U.S. patent application number 12/320692 was filed with the patent office on 2009-08-27 for methods for increasing stearate content in soybean oil.
Invention is credited to Jean Kridl.
Application Number | 20090214744 12/320692 |
Document ID | / |
Family ID | 22462531 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090214744 |
Kind Code |
A1 |
Kridl; Jean |
August 27, 2009 |
Methods for increasing stearate content in soybean oil
Abstract
This invention relates to a method for increasing stearate as a
component of total triglycerides found in soybean seed. The method
generally comprises growing a soybean plant having integrated into
its genome a DNA construct comprising, in the 5' to 3' direction of
transcription, a promoter functional in a soybean plant seed cell,
a DNA sequence encoding an acyl-ACP thioesterase protein having
substantial activity on C18:0 acyl-ACP substrates, and a
transcription termination region functional in a plant cell. The
present invention also provides a soybean seed with about 33 weight
percent or greater stearate as a component of total fatty acids
found in seed triglycerides.
Inventors: |
Kridl; Jean; (Davis,
CA) |
Correspondence
Address: |
ARNOLD & PORTER LLP
555 TWELFTH STREET, N.W., ATTN: IP DOCKETING
WASHINGTON
DC
20004
US
|
Family ID: |
22462531 |
Appl. No.: |
12/320692 |
Filed: |
February 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11305021 |
Dec 19, 2005 |
7504563 |
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12320692 |
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10061280 |
Feb 4, 2002 |
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11305021 |
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09359070 |
Jul 22, 1999 |
6380462 |
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10061280 |
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09134262 |
Aug 14, 1998 |
6365802 |
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09359070 |
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Current U.S.
Class: |
426/601 ;
47/58.1FV; 554/9; 56/327.1 |
Current CPC
Class: |
C12N 15/8247 20130101;
C12N 9/16 20130101 |
Class at
Publication: |
426/601 ; 554/9;
47/58.1FV; 56/327.1 |
International
Class: |
A23D 9/00 20060101
A23D009/00; C11B 1/00 20060101 C11B001/00; A01G 1/00 20060101
A01G001/00; A01D 45/22 20060101 A01D045/22 |
Claims
1-53. (canceled)
54. Soybean seed oil comprising greater than about 20% stearate and
less than about 53% stearate.
55. The soybean seed oil of claim 54, further comprising about 50
weight percent or greater stearate expressed as a component of
total fatty acids contained in said oil.
56. The soybean seed oil of claim 54, further comprising about 25
percent or greater of said total fatty acids in a
saturated-unsaturated-saturated form.
57. The soybean seed oil of claim 54, further comprising about 25
percent or greater stearate as the fatty acid found at the sn-1 and
sn-3 positions of seed triglycerides.
58. The soybean seed oil of claim 57, further comprising oleic acid
as the predominant fatty acid at the sn-2 position of seed
triglycerides.
59. The soybean seed oil of claim 54, wherein said oil without
chemical modification comprises about 24% or greater stearate and
less than 3% linoleate.
60. The soybean seed oil of claim 54, wherein said oil without
chemical modification has a melting point below room
temperature.
61. A food product comprising the soybean seed oil of claim 54,
wherein said food product is selected from the group consisting of
salad oil, cooking oil, margarine, shortening, soymilk, tofu, and
soy flour.
62. A method of producing a soybean seed having above 15% stearate
comprising the steps of: (a) growing an oilseed crop having
inserted into its genome an exogenous DNA encoding a plant FatA
thioesterase; and (b) harvesting said oilseed crop, wherein the
seed of said oilseed crop comprises an endogenous oil comprising
greater than 15% stearate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/305,021, filed Dec. 19, 2005, which is a continuation of
U.S. application Ser. No. 10/061,280, filed Feb. 4, 2002, which is
a continuation of Ser. No. 09/359,070, filed Jul. 22, 1999, now
U.S. Pat. No. 6,380,462, which is a continuation-in-part of U.S.
application Ser. No. 09/134,262, filed Aug. 14, 1998, now U.S. Pat.
No. 6,365,802. The entirety of these applications are hereby
incorporated by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] A paper copy of the Sequence Listing is submitted herewith
electronically via EFS web. A computer-readable form of the
Sequence Listing is also submitted herewith electronically via EFS
web and contains the file named "17030-0004_corr_seqlist.txt",
which is 5,153 bytes in size (measured in MS-DOS) and which was
created on Apr. 1, 2009. The sequence listings submitted herewith
as paper copy and computer-readable form are herein incorporated by
reference.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The invention relates to genetic modification of plants,
plant cells and seeds, particularly altering fatty acid
composition.
[0005] 2. Background
[0006] Soybean (Glycine max) is one of the highest value crops
currently grown in the United States (.apprxeq.$16 billion in
1996). Ranking close to corn (25%) and wheat (22%), soybean
accounted for 19% of the United States crop acres planted in 1994.
Often referred to as a "miracle crop", soybean offers tremendous
value through the oil, protein and whole soybean products.
Agronomic traits, food quality traits related to oils and protein
quality are all important for the soybean industry.
[0007] More soybeans are grown in the United States than anywhere
else in the world (2.4 billion bushels in 1996, 50% of world
production). A bushel of soybean (60 pounds) is comprised of 48
pounds of protein meal and 11 pounds of oil. While protein meal is
the major component in soybean, oil, lecithin, tocopherols,
isoflavones, etc. are all co-products and add value to the bean.
Soybean oil is the major edible oil used in the world (40% of the
59.4 million metric tons in 1993). It also accounts for 70% of the
14 billion pounds of edible vegetable oil in the United States. The
primary food applications where the oil is used extensively are for
baking and frying (40-45%), salad and cooking oil (40-45%),
margarine and shortening (15-20%) and a wide spectrum of processed
foods. Development of other vegetable oils for specialty uses has
recently affected the acreage and production of soybean. The low
cost and ready availability of soybean oil provide an excellent
opportunity to upgrade this commodity item for specialty uses.
[0008] Food fats and oils are chemically composed of triesters of
glycerol containing straight chain, normal aliphatic fatty acids,
also referred to herein as triacylglycerols or triglycerides (TAG).
The properties of food fats and oils are a reflection of the fatty
acids contained in the TAG and their distribution on the glycerol
backbone. When the melting point of the TAG is below room
temperature, the TAG is referred to as an "oil". Triglycerides that
melt above room temperature are referred to as "fat". Gradients
between fluidity and solidity exist. Partially solidified,
non-pourable triglycerides are often referred to as "plastic
fats".
[0009] Fatty acids are organic acids having a hydrocarbon chain
ranging in length from about 4 to 24 carbons. Fatty acids differ
from each other in chain length, and in the presence, number and
position of double bonds. In cells, fatty acids typically exist in
covalently bound forms, the carboxyl portion being referred to as a
fatty acyl group. The chain length and degree of saturation of
these molecules is often depicted by the formula CX:Y, where "X"
indicates number of carbons and "Y" indicates number of double
bonds.
[0010] Typically, oil derived from commercial soybean varieties is
composed of approximately 11% palmitic (C16:0), 4% stearic acid
(C18:0), 21% oleic acid (C18:1), 56% linoleic acid (C18:2), and 10%
linolenic acid (C18:3). The fatty acid composition of soybean oil,
as well as all oils, largely determines its physical and chemical
properties, and thus its uses.
[0011] Fatty acid biosynthesis has been the subject of research
efforts in a number of organisms. For reviews of fatty acid
biosynthesis in plants, see Ohlrogge et al., (1995) Plant Cell,
7:957-970, Ohlrogge et al., (1997) Annu Rev Plant Physiol Plant Mol
Biol, 48:109-136 and Sommerville et al. (1991) Science,
252:80-87.
[0012] As mentioned previously, the fatty acid composition of an
oil determines its physical and chemical properties, and thus its
uses. Plants, especially plant species which synthesize large
amounts of oils in plant seeds, for example soybean, are an
important source of oils both for edible and industrial uses.
Various combinations of fatty acids in the different positions in
the triglyceride will alter the properties of the triglyceride. For
example, if the fatty acyl groups are mostly saturated fatty acids,
then the triglyceride will be solid at room temperature. In
general, however, vegetable oils tend to be mixtures of different
triglycerides. The triglyceride oil properties are therefore a
result of the combination of triglycerides which make up the oil,
which are in turn influenced by their respective fatty acid
compositions.
[0013] Plant breeders have successfully modified the yield and
fatty acid composition of various plant seed oils by introducing
desired traits through plant crosses and selection of progeny
carrying the desired trait forward. Application of this technique
thus is limited to traits which are found within the same plant
species. Alternatively, exposure to mutagenic agents can also
introduce traits which may produce changes in the composition of a
plant seed oil. However, it is important to note that Fatty Acid
Synthesis (FAS) occurs in most tissues of the plant including leaf
(chloroplasts) and seed tissue (proplastids). Thus, although a
mutagenesis approach can sometimes result in a desired modification
of the composition of a plant seed oil, it is difficult to effect a
change which will not alter FAS in other tissues of the plant.
[0014] A wide range of novel vegetable oils compositions and/or
improved means to obtain or manipulate fatty acid compositions,
from biosynthetic or natural plant sources, are needed. Plant
breeding, even with mutagenesis, cannot sufficiently meet this need
and provide for the introduction of novel oil.
[0015] For example, cocoa-butter has certain desirable qualities
(mouthfeel, sharp melting point, etc.) which are a function of its
triglyceride composition. Cocoa-butter contains approximately 24.4%
palmitate (16:0), 34.5% stearate (18:0), 39.1% oleate (18:1) and 2%
linoleate (18:2). Thus, in cocoa butter, palmitate-oleate-stearate
(POS) comprises almost 46% of triglyceride composition, with
stearate-oleate-stearate (SOS) and palmitate-oleate-palmitate (POP)
comprising the major portion of the balance at 33% and 16%,
respectively, of the triglyceride composition. Other novel oils
compositions of interest might include trierucin (three erucic) or
a triglyceride with medium chain fatty acids in each position of
the triglyceride molecule.
[0016] Plant seed oils contain fatty acids acylated at the sn-1,
sn-2, and sn-3 positions of a glycerol backbone, referred to as a
triacylglycerol (TAG). The structure of the TAG, as far as
positional specificity of fatty acids, is determined by the
specificity of enzymes involved in acylating the fatty acyl CoA
substrates to the glycerol backbone. For example, there is a
tendency for such enzymes from many temperate and tropical crop
species to allow either a saturated or an unsaturated fatty acid at
the sn-1 or the sn-3 position, but only an unsaturated fatty acid
at the sn-2 in the seed TAGs. In some species such as cocoa, TAG
compositions suggest that this tendency is carried further in that
there is an apparent preference for acylation of the sn-3 position
with a saturated fatty acid, if the sn-1 position is esterified to
a saturated fatty acid. Thus, there is a higher percentage of
structured TAG of the form Sat-Un-Sat (where Sat=saturated fatty
acid and Un=unsaturated fatty acid).
[0017] Of particular interest are triglyceride molecules in which
stearate is esterified at the sn-1 and sn-3 positions of a
triglyceride molecule with unsaturates in the sn-2 position
particularly oleate. Vegetable oils rich in such SOS
(Stearate-Oleate-Stearate) molecules share certain desirable
qualities with cocoa butter yet have a degree of additional
hardness when blended with other structured lipids. SOS-containing
vegetable oils are currently extracted from relatively expensive
oilseeds from certain trees grown in tropical areas such as Sal,
Shea, and Illipe trees from India, Africa, and Indonesia
respectively. Cheaper and more conveniently grown sources for
SOS-type vegetable oils are desirable.
[0018] In addition, vegetable oils rich in stearate fatty acid
content tend to be solid at room temperature. Such vegetable fats
can be used directly in shortenings, margarine and other food
"spread" products, obviating the need for chemical hydrogenation.
Hydrogenation is a process whereby molecular hydrogen is reacted
with the unsaturated fatty acid triglyceride until the desired
degree of solidity is obtained. The solidity is commonly determined
by the solid fat index (SFI, Official and Tentative Methods,
American Oil Chemists' Society, Cd 10-57(93), Champaign, Ill.).
Values are determined by dilatometry (expansion in volume) over a
defined temperature range of 50.degree., 70.degree., 80.degree.,
92.degree. and 100.degree. or 104.degree. F. The hydrogenation
process converts unsaturated fatty acids to partially or fully
saturated fatty acids, and increases the heat and oxidative
stability of the product. The iodine value (IV) measures the degree
of unsaturation of a fat. Lower values indicate greater saturation.
The oxidative stability may be measured by an oil stability index
(Official and Tentative Methods, American Oil Chemists' Society, Cd
1b-87, Champaign, Ill.) and active oxygen method (AOM, Official and
Tentative Methods, American Oil Chemists' Society, Cd 12h-92,
Champaign, Ill.). The cost and any other factors associated with
chemical hydrogenation, such as the production of trans fatty
acids, can be avoided if the vegetable oil is engineered to be
stearate rich in the plant seed.
[0019] Moreover, some plant tissues use 18 carbon fatty acids as
precursors to make other compounds. These include saturated long
chain fatty acids longer than 18 carbons in length. Since very
little stearate typically accumulates in soybean plants, it may be
necessary to increase stearate accumulation if one wants to
increase production of compounds which depend upon supply of
stearate fatty acids for synthesis.
[0020] The fatty acid composition of soybean oil described above is
often considered less than optimal in terms of oil functionality.
While the limitations of the fatty acid composition may be partly
overcome by chemical hydrogenation, the trans fatty acids produced
as a result of the hydrogenation process are Sat-Un-Satpected of
having unfavorable health effects (Mensink, et al. (1990) N. Eng.
J. Med. 323:439-445).
[0021] Through the efforts of traditional plant breeding
techniques, the fatty acid composition of soybeans has been
improved. For example, using mutagenesis, plant breeders have been
able to increase the amount of stearate (C18:0) produced in the
soybean oil. In such high stearate lines, designated as A6 (ATCC
Accession No. 97392, Hammond and Fehr, (1983) Crop Science 23:192),
stearate levels of up to about 25 weight percent of the total fatty
acid composition have been achieved. Such high stearate containing
lines have been further bred with mutant soybean lines containing
elevated levels of palmitate (16:0). Soybean lines containing the
elevated stearate levels produced by mutagenesis demonstrate a
negative correlation of increased stearate content and seed yield
(Hartmann, et al. (1997) Crop Science 37:124-127). Attempts to
further increase the stearate content and/or improve the seed yield
of such increased stearate lines by breeding have thusfar proven
unsuccessful.
[0022] List, et al. ((1996) J. Am. Oil. Chem. Soc. 73:729-732)
describes the use of genetically modified soybean oils in margarine
formulations. High stearate oil from soybean variety A6 was found
to have an insufficient solid fat index at 24.7.degree. C. and
higher temperatures to make margarine. The soybean oil was blended
with cottonseed or soybean hardstocks to afford mixtures with
sufficient solids content for formulation into margarine.
[0023] While soybean based products are a major food source,
improvements to the nutritional and commercial quality of this
product could add further value to soybean based products.
Alteration of the soybean oil content and composition could result
in products of higher nutritional content and greater stability.
The need for industrial hydrogenation of polyunsaturated oil for
food applications could be reduced by the preparation of soybean
oil with increased concentrations of stearate.
SUMMARY OF THE INVENTION
[0024] The present invention is directed to methods for producing
soybean oil having high levels of stearate (C18:0). The method of
producing a soybean oil containing increased levels of stearate
comprises expression of an acyl-ACP thioesterase capable of
producing C18:0 in the seed tissue of the soybean. In particular,
the acyl-ACP thioesterase has substantial activity toward 18:0
acyl-ACP substrates, and preferably has little or no activity
towards 16:0 acyl-ACP substrates.
[0025] The method generally comprises growing a soybean plant
containing a construct comprising as operably linked components in
the 5' to 3' direction of transcription, a transcription initiation
region functional in a seed tissue and a DNA encoding an acyl-ACP
thioesterase with substantial activity towards 18:0 acyl-ACP
substrates and a transcription termination sequence.
[0026] The stearate content of the soybean oil preferably comprises
greater than about 20%, more preferably greater than about 33% of
the fatty acid moieties in the oil. The oil of the present
invention may be used as a blending source to make a blended oil
product, or it may also be used in the preparation of food.
[0027] In another embodiment of the present invention, a soybean
oil having an increased saturated fatty acid composition is
provided. Soybean oils with saturated fatty acid compositions of
greater than 50 weight percent are exemplified herein.
[0028] In yet another embodiment of the instant invention, the
novel soybean oil, comprising the increased total saturated fatty
acid compositions, provides a novel source of structured TAG of the
Sat-Un-Sat (saturated-unsaturated-saturated) form.
[0029] The present invention further provides food products and
methods for their preparation from a novel soybean [Glycine max]
seed with increased levels of stearic and oleic acids, and
decreased levels of linoleic and linolenic acids under normal
growing conditions. The novel soybean seed is produced by a soybean
plant obtained from cross pollination of a high stearate plant with
a low linolenate plant. There are multiple advantages of a soybean
seed with modified fatty acid content. The soybean oil has
increased levels of stearic acid, normal levels of palmitic and
oleic acids, and decreased levels of linoleic and linolenic acids
relative to common soybean oil. Preferably, the soybean oil has a
stearic acid composition of above about 15%, a linoleic acid
composition below about 45%, and a linolenic acid composition below
about 6%. Oil extracted from the soybean seeds possess increased
stability and superior cooking characteristics than does oil
extracted from standard soybean seeds. The oil has higher levels of
solids than does common soybean oil, making it a more preferred
material for the preparation of food products such as margarine,
tofu, soy flour, soymilk, and shortening. Interesterification of
the oil can further enhance the amount of solids present, and the
oil's utility in the preparation of food products. Food products
prepared from modified soybeans display creamier textures than do
food products prepared from common soybeans. While common and high
stearate soybean oils require the addition of hardstocks for the
formation of margarines and other soy based products, the present
oil may be used without the addition of adjuvants.
[0030] The novel soybean oil as well as the soybean seed containing
the novel oil finds use in many applications.
DESCRIPTION OF THE FIGURES
[0031] FIG. 1. Nucleic acid and translated amino acid sequence of a
mangosteen FatA-type acyl-ACP thioesterase clone (Garm FatA1) is
provided. Garm FatA1 demonstrates primary thioesterase activity on
18:1 acyl-ACP substrate, but also demonstrates substantial activity
on 18:0 substrate (approximately 10-20% of 18:1 activity), as well
as little or no activity on 16:0 substrates.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In accordance with the subject invention, constructs and
methods are provided for the production of soybean plants with an
increased level of stearate (C18:0), as a percentage of the total
fatty acids, in the seed oil. The methods for producing such
soybean plants comprise transforming a soybean plant with
expression constructs comprising a promoter sequence functional in
a plant seed operably linked to a DNA sequence encoding a plant
acyl-ACP thioesterase having substantial activity toward 18:0-ACP
substrates, preferably those with little or no activity toward
16:0-ACP substrates (hereinafter referred to as stearoyl-ACP
thioesterase), and a transcription termination sequence. The
expression constructs provide an increase in the levels of stearate
fatty acids in the seed oil of the transformed soybean plants.
[0033] As described in more detail in the examples that follow, an
acyl-ACP thioesterase coding sequence from mangosteen (Garcinia
mangostana), Garm FatA1 (Hawkins and Kridl (1998) Plant Journal
13(6):743-752; and PCT Patent Application WO 96/36719, the
entireties of which are incorporated herein by reference) is used
in expression constructs to generate transgenic soybean plants with
increased production of the stearoyl-ACP thioesterase in host
cells. In particular the constructs are used to direct the
expression of the Garm FatA1 thioesterase in plant seed cells for
modification of triacylglycerol (TAG) fatty acid composition to
provide increased levels of C18:0 fatty acyl groups. Furthermore,
the constructs of the present invention may find use in plant
genetic engineering applications in conjunction with plants
containing elevated levels of C18:0 (stearate) fatty acids. Such
plants may be obtained by antisense gene regulation of stearoyl-ACP
desaturase using methods described by Knutzon et al (Proc. Nat.
Acad. Sci. (1992) 89:2624-2628), and may also be obtained by
co-suppression using sense expression constructs of the
stearoyl-ACP desaturase gene, or by conventional mutation and plant
breeding programs. In addition, the constructs and methods for
increasing stearate in soybean seed may also find use in plant
genetic engineering applications in conjunction with plants
containing elevated levels of oleate (C 18:1) and/or decreased
levels of linoleate (C18:2) fatty acids and/or linolenate (18:3).
Such plants with elevated levels of oleate and/or with decreased
levels of linoleate and/or linolenate may be obtained through
genetic engineering, or by conventional mutation and plant breeding
programs.
[0034] A plant acyl-ACP thioesterase DNA sequence useful for the
preparation of expression constructs for the alteration of stearate
levels as described herein encodes for amino acids, in the form of
a protein, polypeptide or peptide fragment, which amino acids
demonstrate substantial activity on 18:0 acyl-ACP substrates and
little or no activity on 16:0-ACP to form 18:0 free fatty acid
(i.e., stearate) under plant enzyme reactive conditions. By "enzyme
reactive conditions" is meant that any necessary conditions are
available in an environment (i.e., such factors as temperature, pH,
lack of inhibiting substances) which will permit the enzyme to
function.
[0035] DNA sequences encoding for acyl-ACP thioesterase enzymes
with substantial activity on 18:0 acyl-ACP substrates and little or
no activity on 16:0-ACP to form 18:0 free fatty acid (i.e.,
stearate) are known in the art and are described in Hawkins and
Kridl (1998) supra, and PCT Patent Application WO 96/36719. The
Garm FatA1 DNA sequence described therein and used herein
demonstrates preferential activity on C18:1 acyl-ACP substrate, and
also demonstrates substantial activity (approximately 25% of the
18:1 activity) on C18:0 acyl-ACP substrates. Only a small increase
in C16:0 activity over activity in control cells is observed, and
the 16:0 activity represents only approximately 3% of the 18:1
activity.
[0036] In preparing the expression constructs, the various DNA
fragments may be manipulated, so as to provide for the DNA
sequences in the proper orientation and, as appropriate in the
proper reading frame. Towards this end, adapters or linkers may be
employed to join the DNA fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of
superfluous DNA, removal of restriction sites, or the like. For
this purpose, in vitro mutagenesis, primer repair, restriction,
annealing, resection, ligation, or the like may be employed, where
insertions, deletions or substitutions, e.g. transitions and
transversions, may be involved.
[0037] For the most part, the constructs will involve regulatory
regions functional in plants which provide for modified production
of plant stearoyl-ACP thioesterase, and modification of the fatty
acid composition. The open reading frame, coding for the plant
stearoyl-ACP thioesterase or functional fragment thereof will be
joined at its 5' end to a transcription initiation regulatory
region such as the wild-type sequence naturally found 5' upstream
to the thioesterase structural gene, or to a heterologous
regulatory region from a gene naturally expressed in plant tissues.
Examples of useful plant regulatory gene regions include those from
T-DNA genes, such as nopaline or octopine synthase, plant virus
genes, such as CaMV 35S, or from native plant genes.
[0038] For such applications when 5' upstream non-coding regions
are obtained from other genes regulated during seed maturation,
those preferentially expressed in plant embryo tissue, such as ACP,
napin and .beta.-conglycinin 7S subunit transcription initiation
control regions, as well as the Lesquerella hydroxylase promoter
(described in Broun, et al. (1998) Plant Journal 13(2):201-210 and
in U.S. patent application Ser. No. 08/898,038) and the
stearoyl-ACP desaturase promoter (Slocombe, et al. (1994) Plant
Physiol. 104:1167-1176), are desired. Such "seed-specific
promoters" may be obtained and used in accordance with the
teachings of U.S. Pat. No. 5,420,034 having a title "Seed-Specific
Transcriptional Regulation" and in Chen et al., (1986), Proc. Natl.
Acad. Sci., 83:8560-8564. Transcription initiation regions which
are preferentially expressed in seed tissue, i.e., which are
undetectable in other plant parts, are considered desirable for
fatty acid modifications in order to minimize any disruptive or
adverse effects of the gene product.
[0039] Regulatory transcript termination regions may be provided in
DNA constructs of this invention as well. Transcript termination
regions may be provided by the DNA sequence encoding the plant
stearoyl-ACP thioesterase or a convenient transcription termination
region derived from a different gene source, for example, the
transcript termination region which is naturally associated with
the transcript initiation region. The skilled artisan will
recognize that any convenient transcript termination region which
is capable of terminating transcription in a plant cell may be
employed in the constructs of the present invention. As described
herein, transcription termination sequences derived from DNA
sequences preferentially expressed in plant seed cells are employed
in the expression constructs of the present invention.
[0040] The method of transformation is not critical to the instant
invention; various methods of plant transformation are currently
available. As newer methods are available to transform crops, they
may be directly applied hereunder. For example, many plant species
naturally susceptible to Agrobacterium infection may be
successfully transformed via tripartite or binary vector methods of
Agrobacterium-mediated transformation. In addition, techniques of
microinjection, DNA particle bombardment, and electroporation have
been developed which allow for the transformation of various
monocot and dicot plant species.
[0041] In developing the DNA construct, the various components of
the construct or fragments thereof will normally be inserted into a
convenient cloning vector which is capable of replication in a
bacterial host, e.g., E. coli. Numerous vectors exist that have
been described in the literature. After each cloning, the plasmid
may be isolated and subjected to further manipulation, such as
restriction, insertion of new fragments, ligation, deletion,
insertion, resection, etc., so as to tailor the components of the
desired sequence. Once the construct has been completed, it may
then be transferred to an appropriate vector for further
manipulation in accordance with the manner of transformation of the
host cell.
[0042] Normally, included with the DNA construct will be a
structural gene having the necessary regulatory regions for
expression in a host and providing for selection of transformant
cells. The gene may provide for resistance to a cytotoxic agent,
e.g. antibiotic, heavy metal, toxin, etc., complementation
providing prototrophy to an auxotrophic host, viral immunity or the
like. Depending upon the number of different host species in which
the expression construct or components thereof are introduced, one
or more markers may be employed, where different conditions for
selection are used for the different hosts. A number of markers
have been developed for use for selection of transformed plant
cells, such as those which provide resistance to various
antibiotics, herbicides, or the like. The particular marker
employed is not essential to this invention, one or another marker
being preferred depending on the particular host and the manner of
construction.
[0043] As mentioned above, the manner in which the DNA construct is
introduced into the plant host is not critical to this invention.
Any method which provides for efficient transformation may be
employed. Various methods for plant cell transformation include the
use of Ti- or Ri-plasmids, microinjection, electroporation, DNA
particle bombardment, liposome fusion, or the like. In many
instances, it will be desirable to have the construct bordered on
one or both sides by T-DNA, particularly having the left and right
borders, more particularly the right border. This is particularly
useful when the construct uses A. tumefaciens or A. rhizogenes as a
mode for transformation, although the T-DNA borders may find use
with other modes of transformation.
[0044] Various methods of transforming cells of soybean have been
previously described. Examples of soybean transformation methods
have been described, for example, by Christou et al. U.S. Pat. No.
5,015,580 and by Hinchee et al. U.S. Pat. No. 5,416,011, the
entireties of which are incorporated herein by reference.
[0045] Once a transgenic plant is obtained which is capable of
producing seed having a modified fatty acid composition,
traditional plant breeding techniques, including methods of
mutagenesis, may be employed to further manipulate the fatty acid
composition. Alternatively, additional foreign fatty acid modifying
DNA sequence may be introduced via genetic engineering to further
manipulate the fatty acid composition.
[0046] One may choose to provide for the transcription or
transcription and translation of one or more other sequences of
interest in concert with the expression of a plant stearoyl-ACP
thioesterase in a plant host cell. In particular, the reduced
expression of stearoyl-ACP desaturase in combination with
expression of a plant stearoyl-ACP thioesterase may be preferred in
some applications.
[0047] When one wishes to provide a plant transformed for the
combined effect of more than one nucleic acid sequence of interest,
typically a separate nucleic acid construct will be provided for
each. The constructs, as described above contain transcriptional or
transcriptional and translational regulatory control regions. The
constructs may be introduced into the host cells by the same or
different methods, including the introduction of such a trait by
the inclusion of two transcription cassettes in a single
transformation vector, the simultaneous transformation of two
expression constructs, retransformation using plant tissue
expressing one construct with an expression construct for the
second gene, or by crossing transgenic plants via traditional plant
breeding methods, so long as the resulting product is a plant
having both characteristics integrated into its genome.
[0048] By decreasing the amount of stearoyl-ACP desaturase, an
increased percentage of saturated fatty acids is provided. Using
anti-sense, transwitch, ribozyme or some other stearoyl-ACP
desaturase reducing technology, a decrease in the amount of
stearoyl-ACP desaturase available to the plant cell is produced,
resulting in a higher percentage of saturates such as one or more
of stearate (C18:0), arachidate (C20:0), behenate (C22:0) and
lignocerate (C24:0). In rapeseed reduced stearoyl-ACP desaturase
results in increased stearate levels and total saturates (Knutzon
et al. (1992) Proc. Nat. Acad. Sci. 89:2264-2628).
[0049] Of special interest is the production of triglycerides
having increased levels of stearate. In addition, the production of
a variety of ranges of stearate is desired. Thus, plant cells
having lower and higher levels of stearate fatty acids are
contemplated. For example, fatty acid compositions, including oils,
having a 10% level of stearate as well as compositions designed to
have up to an approximate 60% level of stearate or other such
modified fatty acid(s) composition are contemplated.
[0050] As described in more detail in the examples that follow,
constructs are prepared to direct the expression of a stearoyl-ACP
thioesterase in plant seed tissue. Such expression constructs allow
for the increase in 18:0 levels in oils obtained from the seeds of
transformed soybean plants.
[0051] Increases in the levels of stearate in soybeans transformed
to express Garm FatA1 range from 4 fold to approximately 13 fold
over the levels obtained in seeds from nontransformed control
plants. Additionally, by decreasing the amount of stearoyl-ACP
desaturase available to the plant FAS complex in conjunction with
an increase of the amount of stearoyl-ACP thioesterase available, a
more marked increased percentage of stearate may be obtained. By
manipulation of various aspects of the DNA constructs (e.g., choice
of promoters, number of copies, etc.) and traditional breeding
methods, one skilled in the art may achieve even greater levels of
stearate. By expression of a plant stearoyl-ACP thioesterase in
seed tissue or a decrease in the expression of stearoyl-ACP
desaturase or a combination of both, an increased percentage of
stearate can be achieved in soybean. In addition, modified
thioesterase encoding DNA sequences may find use for increasing
stearate levels in seed tissue. Such modified thioesterase
sequences may be obtained as described in PCT Patent Application WO
96/36719.
[0052] Surprisingly, in the oil of seeds from T2 soybean lines
transformed to express the Garm FatA1 DNA sequence from the
.beta.-conglycinin 7S subunit promoter, stearate levels of up to 53
percent as a percentage of the total fatty acid composition are
obtained. In addition, in the oil of the initial transformed lines
expressing Garm FatA1 DNA sequence from the .beta.-conglycinin 7S
subunit promoter increases in the levels of stearate obtained from
individual seeds range from about 14 weight percent up to about 53
weight percent. Furthermore, transgenic soybean plants expressing
the Garm FatA1 DNA sequence from the napin derived promoter
accumulate increased levels of stearate ranging from approximately
20 weight percent up to approximately 45 weight percent in
individual seeds of T2 soybean lines. Stearate levels obtained from
the oil of individual seeds of nontransformed control soybeans
range from approximately 4 weight percent to approximately 6 weight
percent. Preferred oil compositions for many applications include
33 weight percent or greater stearate fatty acids as a component of
the soybean oil.
[0053] In addition, transformed soybean lines containing increased
stearate levels of the present invention also demonstrate an
increase in the total levels of saturated fatty acids. Transformed
soybean lines containing elevated stearate levels also contain
increased levels of Arachidic acid (20:0) and Behenic acid (22:0).
Increases in 20:0 range from about 3 fold to about 11 fold over the
levels of 20:0 obtained from the seed oil of nontransgenic control
soybean lines. Increases in 22:0 range from about 2 fold to about 5
fold over the levels of 22:0 obtained from the seed oil of
nontransgenic control soybean lines.
[0054] Thus, in soybean lines transformed to express a stearoyl-ACP
thioesterase in the seed tissue, total saturated fatty acids (16:0,
18:0, 20:0 and 22:0) comprise at least 30 percent of the total
fatty acids as a percentage of weight, preferably above 50 weight
percent. In some cases, total saturated fatty acid levels of above
about 65 weight percent may be obtained.
[0055] The novel soybean oil compositions of the present invention
comprise increased total saturated fatty acids and provide a novel
source of structured TAG of the Sat-Un-Sat form. For oil
compositions having greater than about 33 weight percent stearate
the Sat-Un-Sat form of TAG may comprise 25 percent or greater of
the total TAG composition as a stearate-unsaturated-stearate form
of TAG. It is apparent that by utilizing a high oleic acid soybean
line that one may produce a soybean oil with a high proportion of a
stearate-oleate-stearate form of TAG. An example of such high oleic
acid soybean oil is described in PCT Application WO 97/40698.
[0056] The present invention further provides food products and
methods for their preparation from a novel soybean [Glycine max]
seed with increased levels of stearic and oleic acids, and
decreased levels of linoleic and linolenic acids under normal
growing conditions. The novel soybean seed is produced by a soybean
plant obtained from cross pollination of a high stearate plant with
a low linolenate plant. There are multiple advantages of a soybean
seed with modified fatty acid content. The soybean oil has
increased levels of stearic acid, normal levels of palmitic and
oleic acids, and decreased levels of linoleic and linolenic acids
relative to common soybean oil. Preferably, the soybean oil has a
stearic acid composition of above about 15%, a linoleic acid
composition below about 45%, and a linolenic acid composition below
about 6%. Oil extracted from the soybean seeds possess increased
stability and superior cooking characteristics than does oil
extracted from standard soybean seeds. The oil has higher levels of
solids than does common soybean oil, making it a more preferred
material for the preparation of food products such as margarine,
tofu, soy flour, soymilk, and shortening. Interesterification of
the oil can further enhance the amount of solids present, and the
oil's utility in the preparation of food products. Food products
prepared from modified soybeans display creamier textures than do
food products prepared from common soybeans. While common and high
stearate soybean oils require the addition of hardstocks for the
formation of margarines and other soy based products, the present
oil may be used without the addition of adjuvants.
[0057] The soybean oil compositions of the present invention
containing novel fatty acid compositions may find use in a number
of applications, without the need for chemical modifications prior
to use as described herein, or as described in PCT application
titled "Food Products Containing Structured Triglycerides",
PCT/US97/06037, the entirety of which is incorporated herein by
reference. The soybean oil of the present invention may find use in
the preparation of foods to facilitate cooking or heating
applications.
[0058] The soybean oils produced by the methods of the present
invention may be used in the formation of emulsions comprising
water and soybean oil. The soybean oil may be treated by
interesterification prior to the formation of an emulsion. As used
herein, interesterification refers to the process of rearranging
the glyceride structure of fats. Interesterification is
accomplished by a chemical reaction in which fatty acids are
rearranged on the glycerol molecule without modification of the
fatty acids themselves. An emulsion may preferably comprise between
about 70% and about 90% by volume soybean oil, and between 10% and
about 30% by volume water. The aqueous emulsion may further be
defined as margarine. As used herein, the term "margarine" refers
to an edible emulsion comprising oil and water that is both solid
and spreadable at 25.degree. C.
[0059] Alternatively, the soybean seeds containing the modified
fatty acid compositions of the present invention, may be used to
prepare soymilk. Soymilk may be prepared by the steps of selecting
soybean seeds, contacting the seeds with water to form a mixture,
heating the mixture, grinding the mixture, and removing the solids
to form soymilk. Removal of solids may be accomplished by methods
including, but not limited to, filtration, sedimentation and
centrifugation. The heating step may comprise heating the mixture
to any temperature suitable for the formation of tofu, preferably
to a temperature sufficient to inactivate the trypsin inhibitor in
the liquid at least about 80% as compared to the trypsin inhibitor
activity prior to heating, and most preferably to a temperature
between about 90.degree. and about 100.degree. C. Trypsin inhibitor
activity may be conveniently assayed using colorimetric method
described in Liu and Markakis ((1989), Cereal Chem
66(5):415-422).
[0060] Soybean seeds containing the novel soybean oil compositions
of the present invention may find use in the preparation of tofu.
Tofu may be prepared by the steps of selecting soybean seeds
containing soybean oil having oil compositions of the present
invention, contacting the seeds with water to form a mixture,
heating the mixture, grinding the mixture, removing the solids to
form a filtrate, adding a coagulant, and cooling the filtrate to
form tofu. The coagulant used may be, but is not limited, to
glucono-.delta.-lactone, lemon juice, sea salt, calcium sulfate or
magnesium chloride. Removal of solids may be accomplished by
methods including, but not limited to filtration, sedimentation,
and centrifugation. The heating step may comprise heating the
mixture to any temperature suitable for the formation of tofu,
preferably to a temperature sufficient to inactivate the trypsin
inhibitor in the liquid at least about 80% as compared to the
trypsin inhibitor activity prior to heating, and most preferably to
a temperature between about 90.degree. and about 100.degree. C.
Trypsin inhibitor activity may be conveniently assayed using
colorimetric method described in Liu and Markakis ((1989), Cereal
Chem 66(5):415-422). The cooling step may comprise cooling the
filtrate to any temperature suitable for the formation of tofu, and
more preferably to between about 0.degree. C. and about 25.degree.
C.
[0061] Soy flour may also be prepared from soybean seeds containing
the oil compositions of the present invention. A method for
preparation of soy flour comprises the steps of selecting soybean
seeds and grinding the seeds to produce soy flour. Preferably, the
soybean seeds contain soybean oil having increased levels of
stearate. The grinding step may be performed by any means suitable
for the production of soy flour, including, but not limited to,
grinding with wheels, mortar and pestle, plates and blades.
[0062] The soybean oil of the present invention may be further used
to prepare shortenings. As used herein, the term "shortening"
refers to fats, usually plastic in nature, that provide functional
effects related to structure, texture and the eating qualities of a
variety of food products. The shortening prepared based on the
soybean oil of the present invention may also include the
incorporation of emulsifiers or surfactants (selected from 21
C.F.R. 172), such emulsifiers used to provide additional functional
effects in the final food product. The shortening based on the
soybean oil of the present invention may also include a variety of
hardstocks (fully hydrogenated triglycerides sourced from a variety
of common food oils) that could be used to modify or augment the
SFC of the final product and, hence, the physical properties and
functionality of the final blend. The plasticity and crystal
structure of the final shortening composition based on the soybean
oil of the present invention, whether emulsified or not, may be
further modified through the process of controlled crystallization
and specific gravity reduction known as votation. In this process,
the molten shortening described variously above is fed through a
scraped-surface heat exchanger, and crystallized in a directed
manner-usually in the most functional crystalline form. During this
process, a gas is usually whipped into the solidifying product to
adjust the specific gravity of the shortening product, and hence
its plasticity. This is done, primarily, to enhance the handling
characteristics of the final shortening and to allow it to be
better incorporated into a variety of food product systems. The
emulsifier may generally be any material suitable for the
preparation of shortening, preferably an emulsifier approved as a
food additive per 21 C.F.R. .sctn. 172, and more preferably is a
monoglyceride. The use of the novel soybean oil of the present
invention may allow for a reduced level of emulsifier addition to
achieve the same functional effects as would be required in a
standard soybean oil based shortening arrived at through
hydrogenation to attain equal solids.
[0063] The soybean seeds, oil, and products therefrom may also find
use in a number of additional applications known to the art,
including the use in various animal feed applications.
[0064] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included for purposes of illustration only and are not intended to
limit the present invention.
EXAMPLES
Example 1
Plant Expression Vector Construction
[0065] Plant vectors are constructed to control the expression of a
member of the FatA class of acyl-ACP thioesterases from Garcinia
mangostana (Garm FatA1, Hawkins and Kridl (1998) supra, and PCT
Patent Application WO 96/36719) in seeds of soybean utilizing
different seed enhanced promoters.
[0066] A plant transformation construct, pWRG5374, is prepared to
express Garm FatA1 in the embryo tissue of the soybean seed
utilizing the napin promoter. A DNA fragment containing the napin
5'/Garm FatA1/napin 3', described in Hawkins and Kridl (1998)
supra, is cloned into a vector containing the selectable marker
.beta.-glucuronidase (GUS, Jefferson et al., Proc. Natl. Acad. Sci.
(1986) 83:8447-8451) driven by the CAMV 35S (Gardner, et al. (1981)
Nucleic Acids Res. 9:2871-2888) promoter. The GUS gene contains an
untranslated leader sequence derived from a soybean
ribulose-bis-phosphate carboxylase (RuBisCo) small subunit,
(Grandbastien, et al. (1986) Plant Mol. Biol. 7:451-466), ssuL and
a translational termination sequence derived from the soybean
RuBisCo, (Berry-Lowe (1982) Jour. Mol. Appl. Genet. 1:483-498),
SpA. Examples of vectors utilizing a GUS selectable marker are
described in European Patent 0 301 749 B1, the entirety of which is
incorporated herein by reference. The resulting expression
construct, pWRG5374, contains the napin 5'/Garm FatA1/napin 3'
sequences as well as the 35S-ssuL/GUS/SpA 3' for transgenic
selection by indigo blue staining.
[0067] The soybean transformation construct, pWRG5378, containing
the Garm FatA1 coding sequence expressed from the
.beta.-conglycinin 7S subunit promoter was prepared as follows. The
Garm FatA1 coding sequence and napin 3' poly-A termination
sequences were obtained from plasmid pCGN5253 (described in Hawkins
and Kridl (1998) supra). A soybean expression plasmid pWRG5375 was
constructed by insertion of the Garm FatA1 coding and napin 3'
sequences downstream of a heterologous promoter from the soybean
.alpha.' subunit of .beta.-conglycinin (soy 7s, (Chen et al.,
(1986), Proc. Natl. Acad. Sci., 83:8560-8564)). A 941 bp BamHI-XhoI
fragment containing the soy 7s promoter was ligated with a 5186 bp
fragment from plasmid pCGN5253 produced by partial digestion with
KpnI and complete digestion with SalI. Additionally, an 8 bp
BamHI-KpnI adapter having the DNA sequence 5'-GATCGTAC-3' was used
to fuse the BamHI site from the soy 7s promoter fragment with the
KpnI site from pCGN5253. The resulting plasmid was named pWRG5375.
A 3477 bp SacI fragment from plasmid pWRG5375 containing the soy
7s/Garm FatA1/napin 3' was ligated to a 6135 bp fragment containing
the .beta.-glucuronidase (GUS) marker cassette (described above)
for selection of transgenic soybean plants. The soy 7s/Garm
FatA1/napin 3' cassette was inserted such that the transcription of
the GUS gene was in the same direction as that of the Garm FatA1
coding sequence. The resulting 8329 bp plasmid was designated as
pWRG5378.
Example 2
Soybean Transformation with Garm FatA1 Constructs
[0068] Plasmids pWRG5374 and pWRG5378 were digested with NotI and
linearized fragments containing both the chimeric Garm FatA1 coding
sequence and GUS expression cassettes were purified by HPLC. The
linear DNA fragments were stably introduced into soybean (Asgrow
variety A5403) by the method of McCabe, et. al. (1988)
Bio/Technology 6:923-926.
[0069] Transformed soybean plants are identified by indigo blue
staining of seed tissue with 1 mM X-Gluc (Clontech), 0.1M
NaPO.sub.4 (pH 7.0), 0.5 mM potassium ferrocyanide.
Example 3
Fatty Acid Compositional Analysis
[0070] Fatty acid compositions were analyzed from seed of soybean
lines transformed with pWRG5374 or pWRG5378. One to five seeds of
each of the transgenic and control soybean lines were ground
individually using a tissue homogenizer (Pro Scientific) for oil
extraction. Oil from ground soybean seed was extracted overnight in
1.5 ml heptane containing triheptadecanoin (0.50 mg/ml). Aliquots
of 200 .mu.l of the extracted oil was derivatized to methyl esters
with the addition of 500 .mu.l sodium methoxide in absolute
methanol. The derivatization reaction was allowed to progress for
20 minutes at 50.degree. C. The reaction was stopped by the
simultaneous addition of 500 .mu.l 10% (w/v) sodium chloride and
400 .mu.l heptane. The resulting fatty acid methyl esters extracted
in hexane were resolved by gas chromatography (GC) on a Hewlett
Packard model 6890 GC. The GC was fitted with a Supelcowax 250
column (30 m, 0.25 mm id, 0.25 micron film thickness) (Supelco,
Bellefonte, Pa.). Column temperature was 175.degree. C. at
injection and the temperature programmed from 175.degree. C. to
245.degree. C. to 175.degree. C. at 40.degree. C./min. Injector and
detector temperatures were 250.degree. C. and 270.degree. C.,
respectively.
[0071] The results of the fatty acid compositional analysis from
seed oil of the initial transformed 5374 soybean lines is provided
in Table 1. Averages are provided where oil compositional analysis
was performed on more than one seed from the initial transformant.
In seed of transgenic soybean plants expressing Garm FatA1 from the
napin promoter, stearate (C18:0) levels were significantly
increased over the levels obtained from the seed oil of
nontransformed control plants. The increase in stearate is
primarily at the expense of oleate, and to a lesser degree
linoleate and palmitic all of which were decreased in the
transgenic lines. In addition, increases in all saturates examined
greater than C18:0 were observed.
TABLE-US-00001 TABLE 1 STRAIN ID GUS %16:0 %18:0 %18:1 %18:2 %18:3
%20:0 %22:0 5374-A5403-3 + 6.71 24.88 14.53 43.73 6.99 1.84 1.03
5374-A5403-3 + 6.62 26.52 11.9 44.89 7.15 1.8 0.9 5374-A5403-3 +
7.59 22.99 13.17 45.32 8.19 1.63 0.84 5374-A5403-3 + 7.28 23.1 13
43.65 9.74 1.88 1.13 5374-A5403-3 + 7.39 26.54 8.4 41.95 12.38 1.9
1.07 AVERAGE 7.12 24.81 12.2 43.91 8.89 1.81 0.99 5374-A5403-4 +
7.38 20.81 16.45 46.61 5.99 1.57 0.87 5374-A5403-4 + 7.96 18.28
14.69 47.93 8.29 1.54 0.96 5374-A5403-4 + 10.02 9.64 22.78 49.41
6.02 0.91 0.79 5374-A5403-4 + 9.24 12.54 23.06 45.97 5.9 1.17 0.8
5374-A5403-4 + 7.41 20.07 13.42 45.91 10.32 1.55 0.97 AVERAGE 8.40
16.27 18.08 47.17 7.29 1.35 0.88 5374-A5403-14 + 7.96 38.39 7.82
37.34 6.23 2.41 1.07 5374-A5403-35 9.24 33.53 11.14 38.15 7.54 1.97
0.9 5374-A5403-36 + 8.18 19.37 13.92 47.05 8.47 1.6 1.06
5374-A5403-36 + 7.5 19.99 13.49 47.15 8.99 1.59 0.95 5374-A5403-36
+ 7.05 23.44 10.54 46.19 9.64 1.82 0.99 5374-A5403-36 + 7.83 20.06
13.53 47.27 8.45 1.57 0.94 5374-A5403-36 + 7.49 22.9 11.81 46.14
8.54 1.72 1.03 AVERAGE 7.61 21.15 12.66 46.76 8.82 1.66 0.99
5374-A5403-172 + 7.92 15.53 29.9 38.2 5.8 1.36 0.89 5374-A5403-172
+ 7.37 22.45 16.63 44.3 6.58 1.56 0.77 5374-A5403-172 + 8.74 14.38
20.17 47.33 6.94 1.17 0.84 AVERAGE 8.01 17.45 22.23 43.28 6.44 1.36
0.83 Control A5403 - 11.62 4.3 24.26 49.84 7.47 0.48 0.57 A5403 -
12.32 4.24 21.93 52.32 7.49 0.44 0.46 A5403 - 12.64 4.25 20.49
53.42 7.81 0.43 0.51 A5403 - 12.17 4.22 21.56 .52.48 8.15 0.44 0.51
A5403 - 11.68 4.32 25.68 49.67 7.06 0.48 0.54 AVERAGE 12.09 4.27
22.78 51.55 7.60 0.45 0.52
[0072] Selected T2 lines also show the trends of increased
stearate, and decreased palmitate, oleate and linoleate levels in
the seed oil (Table 2). Furthermore, in seed of T2 5374 soybean
lines (T3 seed), stearate levels as high as approximately 45% of
the fatty acid methyl esters are observed. These levels are
increased from approximately 34% in the T1 generation. While null
progeny which do not contain the Garm FatA1 transgene contain
approximately 4.5% of the fatty acid methyl esters as stearate.
TABLE-US-00002 TABLE 2 STRAIN ID GUS 16:0 18:0 18:1 18:2 18:3 20:0
22:0 5374-A5403-3-417 + 6.57 37.18 7.39 35.38 9.36 2.68 1.2
5374-A5403-3-417 + 6.52 38.66 8.26 33.82 8.43 2.78 1.29
5374-A5403-3-417 + 6.23 39.26 7.26 35.11 7.84 2.8 1.26
5374-A5403-3-417 + 6.75 33.55 8.91 37.69 9.13 2.51 1.19
5374-A5403-3-417 + 6.18 42.21 5.88 33.36 7.99 2.94 1.23 average
6.45 38.17 7.54 35.07 8.55 2.74 1.23 5374-A5403-35-483 + 5.78 45.64
6.3 31.86 6.15 2.95 1.16 5374-A5403-35-483 + 5.82 38.21 7.83 37.06
7.22 2.54 1.12 5374-A5403-35-483 + 5.84 38.37 7.44 37.91 6.43 2.59
1.22 5374-A5403-35-483 + 5.74 41.56 6.31 35.4 6.98 2.71 1.12
5374-A5403-35-483 + 5.58 40.35 7.06 36.91 6.11 2.63 1.16 average
5.75 40.83 6.99 35.83 6.58 2.68 1.16 5374-A5403-172-401 + 6.73
23.12 15.02 46.04 6.05 1.77 0.97 5374-A5403-172-401 + 6.92 21.96
14.85 46.47 6.7 1.78 1.02 5374-A5403-172-401 + 6.49 24.15 14.11
45.74 6.46 1.83 0.96 5374-A5403-172-401 + 6.83 23.09 13.64 46.56
6.85 1.79 0.96 5374-A5403-172-401 + 8.32 20.32 11.94 47.05 9.36
1.69 1.02 average 7.06 22.53 13.91 46.37 7.08 1.77 0.99
5374-A5403-36-353 + 6.18 30.73 11.36 41.3 7.3 1.92 0.89
5374-A5403-36-353 + 6.42 30.82 11.14 41.03 7.53 1.85 0.85
5374-A5403-36-353 + 6.43 30.03 11.61 40.84 8.12 1.84 0.84
5374-A5403-36-353 + 6.66 29.27 13.98 40.82 6.26 1.77 0.83
5374-A5403-36-353 + 6.15 30.32 13.67 40.76 5.95 1.92 0.89 average
6.37 30.23 12.35 40.95 7.03 1.86 0.86 5374-A5403-36-489 + 6.57
34.87 10.56 37.1 7.23 2.34 1.09 5374-A5403-36-489 + 6.25 37.1 8.43
37.65 7.05 2.33 1 5374-A5403-36-489 + 6.36 36.22 10.68 36.18 6.88
2.39 1.08 5374-A5403-36-489 + 6.29 36.28 8.69 38.06 7.08 2.33 1.04
5374-A5403-36-489 + 6.26 36.6 8.33 37.79 7.25 2.44 1.11 average
6.35 36.21 9.34 37.36 7.10 2.37 1.06 Control 5374-A5403-36-341 -
10.7 6 24.46 50.9 6.29 0.53 0.61 5374-A5403-36-341 - 11.2 4.92
20.68 54.35 7.37 0.47 0.57 5374-A5403-36-341 - 11.27 4.27 23.71
52.72 6.55 0.43 0.53 5374-A5403-36-341 - 11.33 4.78 20.4 54.38 7.58
0.46 0.55 5374-A5403-36-341 - 11.55 4.52 18.59 55.07 8.69 0.46 0.56
null segregant Ave 11.21 4.90 21.57 53.48 7.30 0.47 0.56
[0073] The results of the fatty acid compositional analysis for
transformed 5378 soybean plants are shown in Table 3. Seeds of
soybean plants transformed to express Garm FatA1 from the 7S
promoter produced increased levels of stearate over those levels
observed in seeds of nontransformed control plants. In the seed oil
of some T1 5378 transgenic soybean, stearate levels of as high as
approximately 53% of the fatty acids were obtained, while levels of
approximately 4% were observed in nontransformed control
plants.
TABLE-US-00003 TABLE 3 STRAIN ID GUS %16:0 %18:0 %18:1 %18:2 %18:3
%20:0 %22:0 5378-A5403-28 + 6.27 41.81 8.64 34.22 5.13 2.62 1
5378-A5403-28 + 6.3 42.63 10.15 32.76 4.16 2.55 0.87 5378-A5403-28
+ 6.48 43.11 7.47 33.72 5.3 2.61 1.04 5378-A5403-28 + 6.48 43.12
9.32 32.97 4.14 2.64 1.02 AVERAGE 6.38 42.67 8.90 33.42 4.68 2.61
0.98 5378-A5403-48 + 8.19 25.51 12.37 44.21 6.85 1.62 0.88
5378-A5403-48 + 7.74 33.77 12.13 36.33 6.41 2.23 1.05 5378-A5403-48
+ 7.42 40.06 9.82 30.97 7.26 2.84 1.23 5378-A5403-48 + 7.89 45.26
5.73 30.23 6.8 2.71 1.06 5378-A5403-48 + 7.04 47.2 5.69 29.58 6.13
2.86 1.2 AVERAGE 7.66 38.36 9.15 34.26 6.69 2.45 1.08 5378-A5403-59
+ 9.96 47.1 9.17 22.11 4.12 4.78 2.3 5378-A5403-59 + 7.06 47.3
4.44. 29.02 7.17 3.31 1.44 5378-A5403-59 + 11.72 50.5 4.69 20.86
5.35 4.34 1.93 5378-A5403-59 + 7.55 51.95 4.99 24.53 5.02 4 1.72
AVERAGE 9.07 49.21 5.82 24.13 5.42 4.11 1.85 5378-A5403-60 + 7.7
35.2 6.09 37.85 8.77 2.74 1.34 5378-A5403-60 + 7.19 35.53 5.86
38.36 8.57 2.79 1.41 5378-A5403-60 + 7.27 36.4 5.51 37.78 8.61 2.78
1.35 5378-A5403-60 + 9.01 52.21 1.71 23.14 7.62 4.41 1.59
5378-A5403-60 + 9.83 52.94 1.77 21.85 7.59 4.28. 1.42 AVERAGE 8.2
42.46 4.19 31.80 8.23 3.40 1.42 5378-A5403-69 + 11.67 4.67 18.46
55.85 7.93 0.43 0.47 5378-A5403-69 + 8.69 13.65 19.02 48.86 7.19
1.13 0.9 5378-A5403-69 + 8.13 18.5 14.7 48.52 7.4 1.41 0.83
5378-A5403-69 + 7.1 21.26 12.86 48.93 7.1 1.43 0.89 5378-A5403-69 +
8.04 44.02 8.09 28.04 7.34 2.96 1.16 AVERAGE 8.73 20.42 14.63 46.04
7.39 1.47 0.85 5378-A5403-103 + 9.31 40.01 8.73 30.52 7.08 2.85
1.04 5378-A5403-113 + 7.41 49.06 4.91 28.83 5.45 2.91 1.06
5378-A5403-113 + 11.01 49.44 4.88 22.53 7.15 3.24 1.1
5378-A5403-113 + 7.03 49.79 4.09 29.08 5.9 2.79 1 5378-A5403-113 +
8.32 51.04 4.29 27.06 5.1 2.91 0.92 5378-A5403-113 + 8.52 52.59
3.69 26.35 4.64 2.93 0.86 AVERAGE 8.46 50.38 4.37 26.77 5.65 2.96
0.99 Control A5403 - 11..62 4.3 24.26 49.84 7.47 0.48 0.57 A5403 -
12.32 4.24 21.93 52.32 7.49 0.44 0.46 A5403 - 12.64 4.25 20.49
53.42 7.81 0.43 0.51 A5403 - 12.17 4.22 21.56 52.48 8.15 0.44 0.51
A5403 - 11.68 4.32 25.68 49.67 7.06 0.48 0.54 AVERAGE 12.09 4.27
22.78 51.55 7.60 0.45 0.52
[0074] In T3 seed of selected T2 soybean lines, increases in
stearate of as high as approximately 53% of the total fatty acid
composition were obtained (Table 4), similar to those levels
obtained from seed oil from T1 5378 soybean lines. Furthermore,
similar to the 5374 soybean plants, decreases in palmitate, oleate
and linoleate were observed in both the T2 and T3 seed oil. In
addition, increases in saturates greater than C18:0 are also
obtained in both the T2 and T3 generations.
TABLE-US-00004 TABLE 4 STRAIN ID GUS 16:0 18:0 18:1 18:2 18:3 20:0
22:0 5378-A5403-48-269 + 8.63 50.62 5.5 23.07 7.4 3.34 1.14
5378-A5403-48-269 + 8.27 53.31 4.54 23.14 6.2 3.26 1.09
5378-A5403-48-269 + 8.6 51.92 4.84 22.65 7.27 3.43 1.01
5378-A5403-48-269 + 8.62 51.62 4.39 23.2 7.43 3.46 1.06
5378-A5403-48-269 + 9.07 50.57 4.77 22.61 7.97 3.52 1.22 average
8.64 51.61 4.81 22.93 7.25 3.40 1.10 5378-A5403-113-304 + 6.99 49.4
4.23 29.37 6.12 2.73 0.98 5378-A5403-113-304 + 6.79 50.25 3.77
28.53 6.76 2.74 0.99 5378-A5403-113-304 + 6.79 50.19 3.73 28.8 6.61
2.75 0.98 5378-A5403-113-304 + 6.34 47.67 4.08 30.03 7.96 2.77 1.01
5378-A5403-113-304 + 6.81 49.89 3.93 29.16 6.43 2.63 0.92 average
6.74 49.48 3.95 29.18 6.78 2.72 0.98 5374-A5403-36-341 - 10.7 6
24.46 50.9 6.29 0.53 0.61 5374-A5403-36-341 - 11.2 4.92 20.68 54.35
7.37 0.47 0.57 5374-A5403-36-341 - 11.27 4.27 23.71 52.72 6.55 0.43
0.53 5374-A5403-36-341 - 11.33 4.78 20.4 54.38 7.58 0.46 0.55
5374-A5403-36-341 - 11.55 4.52 18.59 55.07 8.69 0.46 0.56 null
segregant Ave 11.21 4.90 21.57 53.48 7.30 0.47 0.56
[0075] The above results demonstrate that by expression of an
acyl-ACP thioesterase with substantial activity towards 18:0
acyl-ACP substrates, and capable of producing C18:0 in seed tissue
of soybean plants, it is now possible to increase the levels of
stearate in the seed oil of soybean.
Example 4
Composition of High Stearate Soybean Oil
[0076] In this particular case, soybean variety Hartz H4152 (also
known as HS-2) was developed with a unique fatty acid composition
of about 24% or above stearate and below about 3%, preferably about
2.5% linolenate. H4152 was derived from the cross between a soybean
line with high stearate content (H90-127-113, also known as HS-1)
and a line with low linolenic content (N85-2176). H90-127-113 is a
Hartz variety derived from a cross between Hartz variety H5668 and
soybean line A6. A6 is a soybean mutant with high stearate seed
content (28.1%) released in 1981 by Iowa State University (Hammond,
E. G. and W. R. Fehr. 1983. Registration of A6 germplasm line of
soybean. Crop Sci. 23: 192-193). The high stearate content in line
A6 has been determined to be conditioned by a single recessive
gene, fas-a (Graef, G. L., W. R. Fehr, and E. G. Hammond. 1985.
Inheritance of three stearic acid mutants of soybean. Crop Sci. 25:
1076-1079). N85-2176 is a release from North Carolina State
University selected for its low linolenate seed content.
[0077] The F.sub.1 seeds from the cross between H90-127-113 and
N85-2176 were grown in the greenhouse in November and December. In
February, small portions of the F.sub.2 seeds opposite the embryo
were removed and analyzed in the laboratories of Hartz Seed Co. for
fatty acid composition using a gas chromatography. F.sub.2 seed
number 27 was selected for its high stearate and low linolenate
levels and was grown in the greenhouse. The F.sub.3 seed from plant
#27 were planted in the field at Stuttgart, AR in the following
summer. Ten agronomically desirable F.sub.3 plants with desirable
fatty acid composition were selected, and F.sub.4 progeny rows from
those plants were planted at Stuttgart in the summer. Twenty
agronomically desirable, uniform single plants were selected from
row number 4. The F.sub.5 progeny of those 20 plants was planted in
single rows in a winter nursery in Santa Isabel, Puerto Rico. Ten
uniform, single rows were harvested and bulked. The resulting seed
was grown in a 0.2 acre breeder increase at Stuttgart, AR during
the following summer, forming the foundation for Hartz variety
H4152.
TABLE-US-00005 TABLE 5 Fatty acid distributions of soybean oil
Palmitic Stearic Oleic Linoleic Linolenic Soybean oil C16:0 C18:0
C18:1 C18:2 C18:3 Common 11 4 23 53 8 High stearate 10 21 22 41 3
(H4152)
[0078] The composition of the high stearate soybean oil is unique
in that it has low linoleic and linolenic and high oleic and
stearic fatty acids. This improves the stability of the oil to
oxidative degradation and also changes the triglyceride
composition, resulting in the formation of compounds that have a
higher melting point than those found in common soybean oil. The
melting point of high stearate soybean oil is below room
temperature, but solid fats are present that crystallize when the
oil is stored at room temperature.
Example 5
Stability of High Stearate Soybean Oil
TABLE-US-00006 [0079] TABLE 6 Stability assays of soybean oil
Common soybean High stearate soybean Stability criteria oil oil
Inherent stability 7.0 3.9 Calculated iodine value 132 101 Active
oxygen method 8-10 40
[0080] Inherent stability is a calculated relative reactivity with
oxygen, with higher values denoting a greater predisposition to
oxidation (M. Erickson and N. Frey, Food Technology, 50: 63-68
(1994)). Iodine values represent calculated reactivities with
elemental iodine, with higher values indicating greater
reactivities (Official and Tentative Methods, American Oil
Chemists' Society, Cd 1b-87, Champaign, Ill.). The active oxygen
method assay simulates thermal breakdown encountered during
cooking, with higher values representing greater thermal stability
(Official and Tentative Methods, American Oil Chemists' Society, Cd
12-57(93), Champaign, Ill.). The increase in saturated and
monounsaturated fatty acids, and the decrease in polyunsaturated
fatty acids in high stearate soybean oil results in a greater
thermal stability when compared to common soybean oil. The greater
thermal stability of high stearate soybean oil when compared to
common soybean oil agrees with predictions based upon the
calculated inherent stabilities and iodine values.
Example 6
Solids Profile of Soybean Oils
TABLE-US-00007 [0081] TABLE 7 Solids present at various
temperatures Solids present at temperature Oil type 50.degree. F.
70.degree. F. 80.degree. F. 92.degree. F. 104.degree. F. Common
soybean oil -- -- -- -- -- High stearate (H4152) 9.8 -- -- -- --
Interesterified high 7.0 3.5 3.0 2.0 0.7 stearate
[0082] Solids were evaluated by the solids fat index using
dilatometry. Values represent percent solids in a sample at the
given temperature. Interesterified high stearate oil was prepared
byinteresterification according to the method of Erickson
(Practical Handbook of Soybean Processing and Utilization, American
Oil Chemists' Society Press, Champaign Ill., 1995). In contrast,
soybean oil A6 having high stearic, normal levels of palmitic and
linolenic, and low levels of oleic and linoleic acids, has no
solids present at 24.7.degree. C. and higher temperatures.
Example 7
Application of High Stearate Soybeans in Soy Based Foods
[0083] A. To prepare tofu and soymilk, 50 grams of high stearate
soybeans were soaked in 150 grams water overnight and drained.
Soybeans were rinsed with water. 185 grams of boiling water was
added, and the mixture pureed. 315 grams of water were added, and
the mixture heated to 100.degree. C. for 10 minutes. Okara
(filtered solids) was extracted using the Juiceman Junior machine
(Salton Maxim, Mt. Prospect, Ill.) to dehull the beans. The hot
soymilk should be approximately 8% solids. For firm tofu, 1.5 grams
glucono-.delta.-lactone (Aldrich, Milwaukee, Wis.) was added. The
liquid was allowed to coagulate for 15 to 20 minutes at 90.degree.
C. with light stirring. The liquid was allowed to cool and form
tofu.
[0084] B. To prepare soymilk, 150 grams of high stearate soybeans
were soaked in 500 mL water overnight and drained. Soaked soybeans
were rinsed with water. The beans were equally divided into two
portions. Each portion was ground with 400 mL water in an Oster
blender (Sunbeam, Delray Beach, Fla.) at the highest speed for 1.5
minutes. The combined slurry from the two portions was manually
filtered through cloth. The solid residue was discarded. The
filtrate was heated to 95.degree. C. for 10 minutes to prepare
soymilk. To prepare tofu, the hot soymilk was cooled to about
75.degree. C., and 5 grams of either calcium sulfate or
glucono-.delta.-lactone was added. The mixture was allowed to stand
for 30 minutes to form curd, which became silken tofu. To prepare
firm tofu, the hot curd was broken, placed in a mold, and pressed
to release the whey.
[0085] Tofu prepared from high stearate oil soybeans had a
consistency that was firmer and more creamy than tofu prepared from
standard control soybeans.
Example 8
Applications of Full Fat Soy Flour in Baked Foods
[0086] High stearate soybeans were processed into full fat soy
flour using the standard industry protocol (Practical Handbook of
Soybean Processing and Utilization, American Oil Chemists' Society
Press, Champaign Ill., 1995). The flour can be added to baked
products at high levels (15-20%) to increase the protein content
without affecting the texture of the baked products. Flour obtained
from high stearate soybeans can be used in an array of food
products including candies, gravies, sauces, frozen desserts,
pastas, meat products, and baked goods.
Example 9
Margarine Formulation Using High Stearate Soybean Oil
TABLE-US-00008 [0087] TABLE 8 Margarine composition Ingredient
Weight percent Water 16.85 Whey protein 0.4 Salt 1.9 Lecithin 0.4
Monoglyceride (Super G7, A C Humko Co) 0.45 Sodium benzoate 0.001
Interesterified high stearate oil 80.0 Flavor, color to 100%
(I-1435, Fries & Fries)
[0088] The oil was heated to 65.degree. C. in a microwave oven.
Flavors were withheld and added to the oil phase immediately before
the oil phase was combined with the remaining components which had
been heated to 50.degree. C. in a microwave oven. The two phases
were combined and mixed for 20 minutes at 2000 rpm in a Dispermat
unit (VMA-Getzmann, Germany) maintained at 60.degree. C. The
margarine was filled into one pound tubs and placed at 40.degree.
C. for crystallization. The margarine product was easily spreadable
when removed from refrigeration after one day, and after long term
storage of four weeks. The margarine exhibited good room
temperature stability as well as excellent flavor release and
structure.
Example 10
Formulation of all-Purpose Shortenings with Interesterified High
Stearate Oil
[0089] Commercial shortenings, such as CRISCO (Proctor and Gamble,
Cincinnati, Ohio), are composed of a hydrogenated soybean oil
combined with a fully hydrogenated cottonseed oil component and an
emulsifier such as a monoglyceride. The soybean oil basestocks
generally have a trans fatty acid content of greater than 15%
(w/w), and frequently greater than 25% (w/w). The interesterified
high stearate soybean oil has sufficient solids such that when
combined with the fully hydrogenated cottonseed oil and
monoglyceride, it gives a texture and consistency similar to the
CRISCO product upon votation (rapid chilling and working of fat,
Weiss, T. J., Food oils and Their Uses, Avi Publishing Co.,
Westport, Conn., 1983) and crystallization. Nitrogen is added to
the formulation to modify the final density and solidity of the
product. A 15% overrun of gas corresponds to a 15% reduction in
density of the shortening in comparison to the density before
addition of nitrogen.
TABLE-US-00009 TABLE 9 Shortening formulation Component Percent by
mass Interesterified high stearate oil 89.2 Cottonseed oil (5
iodine value max.) 7.8 Monoglyceride (Super G7, A C Humko Co) 3.0
Nitrogen gas 15%
Example 11
Shelf Life Testing
[0090] Non-hydrogenated oils are preferred over partially
hydrogenated oils due to costs and improved acceptance by the
consumer. However, the short shelf stability of non-hydrogenated
oils severely limit their food applications. The interesterified
high stearate soybean oil was used in a potato chip rancidity assay
to determine the applicability in the preparation of fried
foods.
TABLE-US-00010 TABLE 10 Schaal oven test Oil Days to detect rancid
odor Common soybean oil 2-3 All-purpose shortening (CRISCO) 6-7
Interesterified high stearate oil 11 (all purpose shortening
formulation
[0091] The rancidity assay was performed using a Schaal oven test
at 62.degree. C. (Warner, K. and Eskin, N. M., Methods to Assess
Quality and Stability of Oils and Fat-Containing Foods, American
Oil Chemists' Society Press, Champaign, Ill., 1995).
Interesterified high stearate oil demonstrated a marked increase in
stability, as indicated by the longer duration of time required to
detect an undesirable odor.
[0092] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0093] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
211300DNAGarcinia mangostana 1ccaagatgtt gaagctctct tcttcccgaa
gcccattggc ccgcattccc acccggccca 60ggcccaactc cattcctccc cggataattg
tggtttcctc ctcatccagc aaggttaatc 120cactcaaaac agaggcggtg
gtttcttcgg ggctggctga ccggctccgg ctgggcagct 180tgaccgagga
cgggctttcg tataaggaga agttcatagt gagatgctat gaggttggga
240ttaacaagac cgctactgtt gagactattg ccaacctctt gcaggaggtt
ggatgcaatc 300acgcccaaag cgttggatat tcgacgggtg ggttttcgac
aacccctacc atgagaaaat 360tgcgtctgat atgggttact gctcgcatgc
acatcgaaat ctacaaatat ccagcttgga 420gtgatgtggt ggaaatagag
tcgtggggcc agggtgaagg aaaaatcgga accagacgtg 480attggattct
gagagactat gccactggtc aagttattgg ccgagctact agcaagtggg
540taatgatgaa ccaagacacc aggcgacttc aaaaagtcga tgttgatgtt
cgtgatgagt 600acttggttca ctgtccaaga gaactcagat tggcatttcc
agaggaaaat aatagcagct 660tgaagaaaat ttcaaaactt gaagatcctt
ctcaatattc gaagctgggg cttgtgccta 720gaagagcaga tctggacatg
aatcaacatg ttaataatgt cacctatatt ggatgggtgt 780tggagagcat
gcctcaagaa atcattgata cccatgaact gcaaaccata acattagact
840acagacggga atgccaacat gatgatgtgg ttgattcctt gactagtcca
gagccttctg 900aagatgctga agcagttttc aaccataatg gaacaaatgg
gtctgcaaat gtgagcgcca 960acgaccatgg atgccgcaac tttctgcatc
tactaagatt gtcgggcaat ggacttgaaa 1020tcaaccgtgg tcgtactgag
tggagaaaga aacctacaag atgaggcaat aaagtacatt 1080atgtacttta
tcgttgcttt agccggcttc tggatggtga tttctttctg cattccttct
1140ttcctttttg ttttcctagg gtatccttcg cttcttgcct gtaagagtat
tatgttttcc 1200gtttgccctg aagttgtaaa tttgtcgagg aactcgagtc
attgtttgaa tcgaggatgg 1260tgagaagtgt acttgtttgt tgtattccat
tcttcctgat 13002352PRTGarcinia mangostana 2Met Leu Lys Leu Ser Ser
Ser Arg Ser Pro Leu Ala Arg Ile Pro1 5 10 15Thr Arg Pro Arg Pro Asn
Ser Ile Pro Pro Arg Ile Ile Val Val Ser 20 25 30Ser Ser Ser Ser Lys
Val Asn Pro Leu Lys Thr Glu Ala Val Val Ser 35 40 45Ser Gly Leu Ala
Asp Arg Leu Arg Leu Gly Ser Leu Thr Glu Asp Gly 50 55 60Leu Ser Tyr
Lys Glu Lys Phe Ile Val Arg Cys Tyr Glu Val Gly Ile 65 70 75Asn Lys
Thr Ala Thr Val Glu Thr Ile Ala Asn Leu Leu Gln Glu Val80 85 90
95Gly Cys Asn His Ala Gln Ser Val Gly Tyr Ser Thr Gly Gly Phe Ser
100 105 110Thr Thr Pro Thr Met Arg Lys Leu Arg Leu Ile Trp Val Thr
Ala Arg 115 120 125Met His Ile Glu Ile Tyr Lys Tyr Pro Ala Trp Ser
Asp Val Val Glu 130 135 140Ile Glu Ser Trp Gly Gln Gly Glu Gly Lys
Ile Gly Thr Arg Arg Asp 145 150 155Trp Ile Leu Arg Asp Tyr Ala Thr
Gly Gln Val Ile Gly Arg Ala Thr160 165 170 175Ser Lys Trp Val Met
Met Asn Gln Asp Thr Arg Arg Leu Gln Lys Val 180 185 190Asp Val Asp
Val Arg Asp Glu Tyr Leu Val His Cys Pro Arg Glu Leu 195 200 205Arg
Leu Ala Phe Pro Glu Glu Asn Asn Ser Ser Leu Lys Lys Ile Ser 210 215
220Lys Leu Glu Asp Pro Ser Gln Tyr Ser Lys Leu Gly Leu Val Pro Arg
225 230 235Arg Ala Asp Leu Asp Met Asn Gln His Val Asn Asn Val Thr
Tyr Ile240 245 250 255Gly Trp Val Leu Glu Ser Met Pro Gln Glu Ile
Ile Asp Thr His Glu 260 265 270Leu Gln Thr Ile Thr Leu Asp Tyr Arg
Arg Glu Cys Gln His Asp Asp 275 280 285Val Val Asp Ser Leu Thr Ser
Pro Glu Pro Ser Glu Asp Ala Glu Ala 290 295 300Val Phe Asn His Asn
Gly Thr Asn Gly Ser Ala Asn Val Ser Ala Asn 305 310 315Asp His Gly
Cys Arg Asn Phe Leu His Leu Leu Arg Leu Ser Gly Asn320 325 330
335Gly Leu Glu Ile Asn Arg Gly Arg Thr Glu Trp Arg Lys Lys Pro Thr
340 345 350Arg
* * * * *