophyll and carotenoid compounds. The
refined oil can be hydrogenated resulting in fats with various
melting properties and textures. Winterization (fractionation) may
be used to remove stearine from the hydrogenated oil through
crystallization under carefully controlled cooling conditions.
Deodorization which is principally steam distillation under vacuum,
is the last step and is designed to remove compounds which impart
odor or flavor to the oil. Other valuable byproducts such as
tocopherols and sterols may be removed during the deodorization
process. Deodorized distillate containing these byproducts may be
sold for production of natural vitamin E and other high value
pharmaceutical products. Refined, bleached, (hydrogenated,
fractionated) and deodorized oils and fats may be packaged and sold
directly or further processed into more specialized products. A
more detailed reference to soybean seed processing, soybean oil
production and byproduct utilization can be found in Erickson,
(1995) Practical Handbook of Soybean Processing and Utilization,
The American Oil Chemists' Society and United Soybean Board.
[0118] Hydrogenation is a chemical reaction in which hydrogen is
added to the unsaturated fatty acid double bonds with the aid of a
catalyst such as nickel. High oleic soybean oil contains
unsaturated oleic, linoleic, and linolenic fatty acids and each of
these can be hydrogenated. Hydrogenation has two primary effects.
First, the oxidative stability of the oil is increased as a result
of the reduction of the unsaturated fatty acid content. Second, the
physical properties of the oil are changed because the fatty acid
modifications increase the melting point resulting in a semi-liquid
or solid fat at room temperature.
[0119] There are many variables which affect the hydrogenation
reaction which in turn alter the composition of the final product.
Operating conditions including pressure, temperature, catalyst type
and concentration, agitation and reactor design are among the more
important parameters which can be controlled. Selective
hydrogenation conditions can be used to hydrogenate the more
unsaturated fatty acids in preference to the less unsaturated ones.
Very light or brush hydrogenation is often employed to increase
stability of liquid oils. Further hydrogenation converts a liquid
oil to a physically solid fat. The degree of hydrogenation depends
on the desired performance and melting characteristics designed for
the particular end product. Liquid shortenings, used in the
manufacture of baking products, solid fats and shortenings used for
commercial flying and roasting operations, and base stocks for
margarine manufacture are among the myriad of possible oil and fat
products achieved through hydrogenation. A more detailed
description of hydrogenation and hydrogenated products can be found
in Patterson, H. B. W., (1994) Hydrogenation of Fats and Oils:
Theory and Practice. The American Oil Chemists' Society.
[0120] Interesterification refers to the exchange of the fatty acyl
moiety between an ester and an acid (acidolysis), an ester and an
alcohol (alcoholysis) or an ester and ester (transesterification).
Interesterification reactions are achieved using chemical or
enzymatic processes. Random or directed transesterification
processes rearrange the fatty acids on the triglyceride molecule
without changing the fatty acid composition. The modified
triglyceride structure may result in a fat with altered physical
properties. Directed interesterification reactions using lipases
are becoming of increasing interest for high value specialty
products like cocoa butter substitutes. Products being commercially
produced using interesterification reactions include but are not
limited to shortenings, margarines, cocoa butter substitutes and
structured lipids containing medium chain fatty acids and
polyunsaturated fatty acids. Interesterification is further
discussed in Hui, Y. H., (1996) Bailey's Industrial Oil and Fat
Products, Volume 4, John Wiley & Sons.
[0121] Fatty acids and fatty acid methyl esters are two of the more
important oleochemicals derived from vegetables oils. Fatty acids
are used for the production of many products such as soaps, medium
chain triglycerides, polyol esters, alkanolamides, etc. Vegetable
oils can be hydrolyzed or split into their corresponding fatty
acids and glycerine. Fatty acids produced from various fat
splitting processes may be used crude or more often are purified
into fractions or individual fatty acids by distillation and
fractionation. Purified fatty acids and fractions thereof are
converted into a wide variety of oleochemicals, such as dimer and
trimer acids, diacids, alcohols, amines, amides, and esters. Fatty
acid methyl esters are increasingly replacing fatty acids as
starting materials for many oleochemicals such as fatty alcohols,
alkanolamides, .alpha.-sulfonated methyl esters, diesel oil
components, etc. Glycerine is also obtained by the cleavage of
triglycerides using splitting or hydrolysis of vegetable oils.
Further references on the commercial use of fatty acids and
oleochemicals may be found in Erickson, D. R., (1995) Practical
Handbook of Soybean Processing and Utilization, The American Oil
Chemists' Society, and United Soybean Board; Pryde, E. H., (1979)
Fatty Acids, The American Oil Chemists' Society; and Hui, Y. H.,
(1996) Bailey's Industrial Oil and Fat Products, Volume 4, John
Wiley & Sons.
[0122] In a fifth embodiment, this invention concerns a method of
producing a hypoallergenic soy product from hypoallergenic soybean
seeds which comprises:
[0123] (a) cracking the seeds obtained from a hypoallergenic
soybean plant comprising in its genome at least one of the
recombinant constructs of the invention to remove the meats from
the hulls; and
[0124] (b) flaking the meats obtained in step (a) to obtain the
desired flake thickness.
[0125] In a sixth embodiment, this invention concerns a method for
making a hypoallergenic soybean plant which comprises:
[0126] (a) crossing a first parent soybean which is a soybean plant
comprising in its genome recombinant expression construct to lower
the Gly m Bd 30K (Soybean vacuolar protein P34) content of a
soybean which comprises a beta-conglycinin promoter operably linked
to an isolated Gly m Bd 30K nucleic acid fragment corresponding
substantially to the nucleotide sequence set forth in SEQ ID NO: 1
or a functionally equivalent subfragment thereof, with a second
soybean parent which is substantially free of one or more allergens
selected from the group consisting Gly m Bd 28K, alpha-subunit of
beta-conglycinin, KSTI, Gly m2, Gly m IA, Gly m IB, rGLY m3 and
Glycinin G1; and
[0127] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0128] A hypoallergenic soybean plant made by this method, seeds
obtained therefrom, oil obtained from the seeds, soybean protein
products obtained from such seeds and any food or feed which
incorporates such soybean protein products or oil should be
hypoallergenic with respect to one or more of the following, P34 a
major soybean allergen, and the minor soybean protein allergens Gly
m Bd 28K, alpha-subunit of beta-conglycinin, KSTI, Gly m2, Gly m
IA, Gly m IB, rGLY m3 and glycinin G1.
[0129] In a seventh embodiment, this invention concerns a method
for making a hypoallergenic soybean plant which comprises:
[0130] (a) crossing a first parent soybean which is a soybean plant
comprising in its genome a recombinant expression construct to
lower the Gly m Bd 30K (Soybean vacuolar protein P34) content of a
soybean which comprises a beta-conglycinin promoter operably linked
to an isolated Gly m Bd 30K nucleic acid fragment corresponding
substantially to the nucleotide sequence set forth in SEQ ID NO: 1
or a functionally equivalent subfragment thereof; with a second
soybean parent which is naturally occurring soybean mutant which is
substantially free of Gly m Bd 28K and which is substantially free
of the alpha-subunit of beta-conglycinin; and
[0131] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0132] A hypoallergenic soybean plant made by this method, seeds
obtained therefrom, oil obtained from the seeds, soybean protein
products obtained from such seeds and any food or feed which
incorporates such soybean protein products or oil should be
hypoallergenic with respect to one or more of the following, P34 a
major soybean allergen, and the minor soybean protein allergens Gly
m Bd 28K and the alpha-subunit of beta-conglycinin.
[0133] In an eighth embodiment, this invention concerns a method
for making a hypoallergenic soybean plant which comprises:
[0134] (a) crossing a first parent soybean which is the soybean
plant comprising in its genome at least one of the recombinant
constructs of the invention with a second soybean parent wherein
the second parent is selected from the group consisting of a
soybean plant comprising in its genome a recombinant expression
construct which produces a lower level of the alpha subunit of
beta-conglycinin or a naturally occuring variant thereof; and
[0135] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0136] A hypoallergenic soybean plant made by this method, seeds
obtained therefrom, oil obtained from the seeds, soybean protein
products obtained from such seeds and any food or feed which
incorporates such soybean protein products or oil should be
hypoallergenic with respect to the major soybean allergen P34, and
the minor soybean protein allergens 68-K (the a subunit of
b-conglycinin), KSTI (KTi3), Gly m2, Gly m IA, Gly m IB, rGLY m3,
and glycinin G1.
[0137] In a ninth embodiment, this invention concerns a method for
making a hypoallergenic soybean plant which comprises:
[0138] (a) crossing a first parent soybean which is the soybean
plant comprising in its genome at least one of the recombinant
constructs of the invention with a second soybean parent wherein
the second parent comprises naturally occurring mutant soybean
plants which are substantially free of the KSTI allergen; and
[0139] (b) selecting progeny plants of the cross of step (a) which
are hypoallergenic.
[0140] A hypoallergenic soybean plant made by this method, seeds
obtained therefrom, oil obtained from the seeds, soybean protein
products obtained from such seeds and any food or feed which
incorporates such soybean protein products or oil should be
hypoallergenic with respect to the major soybean allergen P34, and
the minor soybean protein allergens 68-K (the a subunit of
.beta.-conglycinin), KSTI (KTi3), Gly m2, Gly m IA, Gly m IB, rGLY
m3, and glycinin G1.
[0141] Also of interest, are seeds obtained from such plants, oil
obtained from these seeds, soybean protein products obtained from
such seeds and any food or feed which incorporates such soy protein
product or oil as well as any products made from the hydrogenation,
fractionation, interesterification or hydrolysis of oil obtained
from the seeds of such plants.
[0142] In a tenth embodiment, this invention concerns an isolated
nucleic acid fragment comprising a nucleic acid sequence encoding a
soybean Gly m Bd 28K protein. The protein which is encoded by the
nucleic acid fragment can have an amino acid identity of 49% or
greater to the polypeptide sequence set forth in SEQ ID NO:4 or a
functionally equivalent subfragment thereof.
[0143] In another aspect, this isolated nucleic acid fragment can
have a nucleic acid identity of 48% or greater to the sequence set
forth in SEQ ID NO:3.
[0144] Also of interest is any plant protein, similar to Gly m Bd
28K protein, having an amino acid identity of 49% or greater to the
polypeptide encoded by SEQ ID NO:4. Plant proteins of interest
would include, but not be limited to, seed-storage proteins,
proteins exhibiting modifications such as glycosylation, or
allergenic proteins.
[0145] Chimeric genes comprising such nucleic acid fragments or the
reverse complement thereof operably to regulatory sequences are
also of interest as well as hypoallergenic soybean plants
comprising such chimeric genes, seeds obtained from such plants,
oil obtained from such seeds, and products made from the
hydrogenation, fractionation, interesterification or hydrolysis of
oil obtained from the seeds of such plants. In still another
aspect, this invention concerns a hypoallergenic soybean product,
and any food or any feed incorporating this soybean product or
oil.
[0146] Hypoallergenic soybean products are discussed above. Such
products include, but are not limited to, isolates, concentrates,
meal, grits, full fat and defatted flours, textured proteins,
textured flours, textured concentrates and textured isolates.
[0147] In an eleventh embodiment, this invention concerns an
isolated nucleic acid fragment comprising a nucleic acid sequence
encoding a soybean Gly m 2 protein. The protein which is encoded
can have an amino acid identity of 95% or greater to the
polypeptide sequence set forth in SEQ ID NO:6 or a functionally
equivalent subfragment thereof.
[0148] In another aspect, this isolated nucleic acid fragment can
have a nucleic acid identity of 70% or greater to the sequence set
forth in SEQ ID NO:5. Also of interest is any plant Gly m 2 protein
having an amino acid identity of 95% or greater to the polypeptide
sequence set forth in SEQ ID NO:4. Chimeric genes comprising such
nucleic acid fragments or the reverse complement thereof operably
to regulatory sequences are also of interest as well as
hypoallergenic soybean plants comprising such chimeric genes, seeds
obtained from such plants, oil obtained from such seeds, and
products made from the hydrogenation, fractionation,
interesterification or hydrolysis of oil obtained from the seeds of
such plants. In still another aspect, this invention concerns a
hypoallergenic soybean product, and any food or any feed
incorporating this soybean product or oil as is discussed
above.
[0149] In a twelfth embodiment, this invention concerns a
recombinant expression construct to lower Gly m IB content of a
soybean which comprises a promoter operably linked to an isolated
Gly m IB nucleic acid fragment corresponding substantially to the
nucleotide sequence set forth in SEQ ID NO:9 or a functionally
equivalent subfragment thereof.
[0150] In a thirteenth embodiment, this invention concerns a
recombinant expression construct to lower Gly m IB content of a
soybean which comprises a promoter operably linked to an isolated
Gly in IB nucleic acid fragment corresponding substantially to the
nucleotide sequence set forth in SEQ ID NO:9 or a functionally
equivalent subfragment thereof.
[0151] In a fourteenth embodiment, this invention concerns a
recombinant expression construct to lower rGLY m3 content of a
soybean wherein which comprises a promoter operably linked to an
isolated rGly m3 nucleic acid fragment corresponding substantially
to the nucleotide sequence set forth in SEQ ID NOs: 11 and 13 or a
functionally equivalent subfragment thereof.
[0152] In a fifteenth embodiment, this invention concerns a
recombinant expression construct to lower Glycinin G1 (A1aB1b)
content of a soybean wherein which comprises a promoter operably
linked to an isolated Glycinin G1 nucleic acid fragment
corresponding substantially to the nucleotide sequence set forth in
SEQ ID NO: 15 or a functionally equivalent subfragment thereof.
[0153] Also of interest are a hypoallergenic soybean plant
comprising in its genome at least one of the claimed recombinant
expression constructs. Also of interest are seeds obtained from
such plants, oil obtained from these seeds and pro ducts made from
the hydrogenation, fractionation, interesterification or hydrolysis
of oil obtained from the seeds of such plants. In still another
aspect, this invention concerns a hypoallergenic soybean product,
and any food or any feed incorporating this soybean product or
oil.
EXAMPLES
[0154] 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. The disclosures
contained within the references used herein are hereby incorporated
by reference.
EXAMPLE 1
Alteration of Gly m 1 Content of Soybean Somatic Embryos
[0155] The ability to change the Gly m I content of soybean embryos
by gene suppression was tested by preparing transgenic soybean
somatic embryos and assaying the isoflavone levels. The entire
insert from Genbank clone J05560 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:7 and SEQ ID NO:8:
2 5'-GAATTCGCGGCCGCATGGGTTTCCTTGTGT-3' [SEQ ID NO:7]
5'-GAATTCGCGGCCGCTCAAAGAGGAGAGTGA-3' [SEQ ID NO:8]
[0156] The resulting fragment is bound by Not I sites in the primer
sequences (underlined above) and contains a 5' leader sequence, the
coding region for Gly m 1, the untranslated 3' region from SEQ ID
NO: and a stretch of 18 A residues at the 3' end. This fragment was
digested with Not I and ligated to Not I-digested and
phosphatase-treated pKS67. The plasmid pKS67 was prepared from
pRB20 (U.S. Pat. No. 5,846,784) by replacing the 800 bp nopaline
synthase 3' untranslated region (Nos 3') with a shorter 285 bp Nos
3' fragment. Both Nos 3' fragments contain the polyadenylation
signal sequence (Depicker A. et al., (1982) J. Mol. Appl. Genet.
1:561-573). Clones were screened for the sense orientation of the
Gly I m insert fragment by digestion with Bam HI. The resulting
plasmid pKS73, shown in FIG. 4, has the beta-conglycinin promoter
operably linked to the fragment encoding Gly I m followed by the
Nos 3'end. Plasmid pKS73 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 pKS73 also contains the .sup.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.
EXAMPLE 2
Transformation of Somatic Soybean Embryo Cultures
[0157] Soybean embryogenic suspension cultures were maintained in
35 ml liquid media (SB55 or SBP6) on a rotary shaker, 150 rpm, at
28.degree. C. with mixed fluorescent and incandescent lights on a
16:8 h day/night schedule. Cultures were subcultured every four
weeks by inoculating approximately 35 mg of tissue into 35 ml of
liquid medium.
3 TABLE 2 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.20 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 MS FeEDTA 100X Stock Na.sub.2EDTA 3.724 FeSO.sub.4
7H.sub.2O 2.784 B5 Vitamin Stock 10 g m-inositol 100 mg nicotinic
acid 100 mg pyridoxine HCl 1 g thiamine SB55 (per Liter, pH 5.7) 10
mL each MS stocks 1 mL 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) 60 g sucrose 0.667 g
asparagine SBP6 same as SB55 except 0.5 mL 2,4-D SB103 (per Liter,
pH 5.7) 1X MS Salts 6% maltose 750 mg MgCl.sub.2 0.2% Gelrite
SB71-1 (per Liter, pH 5.7) 1X B5 salts 1 ml B5 vitamin stock 3%
sucrose 750 mg MgCl.sub.2 0.2% Gelrite
[0158] Soybean embryogenic suspension cultures were transformed
with pTC3 by the method of particle gun bombardment (Kline et al.
(1987) Nature 327:70). A DuPont Biolistic PDS 1000/HE instrument
(helium retrofit) was used for these transformations. To 50 ml of a
60 mg/ml 1 mm gold particle suspension was added (in order); 5
.mu.l DNA(1 .mu.g/.mu.l), 20 .mu.l spermidine (0.1 M), and 50 .mu.l
CaCl.sub.2 (2.5 M). The particle preparation was agitated for 3
min, spun in a microfuge for 10 sec and the supernatant removed.
The DNA-coated particles were then washed once in 400 .mu.l 70%
ethanol and re suspended in 40 .mu.l of anhydrous ethanol. The
DNA/particle suspension was sonicated three times for 1 sec each.
Five .mu.l of the DNA-coated gold particles were then loaded on
each macro carrier disk.
[0159] Approximately 300-400 mg of a four week old suspension
culture was placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue were
normally bombarded. Membrane rupture pressure was set at 1000 psi
and the chamber was evacuated to a vacuum of 28 inches of mercury.
The tissue was placed approximately 3.5 inches away from the
retaining screen and bombarded three times. Following bombardment,
the tissue was placed back into liquid and cultured as described
above.
[0160] 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.
[0161] Three lines of transformed embryogenic clusters (3/1, 6/1,
and 7/1) were removed from liquid culture and placed on a solid
agar media (SB103) containing no hormones or antibiotics. Embryos
were cultured for four weeks at 26.degree. C. with mixed
fluorescent and incandescent lights on a 16:8 h day/night schedule.
During this period, individual embryos were removed from the
clusters and screened for their lack of allergenic proteins by
protein blot analysis (Example 4).
EXAMPLE 3
The Phenotype of Transgenic Soybean Somatic Embryos is Predictive
of Seed Phenotypes from Resultant Regenerated Plants
[0162] Mature somatic soybean embryos are a good model for zygotic
embryos. While in the globular embryo state in liquid culture,
somatic soybean embryos contain very low amounts of triacylglycerol
or storage proteins typical of maturing, zygotic soybean embryos.
At this developmental stage, the ratio of total triacylglyceride to
total polar lipid (phospholipids and glycolipid) is about 1:4, as
is typical of zygotic soybean embryos at the developmental stage
from which the somatic embryo culture was initiated. At the
globular stage as well, the mRNAs for the prominent seed proteins,
.alpha.' subunit of .beta.-conglycinin, kunitz trypsin inhibitor 3,
and seed lectin are essentially absent. Upon transfer to
hormone-free media to allow differentiation to the maturing somatic
embryo state, triacylglycerol becomes the most abundant lipid
class. As well, mRNAs for .alpha.'-subunit of .beta.-conglycinin,
kunitz trypsin inhibitor 3 and seed lectin become very abundant
messages in the total mRNA population. On this basis somatic
soybean embryo system behaves very similarly to maturing zygotic
soybean embryos in vivo, and is therefore a good and rapid model
system for analyzing the phenotypic effects of modifying the
expression of genes in the fatty acid biosynthesis pathway.
[0163] Most importantly, the model system is also predictive of the
fatty acid composition of seeds from plants derived from transgenic
embryos. This is illustrated with two different antisense
constructs in two different types of experiment that were
constructed following the protocols set forth in the PCT
Publication Nos. WO 93/11245 and WO 94/11516. Liquid culture
globular embryos were transformed with a chimeric gene comprising a
soybean microsomal .DELTA..sup.15 desaturase as described in PCT
Publication No. WO 93/11245 which was published on Jun. 10, 1993,
the disclosure of which is hereby incorporated by reference
(experiment 1,) or a soybean microsomal .DELTA..sup.12 desaturase
as described in PCT Publication No. WO 94/11516 which was published
on May 26, 1994, the disclosure of which is hereby incorporated by
reference (experiment 2). Both gene constructs were introduced in
antisense orientation under the control of a seed-specific promoter
(.beta.-conglycinin promoter) and gave rise to mature embryos. The
fatty acid content of mature somatic embryos from lines transformed
with vector only (control) and the vector containing the antisense
chimeric genes as well as of seeds of plants regenerated from them
was determined.
[0164] In experiment 1, one set of embryos from each line was
analyzed for fatty acid content and another set of embryos from
that same line was regenerated into plants.
[0165] In experiment 2, different lines, containing the same
antisense construct, were used for fatty acid analysis in somatic
embryos and for regeneration into plants. In experiment 1, in all
cases where a reduced 18:3 content was seen in a transgenic embryo
line, compared with the control, a reduced 18:3 content was also
observed in segregating seeds of plants derived from that line,
when compared with the control seed (Table 3).
[0166] In experiment 2, about 55% of the transformed embryo lines
showed an increased 18:1 content when compared with control lines
(Table 4). Soybean seeds, of plants regenerated from different
somatic embryo lines containing the same antisense construct, had a
similar frequency (53%) of high oleate transformants as the somatic
embryos (Table 4). On occasion, an embryo line may be chimeric.
That is, 10-70% of the embroys in a line may not contain the
transgene. The remaining embryos which do contain the transgene,
have been found in all cases to be clonal. In such a case, plants
with both wild type and transgenic phenotypes may be regenerated
from a single, transgenic line, even if most of the embryos
analyzed from that line had a transgenic phenotype. An example of
this is shown in Table 5, in which, of 5 plants regenerated from a
single embryo line, 3 have a high oleic phenotype and two were wild
type. In most cases, all the plants regenerated from a single
transgenic line will have seeds containing the transgene. Thus, it
was concluded that an altered fatty acid phenotype observed in a
transgenic, mature somatic embryo line is predictive of an altered
fatty acid composition of seeds of plants derived from that
line.
4TABLE 3 Percent 18:3 Content Of Embryos and Seeds of Control and
.DELTA..sup.15 Antisense Construct Transgenic Soybean Lines Embryo
Average Seed Average Transformant Line (SD, n = 10) (SD, n = 10)
Control 12.1 (2.6) 8.9 (0.8) .DELTA..sup.15 antisense, line 1 5.6
(1.2) 4.3 (1.6) .DELTA..sup.15 antisense, line 2 8.9 (2.2) 2.5
(1.8) .DELTA..sup.15 antisense, line 3 7.3 (1.1) 4.9 (1.9)
.DELTA..sup.15 antisense, line 4 7.0 (1.9) 2.4 (1.7) .DELTA..sup.15
antisense, line 5 8.5 (1.9) 4.5 (2.2) .DELTA..sup.15 antisense,
line 6 7.6 (1.6) 4.6 (1.6) *[Seeds which were segregating with
wild-type phenotype and without a copy of the transgene are not
included in these averages]
[0167]
5TABLE 4 Oleate Levels in Somatic Embryos and Seeds of Regenerated
Soybeans Transformed With, or Without, .DELTA..sup.12 Desaturase
Antisense Construct # of # of Lines with Average* Vector Lines High
18:1 % 18:1 Somatic embryos: Control 19 0 12.0 .DELTA..sup.12
antisense 20 11 35.3 Seeds of regenerated plants: Control 6 0 18.2
.DELTA..sup.12 antisense 17 9 44.4 *average 18:1 of transgenics is
the average of all embryos or seeds transformed with the
.DELTA..sup.12 antisense construct in which at least one embryo or
seed from that line had an 18:1 content greater than 2 standard
deviations from the control value (12.0 in embryos, 18.2 in seeds).
The control average is the average of embryos or seeds which do not
contain # any transgenic DNA but have been treated in an identical
manner to the transgenics.
[0168]
6TABLE 5 Analysis of Seeds From Five Independent Plants Segregating
From Plant Line 4 Plant # Average seed 18:1% Highest seed 18:1% 1
18.0 26.3 2 33.6 72.1 7 13.6 21.2 9 32.9 57.3 11 24.5 41.7
[0169] Mean of 15-20 seeds from 5 different plants regenerated from
a single embryo line. Only plants # 2, 9 and 11 have seeds with a
high 18:1 phenotype.
EXAMPLE 4
Assay for Gly 1 m Content of Transformed Embryos
[0170] Antibodies to Gly I m were those described in Herman, E. M.,
Melroy, D. L., Buckhout T. J. (1990) Plant Physiol 94:341-349 the
disclosure of which is hereby incorporated by reference.
[0171] Transgenic embryos described in Example 2 were frozen in
liquid nitrogen ground in a mortar with sample buffer (0.125 M
Tris-HCl, Ph 6.8, containing 0.4% SDS, 20% glycerol, 4% SDS, 0.2%
2-mercaptoethanol) at a ratio of 1:5 (w/v). The solubilized
proteins were heated to 70.degree. C. and run on a standard SDS
polyacrylamide gel (Sambrook et al. (1989) "Molecular Cloning" Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The
resolved proteins were transferred to a nitrocellulose membrane by
standard electrotransfer. The membrane was treated with 3% gelatin
solution to block non-specific binding. The filter was then
incubated for 90 min with a 1:5000 dilution of antibody-containing
clarified ascites fluid (described in the Herman reference cited
above) in TBS (Tris-HCl, 2.42 g/l, pH 7.5, NaCl 29.2 g/l) with 1%
gelatin. The membrane was washed with TBS, then incubated with a
1:5000 dilution of anti-mouse IgG-alkaline phosphatase (Sigma). The
membrane was washed with TBS, and finally visualized with 1,2
dioexetane-phosphate luminescent detection as described in Ausubel
et al (1999) Current Protocols in Molecular Biology, Vol. 2. pp
10.8.13 to 10.8.16.
[0172] The results of an assay are shown in FIG. 2. Three
independently transformed soybean embryos (3/1, 6/1, and 7/1),
described in Example 2 above, were tested for the presence of the
Gly 1 m protein. The left panel shows the total protein loaded on
each lane and the right panel shows the results of the antibody
binding. The 7/1 embryo has no detectable Gly 1 m protein, unlike
the other two embryos. A control for the Gly 1 m protein is shown
on the right lane.
EXAMPLE 5
Composition of cDNA Libraries; Isolation and Sequencing of cDNA
Clones
[0173] cDNA libraries representing mRNAs from various soybean
tissues were prepared. The characteristics of the libraries are
described below.
7TABLE 6 cDNA Libraries from Soybean Library Tissue Clone se6
Soybean Embryo, 26 Days After Flowering se6.pk0050.c3 sls1c Soybean
Infected With Sclerotinia sls1c.pk027.a11 sclerotiorum mycelium
[0174] cDNA libraries may be prepared by any one of many methods
available. For example, the cDNAs may be introduced into plasmid
vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR
vectors according to the manufacturer's protocol (Stratagene
Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries
are converted into plasmid libraries according to the protocol
provided by Stratagene. Upon conversion, cDNA inserts will be
contained in the plasmid vector pBluescript. In addition, the cDNAs
may be introduced directly into precut Bluescript II SK(+) vectors
(Stratagene) using T4 DNA ligase (New England Biolabs), followed by
transfection into DH10B cells according to the manufacturer's
protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid
vectors, plasmid DNAs are prepared from randomly picked bacterial
colonies containing recombinant pBluescript plasmids, or the insert
cDNA sequences are amplified via polymerase chain reaction using
primers specific for vector sequences flanking the inserted cDNA
sequences. Amplified insert DNAs or plasmid DNAs are sequenced in
dye-primer sequencing reactions to generate partial cDNA sequences
(expressed sequence tags or "ESTs"; see Adams et al., (1991)
Science 252:1651-1656). The resulting ESTs are analyzed using a
Perkin Elmer Model 377 fluorescent sequencer.
EXAMPLE 6
[0175] Identification of cDNA Clones cDNA clones encoding soybean
allergens were identified by conducting BLAST (Basic Local
Alignment Search Tool; Altschul 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 1 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 and States (1993) Nat. Genet. 3:266-272)
provided by the NCBI. For convenience, the P-value (probability) of
observing a match of a cDNA sequence to a sequence contained in the
searched databases merely by chance as calculated by BLAST are
reported herein as "pLog" values, which represent the negative of
the logarithm of the reported P-value. Accordingly, the greater the
pLog value, the greater the likelihood that the cDNA sequence and
the BLAST "hit" represent homologous proteins.
EXAMPLE 7
Characterization of cDNA Clones Encoding Gly m Bd 28K and Gly
m2
[0176] The BLASTX search using the EST sequences from clones listed
in Table 6 revealed similarity of the polypeptides encoded by the
cDNAs to soybean allergens from Arabidopsis thaliana for Gly mBd
28K (se6.pk0050.c3, SEQ ID NO:4) and cowpea [Vigna unguiculata] for
Gly m2 (s1s1c.pk027.a11, SEQ ID NO:6) (NCBI Accession Nos. gi
4510397 and gi 112671, respectively). Shown in Table 7 are the
BLAST results for the cDNA sequences:
8TABLE 7 BLAST Results for Sequences Encoding Polypeptides
Homologous to Soybean Allergens BLAST pLog Score BLAST pLog Score
Clone gi 4510397 gi 112671 se6.pk0050.c3 115.00 -- sls1c.pk027.a11
-- 38.70
[0177] The data in Table 8 represents a calculation of the percent
identity of the amino acid sequences set forth in SEQ ID NOs:4 and
6 and the Arabidopsis and cowpea proteins (respectively).
9TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the
Nucleotide Sequences of cDNA Clones Encoding Polypeptides
Homologous to Soybean Allergens Percent Identity to Percent
Identity to SEQ ID NO. gi 4510397 gi 112671 4 46.7 -- 6 --
93.3%
[0178] Sequence alignments and percent identity calculations were
performed using the Megalign program of the LASERGENE
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. Sequence alignments and BLAST scores and probabilities
indicate that the nucleic acid fragments comprising the instant
cDNA clones encode a substantial portion of a soybean allergen.
EXAMPLE 8
Coordinated Loss of Both .alpha.- and .alpha.'-Subunits of
Beta-Conglycinin in Co-Suppressed Transgenic Plants
[0179] It is believed that the use of recombinant expression
constructs containing the promoter for the .alpha.'-subunit of beta
conglycinin can result in the co-suppression of the gene encoding
polypeptides for both the .alpha.- and .alpha.'-subunits of
beta-conglycinin (PCT Publication No. WO 97/47731, as cited above).
The construct pKS68 (FIG. 5) carrying the delta-12 desaturase
(Fad2) gene coding region (described in detail by Okuley, J. et al.
(1994) Plant Cell 6:147-158 and in the PCT Publication WO 94/11516,
cited above) is under the control of the same beta-conglycinin
promoter used in Example 1. PKS68 was used to generate soybean
lines co-suppressing the Fad2 locus, according to protocols
outlined in Example 2. Fad2, and its gene product, are responsible
for the synthesis of the polyunsaturated fatty acids found in
soybean oil (see Okuley, and WO 94/11516, cited above). Further
descriptions of Fad2 and its use in altering soybean oil
composition can be found in PCT Publication No. WO 97/40698,
published Nov. 11, 1997, the disclosure of which is hereby
incorporated by reference.
[0180] Protein samples were prepared according to standard methods
from seeds of transformed plants exhibiting a co-suppression
phenotype with respect to Fad2 (i.e., transgenic plants with
altered soybean oil compositions). Protein sample preparation and
SDS polyacrylamide gel protocols were the same as those used in WO
97/47731, cited above. A protein gel of the seed samples is shown
in FIG. 3. Seed protein profiles having reduced levels of .alpha.-
or .alpha.'-subunit polypeptides (lanes 3-7, and 9) always
exhibited a coordinated loss. The loss of the .alpha.'-subunit was
not unexpected due to the use of the promoter for the
.alpha.'-subunit of beta-congylcinin. However, this promoter also
appeared to suppress the accumulation of the .alpha.-subunit
polypeptide as efficiently as the .alpha.'-subunit. Not all of the
altered oil lines showed reduced levels of .alpha. or .alpha.'
subunit (lane 8) even though all contain the beta-conglycinin
promoter. Lanes 1 and 2 are a positive and negative control
(respectively). Therefore, it appears that the use of the promoter
for the .alpha.'-subunit of beta-conglycinin, when used in
recombinant expression constructs, is sufficient to coordinately
suppress both .alpha.- and .alpha.'-subunits of beta-conglycinin in
soybean plants.
Sequence CWU 1
1
16 1 1156 DNA chimeric construct 1 gcggccgcat gggtttcctt gtgttgcttc
ttttctccct cttaggtctc tcttctagtt 60 ccagcatatc aactcatcgt
tccatattgg accttgacct aaccaagttt accacacaga 120 aacaggtgtc
ttcactgttc caactatgga agagtgagca tggacgtgtc taccataacc 180
acgaagaaga ggcaaagaga cttgagattt tcaagaataa ctcgaactat atcagggaca
240 tgaatgcaaa cagaaaatca ccccattctc atcgtttagg attgaacaag
tttgctgaca 300 tcactcctca agagttcagc aaaaagtact tgcaagctcc
caaggatgtg tcgcagcaaa 360 tcaaaatggc caacaagaaa atgaagaagg
aacaatattc ttgtgaccat ccacctgcat 420 catgggattg gaggaaaaaa
ggtgtcatca cccaagtaaa gtaccaaggg ggctgtggaa 480 ggggttgggc
gttttctgcc acgggagcca tagaagcagc acatgcaata gcaacaggag 540
accttgttag cctttctgaa caagaactcg tagactgtgt ggaagaaagc gaaggttctt
600 acaatggatg gcagtatcaa tcgttcgaat gggttttaga acatggtggc
attgccactg 660 atgatgatta tccttacaga gctaaagagg gtagatgcaa
agccaataag atacaagaca 720 aggttacaat tgacggatat gaaactctaa
taatgtcaga tgagagtaca gaatcagaga 780 cagagcaagc gttcttaagc
gccatccttg agcaaccaat tagtgtctca attgatgcaa 840 aagattttca
tttatacacc gggggaattt atgatggaga aaactgtaca agtccgtatg 900
ggattaatca ctttgtttta cttgtgggtt atggttcagc ggatggtgta gattactgga
960 tagcgaaaaa ttcatgggga gaagattggg gagaagatgg ttacatttgg
atccaaagaa 1020 acacgggtaa tttattagga gtgtgtggga tgaattattt
cgcttcatac ccaaccaaag 1080 aggaatcaga aacactggtg tctgctcgcg
ttaaaggtca tcgaagagtt gatcactctc 1140 ctctttgagc ggccgc 1156 2 2970
DNA chimeric construct 2 aagcttgatc catgcccttc atttgccgct
attaattaat ttggtaacag tagtccgtac 60 taatcagtta cttatccttc
ctccatcata attaatcttg gtagtctcga atgccacaac 120 actgactagt
ctcttggatc ataagaaaaa gccaaggaac aaaagaagac aaaacacaat 180
gagagtatcc tttgcatagc aatgtctaag ttcataaaat tcaaacaaaa acgcaatcac
240 acacagtgga catcacttat ccactagctg aatcaggatc gccgcgtcaa
gaaaaaaaaa 300 ctggacccca aaagccatgc acaacaacac gtactcacaa
aggtgtcaat cgagcagccc 360 aaaacattca ccaactcaac ccatcatgag
ccctcacatt tgttgtttct aacccaacct 420 caaactcgta ttctcttccg
ccacctcatt tttgtttatt tcaacacccg tcaaactgca 480 tgccaccccg
tggccaaatg tccatgcatg ttaacaagac ctatgactat aaatatctgc 540
aatctcggcc caggttttca tcatcaagaa ccagttcaat atcctagtac accgtattaa
600 agaatttaag atatactaac agcggccgca tgggtttcct tgtgttgctt
cttttctccc 660 tcttaggtct ctcttctagt tccagcatat caactcatcg
ttccatattg gaccttgacc 720 taaccaagtt taccacacag aaacaggtgt
cttcactgtt ccaactatgg aagagtgagc 780 atggacgtgt ctaccataac
cacgaagaag aggcaaagag acttgagatt ttcaagaata 840 actcgaacta
tatcagggac atgaatgcaa acagaaaatc accccattct catcgtttag 900
gattgaacaa gtttgctgac atcactcctc aagagttcag caaaaagtac ttgcaagctc
960 ccaaggatgt gtcgcagcaa atcaaaatgg ccaacaagaa aatgaagaag
gaacaatatt 1020 cttgtgacca tccacctgca tcatgggatt ggaggaaaaa
aggtgtcatc acccaagtaa 1080 agtaccaagg gggctgtgga aggggttggg
cgttttctgc cacgggagcc atagaagcag 1140 cacatgcaat agcaacagga
gaccttgtta gcctttctga acaagaactc gtagactgtg 1200 tggaagaaag
cgaaggttct tacaatggat ggcagtatca atcgttcgaa tgggttttag 1260
aacatggtgg cattgccact gatgatgatt atccttacag agctaaagag ggtagatgca
1320 aagccaataa gatacaagac aaggttacaa ttgacggata tgaaactcta
ataatgtcag 1380 atgagagtac agaatcagag acagagcaag cgttcttaag
cgccatcctt gagcaaccaa 1440 ttagtgtctc aattgatgca aaagattttc
atttatacac cgggggaatt tatgatggag 1500 aaaactgtac aagtccgtat
gggattaatc actttgtttt acttgtgggt tatggttcag 1560 cggatggtgt
agattactgg atagcgaaaa attcatgggg agaagattgg ggagaagatg 1620
gttacatttg gatccaaaga aacacgggta atttattagg agtgtgtggg atgaattatt
1680 tcgcttcata cccaaccaaa gaggaatcag aaacactggt gtctgctcgc
gttaaaggtc 1740 atcgaagagt tgatcactct cctctttgag cggccgctac
atggccacgt gcatgaagta 1800 tgaactaaaa tgcatgtagg tgtaagagct
catggagagc atggaatatt gtatccgacc 1860 atgtaacagt ataataactg
agctccatct cacttcttct atgaataaac aaaggatgtt 1920 atgatatatt
aacactctat ctatgcacct tattgttcta tgataaattt cctcttatta 1980
ttataaatca tctgaatcgt gacggcttat ggaatgcttc aaatagtaca aaaacaaatg
2040 tgtactataa gactttctaa acaattctaa ctttagcatt gtgaacgaga
cataagtgtt 2100 aagaagacat aacaattata atggaagaag tttgtctcca
tttatatatt atatattacc 2160 cacttatgta ttatattagg atgttaagga
gacataacaa ttataaagag agaagtttgt 2220 atccatttat atattatata
ctacccattt atatattata cttatccact tatttaatgt 2280 ctttataagg
tttgatccat gatatttcta atattttagt tgatatgtat atgaaagggt 2340
actatttgaa ctctcttact ctgtataaag gttggatcat ccttaaagtg ggtctattta
2400 attttattgc ttcttacaga taaaaaaaaa attatgagtt ggtttgataa
aatattgaag 2460 gatttaaaat aataataaat aataaataac atataatata
tgtatataaa tttattataa 2520 tataacattt atctataaaa aagtaaatat
tgtcataaat ctatacaatc gtttagcctt 2580 gctggacgac tctcaattat
ttaaacgaga gtaaacatat ttgacttttt ggttatttaa 2640 caaattatta
tttaacacta tatgaaattt ttttttttta tcagcaaaga aataaaatta 2700
aattaagaag gacaatggtg tgtcccaatc cttatacaac caacttccac aagaaagtca
2760 agtcagagac aacaaaaaaa caagcaaagg aaatttttta atttgagttg
tcttgtttgc 2820 tgcataattt atgcagtaaa acactacaca taaccctttt
agcagtagag caatggttga 2880 ccgtgtgctt agcttctttt attttatttt
tttatcagca aagaataaat aaaataaaat 2940 gagacacttc agggatgttt
caacaagctt 2970 3 1600 DNA Glycine max 3 ggggaaacaa aactaccctt
ttgcttttgc tctttgttct ttgtcatgga gtggccacaa 60 caacaatggc
cttccgtgat gatgagggtg gtgataaaaa gtcaccaaaa agtttgtttt 120
tgatgagcaa ctccacgagg gttttcaaga ctgatgcagg ggaaatgcgt gtgctgaaaa
180 gccatggtgg taggatattt tataggcaca tgcacattgg cttcatctct
atggaaccaa 240 agtccttgtt tgttcctcag tacctcgact ccaatctcat
catattcatc cgtagagggg 300 aagcaaagct gggattcata tatgatgatg
aactagcgga aaggagattg aagacagggg 360 acttgtacat gattccatct
ggttcagcat tctatttggt gaacatagga gaaggtcaga 420 gacttcacgt
tatctgcagc attgacccct ctacaagctt gggattagag accttccagt 480
ccttctatat tgggggagga gccaattcgc actcggtgct ttctggattc gaacctgcca
540 tccttgaaac tgcatttaat gaatcaagaa cggtggtaga ggaaatcttc
tccaaggaac 600 tagatgggcc aattatgttc gtggatgatt ctcatgcacc
tagcttatgg actaaattcc 660 ttcaactgaa gaaggatgac aaagagcaac
agctgaagaa aatgatgcaa gaccaagagg 720 aggatgagga ggagaagcaa
acaagtaggt catggaggaa gctcttggaa accgtatttg 780 ggaaggtgaa
tgagaagata gagaacaaag acactgctgg ttcccctgcc tcttacaacc 840
tctacgatga caaaaaagcc gatttcaaaa acgcttatgg ttggagcaag gcactgcatg
900 gaggcgagta tcctccactc agcgaaccgg atattggagt tttacttgtc
aaactctcag 960 cgggatccat gttggcacct catgtgaatc caatatcaga
tgagtatacc atagtgctga 1020 gtggttatgg tgaactgcat atagggtatc
caaacggaag caaagcaatg aaaactaaaa 1080 tcaaacaagg ggacgtgttt
gttgtgccaa gatacttccc cttctgtcaa gtagcatcaa 1140 gggatggacc
cttagagttc tttggcttct ccacttctgc aaggaagaac aagccacagt 1200
ttctggctgg tgctgcgtcc cttctaagga ccttgatggg gccggagctt tcggcggcgt
1260 tcggagtgag cgaggacacg ttgcggcgcg ctgttgatgc tcagcatgag
gctgtgatac 1320 tgccatcagc atgggctgca ccaccggaaa atgcagggaa
gctgaagatg gaagaagagc 1380 caaatgctat tagaagcttt gccaatgatg
tggttatgga tgttttttaa tttgaacact 1440 tgatttggaa taggggttat
ttggtagtgc tagtgcctag tggaattctg tgttgagttt 1500 tttgttcttt
atatttagtt gagatgtgtg ttgtgttctt gagttgtgaa taaaaatcta 1560
ctttctttgt gcarraaaaa aaaaaaaaaa aaaaaaaaaa 1600 4 454 PRT Glycine
max 4 Met ala Phe Arg Asp Asp Glu Gly Gly Asp Lys Lys Ser Pro Lys
Ser 1 5 10 15 Leu Phe Leu Met Ser Asn Ser Thr Arg Val Phe Lys Thr
Asp Ala Gly 20 25 30 Glu Met Arg Val Leu Lys Ser His Gly Gly Arg
Ile Phe Tyr Arg His 35 40 45 Met His Ile Gly Phe Ile Ser Met Glu
Pro Lys Ser Leu Phe Val Pro 50 55 60 Gln Tyr Leu Asp Ser Asn Leu
Ile Ile Phe Ile Arg Arg Gly Glu Ala 65 70 75 80 Lys Leu Gly Phe Ile
Tyr Asp Asp Glu Leu Ala Glu Arg Arg Leu Lys 85 90 95 Thr Gly Asp
Leu Tyr Met Ile Pro Ser Gly Ser Ala Phe Tyr Leu Val 100 105 110 Asn
Ile Gly Glu Gly Gln Arg Leu His Val Ile Cys Ser Ile Asp Pro 115 120
125 Ser Thr Ser Leu Gly Leu Glu Thr Phe Gln Ser Phe Tyr Ile Gly Gly
130 135 140 Gly Ala Asn Ser His Ser Val Leu Ser Gly Phe Glu Pro Ala
Ile Leu 145 150 155 160 Glu Thr Ala Phe Asn Glu Ser Arg Thr Val Val
Glu Glu Ile Phe Ser 165 170 175 Lys Glu Leu Asp Gly Pro Ile Met Phe
Val Asp Asp Ser His Ala Pro 180 185 190 Ser Leu Trp Thr Lys Phe Leu
Gln Leu Lys Lys Asp Asp Lys Glu Gln 195 200 205 Gln Leu Lys Lys Met
Met Gln Asp Gln Glu Glu Asp Glu Glu Glu Lys 210 215 220 Gln Thr Ser
Arg Ser Trp Arg Lys Leu Leu Glu Thr Val Phe Gly Lys 225 230 235 240
Val Asn Glu Lys Ile Glu Asn Lys Asp Thr Ala Gly Ser Pro Ala Ser 245
250 255 Tyr Asn Leu Tyr Asp Asp Lys Lys Ala Asp Phe Lys Asn Ala Tyr
Gly 260 265 270 Trp Ser Lys Ala Leu His Gly Gly Glu Tyr Pro Pro Leu
Ser Glu Pro 275 280 285 Asp Ile Gly Val Leu Leu Val Lys Leu Ser Ala
Gly Ser Met Leu Ala 290 295 300 Pro His Val Asn Pro Ile Ser Asp Glu
Tyr Thr Ile Val Leu Ser Gly 305 310 315 320 Tyr Gly Glu Leu His Ile
Gly Tyr Pro Asn Gly Ser Lys Ala Met Lys 325 330 335 Thr Lys Ile Lys
Gln Gly Asp Val Phe Val Val Pro Arg Tyr Phe Pro 340 345 350 Phe Cys
Gln Val Ala Ser Arg Asp Gly Pro Leu Glu Phe Phe Gly Phe 355 360 365
Ser Thr Ser Ala Arg Lys Asn Lys Pro Gln Phe Leu Ala Gly Ala Ala 370
375 380 Ser Leu Leu Arg Thr Leu Met Gly Pro Glu Leu Ser Ala Ala Phe
Gly 385 390 395 400 Val Ser Glu Asp Thr Leu Arg Arg Ala Val Asp Ala
Gln His Glu Ala 405 410 415 Val Ile Leu Pro Ser Ala Trp Ala Ala Pro
Pro Glu Asn Ala Gly Lys 420 425 430 Leu Lys Met Glu Glu Glu Pro Asn
Ala Ile Arg Ser Phe Ala Asn Asp 435 440 445 Val Val Met Asp Val Phe
450 5 494 DNA Glycine max unsure (9) n = A, C, G, or T 5 acacagctng
cacatattac atacacgtga atcactaatt aagccatgga gaagaaatca 60
atagctgggt tgtgcttcct cttccttgtt ctctttgttg ctcaagaagt tgtggtgcaa
120 actgaggcaa agacttgcga gaacctggct gatacataca ggggtccatg
cttcaccact 180 ggcagctgcg atgatcactg caagaacaaa gagcacttgc
tcagaggcag atgcagggac 240 gattttcgct gttggtgcac caaaaactgt
taaatggatc cattcactcc aacgtgaaga 300 agatgcatgc agcgctattt
tataaaaaat acaactacta tatactatat ataataagac 360 tgggcgctgc
atcaatgacc ctatgtanta tnntatatat tattaccgat gtcaagaact 420
atagatgcat gtactgtgca taacggctga gttatgtccn tangttanga ataaaaataa
480 agtgctgttg ttgc 494 6 75 PRT Glycine max 6 Met Glu Lys Lys Ser
Ile Ala Gly Leu Cys Phe Leu Phe Leu Val Leu 1 5 10 15 Phe Val Ala
Gln Glu Val Val Val Gln Thr Glu Ala Lys Thr Cys Glu 20 25 30 Asn
Leu Ala Asp Thr Tyr Arg Gly Pro Cys Phe Thr Thr Gly Ser Cys 35 40
45 Asp Asp His Cys Lys Asn Lys Glu His Leu Leu Arg Gly Arg Cys Arg
50 55 60 Asp Asp Phe Arg Cys Trp Cys Thr Lys Asn Cys 65 70 75 7 30
DNA Artificial Sequence Description of Artificial Sequence P34 gene
primer 7 gaattcgcgg ccgcatgggt ttccttgtgt 30 8 30 DNA Glycine max
Description of Artificial Sequence P34 gene primer 8 gaattcgcgg
ccgctcaaag aggagagtga 30 9 701 DNA Glycine max 9 ttaagctttc
aagagacaaa ctgctttgaa aaatgggatc caaggttgtt gcatccgttg 60
cccttctcct ctccatcaac attcttttca tttccatggt tagctccagc agccactacg
120 atccacagcc ccaaccttct cacgtcactg ctcttattac acgacctagt
tgtccggatc 180 tgagtatttg cctcaatatt ttaggcgggt ctctaggaac
cgtggatgat tgttgtgccc 240 tcatcggtgg tcttggtgac attgaagcca
ttgtgtgcct ttgcatccaa ctcagggccc 300 tcggaatatt aaaccttaac
cgtaatttgc agttaatatt aaactcctgt ggacgaagct 360 acccgtcaaa
cgccacttgc ccccgaacct aagaacagaa tatgtatggc actaattacc 420
atattacttc gtatcatggt gtttgtttgt ttgtctgtgt ttaaagttaa ggatgttata
480 cccttcgtgc ctgctacata tatatagtgg gcactataat attaccaata
aattaacgtc 540 catatataag aataataata aataaataaa tatttctata
caaataaagg ttacgtaatg 600 ttgttgttct cgtggatggg gatcttatct
tcctcctcgc tatctttgtt tatcgtattt 660 cagtgaaagt tgttcaataa
aagtcctttg ttcaacaagt g 701 10 119 PRT Glycine max 10 Met Gly Ser
Lys Val Val Ala Ser Val Ala Leu Leu Leu Ser Ile Asn 1 5 10 15 Ile
Leu Phe Ile Ser Met Val Ser Ser Ser Ser His Tyr Asp Pro Gln 20 25
30 Pro Gln Pro Ser His Val Thr Ala Leu Ile Thr Arg Pro Ser Cys Pro
35 40 45 Asp Leu Ser Ile Cys Leu Asn Ile Leu Gly Gly Ser Leu Gly
Thr Val 50 55 60 Asp Asp Cys Cys Ala Leu Ile Gly Gly Leu Gly Asp
Ile Glu Ala Ile 65 70 75 80 Val Cys Leu Cys Ile Gln Leu Arg Ala Leu
Gly Ile Leu Asn Leu Asn 85 90 95 Arg Asn Leu Gln Leu Ile Leu Asn
Ser Cys Gly Arg Ser Tyr Pro Ser 100 105 110 Asn Ala Thr Cys Pro Arg
Thr 115 11 396 DNA Glycine max 11 atgtcgtggc aagcttatgt cgacgatcac
cttctgtgtg gcatcgaagg taaccacctc 60 actcacgctg ctatcatcgg
ccaagacggc agcgtttggc ttcagagtac cgacttccct 120 cagttcaaac
ctgaggagat aactgccatc atgaatgact ttaatgagcc tggatcactt 180
gctccaactg gattgtatct cggtggcacc aaatatatgg tcatccaggg tgaacccggt
240 gctgtcattc gagggaagaa gggtcctggt ggtgttactg tgaagaagac
cggtgcggcc 300 ttgatcattg gcatttatga tgaaccaatg actccaggtc
aatgcaacat ggtagttgaa 360 aggcttggtg attacctcat agatcaaggc tactga
396 12 131 PRT Glycine max 12 Met Ser Trp Gln Ala Tyr Val Asp Asp
His Leu Leu Cys Gly Ile Glu 1 5 10 15 Gly Asn His Leu Thr His Ala
Ala Ile Ile Gly Gln Asp Gly Ser Val 20 25 30 Trp Leu Gln Ser Thr
Asp Phe Pro Gln Phe Lys Pro Glu Glu Ile Thr 35 40 45 Ala Ile Met
Asn Asp Phe Asn Glu Pro Gly Ser Leu Ala Pro Thr Gly 50 55 60 Leu
Tyr Leu Gly Gly Thr Lys Tyr Met Val Ile Gln Gly Glu Pro Gly 65 70
75 80 Ala Val Ile Arg Gly Lys Lys Gly Pro Gly Gly Val Thr Val Lys
Lys 85 90 95 Thr Gly Ala Ala Leu Ile Ile Gly Ile Tyr Asp Glu Pro
Met Thr Pro 100 105 110 Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly
Asp Tyr Leu Ile Asp 115 120 125 Gln Gly Tyr 130 13 396 DNA Glycine
max 13 atgtcctggc aggcgtatgt cgacgatcac cttctgtgtg acatcgaagg
taaccacctc 60 actcacgctg ctatcatcgg ccaagacggc agcgtttggg
ctcagagtac cgacttccct 120 cagttcaaac ctgaggagat aactgccatc
atgaatgact ttaatgagcc tggatcactt 180 gctccaactg gattgtatct
cggtggcacc aaatatatgg tcatccaggg tgaacccggt 240 gctgtcattc
gagggaagaa gggtcctggt ggtgttactg tgaagaagac cggtgcggcc 300
ttgatcattg gcatttatga tgaaccaatg actccaggtc aatgcaacat ggtagttgaa
360 aggcctggtg attacctcat cgaccagggc tactga 396 14 131 PRT Glycine
max 14 Met Ser Trp Gln Ala Tyr Val Asp Asp His Leu Leu Cys Asp Ile
Glu 1 5 10 15 Gly Asn His Leu Thr His Ala Ala Ile Ile Gly Gln Asp
Gly Ser Val 20 25 30 Trp Ala Gln Ser Thr Asp Phe Pro Gln Phe Lys
Pro Glu Glu Ile Thr 35 40 45 Ala Ile Met Asn Asp Phe Asn Glu Pro
Gly Ser Leu Ala Pro Thr Gly 50 55 60 Leu Tyr Leu Gly Gly Thr Lys
Tyr Met Val Ile Gln Gly Glu Pro Gly 65 70 75 80 Ala Val Ile Arg Gly
Lys Lys Gly Pro Gly Gly Val Thr Val Lys Lys 85 90 95 Thr Gly Ala
Ala Leu Ile Ile Gly Ile Tyr Asp Glu Pro Met Thr Pro 100 105 110 Gly
Gln Cys Asn Met Val Val Glu Arg Pro Gly Asp Tyr Leu Ile Asp 115 120
125 Gln Gly Tyr 130 15 1746 DNA Glycine max 15 aaaacaactc
aaacattctc tccattggtc cttaaacact catcagtcat caccatggcc 60
aagctagttt tttccctttg ttttctgctt ttcagtggct gctgcttcgc tttcagttcc
120 agagagcagc ctcagcaaaa cgagtgccag atccaaaaac tcaatgccct
caaaccgggt 180 aaccgtatag agtcagaagg agggctcatt gagacatgga
accctaacaa caagccattc 240 cagtgtgccg gtgttgccct ctctcgctgc
accctcaacc gcaacgccct tcgtagacct 300 tcctacacca acggtcccca
agaaatctac atccaacaag gtaagggtat ttttggcatg 360 atatacccgg
gttgttctag cacatttgaa gagcctcaac aacctcaaca aagaggacaa 420
agcagcagac cacaagaccg tcaccagaag atctataact ccagagaggg tgatttgatc
480 gcagtgccta ctggtgttgc atggtggatg tacaacaatg aagacactcc
tgttgttgcc 540 gtttctatta ttgacaccaa cagcttggag aaccagctcg
accagatgcc taggagattc 600 tatcttgctg ggaaccaaga gcaagagttt
ctaaaatatc agcaagagca aggaggtcat 660 caaagccaga aaggaaagca
tcagcaagaa gaagaaaacg aaggaggcag catattgagt 720 ggcttcaccc
tggaattctt ggaacatgca ttcagcgtgg acaagcagat agcgaaaaac 780
ctacaaggag agaacgaagg ggaagacaag ggagccattg tgacagtgaa aggaggtctg
840 agcgtgataa aaccacccac ggacgagcag caacaaagac cccaggaaga
ggaagaagaa 900 gaagaggatg agaagccaca gtgcaagggt aaagacaaac
actgccaacg cccccgagga 960 agccaaagca
aaagcagaag aaatggcatt gacgagacca tatgcaccat gagacttcgc 1020
cacaacattg gccagacttc atcacctgac atctacaacc ctcaagccgg tagcgtcaca
1080 accgccacca gccttgactt cccagccctc tcgtggctca gactcagtgc
tggatttggg 1140 tctctccgca agaatgcaat gttcgtgcca cactacaacc
tgaacgcgaa cagcataata 1200 tacgcattga atggacgggc attgatacaa
gtggtgaatt gcaacggtga gagagtgttt 1260 gatggagagc tgcaagaggg
acgggtgctg atcgtgccac aaaactttgt ggtggctgca 1320 agatcacaga
gtgacaactt cgagtatgtg tcattcaaga ccaatgatac acccatgatc 1380
ggcactcttg caggggcaaa ctcattgttg aacgcattac cagaggaagt gattcagcac
1440 actttcaacc taaaaagcca gcaggccagg cagataaaga acaacaaccc
tttcaagttc 1500 ctggttccac ctcaggagtc tcagaagaga gctgtggctt
agagcccttt ttgtatgtgc 1560 taccccactt ttgtcttttt ggcaatagtg
ctagcaacca ataaataata ataataataa 1620 tgaataagaa aacaaaggct
ttagcttgcc ttttgttcac tgtaaaataa taatgtaagt 1680 actctctata
atgagtcacg aaacttttgc gggaataaaa ggagaaattc caatgagttt 1740 tctgtt
1746 16 495 PRT Glycine max 16 Met Ala Lys Leu Val Phe Ser Leu Cys
Phe Leu Leu Phe Ser Gly Cys 1 5 10 15 Cys Phe Ala Phe Ser Ser Arg
Glu Gln Pro Gln Gln Asn Glu Cys Gln 20 25 30 Ile Gln Lys Leu Asn
Ala Leu Lys Pro Gly Asn Arg Ile Glu Ser Glu 35 40 45 Gly Gly Leu
Ile Glu Thr Trp Asn Pro Asn Asn Lys Pro Phe Gln Cys 50 55 60 Ala
Gly Val Ala Leu Ser Arg Cys Thr Leu Asn Arg Asn Ala Leu Arg 65 70
75 80 Arg Pro Ser Tyr Thr Asn Gly Pro Gln Glu Ile Tyr Ile Gln Gln
Gly 85 90 95 Lys Gly Ile Phe Gly Met Ile Tyr Pro Gly Cys Ser Ser
Thr Phe Glu 100 105 110 Glu Pro Gln Gln Pro Gln Gln Arg Gly Gln Ser
Ser Arg Pro Gln Asp 115 120 125 Arg His Gln Lys Ile Tyr Asn Ser Arg
Glu Gly Asp Leu Ile Ala Val 130 135 140 Pro Thr Gly Val Ala Trp Trp
Met Tyr Asn Asn Glu Asp Thr Pro Val 145 150 155 160 Val Ala Val Ser
Ile Ile Asp Thr Asn Ser Leu Glu Asn Gln Leu Asp 165 170 175 Gln Met
Pro Arg Arg Phe Tyr Leu Ala Gly Asn Gln Glu Gln Glu Phe 180 185 190
Leu Lys Tyr Gln Gln Glu Gln Gly Gly His Gln Ser Gln Lys Gly Lys 195
200 205 His Gln Gln Glu Glu Glu Asn Glu Gly Gly Ser Ile Leu Ser Gly
Phe 210 215 220 Thr Leu Glu Phe Leu Glu His Ala Phe Ser Val Asp Lys
Gln Ile Ala 225 230 235 240 Lys Asn Leu Gln Gly Glu Asn Glu Gly Glu
Asp Lys Gly Ala Ile Val 245 250 255 Thr Val Lys Gly Gly Leu Ser Val
Ile Lys Pro Pro Thr Asp Glu Gln 260 265 270 Gln Gln Arg Pro Gln Glu
Glu Glu Glu Glu Glu Glu Asp Glu Lys Pro 275 280 285 Gln Cys Lys Gly
Lys Asp Lys His Cys Gln Arg Pro Arg Gly Ser Gln 290 295 300 Ser Lys
Ser Arg Arg Asn Gly Ile Asp Glu Thr Ile Cys Thr Met Arg 305 310 315
320 Leu Arg His Asn Ile Gly Gln Thr Ser Ser Pro Asp Ile Tyr Asn Pro
325 330 335 Gln Ala Gly Ser Val Thr Thr Ala Thr Ser Leu Asp Phe Pro
Ala Leu 340 345 350 Ser Trp Leu Arg Leu Ser Ala Gly Phe Gly Ser Leu
Arg Lys Asn Ala 355 360 365 Met Phe Val Pro His Tyr Asn Leu Asn Ala
Asn Ser Ile Ile Tyr Ala 370 375 380 Leu Asn Gly Arg Ala Leu Ile Gln
Val Val Asn Cys Asn Gly Glu Arg 385 390 395 400 Val Phe Asp Gly Glu
Leu Gln Glu Gly Arg Val Leu Ile Val Pro Gln 405 410 415 Asn Phe Val
Val Ala Ala Arg Ser Gln Ser Asp Asn Phe Glu Tyr Val 420 425 430 Ser
Phe Lys Thr Asn Asp Thr Pro Met Ile Gly Thr Leu Ala Gly Ala 435 440
445 Asn Ser Leu Leu Asn Ala Leu Pro Glu Glu Val Ile Gln His Thr Phe
450 455 460 Asn Leu Lys Ser Gln Gln Ala Arg Gln Ile Lys Asn Asn Asn
Pro Phe 465 470 475 480 Lys Phe Leu Val Pro Pro Gln Glu Ser Gln Lys
Arg Ala Val Ala 485 490 495
* * * * *
References