U.S. patent application number 12/809634 was filed with the patent office on 2011-01-06 for soy protein products having altered characteristics.
Invention is credited to Charles T. Blaisdell, Susan Knowlton.
Application Number | 20110003066 12/809634 |
Document ID | / |
Family ID | 40668454 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110003066 |
Kind Code |
A1 |
Knowlton; Susan ; et
al. |
January 6, 2011 |
SOY PROTEIN PRODUCTS HAVING ALTERED CHARACTERISTICS
Abstract
Soy protein products obtained from high oleic soybeans, wherein
such products, have improved whiteness, reduced viscosity and
reduced gel-strength, are described. Use of such products in food,
beverage and animal feed are also disclosed.
Inventors: |
Knowlton; Susan; (Elkton,
MD) ; Blaisdell; Charles T.; (Odessa, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
40668454 |
Appl. No.: |
12/809634 |
Filed: |
December 19, 2008 |
PCT Filed: |
December 19, 2008 |
PCT NO: |
PCT/US08/87585 |
371 Date: |
June 21, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61015750 |
Dec 21, 2007 |
|
|
|
Current U.S.
Class: |
426/656 ;
426/465; 530/378 |
Current CPC
Class: |
A23K 20/147 20160501;
A23L 11/07 20160801; A23L 2/38 20130101; A23C 11/103 20130101; A23L
2/66 20130101; A23L 33/185 20160801; A23J 3/16 20130101; A23K 40/00
20160501 |
Class at
Publication: |
426/656 ;
530/378; 426/465 |
International
Class: |
A23J 1/14 20060101
A23J001/14; C07K 14/415 20060101 C07K014/415; A23J 3/16 20060101
A23J003/16 |
Claims
1. A soy protein product obtained from a high oleic soybean wherein
said product has at least one characteristic selected from the
group consisting of improved whiteness, reduced gel strength and
reduced viscosity when compared to a soy protein product obtained
from a commodity soybean using the same process as that to obtain
the soy protein product from a high oleic soybean.
2. The soy protein product of claim 1, wherein: a) the whiteness
index is increased by at least 3%; or b) the gel strength is
reduced by at least 25%; or c) the viscosity of an unhydrolyzed soy
protein product is reduced by at least 9%.
3. The soy protein product of anyone of claim 1, or 2 wherein said
protein product has at least 40% protein (N.times.6.25) on a
moisture-free basis.
4. The soy protein product of anyone of claim 1, or 2 wherein said
protein product has at least 65% protein (N.times.6.25) on a
moisture-free basis.
5. The soy protein product of anyone of claim 1, or 2 wherein said
protein product has at least 90% protein (N.times.6.25) on a
moisture-free basis.
6. The soy protein product of anyone of claim 1, or 2 wherein said
product is selected from the group consisting of a soy protein
isolate, a soy protein concentrate, soy meal, full fat flour,
soymilk powder, defatted flour, soymilk, textured proteins,
textured flours, textured concentrates and textured isolates.
7. A food which has incorporated therein the soy protein product of
anyone of claim 1, or 2.
8. A beverage which has incorporated therein the soy protein
product of anyone of claim 1, or 2.
9. Animal feed which has incorporated therein the soy protein
product of anyone of claim 1, or 2.
10. A method for improving drying efficiency of a soy protein
product, comprising feeding at least one soy protein product
obtained from a high oleic soybean seed at higher feed solids to a
pasteurizer or a dryer compared to feeding at least one soy protein
product obtained from a commodity soybean.
11. A method for improving drying efficiency of a soy protein
product, comprising feeding at least one soy protein product
obtained from a high oleic soybean seed at no less than 14% feed
solids to a pasteurizer or a dryer
Description
FIELD OF THE INVENTION
[0001] This invention relates to soy protein products obtained from
high oleic soybeans wherein the protein product(s) have improved
whiteness, reduced viscosity and reduced gel-strength.
BACKGROUND OF THE INVENTION
[0002] Soybeans have the highest protein content of all cereals and
legumes. In particular, soybeans have about 40% protein, while
other legumes have 20-30%, and cereals have about 8-15% protein.
Soybeans also contain about 20% oil with the remaining dry matter
mostly carbohydrate (35%). On a wet basis (as is), soybeans contain
about 35% protein, 17% oil, 31% carbohydrates and 4.4% ash. Soybean
storage protein and lipid bodies are contained in the usable meat
of the soybean called the cotyledon. The complex carbohydrate (or
dietary fiber) is also contained in the cell walls of the
cotyledon. The outer layer of cells (called the seed coat) makes up
about 8% of the soybean's total weight. The raw, dehulled soybean
is, depending on the variety, approximately 18% oil, 15% insoluble
carbohydrates, 14% moisture and ash and 38% protein.
[0003] Plant protein materials are used as functional food
ingredients, and have numerous applications in enhancing desirable
characteristics in food products. Soy protein materials, in
particular, have seen extensive use as functional food ingredients.
Soy protein materials are used as an emulsifier in meats to bind
the meat and give the meat a good texture and a firm bite. Another
common application for soy protein materials as functional food
ingredients is as a thickening agent to provide a creamy viscosity
to the food product.
[0004] In general, soy protein materials include soy flakes, soy
grits, soy meal, soy flour, soy protein concentrates, and soy
protein isolates with a primary difference between these materials
being the degree of refinement relative to whole soybeans.
[0005] Apart from the soy protein content, flavor, gel-strength,
whiteness-index, and viscosity of a soy protein material are also a
relevant criteria for the selection of a soy protein material as a
functional food ingredient. Conventional soy protein material may
have a strong beany, bitter flavor and odor as a result of the
presence of certain volatile compounds and/or an undesired
appearance due to the presence of other relatively low molecular
weight compounds in the soy protein material.
[0006] The present disclosure generally relates to a soy
protein-containing composition having reduced gel-strength, reduced
viscosity, and improved whiteness.
[0007] U.S. Pat. No. 6,599,556 B2, issued to Stark et al. on Jul.
29, 2003, describes confectionery products, which include high
protein content modified oilseed material.
[0008] U.S. Pat. No. 6,716,469 B2, issued to Stark et al. on Apr.
6, 2004, describes frozen dessert products, which include high
protein content modified oilseed material.
[0009] U.S. Pat. No. 6,720,020 B2, issued to Karleskind et al. on
Apr. 13, 2004, describes beverage compositions, which include high
protein content modified oilseed material.
[0010] JP Patent No. 5,168,416 A1, issued to Takeshi et al. on Jul.
2, 1993, describes obtaining a concentrated soybean having improved
taste, flavor and color tone and useful as a food material, etc.,
with simple operation at a low cost without changing the nature of
the protein by washing soybeans, etc., with a water-containing
alcohol under weakly acidic condition in the presence of an
acid.
[0011] JP Patent No. 4,207,159 A1, issued to Hiroko et al on Jul.
29, 1992, describes the title raw material having bright and white
color tone and useful for marine and knead eater-dispersed liquid
of acid-precipitated soybean protein with an alkali metal hydroxide
to control pH.
[0012] WO2007013146A1, published Feb. 1, 2007, describes
compositions for processed soy protein foods.
SUMMARY OF THE INVENTION
[0013] In a first embodiment, the invention concerns a soy protein
product obtained from a high oleic soybean wherein said product has
at least one characteristic selected from the group consisting of
improved whiteness, reduced gel strength and reduced viscosity when
compared to a soy protein product obtained from a commodity soybean
using the same process as that to obtain the soy protein product
from a high oleic soybean.
[0014] In a second embodiment, the invention concerns a soy protein
product derived from high oleic soybeans having an at least 3%
increase in the whiteness index compared to a soy protein product
derived from commodity soybean using the same process as that to
obtain the soy protein product from a high oleic soybean.
[0015] In a third embodiment, the invention concerns an
unhydrolyzed soy protein product derived from high oleic soybeans
having a reduction of viscosity by at least 9% compared to a soy
protein product obtained from a commodity soybean using the same
process as that to obtain the soy protein product from a high oleic
soybean.
[0016] In a fourth embodiment, the invention concerns a soy protein
product derived from high oleic soybeans having reduction in gel
strength by at least 25% compared to a soy protein product obtained
from a commodity soybean using the same process as that to obtain
the soy protein product from a high oleic soybean.
[0017] In a fifth embodiment the invention concerns soy protein
products selected from the group consisting of a soy protein
isolate, a soy protein concentrate, soy meal, full fat flour,
defatted flour, soymilk powder, soymilk, textured proteins,
textured flours, textured concentrates and textured isolates.
[0018] In a sixth embodiment the invention concerns a method for
improving drying efficiency of a soy protein product, comprising
feeding at least one soy protein product obtained from a high oleic
soybean seed at higher feed solids to a pasteurizer or a dryer
compared to feeding at least one soy protein product obtained from
a commodity soybean.
[0019] In a seventh embodiment the invention concerns a method for
improving drying efficiency of a soy protein product, comprising
feeding at least one soy protein product obtained from a high oleic
soybean seed at no less than 14% feed solids to a pasteurizer or a
dryer.
[0020] Additional embodiments of the invention include soy protein
products with at least 40%, 65%, or 90% protein (N.times.6.25) on a
moisture-free basis.
[0021] In other aspects, the soy protein products of the invention
can be used in food, beverages, and animal feed containing the soy
protein product of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0022] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing, which form a part of this application.
[0023] FIG. 1 depicts plasmid pKS210.
[0024] FIG. 2 depicts plasmid PHP17731.
[0025] FIG. 3 depicts plasmid PHP17064.
[0026] FIG. 4 depicts fragment PHP19340A.
[0027] FIG. 5 depicts fragment PHP17752A.
[0028] FIG. 6 depicts plasmid PHP19340.
[0029] FIG. 7 depicts plasmid PHP17752.
[0030] SEQ ID NO:1 sets forth the sequence of the recombinant DNA
fragment PHP21676A
[0031] SEQ ID NO:2 sets forth the sequence of the 1533
polynucleotide fragment comprising 470 nucleotides from the soybean
FAD2-2 gene, 420 nucleotides from the soybean FAD2-1 gene, 643
nucleotides from the soybean FAD3 gene.
[0032] SEQ ID NO:3 sets forth the nucleotide sequence of
oligonucleotide primer BM35 used to amplify an approximately 0.9 Kb
fragment from recombinant DNA fragment KSFAD2-hybrid.
[0033] SEQ ID NO:4 sets forth the nucleotide sequence of
oligonucleotide primer BM39 used to amplify an approximately 0.9 kb
fragment from recombinant DNA fragment KSFAD2-hybrid.
[0034] SEQ ID NO:5 sets forth the nucleotide sequence of
oligonucleotide primer BM40 used to amplify an approximately 0.65
kb DNA fragment from plasmid XF1.
[0035] SEQ ID NO:6 sets forth the nucleotide sequence of
oligonucleotide plasmid BM41 used to amplify an approximately 0.65
kb DNA fragment from plasmid pXF1.
[0036] SEQ ID NO:7 sets forth the nucleotide sequence of
recombinant DNA fragment KSFAD2-hybrid which contains about 470
nucleotides from the soybean FAD2-2 gene and 420 nucleotides from
the soybean FAD2-1 gene.
[0037] SEQ ID NO:8 sets forth the nucleotide sequence of
oligonucleotide primer KS1 used to amplify about 470 nucleotides
from the soybean FAD2-2 gene.
[0038] SEQ ID NO:9 sets forth the nucleotide sequence of
oligonucleotide primer KS2 used to amplify about 470 nucleotides of
the soybean FAD2-2 gene.
[0039] SEQ ID NO:10 sets forth the nucleotide sequence of
oligonucleotide primer KS3 used to amplify about 420 nucleotides of
the soybean FAD2-1 gene.
[0040] SEQ ID NO:11 sets forth the nucleotide sequence of
oligonucleotide primer KS4 used to amplify about 420 nucleotides of
the soybean FAD2-1 gene.
[0041] SEQ ID NO:12 sets forth the nucleotide sequence of the
seed-specific gene expression-silencing cassette from pKS133 which
comprises nucleotides for a Kti3 promoter and terminator bordering
a string of nucleotides that promote formation of a stem structure
which are surrounding a unique Not I restriction endonuclease
site.
[0042] SEQ ID NO:13 sets forth the nucleotide sequence of plasmid
pKS210.
[0043] SEQ ID NO:14 sets forth the nucleotide sequence of plasmid
PHP17731.
[0044] SEQ ID NO:15 sets forth the nucleotide sequence of
recombinant DNA fragment PHP17731A.
[0045] SEQ ID NO:16 sets forth the nucleotide sequence of the ALS
selectable marker recombinant DNA fragment. This recombinant DNA
fragment comprises a promoter operably linked to a nucleotide
fragment encoding a soybean acetolactate synthase to which
mutations have been introduced to make it resistant to treatment
with sulfonylurea herbicides.
[0046] SEQ ID NO:17 sets forth the amino acid sequence of the
soybean herbicide-resistant ALS including mutations in subsequences
B and F.
[0047] SEQ ID NO:18 is the wild type amino acid sequence of
conserved ALS "subsequence B" disclosed in U.S. Pat. No.
5,013,659.
[0048] SEQ ID NO:19 sets forth the wild type amino acid sequence of
conserved ALS "subsequence F" disclosed in U.S. Pat. No.
5,013,659.
[0049] SEQ ID NO:20 sets forth the amino acid sequence of the
additional five amino acids introduced during cloning at the
amino-terminus of the soybean ALS.
[0050] SEQ ID NO:21 sets forth the nucleotide sequence of plasmid
PHP17064
[0051] SEQ ID NO:22 sets forth the nucleotide sequence of
recombinant DNA fragment PHP17064A.
[0052] SEQ ID NO:23 sets forth the nucleotide sequence of fragment
PHP19340A.
[0053] SEQ ID NO:24 sets forth the nucleotide sequence of fragment
PHP17752A.
[0054] SEQ ID NO:25 sets forth the nucleotide sequence of plasmid
PHP19340.
[0055] SEQ ID NO:26 sets forth the nucleotide sequence of plasmid
PHP17752.
[0056] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Res. 13:3021-3030 (1985) and in the
Biochemical J. 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37C.F.R. .sctn.1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0057] All patents, patent applications, and publications cited
herein are incorporated by reference in their entirety.
[0058] In the context of this disclosure, a number of terms shall
be utilized.
[0059] As used herein, "soybean" refers to the species Glycine max,
Glycine soja, or any species that is sexually cross compatible with
Glycine max. A "line" is a group of plants of similar parentage
that display little or no genetic variation between individuals for
a least one trait. Such lines may be created by one or more
generations of self-pollination and selection, or vegetative
propagation from a single parent including by tissue or cell
culture techniques. An "agronomically elite line" or "elite line"
refers to a line with desirable agronomic performance that may or
may not be used commercially. A "variety", "cultivar", "elite
variety", or "elite cultivar" refers to an agronomically superior
elite line that has been extensively tested and is or was being
used for commercial soybean production. "Mutation" refers to a
detectable and heritable genetic change (either spontaneous or
induced) not caused by segregation or genetic recombination.
"Mutant" refers to an individual, or lineage of individuals,
possessing a mutation.
[0060] The "whiteness index" of a soy protein product refers to the
color of the soy-protein-containing composition. Many soy
protein-containing feed compositions will have, to varying degrees,
a yellowish or brownish color. In general, the color of these
compositions can be "improved," i.e., the "whiteness index" of the
product can be increased by the process of the present invention.
In general, the whiteness index is determined using a colorimeter
which provides the L, a, and b color values for the composition
from which the whiteness index may be calculated using a standard
expression of the Whiteness Index (WI), WI=L-3b. The L component
generally indicates the whiteness or, "lightness", of the sample; L
values near 0 indicate a black sample while L values near 100
indicate a white sample. The b value indicates yellow and blue
colors present in the sample; positive b values indicate the
presence of yellow colors while negative b values indicate the
presence of blue colors. The a value, which may be used in other
color measurements, indicates red and green colors; positive values
indicate the presence of red colors while negative values indicate
the presence of green colors. For the b and a values, the absolute
value of the measurement increases directly as the intensity of the
corresponding color increases. Generally, the colorimeter is
standardized using a white standard tile provided with the
colorimeter. A sample is then placed into a glass cell which is
introduced to the colorimeter. The sample cell is covered with an
opaque cover to minimize the possibility of ambient light reaching
the detector through the sample and serves as a constant during
measurement of the sample. After the reading is taken, the sample
cell is emptied and typically refilled as multiple samples of the
same material are generally measured and the whiteness index of the
material expressed as the average of the measurements. Suitable
colorimeters generally include those manufactured by HunterLab
(Reston, Va.) including, for example, Model # DP-9000 with Optical
Sensor D 25.
[0061] Whiteness index measurements of a 5% by weight solids sample
of the suspension before and after treatment are determined using a
HunterLab DP-9000 colorimeter including an optical sensor D-25,
both manufactured by Hunter Associates Laboratory (HunterLab)
(Reston, Va.). For the whiteness index measurement in the large
scale production platform, protein samples are dispersed on a 5%
w/w basis: (5.25 g) is added to deionized water (100 mL). For the
whiteness index measurement in the small scale production platform,
1 g protein sample is dispersed in 19 mL of deionized water on a
w/v basis. The results obtained using the Hunter Colorimeter are
reported in units of L, a, and b. Whiteness Index is calculated
from the L and b scale values using the following:
Whiteness Index=L-3b.
[0062] In addition to the improved color, the soy protein product
produced by the processes in the present disclosure can have a
reduced viscosity.
[0063] Viscosity, gelation and other indicators of structure
formation are important properties of soybean proteins since they
contribute to the overall utility of the product in use. Proteins
contribute to the solidity and elasticity of products by formation
of a three dimensional network of aggregated protein molecules
which entrap water. It is sometimes desirable to have these
properties, for example in the case of meat-like products, or it
may be desired to have less functionality, for example in beverage
applications. For beverage applications, a lower viscosity may be
desirable for sensory, mouthfeel and textural properties of the
beverage. Lower viscosity soy protein-containing compositions may
be intended for use in liquid products (i.e., beverages); and
additionally, in some embodiments, lower viscosity soy
protein-containing compositions may be desired for use in meat
products.
[0064] As used herein, the term "viscosity" means the apparent
viscosity of aqueous slurry or a solution as measured with a
rotating spindle viscometer utilizing a large annulus, where a
particularly preferred rotating spindly viscometer is a Brookfield
viscometer. In another embodiment, the apparent viscosity can be
measured using a Rapid Visco Analyzer (RVA) viscometer, or an
AR-1000 Rheometer.
[0065] In general, the term viscosity refers to the apparent
viscosity of a slurry or a solution as measured with a rotating
spindle viscometer utilizing a large annulus, where a particularly
preferred rotating spindle viscometer is a Brookfield viscometer.
The apparent viscosity of a soy protein material may be measured,
for example, by weighing a sample of the soy material and water to
obtain a known ratio of the soy material to water (preferably 1
part soy material to 9 parts water, by weight), combining and
mixing the soy material and water in a blender or mixer to form a
homogenous slurry of the soy material and water at ambient
temperature and neutral pH, and measuring the apparent viscosity of
the slurry with the rotating spindle viscometer utilizing a large
annulus, operated at approximately 60 revolutions per minute and at
a torque of from 30 to 70%.
[0066] Another important functional characteristic is the gel
forming property of a protein. Protein gelation is important to
obtain desirable sensory and textural structures in foods.
[0067] The formation of a protein gel is a two step process which
initiates through partial denaturation of the protein molecules. As
the proteins denature, the viscosity of the slurry increases as a
result of an increase in the molecular changes associated with the
unfolding proteins. During the second part of the process there is
a large increase in viscosity resulting from protein association
and development of the molecular network.
[0068] Gelation phenomenon requires a driving force to unfold the
native protein structure, followed by an aggregation retaining a
certain degree of order in the matrix formed by association between
protein strands. Protein gelation has been traditionally achieved
by heating, but some physical and chemical processes form protein
gels in an analogous way to heat-induction. A physical means,
besides heat, is high pressure. Chemical means are acidification,
enzymatic cross-linking, and use of salts and urea, causing
modifications in protein-protein and protein-medium interactions.
The characteristics of each gel are different and dependent upon
factors like protein concentration, degree of denaturation caused
by pH, temperature, ionic strength and/or pressure.
[0069] The term "gel-strength" refers to the ability or a measure
of a protein to form gels.
[0070] The term "fatty acids" refers to long-chain aliphatic acids
(alkanoic acids) of varying chain length, from about C.sub.12 to
C.sub.22 (although both longer and shorter chain-length acids are
known). The predominant chain lengths are between C.sub.16 and
C.sub.22. The structure of a fatty acid is represented by a simple
notation system of "X:Y", where X is the total number of C atoms in
the particular fatty acid and Y is the number of double bonds.
[0071] Generally, fatty acids are classified as saturated or
unsaturated. The term "saturated fatty acids" refers to those fatty
acids that have no "double bonds" between their carbon backbone. In
contrast, "unsaturated fatty acids" have "double bonds" along their
carbon backbones (which are most commonly in the
cis-configuration). "Monounsaturated fatty acids" have only one
"double bond" along the carbon backbone (e.g., usually between the
9.sup.th and 10.sup.th carbon atom as for palmitoleic acid (16:1)
and oleic acid (18:1)), while "polyunsaturated fatty acids" (or
"PUFAs") have at least two double bonds along the carbon backbone
(e.g., between the 9.sup.th and 10.sup.th, and 12.sup.th and
13.sup.th carbon atoms for linoleic acid (18:2); and between the
9.sup.th and 10.sup.th, 12.sup.th and 13.sup.th, and 15.sup.th and
16.sup.th for .alpha.-linolenic acid (18:3)).
[0072] The term "total fatty acid content" refers to the sum of the
five major fatty acid components found in soybeans, namely C16:0,
C18:0, C18:1, C18:2, and C18:3. The term "total polyunsaturated
fatty acid content" refers to the total C18:2 plus C18:3
content.
[0073] For the purposes of the present disclosure, the
omega-reference system will be used to indicate the number of
carbons, the number of double bonds and the position of the double
bond closest to the omega carbon, counting from the omega carbon
(which is the terminal carbon of the aliphatic chain and is
numbered 1 for this purpose). This nomenclature is shown below in
Table 1, in the column titled "Shorthand Notation".
TABLE-US-00001 TABLE 1 Nomenclature of Polyunsaturated Fatty Acids
Shorthand Common Name Abbreviation Chemical Name Notation Linoleic
LA cis-9,12- 18:2 .omega.-6 octadecadienoic .alpha.-Linolenic
.alpha.LIN cis-9,12,15- 18:3 .omega.-3 octadecatrienoic
[0074] The term "desaturase" refers to a polypeptide that can
desaturate, i.e., introduce a double bond, in one or more fatty
acids to produce a mono- or polyunsaturated fatty acid or precursor
which is of interest. Despite use of the omega-reference system
throughout the specification in reference to specific fatty acids,
it is more convenient to indicate the activity of a desaturase by
counting from the carboxyl end of the substrate using the
.DELTA.-system.
[0075] The terms "FAD" and fatty acid desaturase are used
interchangeably and refer to membrane bound microsomal oleoyl- and
linoleoyl-phosphatidylcholine desaturases that convert oleic acid
to linoleic acid and linoleic acid to linolenic acid, respectively,
in reactions that reduce molecular oxygen to water and require the
presence of NADH.
[0076] The term "high oleic soybean" refers to soybean seeds that
have an oleic acid content of at least 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, and 95% of the seed by weight. Preferred high
oleic soybean oil starting materials are disclosed in World Patent
Publication WO94/11516, the disclosure of which is hereby
incorporated by reference.
[0077] The term enzyme "activity" refers to the ability of an
enzyme to convert a substrate to a product.
[0078] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", "nucleic acid fragment", and "isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. Nucleotides (usually found in their
5'-monophosphate form) are referred to by a single letter
designation as follows: "A" for adenylate or deoxyadenylate (for
RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate,
"G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for
deoxythymidylate, "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.
[0079] 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.
[0080] Chimeric genes can be designed for use in suppression by
linking a nucleic acid fragment or subfragment thereof, whether or
not it encodes an active enzyme, in the sense or antisense
orientation relative to a plant promoter sequence.
[0081] The terms "homology", "homologous", "substantially similar"
and "corresponding substantially" are used interchangeably herein.
They refer to nucleic acid fragments wherein changes in one or more
nucleotide bases do 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.
[0082] "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 to 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. 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. An "allele" is one of several alternative forms of a
gene occupying a given locus on a chromosome. When all the alleles
present at a given locus on a chromosome are the same that plant is
homozygous at that locus. If the alleles present at a given locus
on a chromosome differ that plant is heterozygous at that locus. A
"codon-optimized gene" is a gene having its frequency of codon
usage designed to mimic the frequency of preferred codon usage of
the host cell.
[0083] "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.
[0084] "Promoter" refers to a region of DNA capable of controlling
the expression of a coding sequence or functional RNA. The promoter
sequence consists of proximal and more distal upstream elements.
These upstream elements are often referred to as enhancers.
Accordingly, an "enhancer" is a DNA sequence that 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.
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. Promoters that 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.
[0085] Any seed-specific promoter can be used in accordance with
the method of the invention. Thus, the origin of the promoter
chosen to drive expression of the recombinant DNA fragment is not
critical as long as it is capable of accomplishing the invention by
transcribing enough RNA from the desired nucleic acid fragment(s)
in the seed.
[0086] A plethora of promoters is described in WO 00/18963,
published on Apr. 6, 2000, the disclosure of which is hereby
incorporated by reference. Examples of seed-specific promoters
include, and are not limited to, the promoter for soybean Kunitz
trypsin inhibitor (Kti3, Jofuku and Goldberg (1989) Plant Cell
1:1079-1093) .beta.-conglycinin (Chen et al. (1989) Dev. Genet. 10:
112-122), the napin promoter, and the phaseolin promoter.
[0087] Specific examples of promoters that may be useful in
expressing the nucleic acid fragments of the invention include, but
are not limited to, the SAM synthetase promoter (PCT Publication
WO00/37662, published Jun. 29, 2000), the CaMV 35S (Odell et al
(1985) Nature 313:810-812), and the promoter described in PCT
Publication WO02/099063 published Dec. 12, 2002.
[0088] The "translation leader sequence" refers to a polynucleotide
sequence located between the promoter sequence of a gene and the
coding sequence. The translation leader sequence is present in the
fully processed mRNA upstream of the translation start sequence.
The translation leader sequence may affect processing of the
primary transcript to mRNA, mRNA stability or translation
efficiency. Examples of translation leader sequences have been
described (Turner and Foster (1995) Mol. Biotechnol.
3:225-236).
[0089] The "3' non-coding sequences" or "transcription
terminator/termination 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.
[0090] 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.
[0091] "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. An RNA transcript is
referred to as the mature RNA when it is an RNA sequence derived
from post-transcriptional processing of the primary transcript.
"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 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.
[0092] 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 regulated by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of regulating 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 a sense or antisense orientation. In
another example, the complementary RNA regions of the invention can
be operably linked, either directly or indirectly, 5' to the target
mRNA, or 3' to the target mRNA, or within the target mRNA, or a
first complementary region is 5' and its complement is 3' to the
target mRNA.
[0093] The term "endogenous RNA" refers to any RNA which is encoded
by any nucleic acid sequence present in the genome of the host
prior to transformation with the recombinant construct of the
present invention, whether naturally-occurring or non-naturally
occurring, i.e., introduced by recombinant means, mutagenesis,
etc.
[0094] The term "non-naturally occurring" means artificial, not
consistent with what is normally found in nature.
[0095] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, 1989.
Transformation methods are well known to those skilled in the art
and are described below.
[0096] "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.
[0097] 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.
[0098] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes that are not part of
the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitates
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0099] The terms "recombinant construct", "expression construct",
"chimeric construct", "construct", and "recombinant DNA construct"
are used interchangeably herein. A recombinant construct comprises
an artificial combination of nucleic acid fragments, e.g.,
regulatory and coding sequences that are not found together in
nature. For example, a chimeric construct 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. Such construct may be used by 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 cells 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, immunoblotting analysis
of protein expression, or phenotypic analysis, among others.
[0100] The term "expression", as used herein, refers to the
production of a functional end-product e.g., a mRNA or a protein
(precursor or mature).
[0101] The term "expression cassette" as used herein, refers to a
discrete nucleic acid fragment into which a nucleic acid sequence
or fragment can be moved.
[0102] "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.
[0103] "Cosuppression" refers to the production of sense RNA
transcripts capable of suppressing the expression of identical or
substantially similar native genes (U.S. Pat. No. 5,231,020, which
issued to Jorgensen et al. on Jul. 27, 1999). Co-suppression
constructs in plants have been previously designed by focusing on
overexpression of a nucleic acid sequence having homology to a
native mRNA, in the sense orientation, which results in the
reduction of all RNA having homology to the overexpressed sequence
(see Vaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000)
Nature 404:804-808). "Antisense inhibition" refers to the
production of antisense RNA transcripts capable of suppressing the
expression of the target protein. Plant viral sequences may be used
to direct the suppression of proximal mRNA encoding sequences (PCT
Publication WO 98/36083 published on Aug. 20, 1998). "Hairpin"
structures that incorporate all, or part, of an mRNA encoding
sequence in a complementary orientation resulting in a potential
"stem-loop" structure for the expressed RNA have been described
(PCT Publication WO 99/53050 published on Oct. 21, 1999). In this
case the stem is formed by polynucleotides corresponding to the
gene of interest inserted in either sense or anti-sense orientation
with respect to the promoter and the loop is formed by some
polynucleotides of the gene of interest, which do not have a
complement in the construct. This increases the frequency of
cosuppression or silencing in the recovered transgenic plants. For
review of hairpin suppression see Wesley et al. (2003) Methods in
Molecular Biology, Plant Functional Genomics: Methods and Protocols
236:273-286. A construct where the stem is formed by at least 30
nucleotides from a gene to be suppressed and the loop is formed by
a random nucleotide sequence has also effectively been used for
suppression (WO 99/61632 published on Dec. 2, 1999). The use of
poly-T and poly-A sequences to generate the stem in the stem-loop
structure has also been described (WO 02/00894 published Jan. 3,
2002). Yet another variation includes using synthetic repeats to
promote formation of a stem in the stem-loop structure. Transgenic
organisms prepared with such recombinant DNA fragment show reduced
levels of the protein encoded by the polynucleotide from which the
nucleotide fragment forming the loop is derived as described in PCT
Publication WO 02/00904, published Jan. 3, 2002. The use of
constructs that result in dsRNA has also been described. In these
constructs convergent promoters direct transcription of
gene-specific sense and antisense RNAs inducing gene suppression
(see for example Shiet al. (2000) RNA 6:1069-1076; Bastinet al.
(2000) J. Cell Sci. 113:3321-3328; Giordanoet al. (2002) Genetics
160:637-648; LaCount and Donelson US patent Application No.
20020182223, published Dec. 5, 2002; Tranet al. (2003) BMC
Biotechnol. 3:21; and Applicant's U.S. Provisional Application No.
60/578,404, filed Jun. 9, 2004).
[0104] Other methods for suppressing an enzyme include, but are not
limited to, use of polynucleotides that may form a catalytic RNA or
may have ribozyme activity (U.S. Pat. No. 4,987,071 issued Jan. 22,
1991), and micro RNA (also called miRNA) interference (Javier et
al. (2003) Nature 425:257-263).
[0105] MicroRNAs (miRNA) are small regulatory RNAs that control
gene expression. miRNAs bind to regions of target RNAs and inhibit
their translation and, thus, interfere with production of the
polypeptide encoded by the target RNA. miRNAs can be designed to be
complementary to any region of the target sequence RNA including
the 3' untranslated region, coding region, etc. miRNAs are
processed from highly structured RNA precursors that are processed
by the action of a ribonuclease III termed DICER. While the exact
mechanism of action of miRNAs is unknown, it appears that they
function to regulate expression of the target gene. See, e.g., U.S.
Patent Publication No. 2004/0268441 AI which was published on Dec.
30, 2004.
[0106] The term "expression", as used herein, refers to the
production of a functional end-product, be it mRNA or translation
of mRNA into a polypeptide.
[0107] "Antisense inhibition" refers to the production of antisense
RNA transcripts capable of suppressing the expression of the target
protein. "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).
[0108] "Overexpression" refers to the production of a functional
end-product in transgenic organisms that exceeds levels of
production when compared to expression of that functional
end-product in a normal, wild type or non-transformed organism.
[0109] "Stable transformation" refers to the transfer of a nucleic
acid fragment into a genome of a host organism, including both
nuclear and organellar genomes, resulting in genetically stable
inheritance. In contrast, "transient transformation" refers to the
transfer of a nucleic acid fragment into the nucleus, or
DNA-containing organelle, of a host organism resulting in gene
expression without integration or stable inheritance. Host
organisms containing the transformed nucleic acid fragments are
referred to as "transgenic" organisms.
[0110] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described by Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989); by
Silhavy et al., Experiments with Gene Fusions, Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel et al.,
Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987). Once the
recombinant construct has been made, it may then be introduced into
a plant cell or yeast cell of choice by methods well known to those
of ordinary skill in the art including, for example, transfection,
transformation and electroporation (see below). Oilseed plant cells
are the preferred plant cells. The transformed plant cell is then
cultured and regenerated under suitable conditions permitting
expression of the recombinant construct which is then recovered and
purified.
[0111] Recombinant constructs may be introduced into one plant cell
or, alternatively, a construct may be introduced into separate
plant cells.
[0112] Expression in a plant cell may be accomplished in a
transient or stable fashion as is described above.
[0113] Plant parts include differentiated and undifferentiated
tissues, including but not limited to: roots, stems, shoots,
leaves, pollen, seeds, tumor tissue, and various forms of cells and
culture such as single cells, protoplasts, embryos, and callus
tissue. The plant tissue may be in plant or in organ, tissue or
cell culture.
[0114] The term "plant organ" refers to plant tissue or group of
tissues that constitute a morphologically and functionally distinct
part of a plant. The term "genome" refers to the following: 1. The
entire complement of genetic material (genes and non-coding
sequences) is present in each cell of an organism, or virus or
organelle. 2. A complete set of chromosomes inherited as a
(haploid) unit from one parent. The term "stably integrated" refers
to the transfer of a nucleic acid fragment into the genome of a
host organism or cell resulting in genetically stable
inheritance.
[0115] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens, and obtaining transgenic plants have
been published, among others, for cotton (U.S. Pat. No. 5,004,863,
U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834, U.S.
Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut
(Cheng et al. (1996) Plant Cell Rep. 15:653-657, McKently et al.
(1995) Plant Cell Rep. 14:699-703); papaya (Ling et al. (1991)
Bio/technology 9:752-758); and pea (Grant et al. (1995) Plant Cell
Rep. 15:254-258). For a review of other commonly used methods of
plant transformation see Newell (2000) Mol. Biotechnol. 16:53-65.
One of these methods of transformation uses Agrobacterium
rhizogenes (Tepfler, and Casse-Delbart (1987) Microbiol. Sci.
4:24-28). Transformation of soybeans using direct delivery of DNA
has been published using PEG fusion (PCT publication WO 92/17598),
electroporation (Chowrira et al. (1995) Mol. Biotechnol. 3:17-23;
Christou et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966),
microinjection, or particle bombardment (McCabe et. al. (1988)
Bio/Technology 6:923; Christou et al. (1988) Plant Physiol.
87:671-674).
[0116] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated. The regeneration, development and
cultivation of plants from single plant protoplast transformants or
from various transformed explants is well known in the art
(Weissbach and Weissbach, (1988) In.: Methods for Plant Molecular
Biology, (Eds.), Academic: San Diego, Calif.). This regeneration
and growth process typically includes the steps of selection of
transformed cells, culturing those individualized cells through the
usual stages of embryonic development through the rooted plantlet
stage. Transgenic embryos and seeds are similarly regenerated. The
resulting transgenic rooted shoots are thereafter planted in an
appropriate plant growth medium such as soil. Preferably, the
regenerated plants are self-pollinated to provide homozygous
transgenic plants. Otherwise, pollen obtained from the regenerated
plants is crossed to seed-grown plants of agronomically important
lines. Conversely, pollen from plants of these important lines is
used to pollinate regenerated plants. A transgenic plant of the
present invention containing a desired polypeptide is cultivated
using methods well known to one skilled in the art.
[0117] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant DNA fragments and
recombinant expression constructs and the screening and isolating
of clones, (see for example, Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor: NY; Maliga et al.
(1995) Methods in ant Molecular Biology, Cold Spring Harbor: NY;
Birren et al. (1998) Genome Analysis: Detecting Genes, 1, Cold
Spring Harbor: NY; Birren et al. (1998) Genome Analysis: Analyzing
DNA, 2, Cold Spring Harbor: NY; Plant Molecular Biology: A
Laboratory Manual, eds. Clark, Springer: NY (1997)).
[0118] In one aspect, the present invention includes protein
products derived from high oleic soybeans.
[0119] The present invention includes a protein product obtained
from high oleic soybeans wherein said product has at least one
characteristic selected from the group consisting of improved
whiteness, reduced gel strength and reduced viscosity when compared
to a soy protein product obtained from a commodity soybean using
the same process as that to obtain the soy protein product from a
high oleic soybean.
[0120] Another embodiment concerns a protein product obtained from
high oleic soybeans, wherein the whiteness index is increased by at
least 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% when compared to a
soy protein product obtained from a commodity soybean using the
same process as that to obtain the soy protein product from a high
oleic soybean.
[0121] An additional embodiment concerns a protein product obtained
from high oleic soybeans, wherein the gel strength is reduced by at
least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
37%, 38%, 39%. 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% when
compared to a soy protein product obtained from a commodity soybean
using the same process as that to obtain the soy protein product
from a high oleic soybean.
[0122] An additional embodiment concerns an unhydrolyzed protein
product obtained from high oleic soybeans, wherein the gel strength
is reduced when compared to a soy protein product obtained from a
commodity soybean using the same process as that to obtain the soy
protein product from a high oleic soybean.
[0123] An additional embodiment concerns an unhydrolyzed protein
product obtained from high oleic soybeans, wherein the gel strength
is reduced by at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,
47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or
60% when compared to a soy protein product obtained from a
commodity soybean using the same process as that to obtain the soy
protein product from a high oleic soybean.
[0124] Yet another embodiment concerns a protein product obtained
from high oleic soybeans, wherein the viscosity is reduced by at
least 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,
47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 58%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, or 87%, when compared to a soy protein product obtained from a
commodity soybean using the same process as that to obtain the soy
protein product from a high oleic soybean.
[0125] One advantage to having reduced viscosity is that it
improves drying efficiency. Currently with commodity soy there is a
limitation in the feed solids concentration that can be fed to the
dryer as a result of the viscosity and propensity to aggregate and
form a gel as a result of exposure to heat. If commodity soy must
be dried at high solids, it is necessary to increase the
temperature of the feed solids to prevent protein aggregation with
resultant gelling; this increased heat is costly and results in
severe damage to the solubility of the protein.
[0126] Reduced viscosity and gel properties allow the operator to
significantly increase the feed solid concentration, because the
slurry can be easily pumped through the equipment at normal
temperatures without gelling. That means that during the drying
process, less water has to be removed for every pound fed to the
dryer. This translates into decreased energy usage and more solids
that can be dried per hour resulting in more protein product for
sale.
[0127] Another embodiment of the invention concerns a soy protein
product selected from the group consisting of a soy protein
isolate, a soy protein concentrate, soy meal, full fat flour,
defatted flour, soymilk, textured proteins, textured flours,
textured concentrates and textured isolates.
[0128] As used herein, "soymilk" refers to an aqueous mixture of
any one or more of the following, finely ground soybeans, soy
flour, soy flakes, soy concentrate, isolated soy protein, soy whey
protein, and aqueous extracts of any one or more of the following,
soybeans, soy flakes and soy flour where insoluble material has
been removed. Soymilk may comprise additional components including
but not limited to fats, carbohydrates, sweeteners, colorants,
stabilizers, thickeners, flavorings, acids, bases.
[0129] One way to prepare soymilk is described below. The
stabilizers (carboxymethylcellulose and carrageenan) are
dry-blended with some sugar and added to 90% water. The mix is
agitated with moderate to high shear for one minute or until no
lumps are observed. Sequestrants agents (potassium citrate, sodium
hexametaphosphate and potassium phosphate) are added mixed for one
minute. The protein is added and dispersed well. The slurry is
heated to 170.degree. F. and hold for 10 minutes. The remaining dry
ingredients are added to the protein slurry and mixed for 5
minutes. The soybean oil is added with constant agitation and mixed
for three minutes. Vitamins and minerals blend is disperse in 10%
water, added to the protein slurry and mixed for 5 minutes. The pH
of the slurry is adjusted to 7.0-7.2 using NaOH as needed. The
slurry is homogenized at 500 psi (second stage) and 2500 psi (first
stage). The slurry is pasteurized by ultra-high temperature (UHT)
processing at 141.degree. C. (286.degree. F.) for 6 seconds. The
mixture is cooled to 31.degree. C. (88.degree. F.) and packaged in
sterilized bottles. The product is stored at refrigerated
temperatures.
[0130] As used herein, "soymilk powder" refers to a dewatered
soymilk. Soymilk may be dewatered by many processes that include
but are not limited to spray drying, tray drying, tunnel drying,
and freeze drying.
[0131] Another embodiment of the invention concerns a method for
improving drying efficiency of a soy protein product, comprising
feeding at least one soy protein product obtained from a high oleic
soybean seed at higher feed solids to a pasteurizer or a dryer
compared to feeding at least one soy protein product obtained from
a commodity soybean to a pasteurizer or dryer.
[0132] An additional embodiment of the invention concerns a method
for improving drying efficiency, comprising feeding high oleic soy
protein products to a pasteurizer or a dryer at no less than 14%,
15%, 15%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,
28%, 29%, or 30% feed solids compared to feeding commodity soy
protein products using the same process as that to obtain the soy
protein product from a high oleic soybean.
[0133] Soy protein products fall into three major groups. These
groups are based on protein content, and range from 40% to over
90%. All three basic soy protein product groups (except full-fat
flours) are derived from defatted flakes. They are the following:
soy flours and grits, soy protein concentrates and soy protein
isolates. These are discussed more fully below.
[0134] As used herein the term "unhydrolyzed protein product",
"unhydrolyzed soy protein product" refers to a protein product that
has not undergone an enzymatic protein hydrolysis step.
[0135] As used herein the term "enzymatic hydrolysis" refers to the
breakdown of proteins or chemical compounds by the addition of
specific enzymes.
[0136] Additional embodiments of the invention include soy protein
products with at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,
49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%,%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% protein
(N.times.6.25) on a moisture-free basis.
[0137] The soy protein products of the invention can be
incorporated into food, beverages, and animal feed.
[0138] The term "animal feed" refers to food that is given to
animals, such as livestock and pets. Some feeds provide a healthy
and nutritious diet, while others may be lacking in nutrients.
Animals are given a wide range of different feeds, but the two
major types of animal feed are processed animal feeds (compound
feed) and fodder.
[0139] Compound feeds are feedstuffs that are blended from various
raw materials and additives. The main ingredients used in
commercially prepared feed are the feed grains, which include corn,
soybeans, sorghum, oats, and barley. These blends are formulated
according to the specific requirements of the target animal
(including different types of livestock and pets). They are
manufactured by feed compounders as meal type, pellets or
crumbles.
[0140] Compound feeds can be complete feeds that provide all the
daily required nutrients, concentrates that provide a part of the
ration (protein, energy) or supplements that only provide
additional micro-nutrients such as minerals and vitamins.
[0141] Oxidation and therefore the shelf life of animal feed
ingredients is a common problem in the industry. Oxidation is an
irreversible chemical reaction in which oxygen reacts with feed and
feed components and can result in decreased animal health and
performance. The negative effects of oxidation can be seen in loss
of palatability, degradation of the oil component, development of
unwanted breakdown products, changes in color, and loss of energy.
Meat obtained from animals grown on oxidized feed has significantly
lower oxidative status compared to animals fed a feed that has not
undergone significant oxidation. Meat from animals fed diets
containing high oleic corn products show extended shelf life and
greater oxidative stability (PCT Publication WO/2006/002052,
published Jan. 5, 2006), particularly when combined with
antioxidants such as tocols. Therefore it is highly desirable to
prevent oxidation of feed and feed ingredients to protect both
nutritional value and organoleptic quality.
[0142] Synthetic antioxidants are used to preserve feed quality by
preventing the oxidation of lipids, which can lead to improved
animal performance. Generally, synthetic antioxidants can act as
free radical scavengers and thereby reduce lipid oxidation.
Synthetic antioxidants can prolong animal feed shelf-life and
protect nutritional and organoleptic quality
[0143] There are multiple methods to test the oxidation status of
solid materials including soybean meal and other soybean protein
products including accelerating aging methods which predict a
material's shelf-life. One test which can be used is to age a
material either at room temperature or elevated temperatures and to
measure the oxidative status of the material at specific time
points. The OSI instrument is useful in this regard in that it
reflects the length of time needed to start the oxidation process
known as the induction time. A longer induction time means that the
material has greater oxidative stability and thereby shelf-life.
Other methods include the measurement of volatiles and color
change.
[0144] Methods for obtaining soy protein products are well known to
those skilled in the art. For example soybean protein products can
be obtained in a variety of ways. Conditions typically used to
prepare soy protein isolates have been described by (Cho, et al,
(1981) U.S. Pat. No. 4,278,597; Goodnight, et al. (1978) U.S. Pat.
No. 4,072,670). Soy protein concentrates are produced 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. 897,574) and Campbell et al. ((1985) in New
Protein Foods, ed. by Altschul and Wilcke, Academic Press, Vol.,
Chapter 10, Seed Storage Proteins, pp 302-338).
[0145] "Soybean-containing products" or "Soy products" can be
defined as those products containing/incorporating a soy protein
product.
[0146] For example, "soy protein products" can include, and are not
limited to, those items listed in Table 2.
TABLE-US-00002 TABLE 2 Soy Protein Products Derived from Soybean
Seeds.sup.a Whole Soybean Products Roasted Soybeans Baked Soybeans
Soy Sprouts Soy Milk Processed Soy Protein Products Full Fat and
Defatted Flours Soy Grits Soy Hypocotyls Soybean Meal Soy Milk Soy
Milk Powder Soy Protein Isolates Specialty Soy Foods/Ingredients
Soy Milk Tofu Tempeh Miso Soy Sauce Hydrolyzed Vegetable Protein
Whipping Protein Soy Protein Concentrates Textured Soy Proteins
Textured Flours and Concentrates Textured Concentrates Textured
Isolates Soy Crisps .sup.aSee Soy Protein Products:
Characteristics, Nutritional Aspects and Utilization (1987). Soy
Protein Council.
[0147] "Processing" refers to any physical and chemical methods
used to obtain the products listed in Table 2 and includes, and is
not limited to, heat conditioning, flaking and grinding, extrusion,
solvent extraction, or aqueous soaking and extraction of whole or
partial seeds. 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).
[0148] Defatted flakes refer to flaked, dehulled cotyledons that
have been defatted and treated with controlled heat to remove the
remaining hexane. This term can also refer to a flour or grit that
has been ground.
[0149] "White" flakes refer to flaked, dehulled cotyledons that
have been defatted and treated with controlled heat to remove the
remaining hexane. This term can also refer to a flour that has been
ground.
[0150] "Grits" refer to defatted, dehulled cotyledons having a U.S.
Standard screen size of between No. 10 and 80.
[0151] "Soy Protein Concentrates" refer to those products produced
from dehulled, defatted soybeans and typically contain 65 wt % to
90 wt % soy protein on a moisture free basis. Soy protein
concentrates are typically manufactured 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).
[0152] As used herein, the term "soy protein isolate" or "isolated
soy protein" refers to a soy protein containing material that
contains at least 90% soy protein by weight on a moisture free
basis.
[0153] "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).
[0154] Residual fatty acid analysis. The commercial process used to
de-fat soy flakes with hexane leaves a residue of fatty acids that
can act as substrate for generation of off-flavor compounds.
Depending on the method of analysis, the residual fat content of
hexane-defatted soy flakes can range from, 0.6-1.0% (W:W) (ether
extractable; AOCS Method 920.39 (Official Methods of Analysis of
the AOAC International (1995), 16.sup.th Edition, Method 920.39C,
Locator #4.2.01 (modified)) to 2.5-3% (W:W) (acid hydrolysable;
AOAC Method 922.06 (Official Methods of Analysis of the AOAC
International (1995), 16.sup.th Edition, Method 922.06, Locator
32.1.13 (modified)). The principle reason for the discrepancy
between these two methods of estimating residual fatty acids is the
chemical nature of the fat classes associated with the protein
matrix after hexane extraction. A small proportion of the residual
fatty acid is in the form of neutral lipid (i.e., triglyceride) and
the remainder is present as polar lipid (e.g., phospholipids,
a.k.a., lecithin). Because of its polar nature the phospholipid is
inaccessible to ether extraction and is only removed from the
protein matrix if acid hydrolysis or some other stringent
extraction protocol is performed. Therefore, the ether extraction
technique gives an estimation of the neutral lipid fraction whereas
the acid hydrolysable method gives a better estimate of the total
residual fatty acid content (i.e., neutral and polar
fractions).
[0155] Both of the AOAC methods described above rely on gravimetric
determinations of the residual fatty acids and, although in
combination they give an indication of the fat classes (neutral vs.
polar), such estimates are crude and are subject to interference
from other hydrophobic materials (e.g. saponins). Further, no
information is obtained on the fatty acid composition and how it
may have been affected by various experimental treatments or by the
genetics of the starting material. AOAC methods for the
determination of the fatty acid composition of residual fatty acids
are available (Official Methods of Analysis of the AOAC
International (2000), 17.sup.th Edition, Method 983.23 Locator
45.4.02, Method 969.33 Locator 41.1.28, Method 996.06 Locator
41.1.28A). These are based on the conversion of residual fatty
acids, extracted by acid hydrolysis, to fatty acid methyl esters
prior to analysis by gas chromatography. Such techniques are rarely
used to assess the residual fatty acid content of food materials in
commercial settings although they are used for fatty acid
evaluations in support of nutritional labeling. A report in which
these methods have been used to determine the residual fatty acid
composition of commercial soy protein isolates has recently been
published (Solina et al. (2005) Volatile aroma components of soy
protein isolate and acid-hydrolysed vegetable protein Food
Chemistry 90: 861-873)
[0156] A facile method for determining the fatty acid composition
of the residual fats in soy protein products is described in
Example 24. The advantage of this method over others is that it
requires no extraction of the residual fats from the matrix prior
to derivatization for GC analysis. Further, the technique is
suitable for all forms of fatty acids i.e., whether they are
initially present as free fatty acids or as fatty acid esters e.g.,
tri-glycerides or phospholipids (Chistie (1989) Gas Chromatography
and Lipids; The Oily Press. Ayr, Scotland). The technique will also
remove fatty acids from the protein matrix even if the polar head
group of the phospholipid is covalently bound to the protein.
[0157] Also, within the scope of this invention are food, food
supplements, food bars, and beverages as well as animal feed (such
as pet foods) that have incorporated therein a soybean protein
product of the invention. The beverage can be in a liquid or in a
dry powdered form.
[0158] The foods to which the soybean protein product of the
invention can be incorporated/added include almost all foods,
beverages and feed (such as pet foods). For example, there can be
mentioned food supplements, food bars, meats such as meat
alternatives, ground meats, emulsified meats, marinated meats, and
meats injected with a soybean protein product of the invention.
Included may be beverages such as nutritional beverages, sports
beverages, protein-fortified beverages, juices, milk, milk
alternatives, and weight loss beverages. Mentioned may also be
cheeses such as hard and soft cheeses, cream cheese, and cottage
cheese. Included may also be frozen desserts such as ice cream, ice
milk, low fat frozen desserts, and non-dairy frozen desserts.
Finally, yogurts, soups, puddings, bakery products, salad
dressings, spreads, and dips (such as mayonnaise and chip dips) may
be included.
[0159] A soy protein product can be added in an amount selected to
deliver a desired amount to a food and/or beverage. The terms
"soybean protein product" and "soy protein product" are used
interchangeably herein.
[0160] Any high oleic soybean seed, whether transgenic or
non-transgenic, can be used as a source of soy protein product.
[0161] Soybeans with decreased levels of saturated fatty acids have
been described resulting from mutation breeding (Erickson et al.
(1994) J. Hered. 79:465-468; Schnebly et al. (1994) Crop Sci.
34:829-833; and Fehr et al. (1991) Crop Sci. 31:88-89) and
transgenic modification (U.S. Pat. No. 5,530,186). Soybeans with
decreased levels of polyunsaturated fatty acids have been described
resulting from mutation breeding and selection. Reduced levels of
linolenic acid have been achieved at relatively constant linoleic
acid (U.S. Pat. No. 5,710,369 and U.S. Pat. No. 5,986,118).
Decreased linoleic and linolenic acids combined have also been
achieved using mutation breeding, genetic crosses and selection
(Rahman, S. M. et al. (2001) Crop Sci. 41:26-29). These methods
produced soybean seeds with oil profiles having linolenic acid
contents of from 1% to 3% of the total fatty acids and total levels
of polyunsaturated fatty acids of about 30 to 35% as compared to
greater than 6% linolenic acid and greater than 50% total
polyunsaturated fatty acids in commodity soybeans.
[0162] The discovery of a method for altering the expression of the
enzymes responsible for introduction of the second (international
patent publication WO 94/11516) and third (international patent
publication WO 93/11245) double bonds into soybean seed storage
lipid in a directed manner has allowed the production of soybeans
with a high mono-unsaturated, very low polyunsaturated fatty acid
content and especially a very low linolenic acid content. The
genetic combination of these two transgene profiles described in
U.S. Pat. No. 6,426,448 leads to a soybean line with minimal
poly-unsaturates and high mono-unsaturates and extreme
environmental stability of the seed fatty acid profile.
[0163] The gene for microsomal delta-12 fatty acid desaturases
described in WO 94/11516, can be used to make a high oleic acid
soybean variety. The resulting high oleic acid soybean variety was
one in which the polyunsaturated fatty acids were reduced from 70%
of the total fatty acids to less than 5%.
[0164] Two soybean fatty acid desaturases, designated FAD2-1 and
FAD2-2, are .DELTA.-12 desaturases that introduce a second double
bond into oleic acid to form linoleic acid, a polyunsaturated fatty
acid. FAD2-1 is expressed only in the developing seed (Heppard et
al. (1996) Plant Physiol. 110:311-319). The expression of this gene
increases during the period of oil deposition, starting around 19
days after flowering, and its gene product is responsible for the
synthesis of the polyunsaturated fatty acids found in soybean oil.
GmFad 2-1 is described in detail by Okuley, J. et al. (1994) Plant
Cell 6:147-158 and in WO94/11516. It is available from the ATCC in
the form of plasmid pSF2-169K (ATCC accession number 69092). FAD
2-2 is expressed in the seed, leaf, root and stem of the soy plant
at a constant level and is the "housekeeping" 12-desaturase gene.
The Fad 2-2 gene product is responsible for the synthesis of
polyunsaturated fatty acids for cell membranes.
[0165] Since FAD2-1 is the major enzyme of this type in soybean
seeds, reduction in the expression of FAD2-1 results in increased
accumulation of oleic acid (18:1) and a corresponding decrease in
polyunsaturated fatty acid content.
[0166] Reduction of expression of FAD2-2 in combination with FAD2-1
leads to a greater accumulation of oleic acid and corresponding
decrease in polyunsaturated fatty acid content.
[0167] FAD3 is a .DELTA.-15 desaturase that introduces a third
double bond into linoleic acid (18:2) to form linolenic acid
(18:3). Reduction of expression of FAD3 in combination with
reduction of FAD2-1 and FAD2-2 leads to a greater accumulation of
oleic acid and corresponding decrease in polyunsaturated fatty acid
content, especially linolenic acid.
[0168] Nucleic acid fragments encoding FAD2-1, FAD2-2, and FAD3
have been described in WO 94/11516 and WO 93/11245. Chimeric
recombinant constructs comprising all or a part of these nucleic
acid fragments or the reverse complements thereof operably linked
to at least one suitable regulatory sequence can be constructed
wherein expression of the chimeric gene results in an altered fatty
acid phenotype. A chimeric recombinant construct can be introduced
into soybean plants via transformation techniques well known to
those skilled in the art.
[0169] Transgenic soybean plants resulting from a transformation
with a recombinant DNA are assayed to select plants with altered
fatty acid profiles. The recombinant construct may contain all or
part of 1) the FAD2-1 gene or 2) the FAD2-2 gene or 3) the FAD3
gene or 4) combinations of all or portions of the FAD2-1, Fad2-2,
or FAD3 genes.
[0170] Recombinant constructs comprising all or part of 1) the
FAD2-1 gene with or without 2) all or part of the Fad2-2 gene with
or without all or part of the FAD3 gene can be used in making a
transgenic soybean plant having a high oleic phenotype. An altered
fatty acid profile, specifically an increase in the proportion of
oleic acid and a decrease in the proportion of the polyunsaturated
fatty acids, indicates that one or more of the soybean seed FAD
genes (FAD2-1, Fad2-2, FAD3) have been suppressed. Assays may be
conducted on soybean somatic embryo cultures and seeds to determine
suppression of FAD2-1, Fad2-2, or FAD3.
[0171] It is well understood by those skilled in the art that
recombinant constructs comprising sequences other than those
specifically exemplified which have similar functions, may be used.
These constructs may include any seed-specific promoter. These
constructs may or may not also include any nucleotides that promote
stem-loop formation. These constructs may contain a polynucleotide
having a nucleotide sequence identical to any portion of the gene
or genes mentioned above inserted in sense or anti-sense
orientation with respect to the promoter. Finally, these constructs
may or may not contain any transcription termination signal.
[0172] Once sufficient transgenic seeds having the desired
phenotype have been obtained, soy protein products such as protein
isolates or whole bean soymilk may be prepared.
EXAMPLES
[0173] The present invention is further defined in the following
Examples, in which 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. Thus, various modifications of the invention
in addition to those shown and described herein will be apparent to
those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the
appended claims.
[0174] The production high oleic soybean lines is described in
detail in Examples 3, 5 and 8, but is not limited to the methods
described therein.
Example 1
Transformation of Soybean (Glycine max)
Embryo Cultures and Regeneration of Soybean Plants.
[0175] Soybean embryogenic suspension cultures are transformed by
the method of particle gun bombardment using procedures known in
the art (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat.
No. 4,945,050; Hazel et al. (1998) Plant Cell. Rep. 17:765-772;
Samoylov et al. (1998) In Vitro Cell Dev. Biol.-Plant 34:8-13). In
particle gun bombardment procedures it is possible to use purified
1) entire plasmid DNA or, 2) DNA fragments containing only the
recombinant DNA expression cassette(s) of interest.
[0176] Stock tissue for transformation experiments are obtained by
initiation from soybean immature seeds. Secondary embryos are
excised from explants after 6 to 8 weeks on culture initiation
medium. The initiation medium is an agar-solidified modified MS
(Murashige and Skoog (1962) Physiol. Plant. 15:473-497) medium
supplemented with vitamins, 2,4-D and glucose. Secondary embryos
are placed in flasks in liquid culture maintenance medium and
maintained for 7-9 days on a gyratory shaker at 26 -/-2.degree. C.
under .about.80 .mu.Em-2s-1 light intensity. The culture
maintenance medium is a modified MS medium supplemented with
vitamins, 2,4-D, sucrose and asparagine. Prior to bombardment,
clumps of tissue are removed from the flasks and moved to an empty
60.times.15 mm petri dish for bombardment. Tissue is dried by
blotting on Whatman #2 filter paper. Approximately 100-200 mg of
tissue corresponding to 10-20 clumps (1-5 mm in size each) are used
per plate of bombarded tissue.
After bombardment, tissue from each bombarded plate is divided and
placed into two flasks of liquid culture maintenance medium per
plate of bombarded tissue. Seven days post bombardment, the liquid
medium in each flask is replaced with fresh culture maintenance
medium supplemented with 100 ng/ml selective agent (selection
medium). For selection of transformed soybean cells the selective
agent used can be a sulfonylurea (SU) compound with the chemical
name, 2-chloro-N-((4-methoxy-6
methyl-1,3,5-triazine-2-yl)aminocarbonyl) benzenesulfonamide
(common names: DPX-W4189 and chlorsulfuron). Chlorsulfuron is the
active ingredient in the DuPont sulfonylurea herbicide, GLEAN.RTM..
The selection medium containing SU is replaced every week for 6-8
weeks. After the 6-8 week selection period, islands of green,
transformed tissue are observed growing from untransformed,
necrotic embryogenic clusters. These putative transgenic events are
isolated and kept in media with SU at 100 ng/ml for another 2-6
weeks with media changes every 1-2 weeks to generate new, clonally
propagated, transformed embryogenic suspension cultures. Embryos
spend a total of around 8-12 weeks in contact with SU. Suspension
cultures are subcultured and maintained as clusters of immature
embryos and also regenerated into whole plants by maturation and
germination of individual somatic embryos.
Example 2
Fatty Acid Analysis of Soybeans
[0177] In order to determine altered fatty acid composition as a
result of suppression of the fatty acid desaturase, the relative
amounts of the fatty acids, palmitic, stearic, oleic, linoleic and
linolenic, can be determined as follows. Fatty acid methyl esters
are prepared from single, mature, somatic soybean embryos or
soybean seed chips by transesterification. One embryo, or a chip
from a seed, is placed in a vial containing 50 .mu.L of
trimethylsulfonium hydroxide and incubated for 30 minutes at room
temperature while shaking. After 30 minutes 0.5 mL of hexane is
added, the sample is mixed and allowed to settle for 15 to 30
minutes to allow the fatty acids to partition into the hexane
phase. Fatty acid methyl esters (5 .mu.L from hexane layer) are
injected, separated, and quantified using a Hewlett-Packard 6890
Gas Chromatograph fitted with an Omegawax 320 fused silica
capillary column (Supelco Inc., Cat#24152). The oven temperature is
programmed to hold at 220.degree. C. for 2.7 minutes, increase to
240.degree. C. at 20.degree. C. per minute, and then hold for an
additional 2.3 minutes. Carrier gas is supplied with a Whatman
hydrogen generator. Retention times were compared to those for
methyl esters of commercially available standards (Nu-Chek Prep,
Inc. catalog #U-99-A).
Example 3
Production of Soybeans with High Levels Oleic Acid and/or High
Levels of Stearic Acid and/or Low Levels of Polyunsaturated Fatty
Acids by Suppression of Fatty Acid Desaturases
[0178] Recombinant DNA fragments were prepared and used in
transformation of soybean for the simultaneous suppression of fatty
acid desaturases FAD2-1 and FAD2-2 and fatty acid desaturase FAD3.
A description of the construction of the recombinant DNA fragments
follows.
A. Recombinant DNA Fragment PHP21676A
[0179] Recombinant DNA fragment PHP21676A contains a gene
expression silencing cassette designed to silence expression of the
FAD2-1 and FAD2-2 genes, and the FAD3 gene, linked in a head to
head configuration to the ALS selectable marker recombinant DNA
fragment of Example 1D below. The nucleotide sequence of
recombinant DNA fragment PHP21676A is shown in SEQ ID NO:1.
Recombinant DNA fragment PHP21676A contains in 5' to 3'
orientation: [0180] a) the complementary strand of the ALS
selectable marker recombinant DNA fragment of Example 1D below,
[0181] b) about 2088 nucleotides of the Kti3 promoter, [0182] c) a
74-nucleotide synthetic sequence, [0183] d) an approximately 1500
polynucleotide fragment comprising about 470 nucleotides from the
soybean FAD2-2 gene, 420 nucleotides from the soybean FAD2-1 gene,
and 643 nucleotides from the soybean FAD3 gene inserted at a unique
Not I restriction endonuclease site, [0184] e) an inverted repeat
of the 74-nucleotide synthetic sequence in c), and [0185] f) about
202 nucleotides of the Kti3 transcription terminator.
[0186] The sequence of the approximately 1500 polynucleotide
fragment of item d) above is shown in SEQ ID NO:2. The
approximately 1500 polynucleotide fragment comprising about 470
nucleotides from the soybean FAD2-2 gene, about 420 nucleotides
from the soybean FAD2-1 gene, about 643 nucleotides from the
soybean FAD3 gene was constructed by PCR amplification as
follows.
[0187] An approximately 0.9 kb DNA fragment, comprising a portion
of the soybean FAD2-2 gene and a portion of the soybean FAD2-1
gene, was obtained by PCR amplification using primers BM35 (SEQ ID
NO:3) and BM39 (SEQ ID NO:4) and using as a template, recombinant
DNA fragment KSFAD2-hybrid, described in Example 1B below.
[0188] An approximately 0.65 kb DNA fragment, comprising a portion
of a FAD3 gene, was obtained by PCR amplification using primers
BM40 (SEQ ID NO:5) and BM41 (SEQ ID NO:6) and using plasmid pXF1 as
template. Plasmid pXF1 comprises a polynucleotide encoding a
soybean delta-15 desaturase (FAD3) and is described in U.S. Pat.
No. 5,952,544 issued on Sep. 14, 1999. Plasmid pXF1 was deposited
with the American Type Culture Collection (ATCC) of Rockville, Md.
on Dec. 3, 1991 under the provisions of the Budapest Treaty, and
bears Accession Number ATCC 68874.
[0189] The approximately 0.9 kb fragment, comprising a portion of
the soybean FAD2-2 gene and a portion of the soybean FAD2-1 gene,
and the approximately 0.65 kb fragment, comprising a portion of a
FAD3 gene, were mixed and used as template for a PCR amplification
with BM35 and BM41 as primers to yield an approximately 1533 bp
fragment that was cloned into the commercially available plasmid
pCR2.1 using the TOPO TA Cloning Kit (Invitrogen).
After digestion with NotI the approximately 1500 bp fragment having
the nucleotide sequence shown in SEQ ID NO:2 was ligated into the
NotI site of plasmid pKS210 (Example 1C below).
B. Recombinant DNA Fragment KSFAD2-Hybrid
[0190] Recombinant DNA Fragment KSFAD2-hybrid contains an
approximately 890 polynucleotide fragment comprising about 470
nucleotides from the soybean FAD2-2 gene and 420 nucleotides from
the soybean FAD2-1 gene. The nucleotide sequence of recombinant DNA
fragment KSFAD2-hybrid is shown in SEQ ID NO:7 Recombinant DNA
Fragment KSFAD2-hybrid was constructed as follows.
[0191] An approximately 0.47 kb DNA fragment comprising a portion
of the soybean FAD2-2 gene was obtained by PCR amplification using
primers KS1 (SEQ ID NO:8) and KS2 (SEQ ID NO:9) and using genomic
DNA purified from leaves of Glycine max cv. Jack as a template.
[0192] An approximately 0.42 kb DNA fragment comprising a portion
of the soybean FAD2-1 gene was obtained by PCR amplification using
primers KS3 (SEQ ID NO:10) and KS4 (SEQ ID NO:11) and using genomic
DNA purified from leaves of Glycine max cv. Jack as a template.
[0193] The 0.47 kb fragment comprising a portion of the soybean
FAD2-2 gene and the 0.42 kb fragment comprising a portion of the
soybean FAD2-1 gene were gel purified using GeneClean (Qbiogene,
Irvine, Calif.), mixed, and used as template for PCR amplification
with KS1 and KS4 as primers to yield an approximately 890 bp
fragment that was cloned into the commercially available plasmid
pGEM-T Easy (Promega, Madison, Wis.) to create a plasmid comprising
recombinant DNA Fragment KSFAD2-hybrid.
C. Preparation of Plasmid pKS210 and Plasmid PHP17731
[0194] Plasmid pKS210 is derived from the commercially available
cloning vector pSP72 (Promega). The beta lactamase coding region
has been replaced by a hygromycin phosphotransferase gene for use
as a selectable marker in E. coli. In addition, a gene expression
silencing cassette linked in a head to head configuration to the
ALS selectable marker recombinant DNA fragment of Example 1B has
been added. The gene expression silencing cassette in plasmid
pKS210 comprises the KTi3 promoter, a 74 nucleotide synthetic
sequence, a unique NotI restriction endonuclease site, an inverted
repeat of the 74 nucleotide synthetic sequence, and the Kti3
terminator region. The gene encoding Kti3 has been described
(Jofuku and Goldberg (1989) Plant Cell 1:1079-1093). The
74-nucleotide synthetic sequences of c) and e) (above) promote
formation of a stem structure. Insertion of a nucleotide fragment
from a desired gene in the unique Not I site has been shown to
result in suppression of the desired gene as described in PCT
Publication WO 02/00904, published 3 Jan. 2002. The nucleotide
sequence of this seed-specific gene expression-silencing cassette
from pKS133 is shown in SEQ ID NO:12. A map of plasmid pKS210 is
shown in FIG. 1 and its nucleotide sequence is disclosed in SEQ ID
NO:13.
[0195] The recombinant plasmid PHP17731, containing gene sequences
for the simultaneous silencing of one of the soybean delta-9
desaturase genes and the soybean delta-12 desaturase gene FAD2-1,
was prepared. The soybean KTI promoter, terminator regions along
with a synthetic inverted repeat sequence were taken from plasmid
KS133 (WO 2002016565A2, A3). A fragment of the FAD2-1 gene was
amplified by PCR using soybean genomic DNA and the sequence in SEQ
ID NO 5 of U.S. Pat. No. 6,372,965 B1 as template to produce the
fragment of base pairs 5423 to 6033 of SEQ ID NO:14 (PHP17731).
Adjacent to that fragment a portion of the coding sequence of copy
3 of the soybean delta-9 desaturase (sequence 1 of WO 2002016565A2,
A3) was placed, which now comprises bases 6054 to 411 of PHP17731.
Fragment PHP17731A (SEQ ID NO:15) was removed from cloning vector
PHP17731 by digestion with restriction endonuclease AscI and
purified as described in section E below. A map of plasmid PHP17731
is shown in FIG. 2.
D. ALS Selectable Marker Recombinant DNA Fragment
[0196] A recombinant DNA fragment comprising a constitutive
promoter directing expression of a mutant soybean acetolactate
synthase (ALS) gene followed by the soybean ALS 3' transcription
terminator was used as a selectable marker for soybean
transformation. The constitutive promoter used is a 1.3-Kb DNA
fragment that functions as the promoter for a soybean
S-adenosylmethionine synthase (SAMS) gene and is described in PCT
publication No. WO 00/37662 published 29 Jun. 2000. The nucleotide
sequence of this recombinant DNA fragment used as a selectable
marker is shown in SEQ ID NO:16. The mutant soybean ALS gene
encodes an enzyme that is resistant to inhibitors of ALS, such as
sulfonylurea herbicides. The deduced amino acid sequence of the
mutant soybean ALS present in the recombinant DNA fragment used as
a selectable marker is shown in SEQ ID NO:17.
[0197] Mutant plant ALS genes encoding enzymes resistant to
sulfonylurea herbicides are described in U.S. Pat. No. 5,013,659.
One such mutant is the tobacco SURB-Hra gene, which encodes a
herbicide-resistant ALS with two substitutions in the amino acid
sequence of the protein. This tobacco herbicide-resistant ALS
contains alanine instead of proline at position 191 in the
conserved "subsequence B" (SEQ ID NO:18) and leucine instead of
tryptophan at position 568 in the conserved "subsequence F" (SEQ ID
NO:19) (U.S. Pat. No. 5,013,659; Lee et al. (1988) EMBO J
7:1241-1248).
[0198] The ALS selectable marker recombinant DNA fragment was
constructed using a polynucleotide for a soybean ALS to which the
two Hra-like mutations were introduced by site directed
mutagenesis. Thus, this recombinant DNA fragment will translate to
a soybean ALS having alanine instead of proline at position 183 and
leucine instead of tryptophan at position 560.
[0199] In addition, during construction of the SAMS promoter-mutant
ALS expression cassette, the coding region of the soybean ALS gene
was extended at the 5'-end by five additional codons, resulting in
five amino acids (M-P-H-N-T; SEQ ID NO:20), added to the
amino-terminus of the ALS protein. These extra amino acids are
adjacent to and presumably removed with the transit peptide during
targeting of the mutant soybean ALS protein to the plastid. A DNA
fragment comprising a polynucleotide encoding the soybean ALS was
digested with KpnI, blunt ended with T4 DNA polymerase, digested
with SaII, and inserted into a plasmid containing the SAMS promoter
which had been previously digested with NcoI and blunt ended by
filling-in with Klenow DNA polymerase.
[0200] A second selectable marker plasmid and subsequent fragment
was prepared by substituting an alternative constitutively
expressed plant promoter for the SAMS promoter described above. The
synthetic promoter SCP1 (U.S. Pat. No. 6,072,050) was placed in
front of the mutant soybean ALS coding sequence to form plasmid
PHP17064 (SEQ ID NO 21 and FIG. 3). For use in soybean
transformation fragment PHP17064A (SEQ ID NO:22) was excised from
its cloning vector using restriction endonuclease XbaI and purified
as described in section E below.
E. Preparation of Recombinant DNA Fragments, PHP21676A, PHP17731A
and PHP17064A, for Soybean Transformation.
[0201] For use in plant transformation experiments, the 7993 bp
recombinant DNA fragment PHP21676A was removed from its cloning
plasmid using restriction endonuclease AscI. Each one of the
recombinant DNA fragments PHP21676A, PHP17731A and PHP17064A was
separated from the remaining plasmid DNA by agarose gel
electrophoresis. Precipitation of the recombinant DNA fragment onto
gold particles and soybean transformation was performed as
described in Example 1. For every eight bombardment
transformations, 30 .mu.l of solution were prepared with 3 mg of
0.6 .mu.m gold particles and 1 to 90 picograms (pg) of DNA fragment
per base pair of DNA fragment.
[0202] Alternatively, mixtures of fragments PHP17064A and PHP17731A
at either equal parts or two parts PHP17731A to PHP17064A were
added to gold particles at the same weight per base pair as
described above and used in transformation to silence the delta-9
and delta-12 desaturase genes.
Example 4
Fatty Acid Analysis of Soybean Transformed with Recombinant DNA
Fragments PHP21676A and with PHP17064A and PHP17731A Combined
[0203] In a soybean transformation experiment using recombinant DNA
fragment PHP21676A as described above, 67 independently transformed
embryogenic suspension cultures found to be resistant to
sulfonylurea herbicide were obtained. An increase in oleic acid as
a percentage of the five major fatty acids, palmitic, stearic,
oleic, linoleic and linolenic, is indicative of suppression of the
FAD2 genes. Thirteen of the 67 herbicide resistant embryogenic
suspension cultures (19%) produced somatic embryos with greater
than 25% oleic acid, compared to about 8% oleic acid for
untransformed embryos.
[0204] Plants were regenerated and T1 seeds were produced from 9 of
the 13 events. Seeds were tested for suppression of fatty acid
desaturases by measuring fatty acid composition of the seed oil as
described in Example 2. Plants derived from 5 transformation events
produced seeds exhibiting the high oleic acid-low polyunsaturated
fatty acid phenotype.
[0205] In a soybean transformation experiment, using the mixture of
recombinant DNA fragments PHP17064A and PHP17731A, transformed
embryogenic suspension cultures found to be resistant to
sulfonylurea herbicide were obtained, screened for the number of
copies of the transgene fragments present by southern analysis and
then by fatty acid profile of the somatic embryo. A rise in the
level of stearic and of oleic acid was taken as indicator of
silencing of the seed expressed delta-9 desaturase and delta-12
desaturase. Thirty-three transformed candidate lines were
regenerated to mature soybean plants and seed from the initial
transformants was analyzed for fatty acid profile. From these lines
further selections were made from seed obtained from selfed plants
in two additional generations. One candidate line was chosen in
which the sum of linoleic and linolenic acid was less than 14% of
total fatty acids and in which the stearic acid content was greater
than 16% of total fatty acids.
Example 5
Genetic Material Used to Produce the High Oleic Trait (Version
1)
[0206] High oleic soybeans were prepared by recombinant
manipulation of the activity of oleoyl 12-desaturase.
[0207] GmFad 2-1 was placed under the control of a strong,
seed-specific promoter derived from the .alpha.'-subunit of the
soybean (Glycine max) .beta.-conglycinin gene. This promoter allows
high level, seed specific expression of the trait gene. It spans
the 606 bp upstream of the start codon of the .alpha.' subunit of
the Glycine max .beta.-congylcinin storage protein. The
.beta.-conglycinin promoter sequence represents an allele of the
published .beta.-conglycinin gene (Doyle et al., (1986) J. Biol.
Chem. 261:9228-9238) having differences at 27 nucleotide positions.
It has been shown to maintain seed specific expression patterns in
transgenic plants (Barker et al., (1988) Proc. Natl. Acad. Sci.
85:458-462 and Beachy et al., (1985) EMBO J. 4:3047-3053).
[0208] The reading frame was terminated with a 3' fragment from the
phaseolin gene of green bean (Phaseolus vulgaris). This is a 1174
bp stretch of sequences 3' of the Phaseolus vulgaris phaseolin gene
stop codon (originated from clone described in Doyle et al.,
1986).
[0209] The GmFad 2-1 open reading frame (ORF) was in a sense
orientation with respect to the promoter so as to produce a gene
silencing of the sense GmFad 2-1 cDNA and the endogenous GmFad 2-1
gene. This phenomenon, known as "sense suppression" is an effective
method for deliberately turning off genes in plants and is
described in U.S. Pat. No. 5,034,323.
[0210] For maintenance and replication of the plasmid in E. coli
the GmFad 2-1 transcriptional unit described above was cloned into
plasmid pGEM-9z(-) (Promega Biotech, Madison Wis., USA).
[0211] For identification of transformed soybean plants the
.beta.-glucuronidase gene (GUS) from E. coli was used. The cassette
used consisted of the three modules; the Cauliflower Mosaic Virus
35S promoter, the .beta.-glucuronidase gene (GUS) from E. coli and
a 0.77 kb DNA fragment containing the gene terminator from the
nopaline synthase (NOS) gene of the Ti-plasmid of Agrobacterium
tumefaciens. The 35S promoter is a 1.4 kb promoter region from CaMV
for constitutive gene expression in most plant tissues (Odell et
al. (1985) Nature 303:810-812), the GUS gene a 1.85 kb fragment
encoding the enzyme .beta.-glucuronidase (Jefferson et al. (1986)
PNAS USA 83:8447-8451) and the NOS terminator a portion of the 3'
end of the nopaline synthase coding region (Fraley et al., (1983)
PNAS US 80:4803-4807). The GUS cassette was cloned into the GmFad
2-1/pGEM-9z(-) construct and was designated pBS43.
[0212] Plasmid pBS43 was transformed into meristems of the elite
soybean line A2396, by the method of particle bombardment as
described in Example 1. Fertile plants were regenerated using
methods well known in the art.
[0213] From the initial population of transformed plants, a plant
was selected which was expressing GUS activity and which was also
positive for the GmFad 2-1 gene (Event 260-05) when evaluated by
PCR. Small chips were taken from a number of R1 seeds of plant
260-05 and screened for fatty acid composition. The chipped seed
was then planted and germinated. Genomic DNA was extracted from the
leaves of the resulting plants and cut with the restriction enzyme
Bam HI. The blots were probed with a phaseolin probe.
[0214] From the DNA hybridization pattern it was clear that in the
original transformation event the GmFad 2-1 construct had become
integrated at two different loci in the soybean genome. At one
locus (Locus A) the GmFad 2-1 construct was causing a silencing of
the endogenous GmFad 2-1 gene, resulting in oleic acid contents as
shown in Table 3. For comparison elite soybean varieties have an
oleic acid content of about 20%. At locus A there were two copies
of pBS43. On the DNA hybridization blot this was seen as two
cosegregating bands. At the other integration locus (Locus B) the
GmFad 2-1 was over-expressing, thus decreasing the oleic acid
content to about 4%.
[0215] Fourth generation segregant lines (R4 plants), generated
from the original transformant, were allowed to grow to maturity.
R4 seeds, which contained only the silencing Locus A (e.g., G94-1)
did not contain any detectable GmFad 2-1 mRNA (when measured by
Northern blotting) in samples recovered 20 days after flowering.
GmFad 2-2 mRNA, although reduced somewhat compared with controls,
was not suppressed. Thus the GmFad 2-1 sense construct had the
desired effect of preventing the expression of the GmFad 2-1 gene
and thus increasing the oleic acid content of the seed. All plants
homozygous for the GmFad 2-1 silencing locus had an identical
Southern blot profile over a number of generations. This indicates
that the insert was stable and at the same position in the genome
over at least four generations.
Example 6
Fatty Acid Analysis High Oleic Trait (Version 1)
[0216] A summary of the oleic acid contents found in the different
generations of recombinant soybean plants and seeds is presented in
Table 7. The Fatty Acid composition was determined as described in
Example 2.
TABLE-US-00003 TABLE 3 Generation Seed Plant ID Planted.sup.a
Analyzed.sup.a Bulk Oleic Acid (%) G253 R0:1 R1:2 84.1% G276 R0:1
R1:2 84.2% G296 R0:1 R1:2 84.1% G313 R0:1 R1:2 83.8% G328 R0:1 R1:2
84.0% G168-187 R1:2 R2:3 84.4% G168-171 R1:2 R2:3 85.2% G168-59-4
R2:3 R3:4 84.0% G168-72-1 R2:3 R3:4 84.1% G168-72-2 R2:3 R3:4 84.5%
G168-72-3 R2:3 R3:4 84.3% G168-72-4 R2:3 R3:4 83.3% .sup.aR0:1
indicates the seed and the plant grown from seed after selfing of
the first generation transformant. R1:2 indicates the seed and the
plant grown from seed after selfing of the second generation
transformant. R2:3 indicates the seed and the plant grown from seed
after selfing of the third generation transformant. R3:4 indicates
the seed and the plant grown from seed after selfing of the fourth
generation transformant.
Example 7
Genetic Material Used to Produce the High Oleic Trait (Version
2)
[0217] A Soybean (Glycine max) event was produced by particle
co-bombardment as described in Example 1 with fragments PHP19340A
(FIG. 4; SEQ ID NO:23) and PHP17752A (FIG. 5; SEQ ID NO:24). These
fragments were obtained by Asc I digestion from a source plasmid.
Fragment PHP19340A was obtained from plasmid PHP19340 (FIG. 6; SEQ
ID NO:25) and fragment PHP17752A was obtained from plasmid PHP17752
(FIG. 7; SEQ ID NO:26). The PHP19340A fragment contains a cassette
with a 597 bp fragment of the soybean microsomal omega-6 desaturase
gene 1 (gm-fad2-1) (Heppard et al., 1996, Plant Physiol. 110:
311-319).
[0218] The presence of the gm-fad2-1 fragment in the expression
cassette acts to suppress expression of the endogenous omega-6
desaturases, resulting in an increased level of oleic acid and
decreased levels of palmitic, linoleic, and linolenic acid levels.
Upstream of the gm-fad2-1 fragment is the promoter region from the
Kunitz trypsin inhibitor gene 3 (KTi3) (Jofuku and Goldberg, 1989,
Plant Cell 1: 1079-1093; Jofuku et al., 1989, Plant Cell 1:
427-435) regulating expression of the transcript. The KTi3 promoter
is highly active in soy embryos and 1000-fold less active in leaf
tissue (Jofuku and Goldberg, 1989, Plant Cell 1: 1079-1093). The 3'
untranslated region of the KTi3 gene (KTi3 terminator) (Jofuku and
Goldberg, 1989, Plant Cell 1: 1079-1093) terminates expression from
this cassette.
[0219] The PHP17752A fragment contains a cassette with a modified
version of the soybean acetolactate synthase gene (gm-hra) encoding
the GM-HRA protein with two amino acid residues modified from the
endogenous enzyme and five additional amino acids at the N-terminal
region of the protein derived from the translation of the soybean
acetolactate synthase gene 5' untranslated region (Falco and Li,
2003, US Patent Application: 2003/0226166). The gm-hra gene encodes
a form of acetolactate synthase, which is tolerant to the
sulfonylurea class of herbicides. The GM-HRA protein is comprised
of 656 amino acids and has a molecular weight of approximately 71
kDa.
[0220] The expression of the gm-hra gene is controlled by the 5'
promoter region of the S-adenosyl-L-methionine synthetase (SAMS)
gene from soybean (Falco and Li, 2003, US Patent Application:
2003/0226166). This 5' region consists of a constitutive promoter
and an intron that interrupts the SAMS 5' untranslated region
(Falco and Li, 2003). The terminator for the gm-hra gene is the
endogenous soybean acetolactate synthase terminator (als
terminator) (Falco and Li, 2003, US Patent Application:
2003/0226166).
Example 8
Transformation and Selection for the Soybean High Oleic Event
(Version 2)
[0221] For transformation of soybean tissue, a linear portion of
DNA, containing the gm-fad2-1 gene sequence and the regulatory
components necessary for expression, was excised from the plasmid
PHP19340 through the use of the restriction enzyme Asc I and
purified using agarose gel electrophoresis. A linear portion of
DNA, containing the gm-hra gene sequences and the regulatory
components necessary for expression, was excised from the plasmid
PHP17752 through the use of the restriction enzyme Asc I and
purified using agarose gel electrophoresis. The linear portion of
DNA containing the gm-fad2-1 gene is designated insert PHP19340A
and is 2924 bp in size. The linear portion of DNA containing the
gm-hra gene is designated insert PHP17752A and is 4511 bp in size.
The only DNA introduced into transformation event DP-305423-1 was
the DNA of the inserts described above.
[0222] The transgenic plants from event DP-305423-1 were obtained
by microprojectile bombardment as described in Example 1.
Embryogenic tissue samples were taken for molecular analysis to
confirm the presence of the gm-fad2-1 and gm-hra transgenes by
Southern analysis. Plants were regenerated from tissue derived from
each unique event and transferred to the greenhouse for seed
production.
Example 9
Southern Analysis of Plants Containing the High Oleic Event Version
2
[0223] Materials and Methods: Genomic DNA was extracted from frozen
soybean leaf tissue of individual plants of the T4 and T5
generations of DP-305423-1 and of control (variety: Jack) using a
standard Urea Extraction Buffer method. Genomic DNA was quantified
on a spectrofluorometer using Pico Green.RTM. reagent (Molecular
Probes, Invitrogen). Approximately 4 .mu.g of DNA per sample was
digested with Hind III or Nco I. For positive control samples,
approximately 3 pg (2 genome copy equivalents) of plasmid PHP19340
or PHP17752 was added to control soybean genomic DNA prior to
digestion. Negative control samples consisted of unmodified soybean
genomic DNA (variety: Jack). DNA fragments were separated by size
using agarose gel electrophoresis.
[0224] Following agarose gel electrophoresis, the separated DNA
fragments were depurinated, denatured, neutralized in situ, and
transferred to a nylon membrane in 20.times.SSC buffer using the
method as described for TURBOBLOTTER.TM. Rapid Downward Transfer
System (Schleicher & Schuell). Following transfer to the
membrane, the DNA was bound to the membrane by UV crosslinking.
[0225] DNA probes for gm-fad2-1 and gm-hra were labeled with
digoxigenin (DIG) by PCR using the PCR DIG Probe Synthesis Kit
(Roche).
[0226] Labeled probes were hybridized to the target DNA on the
nylon membranes for detection of the specific fragments using DIG
Easy Hyb solution (Roche) essentially as described by manufacturer.
Post-hybridization washes were carried out at high stringency.
DIG-labeled probes hybridized to the bound fragments were detected
using the CDP-Star Chemiluminescent Nucleic Acid Detection System
(Roche). Blots were exposed to X-ray film at room temperature for
one or more time points to detect hybridizing fragments. The fatty
Acid composition of the event was determined as described in
Example 2. Oleic acid levels determined in 29 different events (T1
generation) ranged from 61.5-84.6%. Oleic acid level from one event
(T4-T5 generation) ranged from 72-82%.
Example 10
Small Scale Soy Protein Isolate Preparation
[0227] Soy protein isolate preparation is performed as described
below.
a) Production of Yellow Flake:
[0228] Full fat soy flake is prepared in the following manner. A
volume of soybeans is placed in a closed container, with a small
amount of water to prevent drying of the beans during subsequent
microwave heating. Soybeans are heated in a microwave until the
temperature reaches 150.degree. F. and then held for 1 minute. The
beans are quickly cooled to room temperature in a fluid bed cooler
for about 1 minute. The soybeans are then fed through a cracker to
produce 1/2 and 1/4 cracks. Hulls are removed in an aspirator and
the resulting "meats" carried forward to produce flakes. The meats
are placed in a sealed container with a small amount of water and
heated in a microwave until the temperature reaches 150.degree. F.
and then held for 1 minute. The hot meats are then fed through a
flaker to produce soybean flakes that are then cooled quickly to
room temperature in a fluid bed cooler for about 1 minute. Flakes
with a high Protein Dispersibility Index (PDI) are produced with
sufficient character for oil removal by solvent extraction. Flakes
with lower PDI are produced by increasing the amount of water,
temperature and time of exposure during production.
b) Production of White Flake:
[0229] White flake may be produced by contacting yellow flake with
hexane to remove oil. In addition to hexane, flakes are extracted
solely, or in combination with, other solvent systems that have
some degree of oil solubility such as ethanol, ethanol water
mixtures, hexane ethanol mixtures, supercritical CO2 ethanol water
mixtures, etc. Yellow flake is loaded into a batch or a semi
continuous extractor at a solvent:flake ratio, temperature, and
extraction time number, sufficient to remove oil. In a batch
extractor, hexane warmed to 60.degree. C. is added at a 3:1
solvent:flake ratio and circulated through a bed of flake for 45
minutes. The used solvent miscella is removed and the solvent
extraction procedure described above is repeated. The flakes are
given a final one to one rinse with fresh solvent. The semi
continuous extractor uses approximately the same solvent to flake
ratio but fresh solvent is continuously regenerated through the use
of a solid/liquid in-vessel filter followed by vaporization of the
solvent from the oils and recycle of the condensate back to the
extractor. This semi continuous extractor is used to generate any
number of solvent turnovers. In either apparatus, the resulting
hexane-laden white flake is allowed to air dry in a fume hood
overnight. If desired, commercial steam treatment during
desolventization is simulated by adding water to the flake
(typically 5-10% dry flake basis), and placing the wetted flake in
a sealed container and heating for 6 minutes at 100.degree. C. in
the microwave. The hot flakes are then placed in a vacuum oven and
quickly cooled to about 50.degree. C. to produce high PDI flake.
Increasing the amount of water, time, or temperature during this
step produced low PDI flakes. Flakes are milled into flour to a
particle size suitable for efficient protein extraction or this
step may be skipped entirely.
c) Production of Wet Curd:
[0230] A quantity of soy flour is extracted with water (may be
warmed, typically 33.degree. C.) at a water to flour ratio at least
sufficient to make a movable slurry (typically 6:1) in a vessel
capable of imparting good water flake contact and/or further flour
grinding capability (typically a colloid mill). If desired, the
extraction pH may be increased with a base (typically Ca(OH).sub.2
up to a pH of about 9.7) or decreased with an acid to a pH of about
2.0. Defoaming agents at a quantity sufficient to prevent foaming
(typically less than 1% on a flake basis) and sulfite
(Na.sub.2SO.sub.3, typically less than 1% on a flake basis) may be
added at this point to aid extraction. The extract is mixed
(typically in a colloid mill) for approximately 10-15 minutes. The
slurry is fed into a centrifuge (either batch or semi continuous)
at rpm's and time sufficient to separate solids (typically above
Log 4.0 Gsec. at 33.degree. C.). The liquid is decanted and the
solids re-extracted at a water to solids ratio at least sufficient
to make a movable slurry (typically 4 to 1). The slurry (typically
33.degree. C.) is mixed (typically in a colloid mill) and separated
in a centrifuge as described above. If desired, the pH of the
second extract may also be increased or decreased at this point.
Following centrifugation, the liquid is decanted and the spent
flake discarded. The first and second liquid extracts are combined
and carried forward. Additional extractions can be done if desired.
To precipitate the protein, the pH is adjusted to a pH sufficient
to separate the proteins of interest (typically to 4.5) with an
acid (typically 1M HCl) and fed into a semi-continuous or batch
centrifuge at the conditions described above. The liquid is
decanted and discarded and the solids resuspended with fresh water
(typically in a homogenizer). The re-slurry water may be warmed if
desired (typical wash water temperature is about 50-60.degree. C.).
The wash described above may be repeated if desired.
d) Production of Isolated Soy Protein:
[0231] The wet curd is re-suspended to a solids content suitable
for pasteurization of the protein slurry. Typically, the solids
content will be approximately 10-20%. The slurry is mixed
(typically in a colloid mill or homogenizer) and the pH adjusted
with a base (typically NaOH) to approximately 6.8-7.2. The slurry
is pasteurized continuously with steam injection at a temperature
and time sufficient to reduce microbial counts and trypsin
inhibitor activity to safe levels for human ingestion. Typical
conditions may be approximately 120-160.degree. C. for 4-60
seconds. The pasteurized slurry is cooled (typically flash cooled
to 50-60.degree. C. by use of 100-150 mm vacuum). The slurry is fed
into a spray drier at conditions necessary to achieve a dry product
of less than about 5% moisture. Typical conditions include an inlet
temperature of about 250-300.degree. C. and an outlet temperature
of about 90-100.degree. C.
[0232] The conditions set forth in this examples comprise the
process parameters that are used to produce the Supro.RTM. 760,
Supro.RTM. 670 and Supro.RTM. 500E-type protein isolates in the
small scale production platform.
Example 11
Large Scale Defatted Flake Production
[0233] The soy flake material may be formed from soybeans according
to the following process. The soybeans are detrashed by passing the
soybeans through a magnetic separator to remove iron, steel, and
other magnetically susceptible objects, followed by shaking the
soybeans on progressively smaller meshed screens to remove soil
residues, pods, stems, weed seeds, undersized beans, and other
trash. The detrashed soybeans are then cracked by passing the
soybeans through cracking rolls. Cracking rolls are spiral-cut
corrugated cylinders which loosen the hull as the soybeans pass
through the rolls and crack the soybean material into several
pieces. Preferably the cracked soybeans are conditioned to 10% to
11% moisture at 63 to 74.degree. C. to improve the storage quality
retention of the soybean material. The cracked soybeans are then
dehulled, preferably by aspiration. Soy hypocotyls, which are much
smaller than the cotyledons of the soybeans, may be removed by
shaking the dehulled soybeans on a screen of sufficiently small
mesh size to remove the hypocotyls and retain the cotyledons of the
beans. The hypocotyls need not be removed since they comprise only
about 2%, by weight, of the soybeans while the cotyledons comprise
about 90% of the soybeans by weight, however, it is preferred to
remove the hypocotyls since they are associated with the beany
taste of soybeans. The dehulled soybeans, with or without
hypocotyls, are then flaked by passing the soybeans through flaking
rolls. The flaking rolls are smooth cylindrical rolls positioned to
form flakes of the soybeans as they pass through the rolls having a
thickness of from about 0.01 inch to about 0.015 inch.
[0234] The flakes are then defatted. The flakes are defatted by
extracting the flakes with a suitable solvent to remove the oil
from the flakes. Preferably the flakes are extracted with n-hexane
or n-heptane in a countercurrent extraction. The defatted flakes
should contain less than 1.5% fat or oil content, and preferably
less than 0.75%. The solvent-extracted defatted flakes are then
desolventized to remove any residual solvent using conventional
desolventizing methods, including desolventizing with a flash
desolventizer-deodorizer stripper, a vapor desolventizer-vacuum
deodorizer, or desolventizing by down-draft desolventization.
[0235] Preferably, the defatted flakes are comminuted into a soy
flour or a soy grit to improve the protein extraction yield from
the flakes. The flakes are comminuted by grinding the flakes to the
desired particle size using conventional milling and grinding
equipment such as a hammer mill or an air jet mill. Soy flour has a
particle size wherein at least 97%, by weight, of the flour has a
particle size of 150 microns or less (is capable of passing through
a No. 100 mesh U.S. Standard Screen). Soy grits, more coarsely
ground than soy flour, have a particle size greater than soy flour
but smaller than soy flakes. Preferably the soy grit has a particle
size of from 150 microns to about 1000 microns (is capable of
passing though a No. 10-No. 80 U.S. Standard Screen).
Example 12
Large Scale Soy Protein Isolate Preparation
[0236] To produce the soy protein curd material, the HO defatted
soy flour is extracted with water or an aqueous solution having a
pH of from 6.5 to 10 to extract the protein in the flour from
insoluble materials such as fiber. The soy flour is preferably
extracted with an aqueous sodium hydroxide solution having a pH
from about 8 to about 10, although other aqueous alkaline
extractants such as ammonium hydroxide are also effective.
Preferably the weight ratio of the extractant to the soy flour
material is from about 8:1 to about 16:1.
[0237] After extraction, the extract is separated from the
insoluble materials. Preferably the separation is effected by
filtration or by centrifugation and separation of the extract from
the insoluble materials. The pH of the separated extract is then
adjusted to about the isoelectric point of soy protein to
precipitate a soy protein curd so that the soy protein can be
separated from soy solubles including flatulence inducing
oligosaccharides and other water soluble carbohydrates. The pH of
the separated extract is adjusted with a suitable acid to the
isoelectric point of soy protein, preferably to a pH of from about
pH 4 to about pH 5, most preferably from about pH 4.4 to about pH
4.6. Suitable edible acids for adjusting the pH of the extract to
about the isoelectric point of soy protein include hydrochloric
acid, phosphoric acid, sulfuric acid, nitric acid, or acetic acid.
The protein material is precipitated preferably with hydrochloric
acid or phosphoric acid. The precipitated protein material (curd)
is separated from the extract (whey), preferably by centrifugation
or filtration to produce the soy protein curd material. The
separated soy protein curd material is preferably washed with water
to remove residual solubles, preferably at a weight ratio of water
to protein material of about 4:1 to about 10:1. The conditions set
forth in examples 11 and 12 describe essentially the process
parameters that are used to produce the Supro.RTM. 760, Supro.RTM.
1610, Supro.RTM. 651, Supro.RTM. 500E and Supro.RTM. 670-type
protein isolates in the large scale production platform. A detailed
description of the production of Supro.RTM. 760 is described in
Examples 13 and 14.
[0238] The production of Supro.RTM. 670 includes a hydrolization
step that was not applied in the production of the other isolates
and is described In Examples 16-18.
Example 13
Process for Solae Supro.RTM. 760 Type Protein from Defatted HO
Flours
[0239] To produce the Supro.RTM. 760 type protein material, the soy
protein curd material produced as described in Example 12 is first
neutralized to a pH of 6.8 to 7.2 with an aqueous alkaline solution
or an aqueous alkaline earth solution, preferably a sodium
hydroxide solution or a potassium hydroxide solution. The
neutralized soy protein curd material is then heated. Preferably
the neutralized soy curd is heated at a temperature of from about
75.degree. C. to about 160.degree. C. for a period of from about 2
seconds to about 2 hours, where the curd is heated for a longer
time period at lower temperatures and a shorter period at higher
temperatures. More preferably the soy protein curd material is
treated at an elevated temperature and under a positive pressure
greater than atmospheric pressure.
[0240] The preferred method of heating the soy protein curd
material is treating the soy curd at a temperature elevated above
ambient temperatures by injecting pressurized steam into the curd,
hereafter referred to as "jet-cooking." The following description
is a preferred method of jet-cooking the soy protein curd material,
however, the invention is not limited to the described method and
includes any obvious modifications which may be made by one skilled
in the art.
[0241] The soy protein curd material is introduced into a
jet-cooker feed tank where the soy curd is kept in suspension with
a mixer which agitates the soy curd. The curd is directed from the
feed tank to a pump which forces the curd through a reactor tube.
Steam is injected into the curd under pressure as the curd enters
the reactor tube, instantly heating the curd to the desired
temperature. The temperature is controlled by adjusting the
pressure of the injected steam, and preferably is from about
75.degree. C. to about 160.degree. C., more preferably from about
100.degree. C. to about 155.degree. C. The curd is treated at the
elevated temperature for treatment time being controlled by the
flow rate of the slurry through the tube. Preferably the flow rate
is about 18.5 lbs./minute, and the cook time is about 9 seconds at
about 150.degree. C.
[0242] To produce the protein material of the present invention the
heated neutralized curd is then cooled and dried. The curd may be
cooled and dried in any conventional manner known in the art. In a
preferred embodiment of the present invention, the curd is cooled
by flash vaporization. The heated curd is flash vaporized by
introducing the hot curd into a vacuumized chamber having an
internal temperature of from 20.degree. C. to 85.degree. C., which
instantly drops the pressure about the curd to a pressure of from
about 25 mm to about 100 mm Hg, and more preferably to a pressure
of from about 25 mm Hg to about 30 mm Hg. Most preferably the hot
curd is discharged from the reactor tube of the jet-cooker into the
vacuumized chamber, resulting in an instantaneous large pressure
and temperature drop which vaporizes a substantial portion of water
from the curd, instantly cooling the curd to a temperature.
Preferably the vacuumized chamber has an elevated temperature up to
about 85.degree. C. to prevent the gelation of the soy protein curd
material upon introduction of the curd into the vacuumized chamber.
The heat treatment under pressure followed by the rapid pressure
drop and vaporization of water also causes vaporization of
substantial amounts of the volatile components from the soy
material, and thereby improving the taste of the soy material.
[0243] The flash vaporized protein material may then be dried,
preferably by spray drying. Preferably the spray-dryer is a
co-current flow dryer where hot inlet air and the structural
protein material, atomized by being injected into the dryer under
pressure through an atomizer, pass through the dryer in a
co-current flow.
[0244] In a preferred embodiment, the protein material is injected
into the dryer through a nozzle atomizer. Although a nozzle
atomizer is preferred, other spray-dry atomizers, such as a rotary
atomizer, may be utilized. The curd is injected into the dryer
under enough pressure to atomize the slurry. Preferably the slurry
is atomized under a pressure of about 3000 psig to about 5500 psig,
and most preferably about 3500 to 5000 psig. Hot air is injected
into the dryer through a hot air inlet located so the hot air
entering the dryer flows co-currently with the atomized soy curd
sprayed from the atomizer. The hot air has a temperature of about
285.degree. C. to about 315.degree. C., and preferably has a
temperature of about 290.degree. C. to about 300.degree. C.
[0245] The dried soy protein material is collected from the spray
dryer. Conventional means and methods may be used to collect the
soy material, including cyclones, bag filters, electrostatic
precipitators, and gravity collection.
Example 14
Production of the Supro.RTM. 760-Type High Oleic Soy Protein
Isolate
[0246] The SUPRO.RTM. 760-type High Oleic Soy Protein Isolate was
produced from High Oleic Soybeans according to the following
process. 100 lbs of defatted High Oleic soybean flakes were placed
in an extraction tank and extracted with 1,000 lbs of water heated
to 32.degree. C. This provided a weight ratio of water to flakes of
10:1. The flakes were separated from the extract and re-extracted
with 600 lbs of water heated to 32.degree. C. and having sufficient
calcium hydroxide added to adjust the pH to 9.7. This second
extraction step provided a weight ratio of water to flakes of 6:1.
The flakes were removed by centrifugation, and the first and second
extracts were combined and adjusted to a pH of 4.5 with
hydrochloric acid to precipitate a protein curd. The acid
precipitated curd was separated from the extract by centrifugation,
leaving aqueous whey (discarded), and then was washed with water in
a weight amount of seven times that of the starting flake material
to provide an isoelectric protein isolate.
[0247] To produce the SUPRO.RTM. 760-type High Oleic protein
material, water was added to the bioelectric soy protein material
and the pH was adjusted to between 6.9 and 7.3 with an aqueous
solution of sodium hydroxide to produce a neutralized soy protein
slurry and then heated by injecting pressurized steam into the
slurry, hereafter referred to as "jet-cooking." The neutral soy
protein slurry was introduced into a jet-cooker feed tank where it
was kept in suspension with a mixer that agitated the slurry. The
slurry was directed from the feed tank to a pump that forced the
slurry through a reactor tube. Steam was injected into the slurry
under pressure as the slurry entered the reactor tube, instantly
heating the slurry to the desired temperature. The temperature was
controlled by adjusting the pressure of the injected steam. The
heat treatment was about 9 seconds at about 150.degree. C. The
heated neutral slurry was then cooled by flash vaporization, which
vaporized a substantial portion of water from the hot neutral
protein slurry, instantly cooling the neutral protein material.
[0248] The cooled neutral protein slurry was then homogenized and
transferred to a spray dryer wherein most of the moisture was
evaporated by the addition of heat to achieve the final SUPRO.RTM.
760-type soy protein isolate.
Example 15
Process of Production of Solae Supro.RTM. 760 Type Protein from
Defatted HO Flours
[0249] To produce the Supro.RTM. 760-type protein product of the
present invention, 60 lbs of HO soy flour were extracted with 600
lbs of 32.degree. C. water at a 10:1 water to flour ratio in a
mixer which agitates the soy flour for good water-flake contact.
Defoaming agent was added at a quantity sufficient to prevent
foaming and 54.48 g sulfite (Na.sub.2SO.sub.3) was added at this
point. Slurry pH was 6.6. The slurry was mixed 10 minutes. The
slurry was fed into a batch centrifuge at 12.2 lb/min at 8.1%
solids to separate solids from liquid. The liquid was decanted and
the solids were returned to the mixing vessel, and re-extracted at
a 5:1 water to flake ratio at 32.degree. C. The pH of the slurry
was increased with NaOH to 9.7 and the slurry was mixed for 10 min
and separated in a batch centrifuge at 12.2 lb/min with 3.9% feed
solids. Following centrifugation the liquid was decanted and the
spent flake discarded. The first and second liquid extracts were
combined and carried forward. To precipitate the protein, the pH
was adjusted to 4.5 with HCl and held for 10 minutes. The material
was fed into a batch centrifuge at 25 lb/min, 6.0% solids and
55.degree. C. to separate liquid whey from curd. The liquid was
decanted and discarded and the solids reslurried with fresh water
in a 7:1 water to flake ratio and passed through a Dispax grinder
to ensure effective washing. The ground slurry was fed into a batch
centrifuge at 25 lb/min at 59.degree. C. to separate the liquid
wash from the curd. The wet curd was resuspended with water to
11.3% solids suitable for pasteurization of the protein slurry. The
pH of the slurry was increased with NaOH to a pH of 7.1 and the
slurry was homogenized at 550 psig. The slurry was introduced into
a jet-cooker feed tank where the soy curd was kept in suspension
with a mixer which agitated the soy curd. The curd slurry was
directed from the feed tank to a pump which forced the curd through
a reactor tube 0.94 inches in diameter and 33 inches long. Steam
was injected into the curd under pressure as the curd entered the
reactor tube, instantly heating the curd to the desired
temperature. The temperature was controlled by adjusting the
pressure of the injected steam and was 149.degree. C. The curd was
treated at the elevated temperature for 9 seconds. The heated curd
was then cooled and dried. The curd was cooled by flash
vaporization. The heated curd was flash vaporized by introducing
the hot curd into a vacuumized chamber having an internal
temperature of 63.degree. C. which instantly dropped the pressure
to 26 mm Hg. The instantaneous large pressure and temperature drop
vaporized a substantial portion of water from the curd, instantly
cooling the curd to 68.degree. C.
[0250] The flash vaporized protein material was spray dried using a
co-current flow dryer where hot inlet air and protein material was
atomized by being injected into the dryer under pressure through an
atomizer and passed through the dryer in a co-current flow.
[0251] The curd slurry was fed into a homogenizer and homogenized
at 1500 psig at 57.degree. C. The homogenized slurry was injected
into the dryer through a nozzle atomizer at an atomization pressure
of 4000 psig. Hot air was injected into the dryer through a hot air
inlet located so the hot air entering the dryer flowed co-currently
with the atomized soy curd sprayed from the atomizer. The hot air
had a temperature of 265.degree. C. The dryer outlet temperature
was 89.degree. C.
[0252] The dried High Oleic Supro.RTM.760-type isolated soy protein
was collected by gravity collection from the outlet of the spray
dryer.
Example 16
Process for Production of Solae Supro.RTM. 670 Type Protein from
Defatted HO Flours Including an Enzymatic Hydrolization Step
[0253] The Supro.RTM. 670 type protein material is formed from the
soy protein curd material in much the same manner as the
Supro.RTM.670 protein material described in Example 15, however, an
enzymatic protein hydrolysis step is included to hydrolyze the
protein. The soy protein curd material is first diluted to about
12-15% solids and neutralized to a pH of from 7.5 to 8.1 with an
aqueous alkaline solution or an aqueous alkaline earth solution,
preferably a sodium hydroxide solution or a potassium hydroxide
solution. The neutralized soy protein curd is heated and cooled,
preferably by jet cooking and flash cooling, in the same manner as
described above with respect to preparation of the Supro.RTM. 760
protein material. Preferably the curd is cooled to 55.degree. C. to
60.degree. C. after heating.
[0254] After the flash cooling, an enzyme (Bromelain) having an
activity of about 2400 TU/g is added to the solution at about a
0.02% based on curd solids. The enzyme treated solution is allowed
to react for about 15 minutes to 65 minutes preferably 20-45
minutes under continuous mixing. The hydrolysis is terminated by
heating the hydrolyzed soy protein curd material to a temperature
effective to inactivate the enzyme. Most preferably the hydrolyzed
soy protein curd material is jet cooked to inactivate the enzyme,
and flash cooled then dried as described above with respect to
producing the dried Supro.RTM.760 protein material. The dried
hydrolyzed material is the dried Supro.RTM. 670 protein
material.
Example 17
Production of the Supro.RTM. 670-Type High Oleic Soy Protein
Isolate
[0255] The SUPRO.RTM. 670-type high oleic soy protein isolate was
prepared essentially as described in Example 16, but with the
following experimental details. HO soy flour (60 lbs) was extracted
with 600 lbs of 41.degree. C. water at a 10:1 water to flour ratio
in a mixer which agitates the soy flour for good water-flake
contact. Defoaming agent was added at a quantity sufficient to
prevent foaming and 54.48 g sulfite (Na.sub.2SO.sub.3) was added at
this point. Slurry pH was 6.5. The slurry was mixed 10 minutes. The
slurry was fed into a batch centrifuge at 10.0 lb/min at 8.2%
solids to separate solids from liquid. The liquid was decanted and
the solids returned to the mixing vessel, and re-extracted at a 5:1
water to flake ratio at 33.degree. C. The pH of the slurry was
increased with NaOH to 9.7 and the slurry was mixed for 10 min and
separated in a batch centrifuge at 12.0 lb/min with 3.8% feed
solids. Following centrifugation the liquid was decanted and the
spent flake discarded. The first and second liquid extracts were
combined and carried forward. To precipitate the protein, the pH
was adjusted to 4.5 with HCl, held for 10 minutes. The material was
fed into a batch centrifuge at 24 lb/min, 6.0.0% solids and
57.degree. C. to separate liquid whey from curd. The liquid was
decanted and discarded and the solids reslurried with fresh water
in a 7:1 water to flake ratio and passed through a Dispax grinder
to ensure effective washing. The ground slurry was fed into a batch
centrifuge at 14 lb/min at 56.degree. C. to separate the liquid
wash from the curd. The wet curd was resuspended with water to
11.3% solids suitable for pasteurization of the protein slurry. The
pH of the slurry was increased with NaOH to a pH of 7.7 and the
slurry was homogenized at 550 psig. The slurry was introduced into
a jet-cooker feed tank where the soy curd was kept in suspension
with a mixer which agitated the soy curd. The curd slurry was
directed from the feed tank to a pump which forced the curd through
a reactor tube 0.94 inches in diameter and 33 inches long. Steam
was injected into the curd under pressure as the curd entered the
reactor tube, instantly heating the curd to the desired
temperature. The temperature was controlled by adjusting the
pressure of the injected steam and was 129.degree. C. The curd was
treated at the elevated temperature for 9 seconds. The heated curd
was then cooled and dried. The curd was cooled by flash
vaporization. The heated curd was flash vaporized by introducing
the hot curd into a vacuumized chamber having an internal
temperature of 63.degree. C. which instantly dropped the pressure
to 23 mm Hg. The instantaneous large pressure and temperature drop
vaporized a substantial portion of water from the curd, instantly
cooling the curd to 70.degree. C.
[0256] After the flash cooling, an enzyme (Bromelain) having an
activity of about 2400 TU/g was added to the solution at a 0.03%
based on curd solids. The enzyme treated solution was allowed to
react for 35 minutes under continuous mixing. The hydrolysis was
terminated by heating the hydrolyzed soy protein curd material to a
temperature effective to inactivate the enzyme. The hydrolyzed soy
protein curd material was jet cooked at 142.degree. C. for 9
seconds to inactivate the enzyme, and flash cooled. The heated curd
was flash vaporized by introducing the hot curd into a vacuumized
chamber having an internal temperature of 61.degree. C. which
instantly dropped the pressure to 25 mm Hg. The instantaneous large
pressure and temperature drop vaporized a substantial portion of
water from the curd, instantly cooling the curd to 61.degree.
C.
[0257] The flash vaporized protein material was homogenized and
spray dried using a co-current flow dryer where hot inlet air and
protein material were atomized by being injected into the dryer
under pressure through an atomizer and passed through the dryer in
a co-current flow.
[0258] The curd slurry was fed into a homogenizer and homogenized
at 2000 psig at 54.degree. C. The homogenized slurry was injected
into the dryer through a nozzle atomizer at an atomization pressure
of 4000 psig. Hot air was injected into the dryer through a hot air
inlet located so the hot air entering the dryer flowed co-currently
with the atomized soy curd sprayed from the atomizer. The hot air
had a temperature of 307.degree. C. The dryer outlet temperature
was 93.degree. C.
[0259] The dried hydrolyzed High Oleic Supro.RTM.670-type isolated
soy protein was collected by gravity collection from the outlet of
the spray dryer.
Example 18
Production of the Supro.RTM. 670-Type High Oleic Soy Protein
Isolate
[0260] The SUPRO.RTM. 670-type High Oleic Soy Protein Isolate was
produced from High Oleic Soybeans according to the following
process. 100 lbs of defatted High Oleic soybean flakes were placed
in an extraction tank and extracted with 1,000 lbs of water heated
to 32.degree. C. This provided a weight ratio of water to flakes of
10:1. The flakes were separated from the extract and re-extracted
with 600 lbs of water heated to 32.degree. C. and having sufficient
calcium hydroxide added to adjust the pH to 9.7. This second
extraction step provided a weight ratio of water to flakes of 6:1.
The flakes were removed by centrifugation, and the first and second
extracts were combined and adjusted to a pH of 4.5 with
hydrochloric acid to precipitate a protein curd. The acid
precipitated curd was separated from the extract by centrifugation,
leaving aqueous whey (discarded), and then was washed with water in
a weight amount of seven times that of the starting flake material
to provide an isoelectric protein isolate.
[0261] To produce the SUPRO.RTM. 670-type High Oleic protein
material, water was added to the isoelectric soy protein material
and the pH was adjusted to between 7.3 and 7.7 with an aqueous
solution of sodium hydroxide to produce a neutralized soy protein
slurry and then heated by injecting pressurized steam into the
slurry, hereafter referred to as "jet-cooking." The neutral soy
protein slurry was introduced into a jet-cooker feed tank where it
was kept in suspension with a mixer that agitated the slurry. The
slurry was directed from the feed tank to a pump that forced the
slurry through a reactor tube. Steam was injected into the slurry
under pressure as the slurry entered the reactor tube, instantly
heating the slurry to the desired temperature. The temperature was
controlled by adjusting the pressure of the injected steam. The
heat treatment was about 9 seconds at about 130.degree. C. The
heated neutral slurry was then cooled by flash vaporization, which
vaporized a substantial portion of water from the hot neutral
protein slurry, instantly cooling the neutral protein material to
about 61.degree. C. Bromelain enzyme was added to the cooled
protein slurry and allowed to react for a time sufficient to enzyme
hydrolyze the protein to a TNBS value of about 50. The enzyme
treated slurry was then heat treated by jet-cooking to inactivate
the bromelain enzyme. The enzyme treated slurry was cooked in the
jet-cooker for about 9 seconds at about 152.degree. C. The heated
neutral slurry was then cooled by flash vaporization, which
vaporized a substantial portion of water from the hot neutral
protein slurry, instantly cooling the neutral protein material to
about 82.degree. C.
[0262] The cooled neutral protein slurry was then homogenized and
transferred to a spray dryer wherein most of the moisture was
evaporated by the addition of heat to achieve the final SUPRO.RTM.
670-type soy protein isolate.
Example 19
Gel Strength Measurements of High Oleic Protein Soy Protein
Isolates (Small Scale Production Platform)
[0263] The effect of high oleic protein isolates (essentially
prepared as described in Example 10) on gel strength of
refrigerated and pasteurized gels compared to isolates from
commodity or low linolenic (low lin) acid soybeans were analyzed.
The fatty acid composition of the low linolenic acid soybeans used
herein has been dislosed in Table 2 of U.S. Pat. No. 5,981,781,
issued Nov. 9, 1999). Oleic acid levels in the low lin lines are
similar to the levels found in commodity soybeans, whereas
linolenic acid levels are about 3 fold lower.
Low lin samples were used for comparison to high oleic samples in
addition to isolates from commodity soybeans. Gels were prepared by
mixing 75 mL ddH2O and 15 g of protein isolate in a Waring blender
on a mix setting of #2 for 30 seconds (initial hydration). The
mixer was stopped and any residual dry protein was scraped from the
bowl surface.
[0264] In some cases, gels were prepared by adding 0.84 g NaCl at
this point and mixing was resumed for a total of 3 minutes with
additional scraping every 30 seconds. Following preparation, gels
were packed into 5 mL glass vials using a disposable cartridge mini
gun dispenser. Care was taken to eliminate any residual air
bubbles. Vials were sealed with tightly crimped septum and cap.
Sealed vials were either placed immediately in the refrigerator and
stored for 16-24 hours (refrigerated gel) or incubated in an
80.degree. C. bath for 30 minutes, cooled for 30 minutes in a
25.degree. C. water bath prior to refrigeration for 16-24 hrs
(pasteurized gel). Gel strength was measured either on a texture
Analyzer (TAXT.2i, Stable Micro Systems, UK) or an AR-1000
Rheometer (TA Instruments). When gel strength was measured on the
texture Analyzer, gels were removed from the refrigerator and
warmed in a 25.degree. C. Decapped sample vials were centered on
the loading platform and a 3 mm diameter stainless cylinder punch
probe was used for measurement. Gels were penetrated twice in the
center of the vial to a depth of 10 mm and the data recorded using
the instrument manufacturer's software. The area under the positive
portion of the curve was integrated and recorded (labeled area).
Gel preparation and measurements were replicated on a second day
and the data averaged and recorded.
[0265] The results are shown in Table 4. The average gel strength
and standard deviation of the high oleic soybean isolates compared
to non-high oleic soybean isolates was 168.+-.45 g*s and 346.+-.59
g*s, respectively. The reduction in gel strength of high oleic
compared to non-high oleic soybean isolates ranged from 25%-70%
(calculated from the averages).
TABLE-US-00004 TABLE 4 Gel strength of High Oleic isolates compared
to control soybean isolates Commercial Gel texture 1:5 2% Inventory
ID Trait.sup.1 protein type.sup.2 NaCl Past. Area, g*s PPI002385
High Oleic v.1 Supro .RTM. 500E 127 PPI002391 Commodity Supro .RTM.
500E 460 PPI002419 High Oleic v.1 Supro .RTM. 500E 140 PPI002581
High Oleic v.2 Supro .RTM. 760 173 PPI002582 Commodity Supro .RTM.
760 338 PPI002583 High Oleic v.1 Supro .RTM. 760 106 PPI002584 Low
Lin Supro .RTM. 760 318 PPI002588 Low Lin Supro .RTM. 760 315
PPI002589 High Oleic v.2 Supro .RTM. 760 248 PPI002590 Commodity
Supro .RTM. 760 350 PPI002599 High Oleic v.2 Supro .RTM. 760 194
PPI002600 High Oleic v.2/ Supro .RTM. 760 146 High Stearic
PPI002601 High Oleic v.2 Supro .RTM. 760 162 PPI002602 High Oleic
v.2 Supro .RTM. 760 195 PPI006508 Commodity Supro .RTM. 760 294
.sup.1High Oleic, High Oleic v.1 (version 1) and High Oleic v.2
(version 2) soybean protein isolates were prepared as described in
Examples 3, 5, and 8. Commodity and Low Lin (see Table 8) soybean
isolates were used as controls and are referred to as "commodity"
lines for the purpose of this invention. Resulting numbers for gel
strength were rounded up or down after the decimal point. .sup.2the
commercial name refers to the specific process parameters by which
the isolates were made.
Example 20
Hunter Color Determination High Oleic Soybean (Protein) Products
(Small Scale Production Platform)
[0266] Color measurements were made on 5% protein isolate slurries
(essentially prepared as described in Example 10) on a Hunter
Colorflex 45/0 LAV instrument with an instrument setting of D65/10.
A custom ring provided by the manufacturer for small volume
measurements was used to reduce the amount of sample necessary for
analysis by placement within the sample cup. Either 14 mL (for 10
mm ring) or 8 mL (for 5 mm ring) of the 5% isolate slurry was
dispensed by pipette into the center of the ring reaching a fluid
level just above the top of the ring. The sample cup was placed on
the instrument and topped with the white or black disk when
prompted by the software provided by the instrument manufacturer.
Data for L value and Difference from White were calculated by the
software and recorded. The L, a, and b scale values obtained for
the samples are reported as the sample color. Whiteness Index is
calculated from the L and b scale values using the following:
Whiteness Index=L-3b
[0267] The Color L Value, Color difference from white and the
Whiteness index of High Oleic, Low Lin and Commodity soybean
samples are listed in Table 5. The values were measured in 5%
protein slurries. High oleic samples have higher L values, a lower
difference from white value and increased whiteness indices. The
average whiteness index and standard deviation of the high oleic
soybean samples was 45.+-.4.4 and that of non-high oleic soybean
samples 37.+-.4.9. An increase ranging from 3%-35% (calculated from
the averages) in the whiteness index in high oleic samples compared
to non-high oleic samples was observed.
TABLE-US-00005 TABLE 5 Color Whiteness index Commercial Difference
(defined by Solae Inventory ID Trait.sup.1 protein type.sup.2 L
value from White as L-3b) PPI002385 High Oleic Supro .RTM. 500E 74
27 51 PPI002391 Commodity Supro .RTM. 500E 56 45 28 PPI002419 High
Oleic Supro .RTM. 500E 72 29 47 PPI002581 High Oleic Supro .RTM.
760 71 30 44 v.2 PPI002582 Commodity Supro .RTM. 760 64 38 33
PPI002583 High Oleic Supro .RTM. 760 68 34 36 v.1 PPI002584 Low Lin
Supro .RTM. 760 65 36 38 PPI002588 Low Lin Supro .RTM. 760 65 36 41
PPI002589 High Oleic Supro .RTM. 760 67 34 42 v.2 PPI002590
Commodity Supro .RTM. S760 65 35 43 PPI002599 High Oleic Supro
.RTM. S760 70 31 45 v.2 PI002600 High Oleic Supro .RTM.760 73 29 44
v.2/High Stearic PPI002601 High Oleic Supro .RTM. 760 73 28 50 v.2
PPI002602 High Oleic Supro .RTM. 760 71 30 45 v.2 PPI006492
Commodity Supro .RTM. 760 62 39 36 PPI006493 Low Lin Supro .RTM.
760 62 39 36 PPI006495 High Oleic Supro .RTM. 760 70 31 46 v.2
PPI006508 Commodity Supro .RTM. 760 67 34 41
Example 21
Pasteurizer Feed Viscosity Measurements (Small Scale Production
Platform)
[0268] A small sample of pasteurizer feed was collected during the
preparation of protein isolates (essentially prepared as described
in Example 10) following adjustment of the solids concentration and
pH. To prepare a sample for viscosity measurements, the sample was
loaded onto the platform of an AR-1000 Rheometer (TA Instruments)
with a disposable pipette and the head lowered to 1500 mm. Excess
sample was cleaned from around the edge of the geometry and the
cover placed over the geometry in preparation for measurement. The
viscosity was measured 60 minutes post pasteurizer feed preparation
using a 40 mm flat plate geometry at a gap setting of 1000 .mu.m.
Viscosity (measured in centipoises) was recorded and analyzed using
the Rheology Advantage Data Analysis software supplied by the
instrument manufacturer.
High Oleic samples have lower Pasteurizer feed Viscosity compared
to Low Lin or commodity samples (Table 6).
[0269] The average viscosity and standard deviation of the high
oleic soybean samples compared to non-high oleic soybean isolates
were 110.+-.57.8 cp and 449.+-.125 cp, respectively, with a %
reduction (calculated from the averages) in viscosity ranging from
around 9% to 52% for high oleic samples compared to non-high oleic
samples.
TABLE-US-00006 TABLE 6 Viscosity Measurements of High Oleic Soybean
Isolates Pasteurizer Feed Commercial Viscosity-AR1000- Inventory ID
Trait.sup.1 protein type.sup.2 Viscosity at 30/s PPI002385 High
Oleic Supro .RTM. 500E 135 PPI002391 Commodity Supro .RTM. 500E 566
PPI002419 High Oleic Supro .RTM. 500E 24 PPI002588 Low Lin Supro
.RTM. 760 465 PPI002589 High Oleic Supro .RTM. 760 155 v.2
PPI002590 Commodity Supro .RTM. 760 317 PPI002601 High Oleic Supro
.RTM. 760 79 v.2 PPI002602 High Oleic Supro .RTM. 760 158 v.2
Example 22
Improvement of Drying Efficiency by Using High Oleic Soybeans
[0270] High Oleic protein products were fed to a pasteurizer or a
dryer at higher feed solids (above 14%) compared to commodity soy
protein products (Table 7 & Table 8 in Example 23). This can be
explained by the reduced viscosity of high oleic soy protein
products. When protein products are fed to a pasteurizer or a dryer
at increased feed solids, less water has to be removed in every
pound fed to the dryer resulting in decreased energy costs, and
more solids can be dried per hour resulting in better capital
utilization as well as higher production quantities.
TABLE-US-00007 TABLE 7 Measured solid content of slurry to the
Spray dryer for High Oleic vs Commodity beans Slurry Solid
concentration content of measured Dryer feed Defatted flake
Commercial type by CEM by CEM Commodity Supro .RTM. 760-type 13.43
11.99 Commodity Supro .RTM. 760-type 13.28 11.84 Commodity Supro
.RTM. 670-type 12.22 9.69 Commodity Supro .RTM. 670-type 12.01
10.38 High Oleic Supro .RTM. 760-type 13.49 11.77 High Oleic Supro
.RTM. 760-type 14.01 12.28 High Oleic Supro .RTM. 760-type 16.56
14.62 High Oleic Supro .RTM. 760-type 17.12 14.89 High Oleic Supro
.RTM. 760-type 19.40 15.54 High Oleic Supro .RTM. 760-type 18.89
16.09 High Oleic Supro .RTM. 670-type 13.41 10.27 High Oleic Supro
.RTM. 670-type 13.55 11.38 High Oleic Supro .RTM. 670-type 17.02
14.18 High Oleic Supro .RTM. 670-type 17.02 15.31 High Oleic Supro
.RTM. 670-type 18.34 15.19 High Oleic Supro .RTM. 670-type 17.51
16.18
Example 23
Gel Strength Measurement of High Oleic Soy Protein Products
[0271] High Oleic soy protein products have a gel strength
comparable to the gel strength of commodity soy protein products
when fed at no less than 14% feed solids to a dryer.
[0272] Gel strength was measured on the AR-1000 Rheometer, a sample
of the gel was placed on the rheometer platform using a metal
spatula and the head lowered to 1500 .mu.m. Excess gel was trimmed
from the edge of the geometry and the cover placed on top. A 40 mm
cross-hatched geometry a to gap of 1400 .mu.m was used for
measurement in an oscillatory mode controlled by the instrument
software. The G' (labeled gel elasticity, expressed in units of
Pascals [Pa]), from 2 replicates per sample was recorded (Table
8).
TABLE-US-00008 TABLE 8 Measured Slurry Solid Concentration and %
Protein in the Final Product of High Oleic Soybeans compared to
Commodity Soybeans Defatted Commercial Slurry concentration Prod. %
Protein by Average of Average of Sample flake protein measured by
oven Comb. Leco (as Refrigerated Pasteurized No. source type (%)
is) N*6.25 Gel Elasticity Gel Elasticity a2122 Commodity Supro
.RTM. 13.1 91.67 1129 3204 500E type b2123 Commodity Supro .RTM.
13.2 91.88 698 3340 500E type c2121 Commodity Supro .RTM. 13.6
91.07 972 2897 500E type d2131 Commodity Supro .RTM. 13.7 92.21 700
3075 500E type e2124 HO Supro .RTM. 12.6 91.98 26 591 500E type
f2137 HO Supro .RTM. 15.8 92.78 68 697 500E type g2136 HO Supro
.RTM. 16.1 92.71 64 649 500E type h2128 HO Supro .RTM. 19 92 172
1698 500E type i2135 HO Supro .RTM. 20.5 92.32 288 2243 500E type
i2134 HO Supro .RTM. 20.9 93.24 269 1982 500E type k2133 HO Supro
.RTM. 24.7 91.87 664 2077 500E type l2138 HO Supro .RTM. 24.8 91.08
637 1836 500E type
Example 24
Residual Fatty Acid Analysis by Acid Methanolysis
[0273] Triplicate samples (approximately 100 mg) were weighed, to a
precision of 0.1 mg, into 13.times.100 mm screw capped (PTFE
liners) tubes. After addition of C17:0 triacylglycerol internal
standard (10 .mu.l, 5% W:V stock in toluene), 1 ml of fresh
methanolysis solution (5% sulfuric acid in anhydrous methanol) was
added to each tube. The tubes were capped, vortex mixed and heated
at 80.degree. C. for 30 min, with vortex mixing every 10 minutes.
The samples were cooled to room temperature and 1 ml of saline
solution (25% sodium chloride in water), followed by 1 ml heptane,
was added to each tube. After vortex mixing, the phases were
separated by centrifugation (3000.times.g for 10 min) and the
upper, organic phases, were transferred to GC sample vials. Fatty
acid analysis was performed on an Agilent 6890 with FID detector.
The GC was fitted with an OmegaWax-320, 30 m.times.0.32
mm.times.0.25 um column (Supelco, Bellefonte, Pa.). The carrier gas
was hydrogen (28 cm/sec linear velocity) and the following
temperature profile was used; 220.degree. C. for 2.6 min, ramp at
10.degree. C. to 240.degree. C., hold for 1.4 min. Peak areas of
the individual fatty acids were integrated, individual fatty acids
were quantified relative to the C17 internal standard and fatty
acid compositions were estimated based on these values. The
assumption was made that the detector response for each fatty acid
was the same (Morrison et al. (1980) Methods for the quantitative
analysis of lipids in cereal grains and similar tissues. Journal of
Science Food and Agriculture 31: 329-340).
[0274] Using the above-described technique, the fatty acid profile
of residual fatty acids associated with hexane-extracted soy white
flake flours and soy protein isolates manufactured from them was
determined for commodity soybeans and two genetically altered
soybean varieties, high oleic acid soybeans and low linolenic acid
soybeans. The results are shown in Tables 9. Although it is
recognized that other fatty acids are present in soybean oil and
the residual lipid in soy products, they are only present at trace
levels (<3% of total). For the sake of comparison in this patent
we have restricted our analysis to the most abundant fatty acids
i.e., palmitic (16:0), stearic (18:0), oleic (18:1), linoleic
(18:2) and linolenic (18:3) acids.
[0275] The residual fatty acids associated with the hexane-defatted
white flake flour and soy protein isolate is principally in the
form of phospholipid, and therefore derived from membrane lipids,
while the hexane-extracted soy oil is principally composed of
storage triglycerides. Prior to this work it was not known how
closely the residual fatty acid profile would be related to the
fatty acid profile of hexane-extracted soy oil. From the data shown
in Table 9 it can be seen that the level of palmitic acid increases
in the residual fatty acids present in soy white flake flour and
soy protein isolate compared to hexane-extracted soy oil in the
three genetically different soybean varieties tested. In contrast,
the level of oleic acid decreases in the residual fatty acids
compared to hexane-extracted soy oil significantly in the commodity
and low linolenic acid soybeans, but only marginally in the high
oleic soybeans. The polyunsaturated fatty acids, linoleic and
linolenic, are at similar levels in the residual fatty acids and
hexane-extracted soy oil from the three genetically different
soybean varieties.
[0276] The residual fatty acid content in soy white flake flour and
soy protein isolate from low linolenic acid soybeans is lower in
oxidatively unstable linolenic acid than that of commodity soy
protein products, indicating that soy protein products produced
from low linolenic acid soybeans are less likely to generate
off-flavor compounds. Similarly, the residual fatty acid content in
soy white flake flour and soy protein isolate from high oleic acid
soybeans is lower in both of the polyunsaturated fatty acids,
linoleic and linolenic, than that of commodity soy protein
products, indicating that soy protein products produced from high
oleic acid soybeans are less likely to generate off-flavor
compounds.
TABLE-US-00009 TABLE 9 Fatty acid profiles of soy oils, of residual
fatty acids in flours produced from hexane-defatted soy white
flake, and of soy protein isolates 16:0 18:0 18:1 18:2 18:3 % Total
poly- Sample ID % % % % % unsaturates Commodity Soy 8-13 2-6 18-27
51-59 6-10 57-69 Oil.sup.1 High Oleic Soy Oil 6-7 4-5 79-86 2-4 2-5
4-9 Low Linolenic Soy 10 5 29 53 3 62 Oil.sup.4 High Oleic/High 12
22 60 3 3 6 Saturate Soy Oil.sup.5 High Oleic/High 6 19 62 6 6 12
Stearic Soy Oil Commodity Soy 17-27 5-7 11 49-58 7-9 56-67
WFF.sup.2 Residual Fatty Acids High Oleic Soy 9-10 3-4 78-82 2-4
3-5 5-9 WFF.sup.2 Residual Fatty Acids Low Linolenic Soy 24 7 10 57
3 60 WFF.sup.2 Residual Fatty Acids Commodity Soy 18-24 5-7 14-15
45-55 5-7 50-62 SPI.sup.3 Residual Fatty Acids High Oleic Soy 8-10
3 80-83 2-3 3-4 5-7 SPI.sup.3 Residual Fatty Acids Low Linolenic
Soy 26 6 15 52 2 54 SPI.sup.3 Residual Fatty Acids For this table
fatty acid % relates the individual fatty acid to the sum of the
five major fatty acids indicated. Other fatty acid types that are
sometimes present and represent less than 3% of the total fatty
acids are not considered for purposes of comparison. .sup.1Value
ranges for the five major fatty acids in commodity soy oil are
taken from "The Lipid Handbook" 2.sup.nd ed., (1994) Gunstone, F.
D., Harwood, J. L., Padley, F. B., Chapman & Hall. .sup.2WFF =
White flake flour from hexane-extracted soybeans .sup.3SPI = Soy
protein isolate produced from white flake flour .sup.4Table X U.S.
Pat. No. 5,710,369 .sup.5Table 9 U.S. Pat. No. 6,426,448 16:0 =
palmitic acid, 18:0 = stearic acid, 18:1 = oleic acid, 18:2 =
linoleic acid, 18:3 = linolenic acid
Example 25
Fatty Acid Analysis of High Oleic and Commodity Soybean
Isolates
[0277] Isolates from Supro.RTM.760 type high oleic, Supo.RTM.760
type ver. 2 and commodity soybeans were prepared as described in
Example 13.
[0278] Fatty acid analysis of the isolates was performed as
described below and the results are shown in Table 10.
[0279] The relative amounts of the fatty acids of isolated soy
protein was determined as follows. The isolated soy protein was
extracted by the acid hydrolysis fat method (AOAC 922.06).
Extracted lipid was saponified with alcoholic sodium hydroxide. The
fatty acids was esterified in methanol, with boron trifluoride as a
catalyst, taken up in heptane, and injected on an Agilent 5890 Gas
Chromatograph equipped with a flame ionization detector and cool
on-column injector. Fatty acid methyl esters were separated on a
Supelco SP-2560 column (100 m.times.0.25 mm ID). The column oven
temperature was set to 140.degree. C. for 5 minutes, then heated at
4.degree. C. per minute to a maximum temperature of 240.degree. C.
and held at that temperature to the end of the analysis. The
percent of individual fatty acid methyl esters were calculated from
a set of standards containing known concentrations of prepared
methyl esters of selected fatty acids.
TABLE-US-00010 TABLE 10 Fatty acid analysis of HO Supro .RTM.760
type and Commodity Supro .RTM.760 type Isolates HO Supro .RTM.
Commodity Supro .RTM. Fatty Acid (FA) Analysis 760 type 760 type
Fat Total (%) 4.03 2.07 Saturated FA 0.59 0.82 Monounsaturated FA
2.89 0.44 Trans FA <0.04 <0.04 Fatty Acid Profile (%)
Palmitic 9.99 31.1 Stearic 3.44 7.61 Oleic 73.1 19.5 Vaccenic 1.56
2.19 Linoleic 3.17 32.0 Linolenic 3.59 2.69 Others 5.15 4.91
Example 26
Viscosity Measurements of Soy Protein Slurries Prepared from
Isolates Produced Using Large Scale Production Platform
[0280] Viscosity measurements were made using a Brookfield
Viscometer, Model DV-II+.
[0281] Samples were prepared by weighing out a designated amount of
protein (.+-.0.1 g) into a plastic cup for 5 and 10% protein
slurry.
[0282] Into a 250 mL graduated cylinder, a designated amount of
deionized water of 26.degree..+-.1.degree. C. was measured. The
water was poured into a glass pint blender jar and the protein
sample was carefully added. The jar was immediately caped with the
blade assembly and the sample mix was vigorously shaken for 20
seconds to disperse the protein and keep it from adhering to the
sides of the jar. Subsequently the sample was blended for 1 minute
using the lowest speed of the blender. Then the protein slurry was
added to a 600 mL beaker and three drops of antifoam were added to
the slurry and the mixture was swirled. The beaker was covered and
left standing for 30 minutes, then swirled to dissipate and remove
any remaining foam.
[0283] The Brookfield Viscometer was set up (according to the
manufacturer's instructions), and viscosity of the sample was
measured. Viscosity was measured in centipoises (cps). The
Brookfield spindle number was 1, rotational speed was at 100 rpm
and temperature at which the data was recorded was 22.degree.
C.
[0284] As can be seen in Table 11, viscosity measured in cps for 5%
and 10% dispersion of soy protein from High Oleic protein samples
was substantially reduced compared to the respective sample from
commodity soybean (a 83% reduction in 5% slurries and 87% reduction
in 10% slurries).
TABLE-US-00011 TABLE 11 Brookfield Viscosity measurements of High
Oleic and commodity soybean Supro .RTM. 760 - 5% and 10% protein
slurries Sample % protein slurry Viscosity (cps) HOSupro .RTM. 760
5 8 Commodity Supro .RTM. 760 5 47 HOSupro .RTM. 760 10 82
Commodity Supro .RTM. 760 10 630
Example 27
Viscosity Measurements of Soy Protein Slurries Prepared from
Isolates Produced Using Large Scale Production Platform
[0285] For rheological evaluation, protein isolates were hydrated
by placing 90 g DI water into an 8-ounce plastic blender jar,
followed by addition of 10 g isolate powder to the surface of the
water. The mixture was blended with an Oster blender using the
"blend" setting for 90 seconds. After this time, the mixture was
decanted into a 16-ounce plastic cup. The cup was capped with a
plastic lid, and the slurry was allowed to stand for about 4 hours
at 22.degree. C. to permit a major portion of the foam atop the
fluid to dissipate prior to the rheological measurements.
[0286] Rheological measurements were performed in duplicate on a
combination of Anton Paar MCR-300 and MCR-301 rheometers. Each
rheometer was equipped with a concentric cylinder geometry (Anton
Paar CC27) having an active length of 119.2 mm, a position length
of 72.5 mm, and a gap length of 40 mm. Temperature control was
achieved by circulating 22.degree. C. water from
controlled-temperature baths to a Peltier sample heater that
controlled the temperature of the measuring cell. All measurements
were carried out at 25.+-.0.05.degree. C. Viscosity curves for each
sample were obtained via the following 4-step procedure: (1) A 19
mL sample was loaded into the concentric cylinder cup and
pre-sheared for 30 s at a shear rate of 10 1/s to erase sample
loading history. (2) Immediately after the pre-shear step, the
sample was allowed to equilibrate at 25.degree. C. for 10 minutes.
(3) The sample was then subjected to a 1-100 1/s shear rate ramp
during which 20 logarithmically-spaced data points were recorded at
an interval of 30 s per point. (4) The sample was then immediately
exposed to a downward shear rate ramp which had the same
characteristics as the upward ramp, but was applied in the opposite
direction (100-1 1/s). The resulting viscosity versus shear rate
curves were fit to a 2.sup.nd log polynomial model: viscosity=A
(shear rate) [b+c In (shear rate)], where A, b, and c are fitting
constants. Shear stress versus shear rate curves were also recorded
during these measurements and were fitted to a Herschel-Bulkley
power law model: (shear stress)=K (shear rate) n, where K and n are
the Herschel-Bulkley consistency and flow indices, respectively.
The shear stress hysteresis area (HA) bounded by the upward and
downward shear rate ramps was also recorded during each
measurement.
[0287] The rheological characteristics of high oleic and commodity
SUPRO.RTM. ISP products resulting from these measurements are
compared in Table 10-A. Mean values of A, K, n (from the upward
shear rate ramp only) and hysteresis area are reported in the
table. Mean coefficients of variation for each parameter were 2.3,
1.5, 0.4, and 7.4, respectively. Significant rheological
differences were observed between the commodity and high oleic
variants of SUPRO.RTM. 760, SUPRO.RTM. 1610, and SUPRO.RTM. 651 ISP
products. Reductions in the values of A, K, and hysteresis area
ranged from 67-90%, 41-86%, and 45-90%, respectively for the high
oleic samples versus their commodity analogs. The SUPRO.RTM. 760
and SUPRO.RTM. 651 high oleic ISP samples also exhibited larger
flow indices--indicative of more Newtonian behavior--versus their
commodity variants. In contrast, virtually no rheological
differences were observed between the high oleic and commodity
variants of the SUPRO.RTM. 670 ISP product.
TABLE-US-00012 TABLE 12 Rheological comparison of High Oleic and
commodity SUPRO .RTM. type ISP products - 10 wt % aqueous
dispersions at 25.degree. C. Product A [mPa s] K [mPa] n HA [Pa/s]
SUPRO .RTM. 760 Type Commodity 6978.7 5032.3 0.473 175.00 High
Oleic 2293.3 2278.4 0.548 96.28 SUPRO .RTM. 1610 Type Commodity
7680.9 2811.7 0.564 186.20 High Oleic 1712.3 1667.4 0.492 85.48
SUPRO .RTM. 651 Type Commodity 872.8 637.9 0.658 51.50 High Oleic
91.1 89.8 0.817 5.35 SUPRO .RTM. 670 Type Commodity 10.8 11.3 0.946
0.05 High Oleic 12.0 12.4 0.945 0.00
Example 28
Hunter Color Determination High Oleic Soybean (Protein) Products
(Large Scale Production Platform)
[0288] Color measurements using the Hunter colorimeter were made on
high oleic protein and commodity protein powders and 5% aqueous
slurries. Two units of L value differences can be detected and one
unit of Whiteness index differences can be detected. The whiteness
index was increased by 11% in HO Powder compared to commodity
powder and 34% in HO slurries compared to commodity slurries. The
data are shown in Tables 13 and 14.
[0289] Whiteness index measurements of a 5% by weight solids sample
of the suspension for HO and commodity isolates made were
determined using a HunterLab Labscan XE colorimeter manufactured by
Hunter Associates Laboratory (HunterLab, Reston, Va.). For the
whiteness index measurement, protein samples were dispersed on a 5%
w/w basis: (5.25 g) is added to deionized water (100 mL). The
results obtained using the Hunter Colorimeter are reported in units
of L, a, and b. Whiteness Index is calculated from the L and b
scale values using the following:
Whiteness Index=L-3b.
TABLE-US-00013 TABLE 13 Hunter Color Determination of High Oleic
and Commodity Soybean Samples Whiteness Sample Trait L value index
Supro .RTM. 760 High Oleic 87.9 58.0 powder Supro .RTM.760
Commodity 86.3 51.8 powder Supro .RTM.760 High Oleic 69.9 47.5
slurry Supro .RTM.760 Commodity 68.2 31.2 slurry
TABLE-US-00014 TABLE 14 Hunter Color Determination of High Oleic
and Commodity Soybean Samples Whiteness Sample Trait L value index
Supro .RTM. 1610 Commodity 82.78 40.6 Type, Powder Supro .RTM. 1610
High Oleic 84.34 44.38 Type, Powder Supro .RTM. 1610 Commodity
48.01 25.81 Type, Slurry Supro .RTM. 1610 High Oleic 51.94 28.09
Type, Slurry Supro .RTM. 651 Commodity 84.07 39.52 Type, Powder
Supro .RTM. 651 High Oleic 86.62 47.41 Type, Powder Supro .RTM. 651
Commodity 60.36 24.9 Type, Slurry Supro .RTM. 651 High Oleic 63.27
32.43 Type, Slurry Supro .RTM. 670 Commodity 83.26 40.15 Type,
Powder Supro .RTM. 670 High Oleic 85.5 48.48 Type, Powder Supro
.RTM. 670 Commodity 58.99 29.95 Type, Slurry Supro .RTM. 670 High
Oleic 60.09 38.52 Type, Slurry Supro .RTM. 760 Commodity 83.7 44.64
Type, Powder Supro .RTM. 760 High Oleic 85.7 49.2 Type, Powder
Supro .RTM. 760 Commodity 48.81 29.58 Type, Slurry Supro .RTM. 760
High Oleic 55.45 37.54 Type, Slurry
Example 29
Preparation of Plain Flavored Soymilk
[0290] About 20 pounds of dry, whole soybeans were soaked in an
excess (40 pounds or more) of cold tap water, and then allowed to
sit quiescently overnight. The excess water was then drained of and
discarded. The 40 pounds of rehydrated soybeans were ground through
a mill or grinder. A sufficient amount of water was continuously
added during the grinding to keep the slurry moving through the
mill. Sufficient water to bring the total slurry weight to up to
approximately 180 pounds was then added. The slurry was next
transferred to a pressure cooker and the temperature rose, through
steam injection, to 116.degree. C., and the temperature was held
constant for approximately 40 seconds. The slurry or soymilk was
vented out of the pressure cooker and strained through a coarse
mesh cloth. The soy residue (okara) was pressed in the bag to
remove the trapped soymilk and the okara was then discarded. The
resulting soymilk was strained through a fine mesh cloth and then
into a container. This procedure yielded approximately 200 pounds
of 93.3.degree. C. soymilk. Lecithin (93 g), corn oil (533 g) and
yeast flavor (180 g) were added and the mixture is agitated using a
Tekmar High Speed Shear Mixer for 30 seconds.
Example 30
Preparation of Flavored Soymilk
[0291] Soy milk beverages, including the ingredients as set forth
in the table below, was made from the product described above
(example plain flavored soymilk) and a soy protein isolate
(Supro.RTM. 760).
[0292] 100% of water was heated to 65.6.degree. C. and maintained
at 65.6.degree. C. with agitation until all ingredients were added.
The protein product was added with agitation and mixed until
dissolved. Sucrose, carboxymethylcellulose and carrageenan were dry
blended and added to the protein slurry and mixed until dissolved.
Calcium carbonate and sodium chloride were added and dispersed. The
soybean oil was then added followed by flavors and vitamin premix.
The pH of the system was adjusted between 6.8 and 7.0 using HCl or
NaOH as needed. The products were then processed in an ultra high
temperature short time processor at 143.degree. C. for 10 seconds.
Then the products were homogenized in a 2 stage homogenizer at 2000
and 500 psi, cooled and filled into clean bottles, and stored in a
refrigerator. Whiteness Index and viscosity of samples are shown in
Table 15. The whiteness index and viscosity of HO flavored soymilk
compared to flavored soymilk from commodity soybeans were increased
by 22% and reduced by 40%, respectively.
TABLE-US-00015 TABLE 15 Whiteness Viscosity Sample Trait index
(cps) Supro .RTM.760 High Oleic 27.67 6.25 Flavored soymilk Supro
.RTM.760 Commodity 21.52 10.4 Flavored soymilk
Example 31
Plain Soymilk Physical Characteristics
[0293] Plain soymilk from high oleic and commodity soybean was
prepared as described in Example 30. The whiteness index and
viscosity of the samples are shown in Table 16. The viscosity of HO
soymilk was reduced by 17% compared to commodity soymilk. The
Whiteness Index of HO soymilk was increased by 7.5% compared to
soymilk prepared from commodity soybeans.
TABLE-US-00016 TABLE 16 Whiteness Viscosity Sample Trait index
(cps) Supro .RTM. 760 High Oleic 55.27 3.45 plain soymilk Supro
.RTM. 760 Commodity 51.14 4.15 plain soymilk
Example 32
Solid Phase MicroExtraction (SPME) GC MS Method for Soy Volatile
Analysis
[0294] Samples for the analysis of soy volatile compounds using the
SPME GC MS methods are prepared by weighing out 2.5.+-.0.005 g of
the sample to be analyzed into a weigh boat. Then 47.5.+-.0.1 g of
reverse-osmosis (e.g., Mili-Q or Labcono) water are measured into a
250 mL Waring blender cup. The blender is started at minimum speed
and the weighed-out sample is sprinkled into the water over
approximately 10 seconds. The sample and water are blended into a
slurry using minimal speed to keep foaming to a minimum. Blending
time should be enough to achieve good dispersion of the sample and
should be around 30 seconds and not exceed 60 seconds. This should
be kept consistent for each sample matrix. If foam develops, it
should be scooped off with a spoon and manually stirred back into
the sample mix.
[0295] In order to suspend the slurry and to make it as homogenous
as possible it is briefly stirred with a spoon or scoop. Then 30 g
of the slurry are quickly transferred from the blender cup into a
tared SPME vial (50 mL serum bottle: Supelco p/n 33108-u)
containing 11.1 g NaCl (Omnipur grade, EMD Chemicals).
[0296] Of the 49.2 ppm internal standard stock solution of
4-heptanone, 100 .mu.L are pipetted into the SPME vial to yield 164
ppb internal standard concentration upon mixing. A 1'' ( 1/16''
diameter) Teflon stir bar is dropped into the vial and the vial is
sealed with crimp-top septum cap (Supelco) fitted with a Natural
Teflon/Blue Silicone septum (Microanalytical Supplies) to ensure a
good airtight seal. The bottle is then placed on the center of a
stirplate and stirred for 5 min at 300 rpm to allow thorough mixing
and equilibration of the headspace.
[0297] Sample extraction is performed as follows. The SPME fiber is
preconditioned as recommended by the manufacturer's manual
(Supelco) for 30 min at 250.degree. C. with minimal carrier gas
flow if used for the first time. Re-conditioning of the fiber is
done by inserting it into the rear injection port at 280.degree. C.
in between runs for a minimum of 30 min. Once the headspace is
equilibrated, the sheathed SPME fiber is inserted through the
septum. It has to be ensured that neither sheath nor fiber touches
or immerses into the liquid.
[0298] Then the SPME fiber has to be extended out of its sheath,
and exposed to the headspace for 30 min. The height of the fiber
should be adjusted such that the end is approximately 1/4'' over
the surface of the liquid. Subsequently the SPME fiber is retracted
into its sheath and withdrawn from the septum. The fiber containing
the volatiles should be injected as soon as possible for analysis.
Analysis is carried out on an Agilent 6890N GC with a 7973 MSD
detector and Agilent ChemStation software. The sample is injected
with the sheathed SPME fiber through the injector septum, then
rapidly unsheathing the fiber into the injector body. Separation
begins when GC is started. The SPME should be left unsheathed in
the injector for 1.5 min, after which it is removed and
re-conditioned and retracted back into its sheath.
[0299] The data are analyzed using the Agilent ChemStation Software
and peak areas were calculated by manual integration The calculated
peak areas for each target volatile were converted to ppb by the
following formula: Concentration of volatile in slurry (.mu.g/kg
slurry)=164.times.target peak area/int.std.peak area
Example 33
Volatile Analysis of Soy Isolates
[0300] Sample preparation and analysis was performed as described
in Example 32, and the concentration of volatiles in the
slurry.sup.1 are shown in Table 17.
[0301] As can be seen from Table 17 (hexanal levels in high oleic
samples are substantially lower compared to the hexanal levels in
the respective samples form commodity soybean. It is believed that
lower hexanal levels correspond to improved flavor of soybean
protein products.
TABLE-US-00017 TABLE 17 Protein Product High Oleic Commodity
Commodity High Oleic Commodity High Oleic SUPRO .RTM. SUPRO .RTM.
SUPRO .RTM. SUPRO .RTM. Alpha .TM. Alpha .TM. Volatiles 760 type
760 type 670 type 670 type 5800 type 5800 type Pentenal 2.7.sup.1
11.8 3.9 ND 2.5 ND Hexanal 10.6 171 35.9 3.6 19.6 1.7 2-Heptanone
1.4 26.5 12.4 1.6 8.0 0.34 Heptanal 1.1 2.60 0.57 0.25 0.30 0.45
1-Octen-3-ol 0.25 0.68 0.19 ND 0.13 ND 2-Octanone 0.11 1.5 0.54
0.12 0.27 ND 2-Pentylfuran.sup.2 0.90 59.7 15.7 1.5 2.52 0.12
3-Octen-2-one .sup. ND.sup.2 0.60 0.61 ND ND ND 2-Nonanone 0.93 1.2
0.96 0.96 0.15 0.11 Nonanal 2.3 2.4 0.64 0.65 0.39 0.46 Decanal ND
0.52 ND 0.27 0.08 0.22 .sup.1All concentrations are expressed as
.mu.g volatile/kg 5% slurry relative to the 4-heptanone internal
std. which was present at 164 .mu.g/kg 5% slurry. .sup.2ND = not
detected
Sequence CWU 1
1
2617993DNAartificial sequenceRecombinant DNA Fragment PHP21676A
1cgcgccaagc ttggatccgc gccaagcttg gatcctagaa ctagaaacgt gatgccactt
60gttattgaag tcgattacag catctattct gttttactat ttataacttt gccatttctg
120acttttgaaa actatctctg gatttcggta tcgctttgtg aagatcgagc
aaaagagacg 180ttttgtggac gcaatggtcc aaatccgttc tacatgaaca
aattggtcac aatttccact 240aaaagtaaat aaatggcaag ttaaaaaagg
aatatgcatt ttactgattg cctaggtgag 300ctccaagaga agttgaatct
acacgtctac caaccgctaa aaaaagaaaa acattgatat 360gtaacctgat
tccattagct tttgacttct tcaacagatt ctctacttag atttctaaca
420gaaatattat tactagcaca tcattttcag tctcactaca gcaaaaaatc
caacggcaca 480atacagacaa caggagatat cagactacag agatagatag
atgctactgc atgtagtaag 540ttaaataaaa ggaaaataaa atgtcttgct
accaaaacta ctacagacta tgatgctcac 600cacaggccaa atcctgcaac
taggacagca ttatcttata tatattgtac aaaacaagca 660tcaaggaaca
tttggtctag gcaatcagta cctcgttcta ccatcaccct cagttatcac
720atccttgaag gatccattac tgggaatcat cggcaacaca tgctcctgat
ggggcacaat 780gacatcaaga aggtaggggc caggggtgtc caacattctc
tgaattgccg ctctaagctc 840ttccttcttc gtcactcgcg ctgccggtat
cccacaagca tcagcaaact tgagcatgtt 900tgggaatatc tcgctctcgc
tagacggatc tccaagatag gtgtgagctc tattggactt 960gtagaaccta
tcctccaact gaaccaccat acccaaatgc tgattgttca acaacaatat
1020cttaactggg agattctcca ctcttatagt ggccaactcc tgaacattca
tgatgaaact 1080accatcccca tcaatgtcaa ccacaacagc cccagggtta
gcaacagcag caccaatagc 1140cgcaggcaat ccaaaaccca tggctccaag
accccctgag gtcaaccact gcctcggtct 1200cttgtacttg taaaactgcg
cagcccacat ttgatgctgc ccaaccccag tactaacaat 1260agcatctcca
ttagtcaact catcaagaac ctcgatagca tgctgcggag aaatcgcgtc
1320ctggaatgtc ttgtaaccca atggaaactt gtgtttctgc acattaatct
cttctctcca 1380acctccaaga tcaaacttac cctccactcc tttctcctcc
aaaatcatat taattccctt 1440caaggccaac ttcaaatccg cgcaaaccga
cacgtgcgcc tgcttgttct tcccaatctc 1500ggcagaatca atatcaatgt
gaacaatctt agccctacta gcaaaagcct caagcttccc 1560agtaacacgg
tcatcaaacc ttaccccaaa ggcaagcaac aaatcactat tgtcaacagc
1620atagttagca taaacagtac catgcatacc cagcatctga agggaatatt
catcaccaat 1680aggaaaagtt ccaagaccca ttaaagtgct agcaacggga
ataccagtga gttcaacaaa 1740gcgcctcaat tcagcactgg aattcaaact
gccaccgccg acgtagagaa cgggcttttg 1800ggcctccatg atgagtctga
caatgtgttc caattgggcc tcggcggggg gcctgggcag 1860cctggcgagg
taaccgggga ggttaacggg ctcgtcccaa ttaggcacgg cgagttgctg
1920ctgaacgtct ttgggaatgt cgatgaggac cggaccgggg cggccggagg
tggcgacgaa 1980gaaagcctcg gcgacgacgc gggggatgtc gtcgacgtcg
aggatgaggt agttgtgctt 2040cgtgatggat ctgctcacct ccacgatcgg
ggtttcttgg aaggcgtcgg tgccgatcat 2100ccggcgggcg acctggccgg
tgatggcgac gactgggacg ctgtccatta aagcgtcggc 2160gaggccgctc
acgaggttgg tggcgccggg gccggaggtg gcaatgcaga cgccggggag
2220gccggaggaa cgcgcgtagc cttcggcggc gaagacgccg ccctgctcgt
ggcgcgggag 2280cacgttgcgg atggcggcgg agcgcgtgag cgcctggtgg
atctccatcg acgcaccgcc 2340ggggtacgcg aacaccgtcg tcacgccctg
cctctccagc gcctccacaa ggatgtccgc 2400gcccttgcga ggttcgccgg
aggcgaaccg tgacacgaag ggctccgtgg tcggcgcttc 2460cttggtgaag
ggcgccgccg tggggggttt ggagatggaa catttgattt tgagagcgtg
2520gttgggtttg gtgagggttt gatgagagag agggagggtg gatctagtaa
tgcgtttggg 2580gaaggtgggg tgtgaagagg aagaagagaa tcgggtggtt
ctggaagcgg tggccgccat 2640tgtgttgtgt ggcatggtta tacttcaaaa
actgcacaac aagcctagag ttagtaccta 2700aacagtaaat ttacaacaga
gagcaaagac acatgcaaaa atttcagcca taaaaaaagt 2760tataatagaa
tttaaagcaa aagtttcatt ttttaaacat atatacaaac aaactggatt
2820tgaaggaagg gattaattcc cctgctcaaa gtttgaattc ctattgtgac
ctatactcga 2880ataaaattga agcctaagga atgtatgaga aacaagaaaa
caaaacaaaa ctacagacaa 2940acaagtacaa ttacaaaatt cgctaaaatt
ctgtaatcac caaaccccat ctcagtcagc 3000acaaggccca aggtttattt
tgaaataaaa aaaaagtgat tttatttctc ataagctaaa 3060agaaagaaag
gcaattatga aatgatttcg actagatctg aaagtccaac gcgtattccg
3120cagatattaa agaaagagta gagtttcaca tggatcctag atggacccag
ttgaggaaaa 3180agcaaggcaa agcaaaccag aagtgcaaga tccgaaattg
aaccacggaa tctaggattt 3240ggtagaggga gaagaaaagt accttgagag
gtagaagaga agagaagagc agagagatat 3300atgaacgagt gtgtcttggt
ctcaactctg aagcgatacg agtttagagg ggagcattga 3360gttccaattt
atagggaaac cgggtggcag gggtgagtta atgacggaaa agcccctaag
3420taacgagatt ggattgtggg ttagattcaa ccgtttgcat ccgcggctta
gattggggaa 3480gtcagagtga atctcaaccg ttgactgagt tgaaaattga
atgtagcaac caattgagcc 3540aaccccagcc tttgcccttt gattttgatt
tgtttgttgc atacttttta tttgtcttct 3600ggttctgact ctctttctct
cgtttcaatg ccaggttgcc tactcccaca ccactcacaa 3660gaagattcta
ctgttagtat taaatatttt ttaatgtatt aaatgatgaa tgcttttgta
3720aacagaacaa gactatgtct aataagtgtc ttgcaacatt ttttaagaaa
ttaaaaaaaa 3780tatatttatt atcaaaatca aatgtatgaa aaatcatgaa
taatataatt ttatacattt 3840ttttaaaaaa tcttttaatt tcttaattaa
tatcttaaaa ataatgatta atatttaacc 3900caaaataatt agtatgattg
gtaaggaaga tatccatgtt atgtttggat gtgagtttga 3960tctagagcaa
agcttactag agtcgaccga tccgtcgacg gcgcggatcc tcgaagagaa
4020gggttaataa cacatttttt aacattttta acacaaattt tagttattta
aaaatttatt 4080aaaaaattta aaataagaag aggaactctt taaataaatc
taacttacaa aatttatgat 4140ttttaataag ttttcaccaa taaaaaatgt
cataaaaata tgttaaaaag tatattatca 4200atattctctt tatgataaat
aaaaagaaaa aaaaaataaa agttaagtga aaatgagatt 4260gaagtgactt
taggtgtgta taaatatatc aaccccgcca acaatttatt taatccaaat
4320atattgaagt atattattcc atagccttta tttatttata tatttattat
ataaaagctt 4380tatttgttct aggttgttca tgaaatattt ttttggtttt
atctccgttg taagaaaatc 4440atgtgctttg tgtcgccact cactattgca
gctttttcat gcattggtca gattgacggt 4500tgattgtatt tttgtttttt
atggttttgt gttatgactt aagtcttcat ctctttatct 4560cttcatcagg
tttgatggtt acctaatatg gtccatgggt acatgcatgg ttaaattagg
4620tggccaactt tgttgtgaac gatagaattt tttttatatt aagtaaacta
tttttatatt 4680atgaaataat aataaaaaaa atattttatc attattaaca
aaatcatatt agttaatttg 4740ttaactctat aataaaagaa atactgtaac
attcacatta catggtaaca tctttccacc 4800ctttcatttg ttttttgttt
gatgactttt tttcttgttt aaatttattt cccttctttt 4860aaatttggaa
tacattatca tcatatataa actaaaatac taaaaacagg attacacaaa
4920tgataaataa taacacaaat atttataaat ctagctgcaa tatatttaaa
ctagctatat 4980cgatattgta aaataaaact agctgcattg atactgataa
aaaaatatca tgtgctttct 5040ggactgatga tgcagtatac ttttgacatt
gcctttattt tatttttcag aaaagctttc 5100ttagttctgg gttcttcatt
atttgtttcc catctccatt gtgaattgaa tcatttgctt 5160cgtgtcacaa
atacaattta gntaggtaca tgcattggtc agattcacgg tttattatgt
5220catgacttaa gttcatggta gtacattacc tgccacgcat gcattatatt
ggttagattt 5280gataggcaaa tttggttgtc aacaatataa atataaataa
tgtttttata ttacgaaata 5340acagtgatca aaacaaacag ttttatcttt
attaacaaga ttttgttttt gtttgatgac 5400gttttttaat gtttacgctt
tcccccttct tttgaattta gaacacttta tcatcataaa 5460atcaaatact
aaaaaaatta catatttcat aaataataac acaaatattt ttaaaaaatc
5520tgaaataata atgaacaata ttacatatta tcacgaaaat tcattaataa
aaatattata 5580taaataaaat gtaatagtag ttatatgtag gaaaaaagta
ctgcacgcat aatatataca 5640aaaagattaa aatgaactat tataaataat
aacactaaat taatggtgaa tcatatcaaa 5700ataatgaaaa agtaaataaa
atttgtaatt aacttctata tgtattacac acacaaataa 5760taaataatag
taaaaaaaat tatgataaat atttaccatc tcataagata tttaaaataa
5820tgataaaaat atagattatt ttttatgcaa ctagctagcc aaaaagagaa
cacgggtata 5880tataaaaaga gtacctttaa attctactgt acttccttta
ttcctgacgt ttttatatca 5940agtggacata cgtgaagatt ttaattatca
gtctaaatat ttcattagca cttaatactt 6000ttctgtttta ttcctatcct
ataagtagtc ccgattctcc caacattgct tattcacaca 6060actaactaag
aaagtcttcc atagcccccc aagcggccgg agctggtcat ctcgctcatc
6120gtcgagtcgg cggccggagc tggtcatctc gctcatcgtc gagtcggcgg
ccgccggtcc 6180tctctctttc cgtggcatgg caatctattg ggctgtccag
ggttgcatcc ttactggtgt 6240ttgggtcatt gcccatgagt gtggtcacca
tgcattcagt gactaccagc tgcttgatga 6300tattgttggc cttatcctcc
actccgctct cctagtcccg tacttttcat ggaaatacag 6360ccatcgccgt
caccactcca acactggttc tcttgagcgg gatgaagtat ttgtgccaaa
6420gcagaagtcc tgtatcaagt ggtactctaa ataccttaac aatcctccag
gcagagtcct 6480cactcttgct gtcaccctca cacttggttg gcccttgtac
ttggctttaa atgtttctgg 6540aaggccttat gatagatttg cttgccacta
tgacccatat ggtcccattt actctgatcg 6600tgaacgactt caaatatata
tatcagatgc aggagtactt gcaggactta ctctctctac 6660cgtgttgcaa
ccctgaaagg gttggtttgg ctgctatgtg tttatggggt gcctttgctc
6720attgtgaacg gttttcttgt gactatcaca tatttgcagc acacacactt
tgccttgcct 6780cattacgatt catcagaatg ggactggctg aagggagctt
tggcaactat ggacagagat 6840tatgggattc tgaacaaggt gtttcatcac
ataactgata ctcatgtggc tcaccatctc 6900ttctctacaa tgccacatta
ccatgcaatg gaggcaacca atgcaatcaa gccaatattg 6960ggtgagtact
accaatttga tgacacacca ttttacaagg cactgtggag agaagcgaga
7020gagtgcctct atgtggagcc agatgaagga acatccgaga agggctcctc
caccgtttaa 7080gattgcagaa atcagagctt caataccaaa acattgctgg
gtcaagaatc catggagatc 7140cctcagttat gttctcaggg atgtgcttgt
aattgctgca ttggtggctg cagcaattca 7200cttcgacaac tggcttctct
ggctaatcta ttgccccatt caaggcacaa tgttctgggc 7260tctctttgtt
cttggacatg attgtggcca tggaagcttt tcagatagcc ctttgctgaa
7320tagcctggtg ggacacatct tgcattcctc aattcttgtg ccataccatg
gatggagaat 7380tagccacaga actcaccatc aaaaccatgg acacattgag
aaggatgagt catgggttcc 7440attaacagag aagatttaca agaatctaga
cagcatgaca agactcatta gattcactgt 7500gccatttcca ttgtttgtgt
atccaattta tttgttttca agaagccccg gaaaggaagg 7560ctctcacttc
aatccctaca gcaatctgtt cccacccagt gagagaaaag gaatagcaat
7620atcaacactg tgttgggcta ccatgttttc tctgcttatc tatctctcat
tcataactag 7680tccacttcta gtgctcaagc tctatgggcg gccgccgact
cgacgatgag cgagatgacc 7740agctccggcc gccgactcga cgatgagcga
gatgaccagc tccggccgcg acacaagtgt 7800gagagtacta aataaatgct
ttggttgtac gaaatcatta cactaaataa aataatcaaa 7860gcttatatat
gccttccgct aaggccgaat gcaaagaaat tggttctttc tcgttatctt
7920ttgccacttt tactagtacg tattaattac tacttaatca tctttgttta
cggctcatta 7980tatccgtcga cgg 799321533DNAartificial sequence1533
kb recombinant fragment 2cggtcctctc tctttccgtg gcatggcaat
ctattgggct gtccagggtt gcatccttac 60tggtgtttgg gtcattgccc atgagtgtgg
tcaccatgca ttcagtgact accagctgct 120tgatgatatt gttggcctta
tcctccactc cgctctccta gtcccgtact tttcatggaa 180atacagccat
cgccgtcacc actccaacac tggttctctt gagcgggatg aagtatttgt
240gccaaagcag aagtcctgta tcaagtggta ctctaaatac cttaacaatc
ctccaggcag 300agtcctcact cttgctgtca ccctcacact tggttggccc
ttgtacttgg ctttaaatgt 360ttctggaagg ccttatgata gatttgcttg
ccactatgac ccatatggtc ccatttactc 420tgatcgtgaa cgacttcaaa
tatatatatc agatgcagga gtacttgcag gacttactct 480ctctaccgtg
ttgcaaccct gaaagggttg gtttggctgc tatgtgttta tggggtgcct
540ttgctcattg tgaacggttt tcttgtgact atcacatatt tgcagcacac
acactttgcc 600ttgcctcatt acgattcatc agaatgggac tggctgaagg
gagctttggc aactatggac 660agagattatg ggattctgaa caaggtgttt
catcacataa ctgatactca tgtggctcac 720catctcttct ctacaatgcc
acattaccat gcaatggagg caaccaatgc aatcaagcca 780atattgggtg
agtactacca atttgatgac acaccatttt acaaggcact gtggagagaa
840gcgagagagt gcctctatgt ggagccagat gaaggaacat ccgagaaggg
ctcctccacc 900gtttaagatt gcagaaatca gagcttcaat accaaaacat
tgctgggtca agaatccatg 960gagatccctc agttatgttc tcagggatgt
gcttgtaatt gctgcattgg tggctgcagc 1020aattcacttc gacaactggc
ttctctggct aatctattgc cccattcaag gcacaatgtt 1080ctgggctctc
tttgttcttg gacatgattg tggccatgga agcttttcag atagcccttt
1140gctgaatagc ctggtgggac acatcttgca ttcctcaatt cttgtgccat
accatggatg 1200gagaattagc cacagaactc accatcaaaa ccatggacac
attgagaagg atgagtcatg 1260ggttccatta acagagaaga tttacaagaa
tctagacagc atgacaagac tcattagatt 1320cactgtgcca tttccattgt
ttgtgtatcc aatttatttg ttttcaagaa gccccggaaa 1380ggaaggctct
cacttcaatc cctacagcaa tctgttccca cccagtgaga gaaaaggaat
1440agcaatatca acactgtgtt gggctaccat gttttctctg cttatctatc
tctcattcat 1500aactagtcca cttctagtgc tcaagctcta tgg
1533328DNAartificial sequenceprimer BM35 3gcggccgccg gtcctctctc
tttccgtg 28431DNAartificial sequenceprimer BM39 4taaacggtgg
aggagccctt ctcggatgtt c 31533DNAartificial sequenceprimer BM40
5gaacatccga gaagggctcc tccaccgttt aag 33628DNAartificial
sequenceprimer BM41 6gcggccgccc atagagcttg agcactag
287890DNAartificial sequenceGlycine max KSFAD2 hybrid 7cggtcctctc
tctttccgtg gcatggcaat ctattgggct gtccagggtt gcatccttac 60tggtgtttgg
gtcattgccc atgagtgtgg tcaccatgca ttcagtgact accagctgct
120tgatgatatt gttggcctta tcctccactc cgctctccta gtcccgtact
tttcatggaa 180atacagccat cgccgtcacc actccaacac tggttctctt
gagcgggatg aagtatttgt 240gccaaagcag aagtcctgta tcaagtggta
ctctaaatac cttaacaatc ctccaggcag 300agtcctcact cttgctgtca
ccctcacact tggttggccc ttgtacttgg ctttaaatgt 360ttctggaagg
ccttatgata gatttgcttg ccactatgac ccatatggtc ccatttactc
420tgatcgtgaa cgacttcaaa tatatatatc agatgcagga gtacttgcag
gacttactct 480ctctaccgtg ttgcaaccct gaaagggttg gtttggctgc
tatgtgttta tggggtgcct 540ttgctcattg tgaacggttt tcttgtgact
atcacatatt tgcagcacac acactttgcc 600ttgcctcatt acgattcatc
agaatgggac tggctgaagg gagctttggc aactatggac 660agagattatg
ggattctgaa caaggtgttt catcacataa ctgatactca tgtggctcac
720catctcttct ctacaatgcc acattaccat gcaatggagg caaccaatgc
aatcaagcca 780atattgggtg agtactacca atttgatgac acaccatttt
acaaggcact gtggagagaa 840gcgagagagt gcctctatgt ggagccagat
gaaggaacat ccgagaaggg 890828DNAartificial sequenceprimer KS1
8gcggccgccg gtcctctctc tttccgtg 28930DNAartificial sequenceprimer
KS2 9tagagagagt aagtcctgca agtactcctg 301030DNAartificial
sequenceprimer KS3 10caggagtact tgcaggactt actctctcta
301129DNAartificial sequenceprimer KS4 11gcggccggcc ccttctcgga
tgttccttc 29122460DNAartificial sequencegene expression-silencing
cassette 12ccaagcttgg atcctcgaag agaagggtta ataacacatt ttttaacatt
tttaacacaa 60attttagtta tttaaaaatt tattaaaaaa tttaaaataa gaagaggaac
tctttaaata 120aatctaactt acaaaattta tgatttttaa taagttttca
ccaataaaaa atgtcataaa 180aatatgttaa aaagtatatt atcaatattc
tctttatgat aaataaaaag aaaaaaaaaa 240taaaagttaa gtgaaaatga
gattgaagtg actttaggtg tgtataaata tatcaacccc 300gccaacaatt
tatttaatcc aaatatattg aagtatatta ttccatagcc tttatttatt
360tatatattta ttatataaaa gctttatttg ttctaggttg ttcatgaaat
atttttttgg 420ttttatctcc gttgtaagaa aatcatgtgc tttgtgtcgc
cactcactat tgcagctttt 480tcatgcattg gtcagattga cggttgattg
tatttttgtt ttttatggtt ttgtgttatg 540acttaagtct tcatctcttt
atctcttcat caggtttgat ggttacctaa tatggtccat 600gggtacatgc
atggttaaat taggtggcca actttgttgt gaacgataga atttttttta
660tattaagtaa actattttta tattatgaaa taataataaa aaaaatattt
tatcattatt 720aacaaaatca tattagttaa tttgttaact ctataataaa
agaaatactg taacattcac 780attacatggt aacatctttc caccctttca
tttgtttttt gtttgatgac tttttttctt 840gtttaaattt atttcccttc
ttttaaattt ggaatacatt atcatcatat ataaactaaa 900atactaaaaa
caggattaca caaatgataa ataataacac aaatatttat aaatctagct
960gcaatatatt taaactagct atatcgatat tgtaaaataa aactagctgc
attgatactg 1020ataaaaaaat atcatgtgct ttctggactg atgatgcagt
atacttttga cattgccttt 1080attttatttt tcagaaaagc tttcttagtt
ctgggttctt cattatttgt ttcccatctc 1140cattgtgaat tgaatcattt
gcttcgtgtc acaaatacaa tttagntagg tacatgcatt 1200ggtcagattc
acggtttatt atgtcatgac ttaagttcat ggtagtacat tacctgccac
1260gcatgcatta tattggttag atttgatagg caaatttggt tgtcaacaat
ataaatataa 1320ataatgtttt tatattacga aataacagtg atcaaaacaa
acagttttat ctttattaac 1380aagattttgt ttttgtttga tgacgttttt
taatgtttac gctttccccc ttcttttgaa 1440tttagaacac tttatcatca
taaaatcaaa tactaaaaaa attacatatt tcataaataa 1500taacacaaat
atttttaaaa aatctgaaat aataatgaac aatattacat attatcacga
1560aaattcatta ataaaaatat tatataaata aaatgtaata gtagttatat
gtaggaaaaa 1620agtactgcac gcataatata tacaaaaaga ttaaaatgaa
ctattataaa taataacact 1680aaattaatgg tgaatcatat caaaataatg
aaaaagtaaa taaaatttgt aattaacttc 1740tatatgtatt acacacacaa
ataataaata atagtaaaaa aaattatgat aaatatttac 1800catctcataa
gatatttaaa ataatgataa aaatatagat tattttttat gcaactagct
1860agccaaaaag agaacacggg tatatataaa aagagtacct ttaaattcta
ctgtacttcc 1920tttattcctg acgtttttat atcaagtgga catacgtgaa
gattttaatt atcagtctaa 1980atatttcatt agcacttaat acttttctgt
tttattccta tcctataagt agtcccgatt 2040ctcccaacat tgcttattca
cacaactaac taagaaagtc ttccatagcc ccccaagcgg 2100ccggagctgg
tcatctcgct catcgtcgag tcggcggccg gagctggtca tctcgctcat
2160cgtcgagtcg gcggccgccg actcgacgat gagcgagatg accagctccg
gccgccgact 2220cgacgatgag cgagatgacc agctccggcc gcgacacaag
tgtgagagta ctaaataaat 2280gctttggttg tacgaaatca ttacactaaa
taaaataatc aaagcttata tatgccttcc 2340gctaaggccg aatgcaaaga
aattggttct ttctcgttat cttttgccac ttttactagt 2400acgtattaat
tactacttaa tcatctttgt ttacggctca ttatatccgt cgacggcgcg
2460138966DNAartificial sequencevector pKS210 13gatcctcgaa
gagaagggtt aataacacat tttttaacat ttttaacaca aattttagtt 60atttaaaaat
ttattaaaaa atttaaaata agaagaggaa ctctttaaat aaatctaact
120tacaaaattt atgattttta ataagttttc accaataaaa aatgtcataa
aaatatgtta 180aaaagtatat tatcaatatt ctctttatga taaataaaaa
gaaaaaaaaa ataaaagtta 240agtgaaaatg agattgaagt gactttaggt
gtgtataaat atatcaaccc cgccaacaat 300ttatttaatc caaatatatt
gaagtatatt attccatagc ctttatttat ttatatattt 360attatataaa
agctttattt gttctaggtt gttcatgaaa tatttttttg gttttatctc
420cgttgtaaga aaatcatgtg ctttgtgtcg ccactcacta ttgcagcttt
ttcatgcatt 480ggtcagattg acggttgatt gtatttttgt tttttatggt
tttgtgttat gacttaagtc 540ttcatctctt tatctcttca tcaggtttga
tggttaccta atatggtcca tgggtacatg 600catggttaaa ttaggtggcc
aactttgttg tgaacgatag aatttttttt atattaagta 660aactattttt
atattatgaa ataataataa aaaaaatatt ttatcattat taacaaaatc
720atattagtta atttgttaac tctataataa aagaaatact gtaacattca
cattacatgg 780taacatcttt ccaccctttc atttgttttt tgtttgatga
ctttttttct tgtttaaatt 840tatttccctt cttttaaatt tggaatacat
tatcatcata tataaactaa aatactaaaa 900acaggattac acaaatgata
aataataaca caaatattta taaatctagc tgcaatatat 960ttaaactagc
tatatcgata ttgtaaaata aaactagctg cattgatact gataaaaaaa
1020tatcatgtgc tttctggact gatgatgcag tatacttttg acattgcctt
tattttattt 1080ttcagaaaag ctttcttagt
tctgggttct tcattatttg tttcccatct ccattgtgaa 1140ttgaatcatt
tgcttcgtgt cacaaataca atttagntag gtacatgcat tggtcagatt
1200cacggtttat tatgtcatga cttaagttca tggtagtaca ttacctgcca
cgcatgcatt 1260atattggtta gatttgatag gcaaatttgg ttgtcaacaa
tataaatata aataatgttt 1320ttatattacg aaataacagt gatcaaaaca
aacagtttta tctttattaa caagattttg 1380tttttgtttg atgacgtttt
ttaatgttta cgctttcccc cttcttttga atttagaaca 1440ctttatcatc
ataaaatcaa atactaaaaa aattacatat ttcataaata ataacacaaa
1500tatttttaaa aaatctgaaa taataatgaa caatattaca tattatcacg
aaaattcatt 1560aataaaaata ttatataaat aaaatgtaat agtagttata
tgtaggaaaa aagtactgca 1620cgcataatat atacaaaaag attaaaatga
actattataa ataataacac taaattaatg 1680gtgaatcata tcaaaataat
gaaaaagtaa ataaaatttg taattaactt ctatatgtat 1740tacacacaca
aataataaat aatagtaaaa aaaattatga taaatattta ccatctcata
1800agatatttaa aataatgata aaaatataga ttatttttta tgcaactagc
tagccaaaaa 1860gagaacacgg gtatatataa aaagagtacc tttaaattct
actgtacttc ctttattcct 1920gacgttttta tatcaagtgg acatacgtga
agattttaat tatcagtcta aatatttcat 1980tagcacttaa tacttttctg
ttttattcct atcctataag tagtcccgat tctcccaaca 2040ttgcttattc
acacaactaa ctaagaaagt cttccatagc cccccaagcg gccggagctg
2100gtcatctcgc tcatcgtcga gtcggcggcc ggagctggtc atctcgctca
tcgtcgagtc 2160ggcggccgcc gactcgacga tgagcgagat gaccagctcc
ggccgccgac tcgacgatga 2220gcgagatgac cagctccggc cgcgacacaa
gtgtgagagt actaaataaa tgctttggtt 2280gtacgaaatc attacactaa
ataaaataat caaagcttat atatgccttc cgctaaggcc 2340gaatgcaaag
aaattggttc tttctcgtta tcttttgcca cttttactag tacgtattaa
2400ttactactta atcatctttg tttacggctc attatatccg tcgacggcgc
gcccgatcat 2460ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
cccgtttaga ggccccaagg 2520ggttatgcta gttattgctc agcggtggca
gcagccaact cagcttcctt tcgggctttg 2580ttagcagccg gatcgatcca
agctgtacct cactattcct ttgccctcgg acgagtgctg 2640gggcgtcggt
ttccactatc ggcgagtact tctacacagc catcggtcca gacggccgcg
2700cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
gattgcgtcg 2760catcgaccct gcgcccaagc tgcatcatcg aaattgccgt
caaccaagct ctgatagagt 2820tggtcaagac caatgcggag catatacgcc
cggagccgcg gcgatcctgc aagctccgga 2880tgcctccgct cgaagtagcg
cgtctgctgc tccatacaag ccaaccacgg cctccagaag 2940aagatgttgg
cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc
3000gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
gtgcacgagg 3060tgccggactt cggggcagtc ctcggcccaa agcatcagct
catcgagagc ctgcgcgacg 3120gacgcactga cggtgtcgtc catcacagtt
tgccagtgat acacatgggg atcagcaatc 3180gcgcatatga aatcacgcca
tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg 3240aacccgctcg
tctggctaag atcggccgca gcgatcgcat ccatagcctc cgcgaccggc
3300tgcagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
ctgtgcacgg 3360cgggagatgc aataggtcag gctctcgctg aattccccaa
tgtcaagcac ttccggaatc 3420gggagcgcgg ccgatgcaaa gtgccgataa
acataacgat ctttgtagaa accatcggcg 3480cagctattta cccgcaggac
atatccacgc cctcctacat cgaagctgaa agcacgagat 3540tcttcgccct
ccgagagctg catcaggtcg gagacgctgt cgaacttttc gatcagaaac
3600ttctcgacag acgtcgcggt gagttcaggc ttttccatgg gtatatctcc
ttcttaaagt 3660taaacaaaat tatttctaga gggaaaccgt tgtggtctcc
ctatagtgag tcgtattaat 3720ttcgcgggat cgagatctga tcaacctgca
ttaatgaatc ggccaacgcg cggggagagg 3780cggtttgcgt attgggcgct
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 3840tcggctgcgg
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
3900aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
ggaaccgtaa 3960aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
cctgacgagc atcacaaaaa 4020tcgacgctca agtcagaggt ggcgaaaccc
gacaggacta taaagatacc aggcgtttcc 4080ccctggaagc tccctcgtgc
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4140cgcctttctc
ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag
4200ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
ttcagcccga 4260ccgctgcgcc ttatccggta actatcgtct tgagtccaac
ccggtaagac acgacttatc 4320gccactggca gcagccactg gtaacaggat
tagcagagcg aggtatgtag gcggtgctac 4380agagttcttg aagtggtggc
ctaactacgg ctacactaga aggacagtat ttggtatctg 4440cgctctgctg
aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
4500aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
gcagaaaaaa 4560aggatctcaa gaagatcctt tgatcttttc tacggggtct
gacgctcagt ggaacgaaaa 4620ctcacgttaa gggattttgg tcatgacatt
aacctataaa aataggcgta tcacgaggcc 4680ctttcgtctc gcgcgtttcg
gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 4740gacggtcaca
gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
4800agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
agattgtact 4860gagagtgcac catatggaca tattgtcgtt agaacgcggc
tacaattaat acataacctt 4920atgtatcata cacatacgat ttaggtgaca
ctatagaacg gcgcgccaag cttggatccg 4980cgccaagctt ggatcctaga
actagaaacg tgatgccact tgttattgaa gtcgattaca 5040gcatctattc
tgttttacta tttataactt tgccatttct gacttttgaa aactatctct
5100ggatttcggt atcgctttgt gaagatcgag caaaagagac gttttgtgga
cgcaatggtc 5160caaatccgtt ctacatgaac aaattggtca caatttccac
taaaagtaaa taaatggcaa 5220gttaaaaaag gaatatgcat tttactgatt
gcctaggtga gctccaagag aagttgaatc 5280tacacgtcta ccaaccgcta
aaaaaagaaa aacattgata tgtaacctga ttccattagc 5340ttttgacttc
ttcaacagat tctctactta gatttctaac agaaatatta ttactagcac
5400atcattttca gtctcactac agcaaaaaat ccaacggcac aatacagaca
acaggagata 5460tcagactaca gagatagata gatgctactg catgtagtaa
gttaaataaa aggaaaataa 5520aatgtcttgc taccaaaact actacagact
atgatgctca ccacaggcca aatcctgcaa 5580ctaggacagc attatcttat
atatattgta caaaacaagc atcaaggaac atttggtcta 5640ggcaatcagt
acctcgttct accatcaccc tcagttatca catccttgaa ggatccatta
5700ctgggaatca tcggcaacac atgctcctga tggggcacaa tgacatcaag
aaggtagggg 5760ccaggggtgt ccaacattct ctgaattgcc gctctaagct
cttccttctt cgtcactcgc 5820gctgccggta tcccacaagc atcagcaaac
ttgagcatgt ttgggaatat ctcgctctcg 5880ctagacggat ctccaagata
ggtgtgagct ctattggact tgtagaacct atcctccaac 5940tgaaccacca
tacccaaatg ctgattgttc aacaacaata tcttaactgg gagattctcc
6000actcttatag tggccaactc ctgaacattc atgatgaaac taccatcccc
atcaatgtca 6060accacaacag ccccagggtt agcaacagca gcaccaatag
ccgcaggcaa tccaaaaccc 6120atggctccaa gaccccctga ggtcaaccac
tgcctcggtc tcttgtactt gtaaaactgc 6180gcagcccaca tttgatgctg
cccaacccca gtactaacaa tagcatctcc attagtcaac 6240tcatcaagaa
cctcgatagc atgctgcgga gaaatcgcgt cctggaatgt cttgtaaccc
6300aatggaaact tgtgtttctg cacattaatc tcttctctcc aacctccaag
atcaaactta 6360ccctccactc ctttctcctc caaaatcata ttaattccct
tcaaggccaa cttcaaatcc 6420gcgcaaaccg acacgtgcgc ctgcttgttc
ttcccaatct cggcagaatc aatatcaatg 6480tgaacaatct tagccctact
agcaaaagcc tcaagcttcc cagtaacacg gtcatcaaac 6540cttaccccaa
aggcaagcaa caaatcacta ttgtcaacag catagttagc ataaacagta
6600ccatgcatac ccagcatctg aagggaatat tcatcaccaa taggaaaagt
tccaagaccc 6660attaaagtgc tagcaacggg aataccagtg agttcaacaa
agcgcctcaa ttcagcactg 6720gaattcaaac tgccaccgcc gacgtagaga
acgggctttt gggcctccat gatgagtctg 6780acaatgtgtt ccaattgggc
ctcggcgggg ggcctgggca gcctggcgag gtaaccgggg 6840aggttaacgg
gctcgtccca attaggcacg gcgagttgct gctgaacgtc tttgggaatg
6900tcgatgagga ccggaccggg gcggccggag gtggcgacga agaaagcctc
ggcgacgacg 6960cgggggatgt cgtcgacgtc gaggatgagg tagttgtgct
tcgtgatgga tctgctcacc 7020tccacgatcg gggtttcttg gaaggcgtcg
gtgccgatca tccggcgggc gacctggccg 7080gtgatggcga cgactgggac
gctgtccatt aaagcgtcgg cgaggccgct cacgaggttg 7140gtggcgccgg
ggccggaggt ggcaatgcag acgccgggga ggccggagga acgcgcgtag
7200ccttcggcgg cgaagacgcc gccctgctcg tggcgcggga gcacgttgcg
gatggcggcg 7260gagcgcgtga gcgcctggtg gatctccatc gacgcaccgc
cggggtacgc gaacaccgtc 7320gtcacgccct gcctctccag cgcctccaca
aggatgtccg cgcccttgcg aggttcgccg 7380gaggcgaacc gtgacacgaa
gggctccgtg gtcggcgctt ccttggtgaa gggcgccgcc 7440gtggggggtt
tggagatgga acatttgatt ttgagagcgt ggttgggttt ggtgagggtt
7500tgatgagaga gagggagggt ggatctagta atgcgtttgg ggaaggtggg
gtgtgaagag 7560gaagaagaga atcgggtggt tctggaagcg gtggccgcca
ttgtgttgtg tggcatggtt 7620atacttcaaa aactgcacaa caagcctaga
gttagtacct aaacagtaaa tttacaacag 7680agagcaaaga cacatgcaaa
aatttcagcc ataaaaaaag ttataataga atttaaagca 7740aaagtttcat
tttttaaaca tatatacaaa caaactggat ttgaaggaag ggattaattc
7800ccctgctcaa agtttgaatt cctattgtga cctatactcg aataaaattg
aagcctaagg 7860aatgtatgag aaacaagaaa acaaaacaaa actacagaca
aacaagtaca attacaaaat 7920tcgctaaaat tctgtaatca ccaaacccca
tctcagtcag cacaaggccc aaggtttatt 7980ttgaaataaa aaaaaagtga
ttttatttct cataagctaa aagaaagaaa ggcaattatg 8040aaatgatttc
gactagatct gaaagtccaa cgcgtattcc gcagatatta aagaaagagt
8100agagtttcac atggatccta gatggaccca gttgaggaaa aagcaaggca
aagcaaacca 8160gaagtgcaag atccgaaatt gaaccacgga atctaggatt
tggtagaggg agaagaaaag 8220taccttgaga ggtagaagag aagagaagag
cagagagata tatgaacgag tgtgtcttgg 8280tctcaactct gaagcgatac
gagtttagag gggagcattg agttccaatt tatagggaaa 8340ccgggtggca
ggggtgagtt aatgacggaa aagcccctaa gtaacgagat tggattgtgg
8400gttagattca accgtttgca tccgcggctt agattgggga agtcagagtg
aatctcaacc 8460gttgactgag ttgaaaattg aatgtagcaa ccaattgagc
caaccccagc ctttgccctt 8520tgattttgat ttgtttgttg catacttttt
atttgtcttc tggttctgac tctctttctc 8580tcgtttcaat gccaggttgc
ctactcccac accactcaca agaagattct actgttagta 8640ttaaatattt
tttaatgtat taaatgatga atgcttttgt aaacagaaca agactatgtc
8700taataagtgt cttgcaacat tttttaagaa attaaaaaaa atatatttat
tatcaaaatc 8760aaatgtatga aaaatcatga ataatataat tttatacatt
tttttaaaaa atcttttaat 8820ttcttaatta atatcttaaa aataatgatt
aatatttaac ccaaaataat tagtatgatt 8880ggtaaggaag atatccatgt
tatgtttgga tgtgagtttg atctagagca aagcttacta 8940gagtcgaccg
atccgtcgac ggcgcg 8966146611DNAartificial sequencePlasmid PHP17731
14cgcgcctatg cgggaccatc gcagcggacg agaagcggca cgagaacgcg tactcaagaa
60tcgtggagaa gcttctggaa gtggacccca ccggggcaat ggtggccata gggaacatga
120tggagaagaa gatcacgatg ccggcgcacc ttatgtacga tggggatgac
cccaggctat 180tcgagcacta ctccgctgtg gcgcagcgca taggcgtgta
caccgccaac gactacgcag 240acatcttgga tttctcgttg acggtgaaga
ttggagaagc ttgaaggatt gatgcctgag 300gggaagcggg ccccaggatt
tccgtgtgtg ggttgccccc gaggattagg aggttccaag 360aacgcgctga
tgagcgagcg cgtaagatga agaagcatca tgccgttaag ttcagttgga
420ttttcaataa agaattgctt ttgtgagcgg ccgccgactc gacgatgagc
gagatgacca 480gctccggccg ccgactcgac gatgagcgag atgaccagct
ccggccgcga cacaagtgtg 540agagtactaa ataaatgctt tggttgtacg
aaatcattac actaaataaa ataatcaaag 600cttatatatg ccttccgcta
aggccgaatg caaagaaatt ggttctttct cgttatcttt 660tgccactttt
actagtacgt attaattact acttaatcat ctttgtttac ggctcattat
720atccgtcgac ggcgcgcccg atcatccgga tatagttcct cctttcagca
aaaaacccct 780caagacccgt ttagaggccc caaggggtta tgctagttat
tgctcagcgg tggcagcagc 840caactcagct tcctttcggg ctttgttagc
agccggatcg atccaagctg tacctcacta 900ttcctttgcc ctcggacgag
tgctggggcg tcggtttcca ctatcggcga gtacttctac 960acagccatcg
gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc cgacagtccc
1020ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat
catcgaaatt 1080gccgtcaacc aagctctgat agagttggtc aagaccaatg
cggagcatat acgcccggag 1140ccgcggcgat cctgcaagct ccggatgcct
ccgctcgaag tagcgcgtct gctgctccat 1200acaagccaac cacggcctcc
agaagaagat gttggcgacc tcgtattggg aatccccgaa 1260catcgcctcg
ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca ggacattgtt
1320ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg
cccaaagcat 1380cagctcatcg agagcctgcg cgacggacgc actgacggtg
tcgtccatca cagtttgcca 1440gtgatacaca tggggatcag caatcgcgca
tatgaaatca cgccatgtag tgtattgacc 1500gattccttgc ggtccgaatg
ggccgaaccc gctcgtctgg ctaagatcgg ccgcagcgat 1560cgcatccata
gcctccgcga ccggctgcag aacagcgggc agttcggttt caggcaggtc
1620ttgcaacgtg acaccctgtg cacggcggga gatgcaatag gtcaggctct
cgctgaattc 1680cccaatgtca agcacttccg gaatcgggag cgcggccgat
gcaaagtgcc gataaacata 1740acgatctttg tagaaaccat cggcgcagct
atttacccgc aggacatatc cacgccctcc 1800tacatcgaag ctgaaagcac
gagattcttc gccctccgag agctgcatca ggtcggagac 1860gctgtcgaac
ttttcgatca gaaacttctc gacagacgtc gcggtgagtt caggcttttc
1920catgggtata tctccttctt aaagttaaac aaaattattt ctagagggaa
accgttgtgg 1980tctccctata gtgagtcgta ttaatttcgc gggatcgaga
tctgatcaac ctgcattaat 2040gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc gcttcctcgc 2100tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 2160cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
2220gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
cataggctcc 2280gcccccctga cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga aacccgacag 2340gactataaag ataccaggcg tttccccctg
gaagctccct cgtgcgctct cctgttccga 2400ccctgccgct taccggatac
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 2460aatgctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
2520tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
cgtcttgagt 2580ccaacccggt aagacacgac ttatcgccac tggcagcagc
cactggtaac aggattagca 2640gagcgaggta tgtaggcggt gctacagagt
tcttgaagtg gtggcctaac tacggctaca 2700ctagaaggac agtatttggt
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 2760ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
2820agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg 2880ggtctgacgc tcagtggaac gaaaactcac gttaagggat
tttggtcatg acattaacct 2940ataaaaatag gcgtatcacg aggccctttc
gtctcgcgcg tttcggtgat gacggtgaaa 3000acctctgaca catgcagctc
ccggagacgg tcacagcttg tctgtaagcg gatgccggga 3060gcagacaagc
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
3120atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
tcgttagaac 3180gcggctacaa ttaatacata accttatgta tcatacacat
acgatttagg tgacactata 3240gaacggcgcg ccaagcttgg atcctcgaag
agaagggtta ataacacatt ttttaacatt 3300tttaacacaa attttagtta
tttaaaaatt tattaaaaaa tttaaaataa gaagaggaac 3360tctttaaata
aatctaactt acaaaattta tgatttttaa taagttttca ccaataaaaa
3420atgtcataaa aatatgttaa aaagtatatt atcaatattc tctttatgat
aaataaaaag 3480aaaaaaaaaa taaaagttaa gtgaaaatga gattgaagtg
actttaggtg tgtataaata 3540tatcaacccc gccaacaatt tatttaatcc
aaatatattg aagtatatta ttccatagcc 3600tttatttatt tatatattta
ttatataaaa gctttatttg ttctaggttg ttcatgaaat 3660atttttttgg
ttttatctcc gttgtaagaa aatcatgtgc tttgtgtcgc cactcactat
3720tgcagctttt tcatgcattg gtcagattga cggttgattg tatttttgtt
ttttatggtt 3780ttgtgttatg acttaagtct tcatctcttt atctcttcat
caggtttgat ggttacctaa 3840tatggtccat gggtacatgc atggttaaat
taggtggcca actttgttgt gaacgataga 3900atttttttta tattaagtaa
actattttta tattatgaaa taataataaa aaaaatattt 3960tatcattatt
aacaaaatca tattagttaa tttgttaact ctataataaa agaaatactg
4020taacattcac attacatggt aacatctttc caccctttca tttgtttttt
gtttgatgac 4080tttttttctt gtttaaattt atttcccttc ttttaaattt
ggaatacatt atcatcatat 4140ataaactaaa atactaaaaa caggattaca
caaatgataa ataataacac aaatatttat 4200aaatctagct gcaatatatt
taaactagct atatcgatat tgtaaaataa aactagctgc 4260attgatactg
ataaaaaaat atcatgtgct ttctggactg atgatgcagt atacttttga
4320cattgccttt attttatttt tcagaaaagc tttcttagtt ctgggttctt
cattatttgt 4380ttcccatctc cattgtgaat tgaatcattt gcttcgtgtc
acaaatacaa tttagntagg 4440tacatgcatt ggtcagattc acggtttatt
atgtcatgac ttaagttcat ggtagtacat 4500tacctgccac gcatgcatta
tattggttag atttgatagg caaatttggt tgtcaacaat 4560ataaatataa
ataatgtttt tatattacga aataacagtg atcaaaacaa acagttttat
4620ctttattaac aagattttgt ttttgtttga tgacgttttt taatgtttac
gctttccccc 4680ttcttttgaa tttagaacac tttatcatca taaaatcaaa
tactaaaaaa attacatatt 4740tcataaataa taacacaaat atttttaaaa
aatctgaaat aataatgaac aatattacat 4800attatcacga aaattcatta
ataaaaatat tatataaata aaatgtaata gtagttatat 4860gtaggaaaaa
agtactgcac gcataatata tacaaaaaga ttaaaatgaa ctattataaa
4920taataacact aaattaatgg tgaatcatat caaaataatg aaaaagtaaa
taaaatttgt 4980aattaacttc tatatgtatt acacacacaa ataataaata
atagtaaaaa aaattatgat 5040aaatatttac catctcataa gatatttaaa
ataatgataa aaatatagat tattttttat 5100gcaactagct agccaaaaag
agaacacggg tatatataaa aagagtacct ttaaattcta 5160ctgtacttcc
tttattcctg acgtttttat atcaagtgga catacgtgaa gattttaatt
5220atcagtctaa atatttcatt agcacttaat acttttctgt tttattccta
tcctataagt 5280agtcccgatt ctcccaacat tgcttattca cacaactaac
taagaaagtc ttccatagcc 5340ccccaagcgg ccggagctgg tcatctcgct
catcgtcgag tcggcggccg gagctggtca 5400tctcgctcat cgtcgagtcg
gcggccgctg agtgattgct cacgagtgtg gtcaccatgc 5460cttcagcaag
taccaatggg ttgatgatgt tgtgggtttg acccttcact caacactttt
5520agtcccttat ttctcatgga aaataagcca tcgccgccat cactccaaca
caggttccct 5580tgaccgtgat gaagtgtttg tcccaaaacc aaaatccaaa
gttgcatggt tttccaagta 5640cttaaacaac cctctaggaa gggctgtttc
tcttctcgtc acactcacaa tagggtggcc 5700tatgtattta gccttcaatg
tctctggtag accctatgat agttttgcaa gccactacca 5760cccttatgct
cccatatatt ctaaccgtga gaggcttctg atctatgtct ctgatgttgc
5820tttgttttct gtgacttact ctctctaccg tgttgcaacc ctgaaagggt
tggtttggct 5880gctatgtgtt tatggggtgc ctttgctcat tgtgaacggt
tttcttgtga ctatcacata 5940tttgcagcac acacactttg ccttgcctca
ttacgattca tcagaatggg actggctgaa 6000gggagctttg gcaactatgg
acagagatta agcggccgca tgcctccaga aaagaaagaa 6060attttcaagt
ccttggaggg atgggcctcg gagtgggtcc taccgctgct gaagcccgtg
6120gagcaatgct ggcagccaca aaacttcctc cctgacccct cccttccgca
tgaagagttc 6180agccatcagg tgaaggagct tcgcgaacgc actaaagagt
tacctgatga gtactttgtg 6240gtgctggtgg gtgatatggt caccgaggac
gcgcttccca cttaccagac catgatcaac 6300aaccttgatg gagtgaaaga
tgacagcggc acgagcccga gcccgtgggc cgtgtggacc 6360cgggcctgga
ccgccgagga aaacagacac ggggatctgc tcagaactta tttgtatctc
6420tctgggaggg ttgacatggc taaggtcgaa aagaccgtac attacctcat
ttcagctggc 6480atggaccctg ggacagacaa caacccatat ttggggtttg
tgtacacgtc attccaagag 6540cgagcaacat ttgtggcgca cgggaacacg
gctcggctcg cgaaggaggg cggggatcca 6600gtgctggcgc g
6611154097DNAartificial sequenceRecombinant DNA fragment PHP17731A
15cgcgccaagc ttggatcctc gaagagaagg gttaataaca cattttttaa catttttaac
60acaaatttta gttatttaaa aatttattaa aaaatttaaa ataagaagag gaactcttta
120aataaatcta acttacaaaa tttatgattt ttaataagtt ttcaccaata
aaaaatgtca 180taaaaatatg ttaaaaagta tattatcaat attctcttta
tgataaataa aaagaaaaaa 240aaaataaaag ttaagtgaaa atgagattga
agtgacttta ggtgtgtata aatatatcaa 300ccccgccaac aatttattta
atccaaatat attgaagtat attattccat agcctttatt 360tatttatata
tttattatat aaaagcttta tttgttctag
gttgttcatg aaatattttt 420ttggttttat ctccgttgta agaaaatcat
gtgctttgtg tcgccactca ctattgcagc 480tttttcatgc attggtcaga
ttgacggttg attgtatttt tgttttttat ggttttgtgt 540tatgacttaa
gtcttcatct ctttatctct tcatcaggtt tgatggttac ctaatatggt
600ccatgggtac atgcatggtt aaattaggtg gccaactttg ttgtgaacga
tagaattttt 660tttatattaa gtaaactatt tttatattat gaaataataa
taaaaaaaat attttatcat 720tattaacaaa atcatattag ttaatttgtt
aactctataa taaaagaaat actgtaacat 780tcacattaca tggtaacatc
tttccaccct ttcatttgtt ttttgtttga tgactttttt 840tcttgtttaa
atttatttcc cttcttttaa atttggaata cattatcatc atatataaac
900taaaatacta aaaacaggat tacacaaatg ataaataata acacaaatat
ttataaatct 960agctgcaata tatttaaact agctatatcg atattgtaaa
ataaaactag ctgcattgat 1020actgataaaa aaatatcatg tgctttctgg
actgatgatg cagtatactt ttgacattgc 1080ctttatttta tttttcagaa
aagctttctt agttctgggt tcttcattat ttgtttccca 1140tctccattgt
gaattgaatc atttgcttcg tgtcacaaat acaatttagn taggtacatg
1200cattggtcag attcacggtt tattatgtca tgacttaagt tcatggtagt
acattacctg 1260ccacgcatgc attatattgg ttagatttga taggcaaatt
tggttgtcaa caatataaat 1320ataaataatg tttttatatt acgaaataac
agtgatcaaa acaaacagtt ttatctttat 1380taacaagatt ttgtttttgt
ttgatgacgt tttttaatgt ttacgctttc ccccttcttt 1440tgaatttaga
acactttatc atcataaaat caaatactaa aaaaattaca tatttcataa
1500ataataacac aaatattttt aaaaaatctg aaataataat gaacaatatt
acatattatc 1560acgaaaattc attaataaaa atattatata aataaaatgt
aatagtagtt atatgtagga 1620aaaaagtact gcacgcataa tatatacaaa
aagattaaaa tgaactatta taaataataa 1680cactaaatta atggtgaatc
atatcaaaat aatgaaaaag taaataaaat ttgtaattaa 1740cttctatatg
tattacacac acaaataata aataatagta aaaaaaatta tgataaatat
1800ttaccatctc ataagatatt taaaataatg ataaaaatat agattatttt
ttatgcaact 1860agctagccaa aaagagaaca cgggtatata taaaaagagt
acctttaaat tctactgtac 1920ttcctttatt cctgacgttt ttatatcaag
tggacatacg tgaagatttt aattatcagt 1980ctaaatattt cattagcact
taatactttt ctgttttatt cctatcctat aagtagtccc 2040gattctccca
acattgctta ttcacacaac taactaagaa agtcttccat agccccccaa
2100gcggccggag ctggtcatct cgctcatcgt cgagtcggcg gccggagctg
gtcatctcgc 2160tcatcgtcga gtcggcggcc gctgagtgat tgctcacgag
tgtggtcacc atgccttcag 2220caagtaccaa tgggttgatg atgttgtggg
tttgaccctt cactcaacac ttttagtccc 2280ttatttctca tggaaaataa
gccatcgccg ccatcactcc aacacaggtt cccttgaccg 2340tgatgaagtg
tttgtcccaa aaccaaaatc caaagttgca tggttttcca agtacttaaa
2400caaccctcta ggaagggctg tttctcttct cgtcacactc acaatagggt
ggcctatgta 2460tttagccttc aatgtctctg gtagacccta tgatagtttt
gcaagccact accaccctta 2520tgctcccata tattctaacc gtgagaggct
tctgatctat gtctctgatg ttgctttgtt 2580ttctgtgact tactctctct
accgtgttgc aaccctgaaa gggttggttt ggctgctatg 2640tgtttatggg
gtgcctttgc tcattgtgaa cggttttctt gtgactatca catatttgca
2700gcacacacac tttgccttgc ctcattacga ttcatcagaa tgggactggc
tgaagggagc 2760tttggcaact atggacagag attaagcggc cgcatgcctc
cagaaaagaa agaaattttc 2820aagtccttgg agggatgggc ctcggagtgg
gtcctaccgc tgctgaagcc cgtggagcaa 2880tgctggcagc cacaaaactt
cctccctgac ccctcccttc cgcatgaaga gttcagccat 2940caggtgaagg
agcttcgcga acgcactaaa gagttacctg atgagtactt tgtggtgctg
3000gtgggtgata tggtcaccga ggacgcgctt cccacttacc agaccatgat
caacaacctt 3060gatggagtga aagatgacag cggcacgagc ccgagcccgt
gggccgtgtg gacccgggcc 3120tggaccgccg aggaaaacag acacggggat
ctgctcagaa cttatttgta tctctctggg 3180agggttgaca tggctaaggt
cgaaaagacc gtacattacc tcatttcagc tggcatggac 3240cctgggacag
acaacaaccc atatttgggg tttgtgtaca cgtcattcca agagcgagca
3300acatttgtgg cgcacgggaa cacggctcgg ctcgcgaagg agggcgggga
tccagtgctg 3360gcgcgcgcgc ctatgcggga ccatcgcagc ggacgagaag
cggcacgaga acgcgtactc 3420aagaatcgtg gagaagcttc tggaagtgga
ccccaccggg gcaatggtgg ccatagggaa 3480catgatggag aagaagatca
cgatgccggc gcaccttatg tacgatgggg atgaccccag 3540gctattcgag
cactactccg ctgtggcgca gcgcataggc gtgtacaccg ccaacgacta
3600cgcagacatc ttggatttct cgttgacggt gaagattgga gaagcttgaa
ggattgatgc 3660ctgaggggaa gcgggcccca ggatttccgt gtgtgggttg
cccccgagga ttaggaggtt 3720ccaagaacgc gctgatgagc gagcgcgtaa
gatgaagaag catcatgccg ttaagttcag 3780ttggattttc aataaagaat
tgcttttgtg agcggccgcc gactcgacga tgagcgagat 3840gaccagctcc
ggccgccgac tcgacgatga gcgagatgac cagctccggc cgcgacacaa
3900gtgtgagagt actaaataaa tgctttggtt gtacgaaatc attacactaa
ataaaataat 3960caaagcttat atatgccttc cgctaaggcc gaatgcaaag
aaattggttc tttctcgtta 4020tcttttgcca cttttactag tacgtattaa
ttactactta atcatctttg tttacggctc 4080attatatccg tcgacgg
4097163964DNAartificial sequenceALS selectable marker recombinant
DNA fragment 16ggtcgactct agtaagcttt gctctagatc aaactcacat
ccaaacataa catggatatc 60ttccttacca atcatactaa ttattttggg ttaaatatta
atcattattt ttaagatatt 120aattaagaaa ttaaaagatt ttttaaaaaa
atgtataaaa ttatattatt catgattttt 180catacatttg attttgataa
taaatatatt ttttttaatt tcttaaaaaa tgttgcaaga 240cacttattag
acatagtctt gttctgttta caaaagcatt catcatttaa tacattaaaa
300aatatttaat actaacagta gaatcttctt gtgagtggtg tgggagtagg
caacctggca 360ttgaaacgag agaaagagag tcagaaccag aagacaaata
aaaagtatgc aacaaacaaa 420tcaaaatcaa agggcaaagg ctggggttgg
ctcaattggt tgctacattc aattttcaac 480tcagtcaacg gttgagattc
actctgactt ccccaatcta agccgcggat gcaaacggtt 540gaatctaacc
cacaatccaa tctcgttact taggggcttt tccgtcatta actcacccct
600gccacccggt ttccctataa attggaactc aatgctcccc tctaaactcg
tatcgcttca 660gagttgagac caagacacac tcgttcatat atctctctgc
tcttctcttc tcttctacct 720ctcaaggtac ttttcttctc cctctaccaa
atcctagatt ccgtggttca atttcggatc 780ttgcacttct ggtttgcttt
gccttgcttt ttcctcaact gggtccatct aggatccatg 840tgaaactcta
ctctttcttt aatatctgcg gaatacgcgt tggactttca gatctagtcg
900aaatcatttc ataattgcct ttctttcttt tagcttatga gaaataaaat
cacttttttt 960ttatttcaaa ataaaccttg ggccttgtgc tgactgagat
ggggtttggt gattacagaa 1020ttttagcgaa ttttgtaatt gtacttgttt
gtctgtagtt ttgttttgtt ttcttgtttc 1080tcatacattc cttaggcttc
aattttattc gagtataggt cacaatagga attcaaactt 1140tgagcagggg
aattaatccc ttccttcaaa tccagtttgt ttgtatatat gtttaaaaaa
1200tgaaactttt gctttaaatt ctattataac tttttttatg gctgaaattt
ttgcatgtgt 1260ctttgctctc tgttgtaaat ttactgttta ggtactaact
ctaggcttgt tgtgcagttt 1320ttgaagtata accatgccac acaacacaat
ggcggccacc gcttccagaa ccacccgatt 1380ctcttcttcc tcttcacacc
ccaccttccc caaacgcatt actagatcca ccctccctct 1440ctctcatcaa
accctcacca aacccaacca cgctctcaaa atcaaatgtt ccatctccaa
1500accccccacg gcggcgccct tcaccaagga agcgccgacc acggagccct
tcgtgtcacg 1560gttcgcctcc ggcgaacctc gcaagggcgc ggacatcctt
gtggaggcgc tggagaggca 1620gggcgtgacg acggtgttcg cgtaccccgg
cggtgcgtcg atggagatcc accaggcgct 1680cacgcgctcc gccgccatcc
gcaacgtgct cccgcgccac gagcagggcg gcgtcttcgc 1740cgccgaaggc
tacgcgcgtt cctccggcct ccccggcgtc tgcattgcca cctccggccc
1800cggcgccacc aacctcgtga gcggcctcgc cgacgcttta atggacagcg
tcccagtcgt 1860cgccatcacc ggccaggtcg cccgccggat gatcggcacc
gacgccttcc aagaaacccc 1920gatcgtggag gtgagcagat ccatcacgaa
gcacaactac ctcatcctcg acgtcgacga 1980catcccccgc gtcgtcgccg
aggctttctt cgtcgccacc tccggccgcc ccggtccggt 2040cctcatcgac
attcccaaag acgttcagca gcaactcgcc gtgcctaatt gggacgagcc
2100cgttaacctc cccggttacc tcgccaggct gcccaggccc cccgccgagg
cccaattgga 2160acacattgtc agactcatca tggaggccca aaagcccgtt
ctctacgtcg gcggtggcag 2220tttgaattcc agtgctgaat tgaggcgctt
tgttgaactc actggtattc ccgttgctag 2280cactttaatg ggtcttggaa
cttttcctat tggtgatgaa tattcccttc agatgctggg 2340tatgcatggt
actgtttatg ctaactatgc tgttgacaat agtgatttgt tgcttgcctt
2400tggggtaagg tttgatgacc gtgttactgg gaagcttgag gcttttgcta
gtagggctaa 2460gattgttcac attgatattg attctgccga gattgggaag
aacaagcagg cgcacgtgtc 2520ggtttgcgcg gatttgaagt tggccttgaa
gggaattaat atgattttgg aggagaaagg 2580agtggagggt aagtttgatc
ttggaggttg gagagaagag attaatgtgc agaaacacaa 2640gtttccattg
ggttacaaga cattccagga cgcgatttct ccgcagcatg ctatcgaggt
2700tcttgatgag ttgactaatg gagatgctat tgttagtact ggggttgggc
agcatcaaat 2760gtgggctgcg cagttttaca agtacaagag accgaggcag
tggttgacct cagggggtct 2820tggagccatg ggttttggat tgcctgcggc
tattggtgct gctgttgcta accctggggc 2880tgttgtggtt gacattgatg
gggatggtag tttcatcatg aatgttcagg agttggccac 2940tataagagtg
gagaatctcc cagttaagat attgttgttg aacaatcagc atttgggtat
3000ggtggttcag ttggaggata ggttctacaa gtccaataga gctcacacct
atcttggaga 3060tccgtctagc gagagcgaga tattcccaaa catgctcaag
tttgctgatg cttgtgggat 3120accggcagcg cgagtgacga agaaggaaga
gcttagagcg gcaattcaga gaatgttgga 3180cacccctggc ccctaccttc
ttgatgtcat tgtgccccat caggagcatg tgttgccgat 3240gattcccagt
aatggatcct tcaaggatgt gataactgag ggtgatggta gaacgaggta
3300ctgattgcct agaccaaatg ttccttgatg cttgttttgt acaatatata
taagataatg 3360ctgtcctagt tgcaggattt ggcctgtggt gagcatcata
gtctgtagta gttttggtag 3420caagacattt tattttcctt ttatttaact
tactacatgc agtagcatct atctatctct 3480gtagtctgat atctcctgtt
gtctgtattg tgccgttgga ttttttgctg tagtgagact 3540gaaaatgatg
tgctagtaat aatatttctg ttagaaatct aagtagagaa tctgttgaag
3600aagtcaaaag ctaatggaat caggttacat atcaatgttt ttcttttttt
agcggttggt 3660agacgtgtag attcaacttc tcttggagct cacctaggca
atcagtaaaa tgcatattcc 3720ttttttaact tgccatttat ttacttttag
tggaaattgt gaccaatttg ttcatgtaga 3780acggatttgg accattgcgt
ccacaaaacg tctcttttgc tcgatcttca caaagcgata 3840ccgaaatcca
gagatagttt tcaaaagtca gaaatggcaa agttataaat agtaaaacag
3900aatagatgct gtaatcgact tcaataacaa gtggcatcac gtttctagtt
ctagacccgg 3960gtac 396417656PRTartificial sequenceSoybean ALS
resistant to herbicides 17Met Pro His Asn Thr Met Ala Ala Thr Ala
Ser Arg Thr Thr Arg Phe1 5 10 15Ser Ser Ser Ser Ser His Pro Thr Phe
Pro Lys Arg Ile Thr Arg Ser 20 25 30Thr Leu Pro Leu Ser His Gln Thr
Leu Thr Lys Pro Asn His Ala Leu 35 40 45Lys Ile Lys Cys Ser Ile Ser
Lys Pro Pro Thr Ala Ala Pro Phe Thr 50 55 60Lys Glu Ala Pro Thr Thr
Glu Pro Phe Val Ser Arg Phe Ala Ser Gly65 70 75 80Glu Pro Arg Lys
Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln 85 90 95Gly Val Thr
Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu Ile 100 105 110His
Gln Ala Leu Thr Arg Ser Ala Ala Ile Arg Asn Val Leu Pro Arg 115 120
125His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly Tyr Ala Arg Ser Ser
130 135 140Gly Leu Pro Gly Val Cys Ile Ala Thr Ser Gly Pro Gly Ala
Thr Asn145 150 155 160Leu Val Ser Gly Leu Ala Asp Ala Leu Met Asp
Ser Val Pro Val Val 165 170 175Ala Ile Thr Gly Gln Val Ala Arg Arg
Met Ile Gly Thr Asp Ala Phe 180 185 190Gln Glu Thr Pro Ile Val Glu
Val Ser Arg Ser Ile Thr Lys His Asn 195 200 205Tyr Leu Ile Leu Asp
Val Asp Asp Ile Pro Arg Val Val Ala Glu Ala 210 215 220Phe Phe Val
Ala Thr Ser Gly Arg Pro Gly Pro Val Leu Ile Asp Ile225 230 235
240Pro Lys Asp Val Gln Gln Gln Leu Ala Val Pro Asn Trp Asp Glu Pro
245 250 255Val Asn Leu Pro Gly Tyr Leu Ala Arg Leu Pro Arg Pro Pro
Ala Glu 260 265 270Ala Gln Leu Glu His Ile Val Arg Leu Ile Met Glu
Ala Gln Lys Pro 275 280 285Val Leu Tyr Val Gly Gly Gly Ser Leu Asn
Ser Ser Ala Glu Leu Arg 290 295 300Arg Phe Val Glu Leu Thr Gly Ile
Pro Val Ala Ser Thr Leu Met Gly305 310 315 320Leu Gly Thr Phe Pro
Ile Gly Asp Glu Tyr Ser Leu Gln Met Leu Gly 325 330 335Met His Gly
Thr Val Tyr Ala Asn Tyr Ala Val Asp Asn Ser Asp Leu 340 345 350Leu
Leu Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu 355 360
365Glu Ala Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser
370 375 380Ala Glu Ile Gly Lys Asn Lys Gln Ala His Val Ser Val Cys
Ala Asp385 390 395 400Leu Lys Leu Ala Leu Lys Gly Ile Asn Met Ile
Leu Glu Glu Lys Gly 405 410 415Val Glu Gly Lys Phe Asp Leu Gly Gly
Trp Arg Glu Glu Ile Asn Val 420 425 430Gln Lys His Lys Phe Pro Leu
Gly Tyr Lys Thr Phe Gln Asp Ala Ile 435 440 445Ser Pro Gln His Ala
Ile Glu Val Leu Asp Glu Leu Thr Asn Gly Asp 450 455 460Ala Ile Val
Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln465 470 475
480Phe Tyr Lys Tyr Lys Arg Pro Arg Gln Trp Leu Thr Ser Gly Gly Leu
485 490 495Gly Ala Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ala
Val Ala 500 505 510Asn Pro Gly Ala Val Val Val Asp Ile Asp Gly Asp
Gly Ser Phe Ile 515 520 525Met Asn Val Gln Glu Leu Ala Thr Ile Arg
Val Glu Asn Leu Pro Val 530 535 540Lys Ile Leu Leu Leu Asn Asn Gln
His Leu Gly Met Val Val Gln Leu545 550 555 560Glu Asp Arg Phe Tyr
Lys Ser Asn Arg Ala His Thr Tyr Leu Gly Asp 565 570 575Pro Ser Ser
Glu Ser Glu Ile Phe Pro Asn Met Leu Lys Phe Ala Asp 580 585 590Ala
Cys Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Glu Glu Leu Arg 595 600
605Ala Ala Ile Gln Arg Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp
610 615 620Val Ile Val Pro His Gln Glu His Val Leu Pro Met Ile Pro
Ser Asn625 630 635 640Gly Ser Phe Lys Asp Val Ile Thr Glu Gly Asp
Gly Arg Thr Arg Tyr 645 650 655184PRTartificial sequencetobacco
wild type ALS subsequence B 18Gly Gln Val Pro11910PRTartificial
sequencetobacco wild type ALS subsequence F 19Gly Met Val Xaa Gln
Trp Glu Asp Arg Phe1 5 10205PRTartificial sequenceN-terminal amino
acids added to ALS 20Met Pro His Asn Thr1 5216547DNAartificial
sequencePlasmid PHP17064 21gggcgaattg ggttacccgg accggaattc
gggatctgag tctagaaatc cgtcaacatg 60gtggagcacg acactctcgt ctactccaag
aatatcaaag atacagtctc agaagaccaa 120agggctattg agacttttca
acaaagggta atatcgggaa acctcctcgg attccattgc 180ccagctatct
gtcacttcat caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc
240catcattgcg ataaaggaaa ggctatcgtt caagatgcct ctgccgacag
tggtcccaaa 300gatggacccc cacccacgag gagcatcgtg gaaaaagaag
acgttccaac cacgtcttca 360aagcaagtgg attgatgtga tgatcctatg
cgtatggtat gacgtgtgtt caagatgatg 420acttcaaacc tacctatgac
gtatggtatg aacgtgtgtc gactgatgac ttagatccac 480tcgagcggct
ataaatacgt acctacgcac cctgcgctac catccctaga gctgcagctt
540atttttacaa caattaccaa caacaacaaa caacaaacaa cattacaatt
actatttaca 600attacagtcg acccgtaccc acacaacaca atggcggcca
ccgcttccag aaccacccga 660ttctcttctt cctcttcaca ccccaccttc
cccaaacgca ttactagatc caccctccct 720ctctctcatc aaaccctcac
caaacccaac cacgctctca aaatcaaatg ttccatctcc 780aaacccccca
cggcggcgcc cttcaccaag gaagcgccga ccacggagcc cttcgtgtca
840cggttcgcct ccggcgaacc tcgcaagggc gcggacatcc ttgtggaggc
gctggagagg 900cagggcgtga cgacggtgtt cgcgtacccc ggcggtgcgt
cgatggagat ccaccaggcg 960ctcacgcgct ccgccgccat ccgcaacgtg
ctcccgcgcc acgagcaggg cggcgtcttc 1020gccgccgaag gctacgcgcg
ttcctccggc ctccccggcg tctgcattgc cacctccggc 1080cccggcgcca
ccaacctcgt gagcggcctc gccgacgctt taatggacag cgtcccagtc
1140gtcgccatca ccggccaggt cgcccgccgg atgatcggca ccgacgcctt
ccaagaaacc 1200ccgatcgtgg aggtgagcag atccatcacg aagcacaact
acctcatcct cgacgtcgac 1260gacatccccc gcgtcgtcgc cgaggctttc
ttcgtcgcca cctccggccg ccccggtccg 1320gtcctcatcg acattcccaa
agacgttcag cagcaactcg ccgtgcctaa ttgggacgag 1380cccgttaacc
tccccggtta cctcgccagg ctgcccaggc cccccgccga ggcccaattg
1440gaacacattg tcagactcat catggaggcc caaaagcccg ttctctacgt
cggcggtggc 1500agtttgaatt ccagtgctga attgaggcgc tttgttgaac
tcactggtat tcccgttgct 1560agcactttaa tgggtcttgg aacttttcct
attggtgatg aatattccct tcagatgctg 1620ggtatgcatg gtactgttta
tgctaactat gctgttgaca atagtgattt gttgcttgcc 1680tttggggtaa
ggtttgatga ccgtgttact gggaagcttg aggcttttgc tagtagggct
1740aagattgttc acattgatat tgattctgcc gagattggga agaacaagca
ggcgcacgtg 1800tcggtttgcg cggatttgaa gttggccttg aagggaatta
atatgatttt ggaggagaaa 1860ggagtggagg gtaagtttga tcttggaggt
tggagagaag agattaatgt gcagaaacac 1920aagtttccat tgggttacaa
gacattccag gacgcgattt ctccgcagca tgctatcgag 1980gttcttgatg
agttgactaa tggagatgct attgttagta ctggggttgg gcagcatcaa
2040atgtgggctg cgcagtttta caagtacaag agaccgaggc agtggttgac
ctcagggggt 2100cttggagcca tgggttttgg attgcctgcg gctattggtg
ctgctgttgc taaccctggg 2160gctgttgtgg ttgacattga tggggatggt
agtttcatca tgaatgttca ggagttggcc 2220actataagag tggagaatct
cccagttaag atattgttgt tgaacaatca gcatttgggt 2280atggtggttc
agttggagga taggttctac aagtccaata gagctcacac ctatcttgga
2340gatccgtcta gcgagagcga gatattccca aacatgctca agtttgctga
tgcttgtggg 2400ataccggcag cgcgagtgac gaagaaggaa gagcttagag
cggcaattca gagaatgttg 2460gacacccctg gcccctacct tcttgatgtc
attgtgcccc atcaggagca tgtgttgccg 2520atgattccca gtaatggatc
cttcaaggat gtgataactg agggtgatgg tagaacgagg 2580tactgattgc
ctagaccaaa tgttccttga tgcttgtttt gtacaatata tataagataa
2640tgctgtccta gttgcaggat ttggcctgtg gtgagcatca tagtctgtag
tagttttggt 2700agcaagacat tttattttcc ttttatttaa cttactacat
gcagtagcat ctatctatct 2760ctgtagtctg atatctcctg ttgtctgtat
tgtgccgttg gattttttgc tgtagtgaga 2820ctgaaaatga tgtgctagta
ataatatttc tgttagaaat ctaagtagag
aatctgttga 2880agaagtcaaa agctaatgga atcaggttac atatcaatgt
ttttcttttt ttagcggttg 2940gtagacgtgt agattcaact tctcttggag
ctcacctagg caatcagtaa aatgcatatt 3000ccttttttaa cttgccattt
atttactttt agtggaaatt gtgaccaatt tgttcatgta 3060gaacggattt
ggaccattgc gtccacaaaa cgtctctttt gctcgatctt cacaaagcga
3120taccgaaatc cagagatagt tttcaaaagt cagaaatggc aaagttataa
atagtaaaac 3180agaatagatg ctgtaatcga cttcaataac aagtggcatc
acgtttctag ttctagaccc 3240gggtctagag tcgacctgca ggcatgcccg
cggatatcga tgggccccgg ccgaagcttc 3300ggtccgggtc acccagcttg
agtattctat agtgtcacct aaatagcttg gcgtaatcat 3360ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
3420ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
acattaattg 3480cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg cattaatgaa 3540tcggccaacg cgcggggaga ggcggtttgc
gtattgggcg ctcttccgct tcctcgctca 3600ctgactcgct gcgctcggtc
gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 3660taatacggtt
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
3720agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttcgat
aggctccgcc 3780cccctgacga gcatcacaaa aatcgacgct caagtcagag
gtggcgaaac ccgacaggac 3840tataaagata ccaggcgttt ccccctggaa
gctccctcgt gcgctctcct gttccgaccc 3900tgccgcttac cggatacctg
tccgcctttc tcccttcggg aagcgtggcg ctttctcata 3960gctcacgctg
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
4020acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
cttgagtcca 4080acccggtaag acacgactta tcgccactgg cagcagccac
tggtaacagg attagcagag 4140cgaggtatgt aggcggtgct acagagttct
tgaagtggtg gcctaactac ggctacacta 4200gaaggacagt atttggtatc
tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4260gtagctcttg
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
4320agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt 4380ctgacgctca gtggaacgaa aactcacgtt aagggatttt
ggtcatggag ccacgttgtg 4440tctcaaaatc tctgatgtta cattgcacaa
gataaaaata tatcatcatg aacaataaaa 4500ctgtctgctt acataaacag
taatacaagg ggtgttatga gccatattca acgggaaacg 4560tcttgctcga
ggccgcgatt aaattccaac atggatgctg atttatatgc ctataaatgg
4620gctcgcgata atgtcggcca atcaggtccg acaatctatc gattgtatgg
gaagcccgat 4680gcgccagact tgtttctgaa acatggcaaa ggtagccttg
ccaatgatgt tacagatgag 4740atggtcagac taaactgcct gacggaattt
atgcctcttc cgaccatcaa gcattttatc 4800cgtactcctg atgatgcatg
gttactcacc actgcgatcc cngggaaaac agcattccag 4860gtattagaag
aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg
4920cgccggttgc attcgattcc tctttgtaat tgtcctttta acagcgatcg
cgtatttcgt 4980ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
atgcgagtga ttttgatgac 5040gagcgtaatg gctggcctgt tgaacaagtc
tggaaagaaa tgcataanct tttgccattc 5100tcaccggatt cagtcgtcac
tcatggtgat ttctcacttg ataaccttat ttttgaccag 5160gcgaaattaa
taggttgtat tgatcttcga cgagtcggaa tcgcagaccg ataccaggat
5220cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
acggcttttt 5280caaaaatatg gtattgataa tcctgatatg aataaattgc
agtttcattt gatcctcgat 5340gagtttttct aatcagaatt ggttaattgg
ttgtaacact ggcagagcat tacgctgact 5400tgacgggacg gcggctttgt
tgaataaatc gaacttttgc tgacttgaag gatcagatca 5460cgcatcttcc
cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact
5520ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg
ctggatgatg 5580gggcgattca ggcctggtat gagtcagcaa caccttcttc
acgagccatg acattaacct 5640ataaaaatag gcgtatcacg aggccctttc
gtctcgcgcg tttcggtgat gacggtgaaa 5700acctctgaca catgcagctc
ccggagacgg tcacagcttg tctgtaagcg gatgccggga 5760gcagacaagc
ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
5820atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
ataccgcaca 5880gatgcgtaag gagaaaatac cgcatcaggc gaaattgtaa
acgttaatat tttgttaaaa 5940ttcgcgttaa atatttgtta aatcagctca
ttttttaacc aataggccga aatcggcaaa 6000atcccttata aatcaaaaga
atagaccgag atagggttga gtgttgttcc agtttggaac 6060aagagtccac
tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
6120ggcgatggcc cactacgtga accatcaccc aaatcaagtt ttttgcggtc
gaggtgccgt 6180aaagctctaa atcggaaccc taaagggagc ccccgattta
gagcttgacg gggaaagccg 6240gcgaacgtgg cgagaaagga agggaagaaa
gcgaaaggag cgggcgctag ggcgctggca 6300agtgtagcgg tcacgctgcg
cgtaaccacc acacccgccg cgcttaatgc gccgctacag 6360ggcgcgtcca
ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct
6420cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
agttgggtaa 6480cgccagggtt ttcccagtca cgacgttgta aaacgacggc
cagtgaattg taatacgact 6540cactata 6547223191DNAartificial
sequenceRecombinant DNA fragment PHP17064A 22ctagaaatcc gtcaacatgg
tggagcacga cactctcgtc tactccaaga atatcaaaga 60tacagtctca gaagaccaaa
gggctattga gacttttcaa caaagggtaa tatcgggaaa 120cctcctcgga
ttccattgcc cagctatctg tcacttcatc aaaaggacag tagaaaagga
180aggtggcacc tacaaatgcc atcattgcga taaaggaaag gctatcgttc
aagatgcctc 240tgccgacagt ggtcccaaag atggaccccc acccacgagg
agcatcgtgg aaaaagaaga 300cgttccaacc acgtcttcaa agcaagtgga
ttgatgtgat gatcctatgc gtatggtatg 360acgtgtgttc aagatgatga
cttcaaacct acctatgacg tatggtatga acgtgtgtcg 420actgatgact
tagatccact cgagcggcta taaatacgta cctacgcacc ctgcgctacc
480atccctagag ctgcagctta tttttacaac aattaccaac aacaacaaac
aacaaacaac 540attacaatta ctatttacaa ttacagtcga cccgtaccca
cacaacacaa tggcggccac 600cgcttccaga accacccgat tctcttcttc
ctcttcacac cccaccttcc ccaaacgcat 660tactagatcc accctccctc
tctctcatca aaccctcacc aaacccaacc acgctctcaa 720aatcaaatgt
tccatctcca aaccccccac ggcggcgccc ttcaccaagg aagcgccgac
780cacggagccc ttcgtgtcac ggttcgcctc cggcgaacct cgcaagggcg
cggacatcct 840tgtggaggcg ctggagaggc agggcgtgac gacggtgttc
gcgtaccccg gcggtgcgtc 900gatggagatc caccaggcgc tcacgcgctc
cgccgccatc cgcaacgtgc tcccgcgcca 960cgagcagggc ggcgtcttcg
ccgccgaagg ctacgcgcgt tcctccggcc tccccggcgt 1020ctgcattgcc
acctccggcc ccggcgccac caacctcgtg agcggcctcg ccgacgcttt
1080aatggacagc gtcccagtcg tcgccatcac cggccaggtc gcccgccgga
tgatcggcac 1140cgacgccttc caagaaaccc cgatcgtgga ggtgagcaga
tccatcacga agcacaacta 1200cctcatcctc gacgtcgacg acatcccccg
cgtcgtcgcc gaggctttct tcgtcgccac 1260ctccggccgc cccggtccgg
tcctcatcga cattcccaaa gacgttcagc agcaactcgc 1320cgtgcctaat
tgggacgagc ccgttaacct ccccggttac ctcgccaggc tgcccaggcc
1380ccccgccgag gcccaattgg aacacattgt cagactcatc atggaggccc
aaaagcccgt 1440tctctacgtc ggcggtggca gtttgaattc cagtgctgaa
ttgaggcgct ttgttgaact 1500cactggtatt cccgttgcta gcactttaat
gggtcttgga acttttccta ttggtgatga 1560atattccctt cagatgctgg
gtatgcatgg tactgtttat gctaactatg ctgttgacaa 1620tagtgatttg
ttgcttgcct ttggggtaag gtttgatgac cgtgttactg ggaagcttga
1680ggcttttgct agtagggcta agattgttca cattgatatt gattctgccg
agattgggaa 1740gaacaagcag gcgcacgtgt cggtttgcgc ggatttgaag
ttggccttga agggaattaa 1800tatgattttg gaggagaaag gagtggaggg
taagtttgat cttggaggtt ggagagaaga 1860gattaatgtg cagaaacaca
agtttccatt gggttacaag acattccagg acgcgatttc 1920tccgcagcat
gctatcgagg ttcttgatga gttgactaat ggagatgcta ttgttagtac
1980tggggttggg cagcatcaaa tgtgggctgc gcagttttac aagtacaaga
gaccgaggca 2040gtggttgacc tcagggggtc ttggagccat gggttttgga
ttgcctgcgg ctattggtgc 2100tgctgttgct aaccctgggg ctgttgtggt
tgacattgat ggggatggta gtttcatcat 2160gaatgttcag gagttggcca
ctataagagt ggagaatctc ccagttaaga tattgttgtt 2220gaacaatcag
catttgggta tggtggttca gttggaggat aggttctaca agtccaatag
2280agctcacacc tatcttggag atccgtctag cgagagcgag atattcccaa
acatgctcaa 2340gtttgctgat gcttgtggga taccggcagc gcgagtgacg
aagaaggaag agcttagagc 2400ggcaattcag agaatgttgg acacccctgg
cccctacctt cttgatgtca ttgtgcccca 2460tcaggagcat gtgttgccga
tgattcccag taatggatcc ttcaaggatg tgataactga 2520gggtgatggt
agaacgaggt actgattgcc tagaccaaat gttccttgat gcttgttttg
2580tacaatatat ataagataat gctgtcctag ttgcaggatt tggcctgtgg
tgagcatcat 2640agtctgtagt agttttggta gcaagacatt ttattttcct
tttatttaac ttactacatg 2700cagtagcatc tatctatctc tgtagtctga
tatctcctgt tgtctgtatt gtgccgttgg 2760attttttgct gtagtgagac
tgaaaatgat gtgctagtaa taatatttct gttagaaatc 2820taagtagaga
atctgttgaa gaagtcaaaa gctaatggaa tcaggttaca tatcaatgtt
2880tttctttttt tagcggttgg tagacgtgta gattcaactt ctcttggagc
tcacctaggc 2940aatcagtaaa atgcatattc cttttttaac ttgccattta
tttactttta gtggaaattg 3000tgaccaattt gttcatgtag aacggatttg
gaccattgcg tccacaaaac gtctcttttg 3060ctcgatcttc acaaagcgat
accgaaatcc agagatagtt ttcaaaagtc agaaatggca 3120aagttataaa
tagtaaaaca gaatagatgc tgtaatcgac ttcaataaca agtggcatca
3180cgtttctagt t 3191232924DNAartificial sequencefragment PHP19340A
23cgcgccaagc ttggatcctc gaagagaagg gttaataaca cactttttta acatttttaa
60cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga ggaactcttt
120aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat
aaaaaatgtc 180ataaaaatat gttaaaaagt atattatcaa tattctcttt
atgataaata aaaagaaaaa 240aaaaataaaa gttaagtgaa aatgagattg
aagtgacttt aggtgtgtat aaatatatca 300accccgccaa caatttattt
aatccaaata tattgaagta tattattcca tagcctttat 360ttatttatat
atttattata taaaagcttt atttgttcta ggttgttcat gaaatatttt
420tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc
actattgcag 480ctttttcatg cattggtcag attgacggtt gattgtattt
ttgtttttta tggttttgtg 540ttatgactta agtcttcatc tctttatctc
ttcatcaggt ttgatggtta cctaatatgg 600tccatgggta catgcatggt
taaattaggt ggccaacttt gttgtgaacg atagaatttt 660ttttatatta
agtaaactat ttttatatta tgaaataata ataaaaaaaa tattttatca
720ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa
tactgtaaca 780ttcacattac atggtaacat ctttccaccc tttcatttgt
tttttgtttg atgacttttt 840ttcttgttta aatttatttc ccttctttta
aatttggaat acattatcat catatataaa 900ctaaaatact aaaaacagga
ttacacaaat gataaataat aacacaaata tttataaatc 960tagctgcaat
atatttaaac tagctatatc gatattgtaa aataaaacta gctgcattga
1020tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact
tttgacattg 1080cctttatttt atttttcaga aaagctttct tagttctggg
ttcttcatta tttgtttccc 1140atctccattg tgaattgaat catttgcttc
gtgtcacaaa tacaatttag ntaggtacat 1200gcattggtca gattcacggt
ttattatgtc atgacttaag ttcatggtag tacattacct 1260gccacgcatg
cattatattg gttagatttg ataggcaaat ttggttgtca acaatataaa
1320tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt
tttatcttta 1380ttaacaagat tttgtttttg tttgatgacg ttttttaatg
tttacgcttt cccccttctt 1440ttgaatttag aacactttat catcataaaa
tcaaatacta aaaaaattac atatttcata 1500aataataaca caaatatttt
taaaaaatct gaaataataa tgaacaatat tacatattat 1560cacgaaaatt
cattaataaa aatattatat aaataaaatg taatagtagt tatatgtagg
1620aaaaaagtac tgcacgcata atatatacaa aaagattaaa atgaactatt
ataaataata 1680acactaaatt aatggtgaat catatcaaaa taatgaaaaa
gtaaataaaa tttgtaatta 1740acttctatat gtattacaca cacaaataat
aaataatagt aaaaaaaatt atgataaata 1800tttaccatct cataagatat
ttaaaataat gataaaaata tagattattt tttatgcaac 1860tagctagcca
aaaagagaac acgggtatat ataaaaagag tacctttaaa ttctactgta
1920cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt
taattatcag 1980tctaaatatt tcattagcac ttaatacttt tctgttttat
tcctatccta taagtagtcc 2040cgattctccc aacattgctt attcacacaa
ctaactaaga aagtcttcca tagcccccca 2100agcggccgct gagtgattgc
tcacgagtgt ggtcaccatg ccttcagcaa gtaccaatgg 2160gttgatgatg
ttgtgggttt gacccttcac tcaacacttt tagtccctta tttctcatgg
2220aaaataagcc atcgccgcca tcactccaac acaggttccc ttgaccgtga
tgaagtgttt 2280gtcccaaaac caaaatccaa agttgcatgg ttttccaagt
acttaaacaa ccctctagga 2340agggctgttt ctcttctcgt cacactcaca
atagggtggc ctatgtattt agccttcaat 2400gtctctggta gaccctatga
tagttttgca agccactacc acccttatgc tcccatatat 2460tctaaccgtg
agaggcttct gatctatgtc tctgatgttg ctttgttttc tgtgacttac
2520tctctctacc gtgttgcaac cctgaaaggg ttggtttggc tgctatgtgt
ttatggggtg 2580cctttgctca ttgtgaacgg ttttcttgtg actatcacat
atttgcagca cacacacttt 2640gccttgcctc attacgattc atcagaatgg
gactggctga agggagcttt ggcaactatg 2700gacagagatt aagcggccgc
gacacaagtg tgagagtact aaataaatgc tttggttgta 2760cgaaatcatt
acactaaata aaataatcaa agcttatata tgccttccgc taaggccgaa
2820tgcaaagaaa ttggttcttt ctcgttatct tttgccactt ttactagtac
gtattaatta 2880ctacttaatc atctttgttt acggctcatt atatccgtcg acgg
2924244511DNAartificial sequencefragment PHP17752 24cgcgccaagc
ttggatcccc cctcgaggtc gacggtatcg ataagcttct gcaggaattc 60tgagctagcg
aagttcctat tccgaagttc ctattcttca aaaagtatag gaacttcaga
120cgtcctcgag tccgtcctgt agaaacccca acccgtgaaa tcaaaaaact
cgacggcctg 180tgggcattca gtctggatcg cgaaaactgt ggaattgatc
cagaattcgc tagcgaagtt 240cctattccga agttcctatt ctctagaaag
tataggaact tcagatccag aattcggtcc 300gggccatcgt ggcctcttgc
tcttcaggat gaagagctat gtttcgcgcc aagcttggat 360cctagaacta
gaaacgtgat gccacttgtt attgaagtcg attacagcat ctattctgtt
420ttactattta taactttgcc atttctgact tttgaaaact atctctggat
ttcggtatcg 480ctttgtgaag atcgagcaaa agagacgttt tgtggacgca
atggtccaaa tccgttctac 540atgaacaaat tggtcacaat ttccactaaa
agtaaataaa tggcaagtta aaaaaggaat 600atgcatttta ctgattgcct
aggtgagctc caagagaagt tgaatctaca cgtctaccaa 660ccgctaaaaa
aagaaaaaca ttgatatgta acctgattcc attagctttt gacttcttca
720acagattctc tacttagatt tctaacagaa atattattac tagcacatca
ttttcagtct 780cactacagca aaaaatccaa cggcacaata cagacaacag
gagatatcag actacagaga 840tagatagatg ctactgcatg tagtaagtta
aataaaagga aaataaaatg tcttgctacc 900aaaactacta cagactatga
tgctcaccac aggccaaatc ctgcaactag gacagcatta 960tcttatatat
attgtacaaa acaagcatca aggaacattt ggtctaggca atcagtacct
1020cgttctacca tcaccctcag ttatcacatc cttgaaggat ccattactgg
gaatcatcgg 1080caacacatgc tcctgatggg gcacaatgac atcaagaagg
taggggccag gggtgtccaa 1140cattctctga attgccgctc taagctcttc
cttcttcgtc actcgcgctg ccggtatccc 1200acaagcatca gcaaacttga
gcatgtttgg gaatatctcg ctctcgctag acggatctcc 1260aagataggtg
tgagctctat tggacttgta gaacctatcc tccaactgaa ccaccatacc
1320caaatgctga ttgttcaaca acaatatctt aactgggaga ttctccactc
ttatagtggc 1380caactcctga acattcatga tgaaactacc atccccatca
atgtcaacca caacagcccc 1440agggttagca acagcagcac caatagccgc
aggcaatcca aaacccatgg ctccaagacc 1500ccctgaggtc aaccactgcc
tcggtctctt gtacttgtaa aactgcgcag cccacatttg 1560atgctgccca
accccagtac taacaatagc atctccatta gtcaactcat caagaacctc
1620gatagcatgc tgcggagaaa tcgcgtcctg gaatgtcttg taacccaatg
gaaacttgtg 1680tttctgcaca ttaatctctt ctctccaacc tccaagatca
aacttaccct ccactccttt 1740ctcctccaaa atcatattaa ttcccttcaa
ggccaacttc aaatccgcgc aaaccgacac 1800gtgcgcctgc ttgttcttcc
caatctcggc agaatcaata tcaatgtgaa caatcttagc 1860cctactagca
aaagcctcaa gcttcccagt aacacggtca tcaaacctta ccccaaaggc
1920aagcaacaaa tcactattgt caacagcata gttagcataa acagtaccat
gcatacccag 1980catctgaagg gaatattcat caccaatagg aaaagttcca
agacccatta aagtgctagc 2040aacgggaata ccagtgagtt caacaaagcg
cctcaattca gcactggaat tcaaactgcc 2100accgccgacg tagagaacgg
gcttttgggc ctccatgatg agtctgacaa tgtgttccaa 2160ttgggcctcg
gcggggggcc tgggcagcct ggcgaggtaa ccggggaggt taacgggctc
2220gtcccaatta ggcacggcga gttgctgctg aacgtctttg ggaatgtcga
tgaggaccgg 2280accggggcgg ccggaggtgg cgacgaagaa agcctcggcg
acgacgcggg ggatgtcgtc 2340gacgtcgagg atgaggtagt tgtgcttcgt
gatggatctg ctcacctcca cgatcggggt 2400ttcttggaag gcgtcggtgc
cgatcatccg gcgggcgacc tggccggtga tggcgacgac 2460tgggacgctg
tccattaaag cgtcggcgag gccgctcacg aggttggtgg cgccggggcc
2520ggaggtggca atgcagacgc cggggaggcc ggaggaacgc gcgtagcctt
cggcggcgaa 2580gacgccgccc tgctcgtggc gcgggagcac gttgcggatg
gcggcggagc gcgtgagcgc 2640ctggtggatc tccatcgacg caccgccggg
gtacgcgaac accgtcgtca cgccctgcct 2700ctccagcgcc tccacaagga
tgtccgcgcc cttgcgaggt tcgccggagg cgaaccgtga 2760cacgaagggc
tccgtggtcg gcgcttcctt ggtgaagggc gccgccgtgg ggggtttgga
2820gatggaacat ttgattttga gagcgtggtt gggtttggtg agggtttgat
gagagagagg 2880gagggtggat ctagtaatgc gtttggggaa ggtggggtgt
gaagaggaag aagagaatcg 2940ggtggttctg gaagcggtgg ccgccattgt
gttgtgtggc atggttatac ttcaaaaact 3000gcacaacaag cctagagtta
gtacctaaac agtaaattta caacagagag caaagacaca 3060tgcaaaaatt
tcagccataa aaaaagttat aatagaattt aaagcaaaag tttcattttt
3120taaacatata tacaaacaaa ctggatttga aggaagggat taattcccct
gctcaaagtt 3180tgaattccta ttgtgaccta tactcgaata aaattgaagc
ctaaggaatg tatgagaaac 3240aagaaaacaa aacaaaacta cagacaaaca
agtacaatta caaaattcgc taaaattctg 3300taatcaccaa accccatctc
agtcagcaca aggcccaagg tttattttga aataaaaaaa 3360aagtgatttt
atttctcata agctaaaaga aagaaaggca attatgaaat gatttcgact
3420agatctgaaa gtccaacgcg tattccgcag atattaaaga aagagtagag
tttcacatgg 3480atcctagatg gacccagttg aggaaaaagc aaggcaaagc
aaaccagaag tgcaagatcc 3540gaaattgaac cacggaatct aggatttggt
agagggagaa gaaaagtacc ttgagaggta 3600gaagagaaga gaagagcaga
gagatatatg aacgagtgtg tcttggtctc aactctgaag 3660cgatacgagt
ttagagggga gcattgagtt ccaatttata gggaaaccgg gtggcagggg
3720tgagttaatg acggaaaagc ccctaagtaa cgagattgga ttgtgggtta
gattcaaccg 3780tttgcatccg cggcttagat tggggaagtc agagtgaatc
tcaaccgttg actgagttga 3840aaattgaatg tagcaaccaa ttgagccaac
cccagccttt gccctttgat tttgatttgt 3900ttgttgcata ctttttattt
gtcttctggt tctgactctc tttctctcgt ttcaatgcca 3960ggttgcctac
tcccacacca ctcacaagaa gattctactg ttagtattaa atatttttta
4020atgtattaaa tgatgaatgc ttttgtaaac agaacaagac tatgtctaat
aagtgtcttg 4080caacattttt taagaaatta aaaaaaatat atttattatc
aaaatcaaat gtatgaaaaa 4140tcatgaataa tataatttta tacatttttt
taaaaaatct tttaatttct taattaatat 4200cttaaaaata atgattaata
tttaacccaa aataattagt atgattggta aggaagatat 4260ccatgttatg
tttggatgtg agtttgatct agagcaaagc ttactagagt cgaccgatcc
4320gtcgacggcg cgcgcgcctc tagttgaaga cacgttcatg tcttcatcgt
aagaagacac 4380tcagtagtct tcggccagaa tggcccggac cgaagcttct
gcaggaattc tgagctagcg 4440aagttcctat tccgaagttc ctattctcta
gaaagtatag gaacttcaga tccactagga 4500tccgtcgacg g
4511255437DNAartificial sequenceplasmid PHP19340 25ggccgcgaca
caagtgtgag agtactaaat aaatgctttg gttgtacgaa atcattacac 60taaataaaat
aatcaaagct tatatatgcc ttccgctaag gccgaatgca aagaaattgg
120ttctttctcg ttatcttttg ccacttttac tagtacgtat taattactac
ttaatcatct 180ttgtttacgg ctcattatat ccgtcgacgg cgcgcccgat
catccggata tagttcctcc 240tttcagcaaa aaacccctca agacccgttt
agaggcccca aggggttatg ctagttattg 300ctcagcggtg gcagcagcca
actcagcttc ctttcgggct ttgttagcag ccggatcgat 360ccaagctgta
cctcactatt cctttgccct cggacgagtg
ctggggcgtc ggtttccact 420atcggcgagt acttctacac agccatcggt
ccagacggcc gcgcttctgc gggcgatttg 480tgtacgcccg acagtcccgg
ctccggatcg gacgattgcg tcgcatcgac cctgcgccca 540agctgcatca
tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa gaccaatgcg
600gagcatatac gcccggagcc gcggcgatcc tgcaagctcc ggatgcctcc
gctcgaagta 660gcgcgtctgc tgctccatac aagccaacca cggcctccag
aagaagatgt tggcgacctc 720gtattgggaa tccccgaaca tcgcctcgct
ccagtcaatg accgctgtta tgcggccatt 780gtccgtcagg acattgttgg
agccgaaatc cgcgtgcacg aggtgccgga cttcggggca 840gtcctcggcc
caaagcatca gctcatcgag agcctgcgcg acggacgcac tgacggtgtc
900gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata
tgaaatcacg 960ccatgtagtg tattgaccga ttccttgcgg tccgaatggg
ccgaacccgc tcgtctggct 1020aagatcggcc gcagcgatcg catccatagc
ctccgcgacc ggctgcagaa cagcgggcag 1080ttcggtttca ggcaggtctt
gcaacgtgac accctgtgca cggcgggaga tgcaataggt 1140caggctctcg
ctgaattccc caatgtcaag cacttccgga atcgggagcg cggccgatgc
1200aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat
ttacccgcag 1260gacatatcca cgccctccta catcgaagct gaaagcacga
gattcttcgc cctccgagag 1320ctgcatcagg tcggagacgc tgtcgaactt
ttcgatcaga aacttctcga cagacgtcgc 1380ggtgagttca ggcttttcca
tgggtatatc tccttcttaa agttaaacaa aattatttct 1440agagggaaac
cgttgtggtc tccctatagt gagtcgtatt aatttcgcgg gatcgagatc
1500tgatcaacct gcattaatga atcggccaac gcgcggggag aggcggtttg
cgtattgggc 1560gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
cgttcggctg cggcgagcgg 1620tatcagctca ctcaaaggcg gtaatacggt
tatccacaga atcaggggat aacgcaggaa 1680agaacatgtg agcaaaaggc
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 1740cgtttttcca
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
1800ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
agctccctcg 1860tgcgctctcc tgttccgacc ctgccgctta ccggatacct
gtccgccttt ctcccttcgg 1920gaagcgtggc gctttctcaa tgctcacgct
gtaggtatct cagttcggtg taggtcgttc 1980gctccaagct gggctgtgtg
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 2040gtaactatcg
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
2100ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
ttgaagtggt 2160ggcctaacta cggctacact agaaggacag tatttggtat
ctgcgctctg ctgaagccag 2220ttaccttcgg aaaaagagtt ggtagctctt
gatccggcaa acaaaccacc gctggtagcg 2280gtggtttttt tgtttgcaag
cagcagatta cgcgcagaaa aaaaggatct caagaagatc 2340ctttgatctt
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
2400tggtcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
ctcgcgcgtt 2460tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
ggagacggtc acagcttgtc 2520tgtaagcgga tgccgggagc agacaagccc
gtcagggcgc gtcagcgggt gttggcgggt 2580gtcggggctg gcttaactat
gcggcatcag agcagattgt actgagagtg caccatatgg 2640acatattgtc
gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
2700gatttaggtg acactataga acggcgcgcc aagcttggat cctcgaagag
aagggttaat 2760aacacatttt ttaacatttt taacacaaat tttagttatt
taaaaattta ttaaaaaatt 2820taaaataaga agaggaactc tttaaataaa
tctaacttac aaaatttatg atttttaata 2880agttttcacc aataaaaaat
gtcataaaaa tatgttaaaa agtatattat caatattctc 2940tttatgataa
ataaaaagaa aaaaaaaata aaagttaagt gaaaatgaga ttgaagtgac
3000tttaggtgtg tataaatata tcaaccccgc caacaattta tttaatccaa
atatattgaa 3060gtatattatt ccatagcctt tatttattta tatatttatt
atataaaagc tttatttgtt 3120ctaggttgtt catgaaatat ttttttggtt
ttatctccgt tgtaagaaaa tcatgtgctt 3180tgtgtcgcca ctcactattg
cagctttttc atgcattggt cagattgacg gttgattgta 3240tttttgtttt
ttatggtttt gtgttatgac ttaagtcttc atctctttat ctcttcatca
3300ggtttgatgg ttacctaata tggtccatgg gtacatgcat ggttaaatta
ggtggccaac 3360tttgttgtga acgatagaat tttttttata ttaagtaaac
tatttttata ttatgaaata 3420ataataaaaa aaatatttta tcattattaa
caaaatcata ttagttaatt tgttaactct 3480ataataaaag aaatactgta
acattcacat tacatggtaa catctttcca ccctttcatt 3540tgttttttgt
ttgatgactt tttttcttgt ttaaatttat ttcccttctt ttaaatttgg
3600aatacattat catcatatat aaactaaaat actaaaaaca ggattacaca
aatgataaat 3660aataacacaa atatttataa atctagctgc aatatattta
aactagctat atcgatattg 3720taaaataaaa ctagctgcat tgatactgat
aaaaaaatat catgtgcttt ctggactgat 3780gatgcagtat acttttgaca
ttgcctttat tttatttttc agaaaagctt tcttagttct 3840gggttcttca
ttatttgttt cccatctcca ttgtgaattg aatcatttgc ttcgtgtcac
3900aaatacaatt tagntaggta catgcattgg tcagattcac ggtttattat
gtcatgactt 3960aagttcatgg tagtacatta cctgccacgc atgcattata
ttggttagat ttgataggca 4020aatttggttg tcaacaatat aaatataaat
aatgttttta tattacgaaa taacagtgat 4080caaaacaaac agttttatct
ttattaacaa gattttgttt ttgtttgatg acgtttttta 4140atgtttacgc
tttccccctt cttttgaatt tagaacactt tatcatcata aaatcaaata
4200ctaaaaaaat tacatatttc ataaataata acacaaatat ttttaaaaaa
tctgaaataa 4260taatgaacaa tattacatat tatcacgaaa attcattaat
aaaaatatta tataaataaa 4320atgtaatagt agttatatgt aggaaaaaag
tactgcacgc ataatatata caaaaagatt 4380aaaatgaact attataaata
ataacactaa attaatggtg aatcatatca aaataatgaa 4440aaagtaaata
aaatttgtaa ttaacttcta tatgtattac acacacaaat aataaataat
4500agtaaaaaaa attatgataa atatttacca tctcataaga tatttaaaat
aatgataaaa 4560atatagatta ttttttatgc aactagctag ccaaaaagag
aacacgggta tatataaaaa 4620gagtaccttt aaattctact gtacttcctt
tattcctgac gtttttatat caagtggaca 4680tacgtgaaga ttttaattat
cagtctaaat atttcattag cacttaatac ttttctgttt 4740tattcctatc
ctataagtag tcccgattct cccaacattg cttattcaca caactaacta
4800agaaagtctt ccatagcccc ccaagcggcc gctgagtgat tgctcacgag
tgtggtcacc 4860atgccttcag caagtaccaa tgggttgatg atgttgtggg
tttgaccctt cactcaacac 4920ttttagtccc ttatttctca tggaaaataa
gccatcgccg ccatcactcc aacacaggtt 4980cccttgaccg tgatgaagtg
tttgtcccaa aaccaaaatc caaagttgca tggttttcca 5040agtacttaaa
caaccctcta ggaagggctg tttctcttct cgtcacactc acaatagggt
5100ggcctatgta tttagccttc aatgtctctg gtagacccta tgatagtttt
gcaagccact 5160accaccctta tgctcccata tattctaacc gtgagaggct
tctgatctat gtctctgatg 5220ttgctttgtt ttctgtgact tactctctct
accgtgttgc aaccctgaaa gggttggttt 5280ggctgctatg tgtttatggg
gtgcctttgc tcattgtgaa cggttttctt gtgactatca 5340catatttgca
gcacacacac tttgccttgc ctcattacga ttcatcagaa tgggactggc
5400tgaagggagc tttggcaact atggacagag attaagc
5437267025DNAartificial sequenceplasmid PHP17752 26gatccgtcga
cggcgcgccc gatcatccgg atatagttcc tcctttcagc aaaaaacccc 60tcaagacccg
tttagaggcc ccaaggggtt atgctagtta ttgctcagcg gtggcagcag
120ccaactcagc ttcctttcgg gctttgttag cagccggatc gatccaagct
gtacctcact 180attcctttgc cctcggacga gtgctggggc gtcggtttcc
actatcggcg agtacttcta 240cacagccatc ggtccagacg gccgcgcttc
tgcgggcgat ttgtgtacgc ccgacagtcc 300cggctccgga tcggacgatt
gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat 360tgccgtcaac
caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga
420gccgcggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc
tgctgctcca 480tacaagccaa ccacggcctc cagaagaaga tgttggcgac
ctcgtattgg gaatccccga 540acatcgcctc gctccagtca atgaccgctg
ttatgcggcc attgtccgtc aggacattgt 600tggagccgaa atccgcgtgc
acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca 660tcagctcatc
gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc
720agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta
gtgtattgac 780cgattccttg cggtccgaat gggccgaacc cgctcgtctg
gctaagatcg gccgcagcga 840tcgcatccat agcctccgcg accggctgca
gaacagcggg cagttcggtt tcaggcaggt 900cttgcaacgt gacaccctgt
gcacggcggg agatgcaata ggtcaggctc tcgctgaatt 960ccccaatgtc
aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat
1020aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat
ccacgccctc 1080ctacatcgaa gctgaaagca cgagattctt cgccctccga
gagctgcatc aggtcggaga 1140cgctgtcgaa cttttcgatc agaaacttct
cgacagacgt cgcggtgagt tcaggctttt 1200ccatgggtat atctccttct
taaagttaaa caaaattatt tctagaggga aaccgttgtg 1260gtctccctat
agtgagtcgt attaatttcg cgggatcgag atctgatcaa cctgcattaa
1320tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
cgcttcctcg 1380ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
cggtatcagc tcactcaaag 1440gcggtaatac ggttatccac agaatcaggg
gataacgcag gaaagaacat gtgagcaaaa 1500ggccagcaaa aggccaggaa
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1560cgcccccctg
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
1620ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
tcctgttccg 1680accctgccgc ttaccggata cctgtccgcc tttctccctt
cgggaagcgt ggcgctttct 1740caatgctcac gctgtaggta tctcagttcg
gtgtaggtcg ttcgctccaa gctgggctgt 1800gtgcacgaac cccccgttca
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1860tccaacccgg
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
1920agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
ctacggctac 1980actagaagga cagtatttgg tatctgcgct ctgctgaagc
cagttacctt cggaaaaaga 2040gttggtagct cttgatccgg caaacaaacc
accgctggta gcggtggttt ttttgtttgc 2100aagcagcaga ttacgcgcag
aaaaaaagga tctcaagaag atcctttgat cttttctacg 2160gggtctgacg
ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gacattaacc
2220tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
tgacggtgaa 2280aacctctgac acatgcagct cccggagacg gtcacagctt
gtctgtaagc ggatgccggg 2340agcagacaag cccgtcaggg cgcgtcagcg
ggtgttggcg ggtgtcgggg ctggcttaac 2400tatgcggcat cagagcagat
tgtactgaga gtgcaccata tggacatatt gtcgttagaa 2460cgcggctaca
attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
2520agaacggcgc gccaagcttg gatcccccct cgaggtcgac ggtatcgata
agcttctgca 2580ggaattctga gctagcgaag ttcctattcc gaagttccta
ttcttcaaaa agtataggaa 2640cttcagacgt cctcgagtcc gtcctgtaga
aaccccaacc cgtgaaatca aaaaactcga 2700cggcctgtgg gcattcagtc
tggatcgcga aaactgtgga attgatccag aattcgctag 2760cgaagttcct
attccgaagt tcctattctc tagaaagtat aggaacttca gatccagaat
2820tcggtccggg ccatcgtggc ctcttgctct tcaggatgaa gagctatgtt
tcgcgccaag 2880cttggatcct agaactagaa acgtgatgcc acttgttatt
gaagtcgatt acagcatcta 2940ttctgtttta ctatttataa ctttgccatt
tctgactttt gaaaactatc tctggatttc 3000ggtatcgctt tgtgaagatc
gagcaaaaga gacgttttgt ggacgcaatg gtccaaatcc 3060gttctacatg
aacaaattgg tcacaatttc cactaaaagt aaataaatgg caagttaaaa
3120aaggaatatg cattttactg attgcctagg tgagctccaa gagaagttga
atctacacgt 3180ctaccaaccg ctaaaaaaag aaaaacattg atatgtaacc
tgattccatt agcttttgac 3240ttcttcaaca gattctctac ttagatttct
aacagaaata ttattactag cacatcattt 3300tcagtctcac tacagcaaaa
aatccaacgg cacaatacag acaacaggag atatcagact 3360acagagatag
atagatgcta ctgcatgtag taagttaaat aaaaggaaaa taaaatgtct
3420tgctaccaaa actactacag actatgatgc tcaccacagg ccaaatcctg
caactaggac 3480agcattatct tatatatatt gtacaaaaca agcatcaagg
aacatttggt ctaggcaatc 3540agtacctcgt tctaccatca ccctcagtta
tcacatcctt gaaggatcca ttactgggaa 3600tcatcggcaa cacatgctcc
tgatggggca caatgacatc aagaaggtag gggccagggg 3660tgtccaacat
tctctgaatt gccgctctaa gctcttcctt cttcgtcact cgcgctgccg
3720gtatcccaca agcatcagca aacttgagca tgtttgggaa tatctcgctc
tcgctagacg 3780gatctccaag ataggtgtga gctctattgg acttgtagaa
cctatcctcc aactgaacca 3840ccatacccaa atgctgattg ttcaacaaca
atatcttaac tgggagattc tccactctta 3900tagtggccaa ctcctgaaca
ttcatgatga aactaccatc cccatcaatg tcaaccacaa 3960cagccccagg
gttagcaaca gcagcaccaa tagccgcagg caatccaaaa cccatggctc
4020caagaccccc tgaggtcaac cactgcctcg gtctcttgta cttgtaaaac
tgcgcagccc 4080acatttgatg ctgcccaacc ccagtactaa caatagcatc
tccattagtc aactcatcaa 4140gaacctcgat agcatgctgc ggagaaatcg
cgtcctggaa tgtcttgtaa cccaatggaa 4200acttgtgttt ctgcacatta
atctcttctc tccaacctcc aagatcaaac ttaccctcca 4260ctcctttctc
ctccaaaatc atattaattc ccttcaaggc caacttcaaa tccgcgcaaa
4320ccgacacgtg cgcctgcttg ttcttcccaa tctcggcaga atcaatatca
atgtgaacaa 4380tcttagccct actagcaaaa gcctcaagct tcccagtaac
acggtcatca aaccttaccc 4440caaaggcaag caacaaatca ctattgtcaa
cagcatagtt agcataaaca gtaccatgca 4500tacccagcat ctgaagggaa
tattcatcac caataggaaa agttccaaga cccattaaag 4560tgctagcaac
gggaatacca gtgagttcaa caaagcgcct caattcagca ctggaattca
4620aactgccacc gccgacgtag agaacgggct tttgggcctc catgatgagt
ctgacaatgt 4680gttccaattg ggcctcggcg gggggcctgg gcagcctggc
gaggtaaccg gggaggttaa 4740cgggctcgtc ccaattaggc acggcgagtt
gctgctgaac gtctttggga atgtcgatga 4800ggaccggacc ggggcggccg
gaggtggcga cgaagaaagc ctcggcgacg acgcggggga 4860tgtcgtcgac
gtcgaggatg aggtagttgt gcttcgtgat ggatctgctc acctccacga
4920tcggggtttc ttggaaggcg tcggtgccga tcatccggcg ggcgacctgg
ccggtgatgg 4980cgacgactgg gacgctgtcc attaaagcgt cggcgaggcc
gctcacgagg ttggtggcgc 5040cggggccgga ggtggcaatg cagacgccgg
ggaggccgga ggaacgcgcg tagccttcgg 5100cggcgaagac gccgccctgc
tcgtggcgcg ggagcacgtt gcggatggcg gcggagcgcg 5160tgagcgcctg
gtggatctcc atcgacgcac cgccggggta cgcgaacacc gtcgtcacgc
5220cctgcctctc cagcgcctcc acaaggatgt ccgcgccctt gcgaggttcg
ccggaggcga 5280accgtgacac gaagggctcc gtggtcggcg cttccttggt
gaagggcgcc gccgtggggg 5340gtttggagat ggaacatttg attttgagag
cgtggttggg tttggtgagg gtttgatgag 5400agagagggag ggtggatcta
gtaatgcgtt tggggaaggt ggggtgtgaa gaggaagaag 5460agaatcgggt
ggttctggaa gcggtggccg ccattgtgtt gtgtggcatg gttatacttc
5520aaaaactgca caacaagcct agagttagta cctaaacagt aaatttacaa
cagagagcaa 5580agacacatgc aaaaatttca gccataaaaa aagttataat
agaatttaaa gcaaaagttt 5640cattttttaa acatatatac aaacaaactg
gatttgaagg aagggattaa ttcccctgct 5700caaagtttga attcctattg
tgacctatac tcgaataaaa ttgaagccta aggaatgtat 5760gagaaacaag
aaaacaaaac aaaactacag acaaacaagt acaattacaa aattcgctaa
5820aattctgtaa tcaccaaacc ccatctcagt cagcacaagg cccaaggttt
attttgaaat 5880aaaaaaaaag tgattttatt tctcataagc taaaagaaag
aaaggcaatt atgaaatgat 5940ttcgactaga tctgaaagtc caacgcgtat
tccgcagata ttaaagaaag agtagagttt 6000cacatggatc ctagatggac
ccagttgagg aaaaagcaag gcaaagcaaa ccagaagtgc 6060aagatccgaa
attgaaccac ggaatctagg atttggtaga gggagaagaa aagtaccttg
6120agaggtagaa gagaagagaa gagcagagag atatatgaac gagtgtgtct
tggtctcaac 6180tctgaagcga tacgagttta gaggggagca ttgagttcca
atttataggg aaaccgggtg 6240gcaggggtga gttaatgacg gaaaagcccc
taagtaacga gattggattg tgggttagat 6300tcaaccgttt gcatccgcgg
cttagattgg ggaagtcaga gtgaatctca accgttgact 6360gagttgaaaa
ttgaatgtag caaccaattg agccaacccc agcctttgcc ctttgatttt
6420gatttgtttg ttgcatactt tttatttgtc ttctggttct gactctcttt
ctctcgtttc 6480aatgccaggt tgcctactcc cacaccactc acaagaagat
tctactgtta gtattaaata 6540ttttttaatg tattaaatga tgaatgcttt
tgtaaacaga acaagactat gtctaataag 6600tgtcttgcaa cattttttaa
gaaattaaaa aaaatatatt tattatcaaa atcaaatgta 6660tgaaaaatca
tgaataatat aattttatac atttttttaa aaaatctttt aatttcttaa
6720ttaatatctt aaaaataatg attaatattt aacccaaaat aattagtatg
attggtaagg 6780aagatatcca tgttatgttt ggatgtgagt ttgatctaga
gcaaagctta ctagagtcga 6840ccgatccgtc gacggcgcgc gcgcctctag
ttgaagacac gttcatgtct tcatcgtaag 6900aagacactca gtagtcttcg
gccagaatgg cccggaccga agcttctgca ggaattctga 6960gctagcgaag
ttcctattcc gaagttccta ttctctagaa agtataggaa cttcagatcc 7020actag
7025
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