U.S. patent application number 12/318591 was filed with the patent office on 2009-05-07 for nuleic acid constructs and methods for producing altered seed oil compositions.
Invention is credited to Neal A. Bringe, Katayoon Dehesh, Joanne J. Fillatti.
Application Number | 20090119805 12/318591 |
Document ID | / |
Family ID | 28457116 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090119805 |
Kind Code |
A1 |
Fillatti; Joanne J. ; et
al. |
May 7, 2009 |
Nuleic acid constructs and methods for producing altered seed oil
compositions
Abstract
The present invention is in the field of plant genetics and
provides recombinant nucleic acid molecules, constructs, and other
agents associated with the coordinate manipulation of multiple
genes in the fatty acid synthesis pathway. In particular, the
agents of the present invention are associated with the
simultaneous enhanced expression of certain genes in the fatty acid
synthesis pathway and suppressed expression of certain other genes
in the sane pathway. Also provided are plants incorporating such
agents, and in particular plants incorporating such constructs
where the plants exhibit altered seed oil compositions.
Inventors: |
Fillatti; Joanne J.; (Davis,
CA) ; Bringe; Neal A.; (St. Charles, MO) ;
Dehesh; Katayoon; (Vacaville, CA) |
Correspondence
Address: |
ARNOLD & PORTER LLP
555 TWELFTH STREET, N.W., ATTN: IP DOCKETING
WASHINGTON
DC
20004
US
|
Family ID: |
28457116 |
Appl. No.: |
12/318591 |
Filed: |
December 31, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10508401 |
Mar 25, 2005 |
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PCT/US03/08610 |
Mar 21, 2003 |
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12318591 |
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60390185 |
Jun 21, 2002 |
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60365794 |
Mar 21, 2002 |
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Current U.S.
Class: |
800/312 |
Current CPC
Class: |
C12N 15/8237 20130101;
C11B 1/00 20130101; C12N 9/0083 20130101; Y02E 50/10 20130101; C12N
15/8247 20130101; C10L 1/026 20130101; Y02E 50/13 20130101; A23K
10/30 20160501; A23K 20/147 20160501; A23D 9/00 20130101; A23K
20/158 20160501 |
Class at
Publication: |
800/312 |
International
Class: |
A01H 5/10 20060101
A01H005/10 |
Claims
1. A soybean seed exhibiting an oil composition comprising 55 to
80% by weight oleic acid, 10 to 40% by weight linoleic acid, 6% or
less by weight linolenic acid, and 2 to 8% by weight saturated
fatty acids.
2-72. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to recombinant nucleic
acid molecules, constructs, and other agents associated with the
coordinate manipulation of multiple genes in the fatty acid
synthesis pathway. In particular, the agents of the present
invention are associated with the simultaneous enhanced expression
of certain genes in the fatty acid synthesis pathway and suppressed
expression of certain other genes in the same pathway. The present
invention is also directed to plants incorporating such agents, and
in particular to plants incorporating such constructs where the
plants exhibit altered seed oil compositions.
BACKGROUND
[0002] Plant oils are used in a variety of applications. Novel
vegetable oil compositions and improved approaches to obtain oil
compositions, from biosynthetic or natural plant sources, are
needed Depending upon the intended oil use, various different fatty
acid compositions are desired. Plants, especially species which
synthesize large amounts of oils in seeds, are an important source
of oils both for edible and industrial uses. Seed oils are composed
almost entirely of triacylglycerols in which fatty acids are
esterified to the three hydroxyl groups of glycerol.
[0003] Soybean oil typically contains about 16-20% saturated fatty
acids: 13-16% palmitate and 34% stearate. See generally Gunstone et
al., The Lipid Handbook, Chapman & Hall, London (1994). Soybean
oils have been modified by various breeding methods to create
benefits for specific markets. However, a soybean oil that is
broadly beneficial to major soybean oil users such as consumers of
salad oil, cooking oil and flying oil, and industrial markets such
as biodiesel and biolube markets, is not available. Prior soybean
oils were either too expensive or lacked an important food quality
property such as oxidative stability, good fried food flavor or
saturated fat content, or an important biodiesel property such as
appropriate nitric oxide emissions or cold tolerance or cold
flow.
[0004] Higher plants synthesize fatty acids via a common metabolic
pathway--the fatty acid synthetase (FAS) pathway, which is located
in the plastids. .beta.-ketoacyl-ACP synthases are important
rate-limiting enzymes in the FAS of plant cells and exist in
several versions. .beta.-ketoacyl-ACP synthase I catalyzes chain
elongation to palmitoyl-ACP (C16:0), whereas .beta.-ketoacyl-ACP
synthase II catalyzes chain elongation to stearoyl-ACP (C18:0).
.beta.-ketoacyl-ACP synthase IV is a variant of .beta.-ketoacyl-ACP
synthase II, and can also catalyze chain elongation to 18:0-ACP. In
soybean, the major products of FAS are 16:0-ACP and 18:0-ACP. The
desaturation of 18:0-ACP to form 18:1-ACP is catalyzed by a
plastid-localized soluble delta-9 desaturase (also referred to as
"stearoyl-ACP desaturase"). See Voelker et al., 52 Annu. Rev. Plant
Physiol. Plant Mol. Biol. 33561 (2001).
[0005] The products of the plastidial FAS and delta-9 desaturase,
16:0-ACP, 18:0-ACP, and 18:1-ACP, are hydrolyzed by specific
thioesterases (FAT). Plant thioesterases can be classified into two
gene families based on sequence homology and substrate preference.
The first family, FATA, includes long chain acyl-ACP thioesterases
having activity primarily on 18:1-ACP. Enzymes of the second
family, FATB, commonly utilize 16:0-ACP (palmitoyl-ACP), 18:0-ACP
(stearoyl-ACP), and 18:1-ACP (oleoyl-ACP). Such thioesterases have
an important role in determining chain length during de novo fatty
acid biosynthesis in plants, and thus these enzymes are useful in
the provision of various modifications of fatty acyl compositions,
particularly with respect to the relative proportions of various
fatty acyl groups that are present in seed storage oils.
[0006] The products of the FATA and FATB reactions, the free fatty
acids, leave the plastids and are converted to their respective
acyl-CoA esters. Acyl-CoAs are substrates for the
lipid-biosynthesis pathway (Kennedy Pathway), which is located in
the endoplasmic reticulum (ER). This pathway is responsible for
membrane lipid formation as well as the biosynthesis of
triacylglycerols, which constitute the seed oil. In the ER there
are additional membrane-bound desaturases, which can further
desaturate 18:1 to polyunsaturated fatty acids. A delta-12
desaturase (FAD2) catalyzes the insertion of a double bond into
18:1, forming linoleic acid (18:2). A delta-15 desaturase (FAD3)
catalyzes the insertion of a double bond into 18:2, forming
linolenic acid (18:3).
[0007] Many complex biochemical pathways have now been manipulated
genetically, usually by suppression or over-expression of single
genes. Further exploitation of the potential for plant genetic
manipulation will require the coordinate manipulation of multiple
genes in a pathway. A number of approaches have been used to
combine transgenes in one plant--including sexual crossing,
retransformation, co-transformation, and the use of linked
transgenes. A chimeric transgene with linked partial gene sequences
can be used to coordinately suppress numerous plant endogenous
genes. Constructs modeled on viral polyproteins can be used to
simultaneously introduce multiple coding genes into plant cells.
For a review, see Halpin et al., Plant Mol. Biol. 47:295-310
(2001).
[0008] Thus, a desired plant phenotype may require the expression
of one or more genes and the concurrent reduction of expression of
another gene or genes. Thus, there exists a need to simultaneously
over-express one or more genes and suppress, or down-regulate, the
expression of a another gene or genes in plants using a single
transgenic construct.
SUMMARY OF THE INVENTION
[0009] The present invention provides a nucleic acid molecule or
molecules, which when introduced into a cell or organism are
capable of suppressing, at least partially reducing, reducing,
substantially reducing, or effectively eliminating the expression
of at least one or more endogenous FAD2, FAD3, or FATB RNAs while
at the same time coexpressing, simultaneously expressing, or
coordinately producing one or more RNAs or proteins transcribed
from or encoded by beta-ketoacyl-ACP synthase 1, beta-ketoacyl-ACP
synthase IV, delta-9 desaturase, or CP4 EPSPS, plant cells and
plants transformed with the same, and seeds, oil, and other
products produced from the transformed plants.
[0010] Also provided by the present invention is a recombinant
nucleic acid molecule comprising a first set of DNA sequences that
is capable, when expressed in a host cell, of suppressing the
endogenous expression of at least one, preferably two, genes
selected from the group consisting of FAD2, FAD3, and FATB genes;
and a second set of DNA sequences that is capable, when expressed
in a host cell, of increasing the endogenous expression of at least
one gene selected from the group consisting of a beta-ketoacyl-ACP
synthase I gene, a beta-ketoacyl-ACP synthase IV gene, and a
delta-9 desaturase gene.
[0011] Further provided by the present invention is a recombinant
nucleic acid molecule comprising a first set of DNA sequences that
is capable, when expressed in a host cell, of forming a dsRNA
construct and suppressing the endogenous expression of at least
one, preferably two, genes selected from the group consisting of
FAD2, FAD3, and FATB genes, where the first set of DNA sequences
comprises a first non-coding sequence that expresses a first RNA
sequence that exhibits at least 90% identity to a non-coding region
of a FAD2 gene, a first antisense sequence that expresses a first
antisense RNA sequence capable of forming a double-stranded RNA
molecule with the first RNA sequence, a second non-coding sequence
that expresses a second RNA sequence that exhibits at least 90%
identity to a non-coding region of a FAD3 gene, and a second
antisense sequence that expresses a second antisense RNA sequence
capable of forming a double-stranded RNA molecule with the second
RNA sequence; and a second set of DNA sequences that is capable,
when expressed in a host cell, of increasing the endogenous
expression of at least one gene selected from the group consisting
of a beta-ketoacyl-ACP synthase I gene, a beta-ketoacyl-ACP
synthase IV gene, and a delta-9 desaturase gene.
[0012] The present invention provides methods of transforming
plants with these recombinant nucleic acid molecules. The methods
include a method of producing a transformed plant having seed with
an increased oleic acid content, reduced saturated fatty acid
content, and reduced polyunsaturated fatty acid content, comprising
(A) transforming a plant cell with a recombinant nucleic acid
molecule which comprises a first set of DNA sequences that is
capable, when expressed in a host cell, of suppressing the
endogenous expression of at least one, preferably two, genes
selected from the group consisting of FAD2, FAD3, and FATB genes,
and a second set of DNA sequences that is capable, when expressed
in a host cell, of increasing the endogenous expression of at least
one gene selected from the group consisting of a beta-ketoacyl-ACP
synthase I gene, a beta-ketoacyl-ACP synthase IV gene, and a
delta-9 desaturase gene; and (B) growing the transformed plant,
where the transformed plant produces seed with an increased oleic
acid content, reduced saturated fatty acid content, and reduced
polyunsaturated fatty acid content relative to seed from a plant
having a similar genetic background but lacking the recombinant
nucleic acid molecule.
[0013] Further provided are methods of transforming plant cells
with the recombinant nucleic acid molecules. The methods include a
method of altering the oil composition of a plant cell comprising
(A) transforming a plant cell with a recombinant nucleic acid
molecule which comprises a first set of DNA sequences that is
capable, when expressed in a host cell, of suppressing the
endogenous expression of at least one, preferably two, genes
selected from the group consisting of FAD2, FAD3, and FATB genes,
and a second set of DNA sequences that is capable, when expressed
in a host cell, of increasing the endogenous expression of at least
one gene selected from the group consisting of a beta-ketoacyl-ACP
synthase I gene, a beta-ketoacyl-ACP synthase IV gene, and a
delta-9 desaturase gene; and (B) growing the plant cell under
conditions where transcription of the first set of DNA sequences
and the second set of DNA sequences is initiated, where the oil
composition is altered relative to a plant cell with a similar
genetic background but lacking the recombinant nucleic acid
molecule.
[0014] The present invention also provides a transformed plant
comprising a recombinant nucleic acid molecule which comprises a
first set of DNA sequences that is capable, when expressed in a
host cell, of suppressing the endogenous expression of at least
one, preferably two, genes selected from the group consisting of
FAD2, FAD3, and FATB genes, and a second set of DNA sequences that
is capable, when expressed in a host cell, of increasing the
endogenous expression of at least one gene selected from the group
consisting of a beta-ketoacyl-ACP synthase I gene, a
beta-ketoacyl-ACP synthase IV gene, and a delta-9 desaturase gene.
Further provided by the present invention is a transformed soybean
plant bearing seed, where the seed exhibits an oil composition
which comprises 55 to 80% by weight oleic acid, 10 to 40% by weight
linoleic acid, 6% or less by weight linolenic acid, and 2 to 8% by
weight saturated fatty acids, and feedstock plant parts, and seed
derived from the plant.
[0015] The present invention provides a soybean seed exhibiting an
oil composition comprising 55 to 80% by weight oleic acid, 10 to
40% by weight linoleic acid, 6% or less by weight linolenic acid,
and 2 to 8% by weight saturated fatty acids, and also provides a
soybean seed exhibiting an oil composition comprising 65 to 80% by
weight oleic acid, 10 to 30% by weight linoleic acid, 6% or less by
weight linolenic acid, and 2 to 8% by weight of saturated fatty
acids. Also provided by the present invention are soyfoods
comprising an oil composition which comprises 69 to 73% by weight
oleic acid, 21 to 24% by weight linoleic acid, 0.5 to 3% by weight
linolenic acid, and 2-3% by weight of saturated fatty acids.
[0016] The crude soybean oil provided by the present invention
exhibits an oil composition comprising 55 to 80% by weight oleic
acid, 10 to 40% by weight linoleic acid, 6% or less by weight
linolenic acid, and 2 to 8% by weight saturated fatty acids.
Another crude soybean oil provided by the present invention
exhibits an oil composition comprising 65 to 80% by weight oleic
acid, 10 to 30% by weight linoleic acid, 6% or less by weight
linolenic acid, and 2 to 8% by weight of saturated fatty acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1-4 each depict exemplary nucleic acid molecule
configurations;
[0018] FIGS. 5 and 6 each depict illustrative configurations of a
first set of DNA sequences; and
[0019] FIGS. 7-15 each depict nucleic acid molecules of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
Description of the Nucleic Acid Sequences
[0020] SEQ ID NO: 1 is a nucleic acid sequence of a FAD2-1A intron
1.
[0021] SEQ ID NO: 2 is a nucleic acid sequence of a FAD2-1B intron
1.
[0022] SEQ ID NO: 3 is a nucleic acid sequence of a FAD2-1B
promoter.
[0023] SEQ ID NO: 4 is a nucleic acid sequence of a FAD2-1A genomic
clone.
[0024] SEQ ID NOs: 5 & 6 are nucleic acid sequences of a
FAD2-1A 3' UTR and 5'UTR, respectively.
[0025] SEQ ID NOs: 7-13 are nucleic acid sequences of FAD3-1A
introns 1, 2, 3A, 4, 5, 3B, and 3C, respectively.
[0026] SEQ ID NO: 14 is a nucleic acid sequence of a FAD3-1C intron
4.
[0027] SEQ ID NO: 15 is a nucleic acid sequence of a partial
FAD3-1A genomic clone.
[0028] SEQ ID NOs: 16 & 17 are nucleic acid sequences of a
FAD3-1A 3'UTR and 5'UTR, respectively.
[0029] SEQ ID NO: 18 is a nucleic acid sequence of a partial
FAD3-1B genomic clone.
[0030] SEQ ID NOs: 19-25 are nucleic acid sequences of FAD3-1B
introns 1, 2, 3A, 3B, 3C, 4, and 5, respectively.
[0031] SEQ ID NOs: 26 & 27 are nucleic acid sequences of a
FAD3-1B 3'UTR and 5'UTR, respectively.
[0032] SEQ ID NO: 28 is a nucleic acid sequence of a FATB genomic
clone.
[0033] SEQ ID NO: 29-35 are nucleic acid sequences of FATB introns
I, II, III, IV, V, VI, and VII, respectively.
[0034] SEQ ID NOs: 36 & 37 are nucleic acid sequences of a FATB
3'UTR and 5'UTR, respectively.
[0035] SEQ ID NO: 38 is a nucleic acid sequence of a Cuphea
pulcherrima KAS IV gene.
[0036] SEQ ID NO: 39 is a nucleic acid sequence of a Cuphea
pulcherrima KAS IV gene.
[0037] SEQ ID NOs: 40 & 41 are nucleic acid sequences of
Ricinus communis and Simmondsia chinensis delta-9 desaturase genes,
respectively.
DEFINITIONS
[0038] "ACP" refers to an acyl carrier protein moiety. "Altered
seed oil composition" refers to a seed oil composition from a
transgenic or transformed plant of the invention which has altered
or modified levels of the fatty acids therein, relative to a seed
oil from a plant having a similar genetic background but that has
not been transformed. "Antisense suppression" refers to
gene-specific silencing that is induced by the introduction of an
antisense RNA molecule.
[0039] "Coexpression of more than one agent such as an mRNA or
protein" refers to the simultaneous expression of an agent in
overlapping time frames and in the same cell or tissue as another
agent. "Coordinated expression of more than one agent" refers to
the coexpression of more than one agent when the production of
transcripts and proteins from such agents is carried out utilizing
a shared or identical promoter. "Complement" of a nucleic acid
sequence refers to the complement of the sequence along its
complete length.
[0040] "Cosuppression" is the reduction in expression levels,
usually at the level of RNA, of a particular endogenous gene or
gene family by the expression of a homologous sense construct that
is capable of transcribing mRNA of the same strandedness as the
transcript of the endogenous gene. Napoli et al., Plant Cell
2:279-289 (1990); van der Krol et al., Plant Cell 2:291-299 (1990).
"Crude soybean oil" refers to soybean oil that has been extracted
from soybean seeds, but has not been refined, processed, or
blended, although it may be degummed.
[0041] When referring to proteins and nucleic acids herein,
"derived" refers to either directly (for example, by looking at the
sequence of a known protein or nucleic acid and preparing a protein
or nucleic acid having a sequence similar, at least in part, to the
sequence of the known protein or nucleic acid) or indirectly (for
example, by obtaining a protein or nucleic acid from an organism
which is related to a known protein or nucleic acid) obtaining a
protein or nucleic acid from a known, protein or nucleic acid.
Other methods of "deriving" a protein or nucleic acid from a known
protein or nucleic acid are known to one of skill in the art.
[0042] "dsRNA", "dsRNAi" and "RNAi" all refer to gene-specific
silencing that is induced by the introduction of a construct
capable of forming a double-stranded RNA molecule. A "dsRNA
molecule" and an "RNAi molecule" both refer to a double-stranded
RNA molecule capable, when introduced into a cell or organism, of
at least partially reducing the level of an mRNA species present in
a cell or a cell of an organism.
[0043] "Exon" refers to the normal sense of the term as meaning a
segment of nucleic acid molecules, usually DNA, that encodes part
of or all of an expressed protein.
[0044] "Fatty acid" refers to free fatty acids and fatty acyl
groups.
[0045] "Gene" refers to a nucleic acid sequence that encompasses a
5' promoter region associated with the expression of the gene
product, any intron and exon regions and 3' or 5' untranslated
regions associated with the expression of the gene product "Gene
silencing" refers to the suppression of gene expression or
down-regulation of gene expression.
[0046] A "gene family" is two or more genes in an organism which
encode proteins that exhibit similar functional attributes, and a
"gene family member" is any gene of the gene family found within
the genetic material of the plant, e.g., a "FAD2 gene family
member" is any FAD2 gene found within the genetic material of the
plant. An example of two members of a gene family are FAD2-1 and
FAD2-2. A gene family can be additionally classified by the
similarity of the nucleic acid sequences. Preferably, a gene family
member exhibits at least 60%, more preferably at least 70%, more
preferably at least 80% nucleic acid sequence identity in the
coding sequence portion of the gene.
[0047] "Heterologous" means not naturally occurring together. A
"high oleic soybean seed" is a seed with oil having greater Man 75%
oleic acid present in the oil composition of the seed.
[0048] A nucleic acid molecule is said to be "introduced" if it is
inserted into a cell or organism as a result of human manipulation,
no matter how indirect Examples of introduced nucleic acid
molecules include, but are not limited to, nucleic acids that have
been introduced into cells via transformation, transfection,
injection, and projection, and those that have been introduced into
an organism via methods including, but not limited to, conjugation,
endocytosis, and phagocytosis.
[0049] "Intron" refers to the normal sense of the term as meaning a
segment of nucleic acid molecules, usually DNA, that does not
encode part of or all of an expressed protein, and which, in
endogenous conditions, is transcribed into RNA molecules, but which
is spliced out of the endogenous RNA before the RNA is translated
into a protein. An "intron dsRNA molecule" and an "intron RNAi
molecule" both refer to a double-stranded RNA molecule capable,
when introduced into a cell or organism, of at least partially
reducing the level of an mRNA species present in a cell or a cell
of an organism where the double-stranded RNA molecule exhibits
sufficient identity to an intron of a gene present in the cell or
organism to reduce the level of an mRNA containing that intron
sequence.
[0050] A "low saturate" oil composition contains between 3.6 and 8
percent saturated fatty acids.
[0051] A "mid-oleic soybean seed" is a seed having between 50% and
85% oleic acid present in the oil composition of the seed.
[0052] The term "non-coding" refers to sequences of nucleic acid
molecules that do not encode part or all of an expressed protein.
Non-coding sequences include but are not limited to introns,
promoter regions, 3' untranslated regions (3'UTRs), and 5'
untranslated regions (5'UTRs).
[0053] A promoter that is "operably linked" to one or more nucleic
acid sequences is capable of driving expression of one or more
nucleic acid sequences, including multiple coding or non-coding
nucleic acid sequences arranged in a polycistronic
configuration.
[0054] "Physically linked" nucleic acid sequences are nucleic acid
sequences that are found on a single nucleic acid molecule. A
"plant" includes reference to whole plants, plant organs (e.g.,
leaves, stems, roots, etc.), seeds, and plant cells and progeny of
the same. The term "plant cell" includes, without limitation, seed
suspension cultures, embryos, meristematic regions, callus tissue,
leaves, roots, shoots, gametophytes, sporophytes, pollen, and
microspores. "Plant promoters," include, without limitation, plant
viral promoters, promoters derived from plants, and synthetic
promoters capable of functioning in a plant cell to promote the
expression of an mRNA.
[0055] A "polycistronic gene" or "polycistronic mRNA" is any gene
or mRNA that contains transcribed nucleic acid sequences which
correspond to nucleic acid sequences of more than one gene targeted
for expression. It is understood that such polycistronic genes or
mRNAs may contain sequences that correspond to introns, 5'UTRs,
3'UTRs, or combinations thereof and that a recombinant
polycistronic gene or mRNA might, for example without limitation,
contain sequences that correspond to one or more UTRs from one gene
and one or more introns from a second gene.
[0056] A "seed-specific promoter" refers to a promoter that is
active preferentially or exclusively in a seed. "Preferential
activity" refers to promoter activity that is substantially greater
in the seed than in other tissues, organs or organelles of the
plant. "Seed-specific" includes without limitation activity in the
aleurone layer, endosperm, and/or embryo of the seed.
[0057] "Sense intron suppression" refers to gene silencing that is
induced by the introduction of a sense intron or fragment thereof.
Sense intron suppression is described by Fillatti in PCT WO
01/14538 A2. "Simultaneous expression" of more than one agent such
as an mRNA or protein refers to the expression of an agent at the
same time as another agent. Such expression may only overlap in
part and may also occur in different tissue or at different
levels.
[0058] "Total oil level" refers to the total aggregate amount of
fatty acid without regard to the type of fatty acid. "Transgene"
refers to a nucleic acid sequence associated with the expression of
a gene introduced into an organism. A transgene includes, but is
not limited to, a gene endogenous or a gene not naturally occurring
in the organism. A "transgenic plant" is any plant that stably
incorporates a transgene in a manner that facilitates transmission
of that transgene from a plant by any sexual or asexual method.
[0059] A "zero saturate" oil composition contains less than 3.6
percent saturated fatty acids.
[0060] When referring to proteins and nucleic acids herein, the use
of plain capitals, e.g., "FAD2", indicates a reference to an
enzyme, protein, polypeptide, or peptide, and the use of italicized
capitals, e.g., "FAD2", is used to refer to nucleic acids,
including without limitation genes, cDNAs, and mRNAs. A cell or
organism can have a family of more than one gene encoding a
particular enzyme, and the capital letter that follows the gene
terminology (A, B, C) is used to designate the family member, i.e.,
FAD2-1A is a different gene family member from FAD2-1B.
[0061] As used herein, any range set forth is inclusive of the end
points of the range unless otherwise stated.
A. Agents
[0062] The agents of the invention will preferably be "biologically
active" with respect to either a structural attribute, such as the
capacity of a nucleic acid molecule to hybridize to another nucleic
acid molecule, or the ability of a protein to be bound by an
antibody (or to compete with another molecule for such binding).
Alternatively, such an attribute may be catalytic and thus involve
the capacity of the agent to mediate a chemical reaction or
response. The agents will preferably be "substantially purified."
The term "substantially purified," as used herein, refers to a
molecule separated from substantially all other molecules normally
associated with it in its native environmental conditions. More
preferably a substantially purified molecule is the predominant
species present in a preparation. A substantially purified molecule
may be greater than 60% free, greater than 75% free, preferably
greater than 90% free, and most preferably greater than 95% free
from the other molecules (exclusive of solvent) present in the
natural mixture. The term "substantially purified" is not intended
to encompass molecules present in their native environmental
conditions.
[0063] The agents of the invention may also be recombinant. As used
herein, the term "recombinant" means any agent (e.g., including but
limited to DNA, peptide), that is, or results, however indirectly,
from human manipulation of a nucleic acid molecule. It is also
understood that the agents of the invention may be labeled with
reagents that facilitate detection of the agent, e.g., fluorescent
labels, chemical labels, and/or modified bases.
[0064] Agents of the invention include nucleic acid molecules that
comprise a DNA sequence which is at least 50%, 60%, or 70%
identical over their entire length to a plant coding region or
non-coding region, or to a nucleic acid sequence that is
complementary to a plant coding or non-coding region. More
preferable are DNA sequences that are, over their entire length, at
least 80% identical; at least 85% identical; at least 90%
identical; at least 95% identical; at least 97% identical; at least
98% identical; at least 99% identical; or 100% identical to a plant
coding region or non-coding region, or to a nucleic acid sequence
that is complementary to a plant coding or non-coding region.
[0065] "Identity," as is well understood in the art, is a
relationship between two or more polypeptide sequences or two or
more nucleic acid molecule sequences, as determined by comparing
the sequences. In the art, "identity" also means the degree of
sequence relatedness between polypeptide or nucleic acid molecule
sequences, as determined by the match between strings of such
sequences. "Identity" can be readily calculated by known methods
including, but not limited to, those described in Computational
Molecular Biology, Lesk, ed., Oxford University Press, New York
1988; Biocomputing: Informatics and Genome Projects, Smith, ed.,
Academic Press, New York 1993; Computer Analysis of Sequence Data,
Part I, Griffin and Griffin, eds., Humana Press, New Jersey 1994;
Sequence Analysis in Molecular Biology, von Heinje, Academic Press
1987; Sequence Analysis Primer, Gribskov and Devereux, eds.,
Stockton Press, New York 1991; and Carillo and Lipman, SIAM J.
Applied Math, 48:1073 1988.
[0066] Methods to determine identity are designed to give the
largest match between the sequences tested. Moreover, methods to
determine identity are codified in publicly available programs.
Computer programs which can be used to determine identity between
two sequences include, but are not limited to, GCG; a suite of five
BLAST programs, three designed for nucleotide sequences queries
(BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence
queries (BLASTP and TBLASTN). The BLASIX program is publicly
available from NCBI and other sources, e.g., BLAST Manual, Altschul
et al., NCBI NLM NIH, Bethesda, Md. 20894; Altschul et al., J. Mol.
Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm
can also be used to determine identity.
[0067] Parameters for polypeptide sequence comparison typically
include the following: Algorithm: Needleman and Wunsch, J. Mol.
Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from
Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919
(1992); Gap Penalty: 12; Gap Length Penalty: 4. A program that can
be used with these parameters is publicly available as the "gap"
program from Genetics Computer Group ("GCG"), Madison, Wis. The
above parameters along with no penalty for end gap are the default
parameters for peptide comparisons.
[0068] Parameters for nucleic acid molecule sequence comparison
include the following: Algorithm: Needleman and Wunsch, J. Mol.
Bio. 48:443-453 (1970); Comparison matrix: matches -+10;
mismatches=0; Gap Penalty: 50; Gap Length Penalty: 3. As used
herein, "% identity" is determined using the above parameters as
the default parameters for nucleic acid molecule sequence
comparisons and the "gap" program from GCG, version 10.2.
[0069] Subsets of the nucleic acid sequences of the present
invention include fragment nucleic acid molecules. "Fragment
nucleic acid molecule" refers to a piece of a larger nucleic acid
molecule, which may consist of significant portion(s) of, or indeed
most of, the larger nucleic acid molecule, or which may comprise a
smaller oligonucleotide having from about 15 to about 400
contiguous nucleotides and more preferably, about 15 to about 45
contiguous nucleotides, about 20 to about 45 contiguous
nucleotides, about 15 to about 30 contiguous nucleotides, about 21
to about 30 contiguous nucleotides, about 21 to about 25 contiguous
nucleotides, about 21 to about 24 contiguous nucleotides, about 19
to about 25 contiguous nucleotides, or about 21 contiguous
nucleotides. Fragment nucleic acid molecules may consist of
significant portion(s) of, or indeed most of, a plant coding or
non-coding region, or alternatively may comprise smaller
oligonucleotides. In a preferred embodiment, a fragment shows 100%
identity to the plant coding or non-coding region. In another
preferred embodiment, a fragment comprises a portion of a larger
nucleic acid sequence. In another aspect, a fragment nucleic acid
molecule has a nucleic acid sequence that has at least 15, 25, 50,
or 100 contiguous nucleotides of a nucleic acid molecule of the
present invention. In a preferred embodiment, a nucleic acid
molecule has a nucleic acid sequence that has at least 15, 25, 50,
or 100 contiguous nucleotides of a plant coding or non-coding
region.
[0070] In another aspect of the present invention, the DNA sequence
of the nucleic acid molecules of the present invention can comprise
sequences that differ from those encoding a polypeptide or fragment
of the protein due to conservative amino acid changes in the
polypeptide; the nucleic acid sequences coding for the polypeptide
can therefore have sequence differences corresponding to the
conservative changes. In a further aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
differ in nucleic acid sequence from those for which a specific
sequence is provided herein because one or more codons have been
replaced with a codon that encodes a conservative substitution of
the amino acid originally encoded.
[0071] Agents of the invention also include nucleic acid molecules
that encode at least about a contiguous 10 amino acid region of a
polypeptide of the present invention, more preferably at least
about a contiguous 25, 40, 50, 100, or 125 amino acid region of a
polypeptide of the present invention. Due to the degeneracy of the
genetic code, different nucleotide codons may be used to code for a
particular amino acid. A host cell often displays a preferred
pattern of codon usage. Structural nucleic acid sequences are
preferably constructed to utilize the codon usage pattern of the
particular host cell. This generally enhances the expression of the
structural nucleic acid sequence in a transformed host cell. Any of
the above-described nucleic acid and amino acid sequences may be
modified to reflect the preferred codon usage of a host cell or
organism in which they are contained. Therefore, a contiguous 10
amino acid region of a polypeptide of the present invention could
be encoded by numerous different nucleic acid sequences.
Modification of a structural nucleic acid sequence for optimal
codon usage in plants is described in U.S. Pat. No. 5,689,052.
[0072] Agents of the invention include nucleic acid molecules. For
example, without limitation, in an aspect of the present invention,
the nucleic acid molecule of the present invention comprises an
intron sequence of SEQ ID NO: 19, 20, 21, 22, 23, 25, 32, 33, 34,
or 35 or fragments thereof or complements thereof. In another
aspect of the invention, the nucleic acid molecule comprises a
nucleic acid sequence, which when introduced into a cell or
organism, is capable of suppressing the production of an RNA or
protein while simultaneously expressing, coexpressing or
coordinately expressing another RNA or protein. In an aspect of the
invention, the nucleic acid molecule comprises a nucleic acid
sequence, which when introduced into a cell or organism is capable
of suppressing, at least partially reducing, reducing,
substantially reducing, or effectively eliminating the expression
of endogenous FAD2, FAD3, and/or FATB RNA while at the same time
coexpressing, simultaneously expressing, or coordinately expressing
a beta-ketoacyl-ACP synthase I, beta-ketoacyl-ACP synthase IV,
delta-9 desaturase, and/or CP4 EPSPS RNA or protein.
[0073] By decreasing the amount of FAD2 and/or FAD3 available in a
plant cell, a decreased percentage of polyunsaturated fatty acids
such as linoleate (C18:2) and linolenate (C18:3) may be provided.
Modifications in the pool of fatty acids available for
incorporation into triacylglycerols may likewise affect the
composition of oils in the plant cell. Thus, a decrease in
expression of FAD2 and/or FAD3 may result in an increased
proportion of mono-unsaturated fatty acids such as oleate (C18:1).
When the amount of FATB is decreased in a plant cell, a decreased
amount of saturated fatty acids such as palmitate and stearate may
be provided. Thus, a decrease in expression of FATB may result in
an increased proportion of unsaturated fatty acids such as oleate
(18:1). The simultaneous suppression of FAD2, FAD3, and FATB
expression thereby results in driving the FAS pathway toward an
overall increase in mono-unsaturated fatty acids of 18-carbon
length, such as oleate (C18:1). See U.S. Pat. No. 5,955,650.
[0074] By increasing the amount of beta-ketoacyl-ACP synthase I
(KAS I) and/or beta-ketoacyl-ACP synthase IV (KAS IV) available in
a plant cell, a decreased percentage of 16:0-ACP may be provided,
leading to an increased percentage of 18:0-ACP. A greater amount of
18:0-ACP in combination with the simultaneous suppression of one or
more of FAD2, FAD3, and FATB, thereby helps drive the oil
composition toward an overall increase in oleate (C18:1). By
increasing the amount of delta-9 desaturase available in a plant
cell, an increased percentage of unsaturated fatty acids may be
provided, resulting in an overall lowering of stearate and total
saturates.
[0075] These combinations of increased and decreased enzyme
expression may be manipulated to produce fatty acid compositions,
including oils, having an increased oleate level, decreased
linoleate, linolenate, stearate, and/or paInitate levels, and a
decreased overall level of saturates. Enhancement of gene
expression in plants may occur through the introduction of extra
copies of coding sequences of the genes into the plant cell or,
preferably, the incorporation of extra copies of coding sequences
of the gene into the plant genome. Over-expression may also occur
though increasing the activities of the regulatory mechanisms that
regulate the expression of genes, i.e., up-regulation of the gene
expression.
[0076] Production of CP4 EPSPS in a plant cell provides the plant
cell with resistance or tolerance to glyphosate, thereby providing
a convenient method for identification of successful transformants
via glyphosate-tolerant selection.
[0077] Suppression of gene expression in plants, also known as gene
silencing, occurs at both the transcriptional level and
post-transcriptional level. There are various methods for the
suppression of expression of endogenous sequences in a host cell,
including, but not limited to, antisense suppression,
co-suppression, ribozymes, combinations of sense and antisense
(double-stranded RNAi), promoter silencing, and DNA binding
proteins such as zinc finger proteins. (See, e.g., WO 98/53083 and
WO 01/14538). Certain of these mechanisms are associated with
nucleic acid homology at the DNA or RNA level. In plants,
double-stranded RNA molecules can induce sequence-specific
silencing. Gene silencing is often referred to as double stranded
RNA ("dsRNAi") in plants, as RNA interference or RNAi in
Caenorhabditis elegans and in animals, and as quelling in
fungi.
[0078] In a preferred embodiment, the nucleic acid molecule of the
present invention comprises (a) a first set of DNA sequences, each
of which exhibits sufficient homology to one or more coding or
non-coding sequences of a plant gene such that when it is
expressed, it is capable of effectively eliminating, substantially
reducing, or at least partially reducing the level of an mRNA
transcript or protein encoded by the gene from which the coding or
non-coding sequence was derived, or any gene which has homology to
the target non-coding sequence, and (b) a second set of DNA
sequences, each of which exhibits sufficient homology to a plant
gene so that when it is expressed, it is capable of at least
partially enhancing, increasing, enhancing, or substantially
enhancing the level of an mRNA transcript or protein encoded by the
gene.
[0079] As used herein, "a reduction" of the level or amount of an
agent such as a protein or mRNA means that the level or amount is
reduced relative to a cell or organism lacking a DNA sequence
capable of reducing the agent. For example, "at least a partial
reduction" refers to a reduction of at least 25%, "a substantial
reduction" refers to a reduction of at least 75%, and "an effective
elimination" refers to a reduction of greater than 95%, all of
which reductions in the level or amount of the agent are relative
to a cell or organism lacking a DNA sequence capable of reducing
the agent.
[0080] As used herein, "an enhanced" or "increased" level or amount
of an agent such as a protein or mRNA means that the level or
amount is higher than the level or amount of agent present in a
cell, tissue or plant with a similar genetic background but lacking
an introduced nucleic acid molecule encoding the protein or mRNA.
For example, an "at least partially enhanced" level refers to an
increase of at least 25%, and a "substantially enhanced" level
refers to an increase of at least 100%, all of which increases in
the level or amount of an agent are relative to the level or amount
of agent that is present in a cell, tissue or plant with a similar
genetic background but lacking an introduced nucleic acid molecule
encoding the protein or mRNA.
[0081] When levels of an agent are compared, such a comparison is
preferably carried out between organisms with a similar genetic
background. Preferably, a similar genetic background is a
background where the organisms being compared share 50% or greater,
more preferably 75% or greater, and, even more preferably 90% or
greater sequence identity of nuclear genetic material. In another
preferred aspect, a similar genetic background is a background
where the organisms being compared are plants, and the plants are
isogenic except for any genetic material originally introduced
using plant transformation techniques. Measurement of the level or
amount of an agent may be carried out by any suitable method,
non-limiting examples of which include comparison of mRNA
transcript levels, protein or peptide levels, and/or phenotype,
especially oil content. As used herein, mRNA transcripts include
processed and non-processed mRNA transcripts, and proteins or
peptides include proteins or peptides with or without any
post-translational modification.
[0082] The DNA sequences of the first set of DNA sequences may be
coding sequences, intron sequences, 3'UTR sequences, 5'UTR
sequences, promoter sequences, other non-coding sequences, or any
combination of the foregoing. The first set of DNA sequences
encodes one or more sequences which, when expressed, are capable of
selectively reducing either or both the protein and the transcript
encoded by a gene selected from the group consisting of FAD2, FAD3,
and FATB. In a preferred embodiment, the first set of DNA sequences
is capable of expressing antisense RNA, in which the individual
antisense sequences may be linked in one transcript, or may be in
unlinked individual transcripts. In a further preferred embodiment,
the first set of DNA sequences are physically linked sequences
which are capable of expressing a single dsRNA molecule. In a
different preferred embodiment, the first set of DNA sequences is
capable of expressing sense cosuppresion RNA, in which the
individual sense sequences may be linked in one transcript, or may
be in unlinked individual transcripts. Exemplary embodiments of the
first set of DNA sequences are described in Part B of the Detailed
Description, and in the Examples.
[0083] The second set of DNA sequences encodes one or more
sequences which, when expressed, are capable of increasing one or
both of the protein and transcript encoded by a gene selected from
the group consisting of beta-ketoacyl-ACP synthase I (KAS I),
beta-ketoacyl-ACP synthase IV (KAS IV), delta-9 desaturase, and CP4
EPSPS. The DNA sequences of the second set of DNA sequences may be
physically linked sequences. Exemplary embodiments of the second
set of DNA sequences are described below in Parts C and D of the
Detailed Description.
[0084] Thus, the present invention provides methods for altering
the composition of fatty acids and compounds containing such fatty
acids, such as oils, waxes, and fats. The present invention also
provides methods for the production of particular fatty acids in
host cell plants. Such methods employ the use of the expression
cassettes described herein for the modification of the host plant
cell's FAS pathway.
B. First Set of DNA Sequences
[0085] In an aspect of the present invention, a nucleic acid
molecule comprises a first set of DNA sequences, which when
introduced into a cell or organism, expresses one or more sequences
capable of effectively eliminating, substantially reducing, or at
least partially reducing the levels of mRNA transcripts or proteins
encoded by one or more genes. Preferred aspects include as a target
an endogenous gene, a plant gene, and a non-viral gene. In an
aspect of the present invention, a gene is a FAD2, FAD3, or FATB
gene.
[0086] In an aspect, a nucleic acid molecule of the present
invention comprises a DNA sequence which exhibits sufficient
homology to one or more coding or non-coding sequences from a plant
gene, which when introduced into a plant cell or plant and
expressed, is capable of effectively eliminating, substantially
reducing, or at least partially reducing the level of an mRNA
transcript or protein encoded by the gene from which the coding or
non-coding sequence(s) was derived. The DNA sequences of the first
set of DNA sequences encode RNA sequences or RNA fragments which
exhibit at least 90%, preferably at least 95%, more preferably at
least 98%, most preferably at least 100% identity to a coding or
non-coding region derived from the gene which is to be suppressed.
Such percent identity may be to a nucleic acid fragment.
[0087] Preferably, the non-coding sequence is a 3' UTR, 5'UTR, or a
plant intron from a plant gene. More preferably, the non-coding
sequence is a promoter sequence, 3'UTR, 5'UTR, or a plant intron
from a plant gene. The intron may be located between exons, or
located within a 5' or 3' UTR of a plant gene.
[0088] The sequence(s) of the first set of DNA sequences may be
designed to express a dsRNA construct, a sense suppression RNA
construct, or an antisense RNA construct or any other suppression
construct in order to achieve the desired effect when introduced
into a plant cell or plant. Such DNA sequence(s) may be fragment
nucleic acid molecules. In a preferred aspect, a dsRNA construct
contains exon sequences, but the exon sequences do not correspond
to a sufficient part of a plant exon to be capable of effectively
eliminating, substantially reducing, or at least partially reducing
the level of an mRNA transcript or protein encoded by the gene from
which the exon was derived.
[0089] A plant intron can be any plant intron from a gene, whether
endogenous or introduced. Nucleic acid sequences of such introns
can be derived from a multitude of sources, including, without
limitation, databases such as EMBL and Genbank which may be found
on the Internet at ebi.ac.uk/swisprot/; expasy.ch/;
embl-heidelberg.de/; and ncbi.nlmnih.gov. Nucleic acid sequences of
such introns can also be derived, without limitation, from sources
such as the GENSCAN program which may be found on the Internet at
genes.mit.edu/GENSCAN.html.
[0090] Additional introns may also be obtained by methods which
include, without limitation, screening a genomic library with a
probe of either known exon or intron sequences, comparing genomic
sequence with its corresponding cDNA sequence, or cloning an intron
such as a soybean intron by alignment to an intron from another
organism, such as, for example, Arabidopsis. In addition, other
nucleic acid sequences of introns will be apparent to one of
ordinary skill in the art. The above-described methods may also be
used to derive and obtain other non-coding sequences, including but
not limited to, promoter sequences, 3'UTR sequences, and 5'UTR
sequences.
[0091] A "FAD2", "A12 desaturase" or "omega-6 desaturase" gene
encodes an enzyme (FAD2) capable of catalyzing the insertion of a
double bond into a fatty acyl moiety at the twelfth position
counted from the carboxyl terminus. The term "FAD2-1" is used to
refer to a FAD2 gene that is naturally expressed in a specific
manner in seed tissue, and the term "FAD2-2" is used to refer a
FAD2 gene that is (a) a different gene from a FAD2-1 gene and (b)
is naturally expressed in multiple tissues, including the seed.
Representative FAD2 sequences include, without limitation, those
set forth in U.S. patent application Ser. No. 10/176,149 filed on
Jun. 21, 2002, and in SEQ ID NOs: 1-6.
[0092] A "FAD3", ".DELTA.15 desaturase" or "omega-3 desaturase"
gene encodes an enzyme (FAD3) capable of catalyzing the insertion
of a double bond into a fatty acyl moiety at the fifteenth position
counted from the carboxyl terminus. The term "FAD3-1" is used to
refer a FAD3 gene family member that is naturally expressed in
multiple tissues, including the seed.
[0093] Representative FAD3 sequences include, without limitation,
those set forth in U.S. patent application Ser. No. 10/176,149
filed on Jun. 21, 2002, and in SEQ ID NOs: 7-27.
[0094] A "FATB" or "palmitoyl-ACP thioesterase" gene encodes an
enzyme (FATB) capable of catalyzing the hydrolytic cleavage of the
carbon-sulfur thioester bond in the panthothene prosthetic group of
palmitoyl-ACP as its preferred reaction. Hydrolysis of other fatty
acid-ACP thioesters may also be catalyzed by this enzyme.
Representative FATB sequences include, without limitation, those
set forth in U.S. Provisional Application No. 60/390,185 filed on
Jun. 21, 2002; U.S. Pat. Nos. 5,955,329; 5,723,761; 5,955,650; and
6,331,664; and SEQ ID NOs: 28-37.
C. Second Set of DNA Sequences
[0095] In an aspect of the present invention, a nucleic acid
molecule comprises a second set of DNA sequences, which when
introduced into a cell or organism, is capable of partially
enhancing, increasing, enhancing, or substantially enhancing the
levels of mRNA transcripts or proteins encoded by one or more
genes. In an aspect of the present invention, a gene is an
endogenous gene. In an aspect of the present invention, a gene is a
plant gene. In another aspect of the present invention, a gene is a
truncated gene where the truncated gene is capable of catalyzing
the reaction catalyzed by the full gene. In an aspect of the
present invention, a gene is a beta-ketoacyl-ACP synthase I,
beta-ketoacyl-ACP synthase IV, delta-9 desaturase, or CP4 EPSPS
gene.
[0096] A gene of the present invention can be any gene, whether
endogenous or introduced. Nucleic acid sequences of such genes can
be derived from a multitude of sources, including, without
limitation, databases such as EMBL and Genbank which may be found
on the Internet at ebi.ac.uk/swisprot/; expasy.ch/;
embl-heidelberg.del; and ncbi.nhn.nih.gov. Nucleic acid sequences
of such genes can also be derived, without limitation, from sources
such as the GENSCAN program which may be found on the Internet at
genes.mit.edu/GENSCAN.html.
[0097] Additional genes may also be obtained by methods which
include, without limitation, screening a genomic library or a cDNA
library with a probe of either known gene sequences, cloning a gene
by alignment to a gene or probe from another organism, such as, for
example, Arabidopsis. In addition, other nucleic acid sequences of
genes will be apparent to one of ordinary skill in the art.
Additional genes may, for example without limitation, be amplified
by polymerase chain reaction (PCR) and used in an embodiment of the
present invention. In addition, other nucleic acid sequences of
genes will be apparent to one of ordinary skill in the art.
[0098] Automated nucleic acid synthesizers may be employed for this
purpose, and to make a nucleic acid molecule that has a sequence
also found in a cell or organism. In lieu of such synthesis,
nucleic acid molecules may be used to define a pair of primers that
can be used with the PCR to amplify and obtain any desired nucleic
acid molecule or fragment of a first gene.
[0099] A "KAS I" or "beta-ketoacyl-ACP synthase I" gene encodes an
enzyme (KAS I) capable of catalyzing the elongation of a fatty acyl
moiety up to palmitoyl-ACP (C16:0). Representative KAS I sequences
include, without limitation, those set forth in U.S. Pat. No.
5,475,099 and PCT Publication WO 94/10189, and in SEQ ID NO:
38.
[0100] A "KAS IV" or "beta-ketoacyl-ACP synthase IV" gene encodes
an enzyme (KAS IV) capable of catalyzing the condensation of medium
chain acyl-ACPs and enhancing the production of 18:0-ACP.
Representative KAS IV sequences include, without limitation, those
set forth in PCT Publication WO 98/46776, and in SEQ ID NO: 39.
[0101] A "delta-9 desaturase" or "stearoyl-ACP desaturase" or
"omega-9 desaturase" gene encodes an enzyme capable of catalyzing
the insertion of a double bond into a fatty acyl moiety at the
ninth position counted from the carboxyl terminus. A preferred
delta-9 desaturase of the present invention is a plant or
cyanobacterial delta-9 desaturase, and more preferably a delta-9
desaturase that is also found in an organism selected from the
group consisting of Cartharmus tinctorius, Ricinus communis,
Simmonsia chinensis, and Brassica campestris. Representative
delta-9 desaturase sequences include, without limitation, those set
forth in U.S. Pat. No. 5,723,595, and SEQ ID NOs: 40-41.
[0102] A "CP4 EPSPS" or "CP4 5-enolpyruvylshikimate-3-phosphate
synthase" gene encodes an enzyme (CP4 EPSPS) capable of conferring
a substantial degree of glyphosate resistance upon the plant cell
and plants generated therefrom. The CP4 EPSPS sequence may be a CP4
EPSPS sequence derived from Agrobacterium tumefaciens sp. CP4 or a
variant or synthetic form thereof, as described in U.S. Pat. No.
5,633,435. Representative CP4 EPSPS sequences include, without
limitation, those set forth in U.S. Pat. Nos. 5,627,061 and
5,633,435.
D. Recombinant Vectors and Constructs
[0103] One or more of the nucleic acid constructs of the invention
may be used in plant transformation or transfection. The levels of
products such as transcripts or proteins may be increased or
decreased throughout an organism such as a plant or localized in
one or more specific organs or tissues of the organism. For example
the levels of products may be increased or decreased in one or more
of the tissues and organs of a plant including without limitation:
roots, tubers, stems, leaves, stalks, fruit, berries, nuts, bark,
pods, seeds and flowers. A preferred organ is a seed. For example,
exogenous genetic material may be transferred into a plant cell and
the plant cell regenerated into a whole, fertile or sterile plant
or plant part.
[0104] "Exogenous genetic material" is any genetic material,
whether naturally occurring or otherwise, from any source that is
capable of being inserted into any organism. Such exogenous genetic
material includes, without limitation, nucleic acid molecules and
constructs of the present invention. Exogenous genetic material may
be transferred into a host cell by the use of a DNA vector or
construct designed for such a purpose. Design of such a vector is
generally within the skill of the art (See, e.g., Plant Molecular
Biology: A Laboratory Manual, Clark (ed.), Springer, N.Y.
(1997)).
[0105] A construct or vector may include a promoter functional in a
plant cell, or a plant promoter, to express a nucleic acid molecule
of choice. A number of promoters that are active in plant cells
have been described in the literature, and the CaMV 35S and FMV
promoters are preferred for use in plants. Preferred promoters are
enhanced or duplicated versions of the CaMV 35S and FMV 35S
promoters. Odell et al., Nature 313: 810-812 (1985); U.S. Pat. No.
5,378,619. Additional promoters that may be utilized are described,
for example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147;
5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435;
and 4,633,436. In addition, a tissue specific enhancer may be
used.
[0106] Particularly preferred promoters can also be used to express
a nucleic acid molecule of the present invention in seeds or
fruits. Indeed, in a preferred embodiment, the promoter used is a
seed specific promoter. Examples of such promoters include the 5'
regulatory regions from such genes as napin (Kridl et al., Seed
Sci. Res. 1:209-219 (1991)), phaseolin, stearoyl-ACP desaturase,
7S.alpha., 7s.alpha.' (Chen et al., Proc. Natl. Acad. Sci.,
83:8560-8564 (1986)), USP, arcelin and oleosin. Preferred promoters
for expression in the seed are 7S.alpha., 7s.alpha.', napin, and
FAD2-1A promoters.
[0107] Constructs or vectors may also include other genetic
elements, including but not limited to, 3' transcriptional
terminators, 3' polyadenylation signals, other untranslated nucleic
acid sequences, transit or targeting sequences, selectable or
screenable markers, promoters, enhancers, and operators. Constructs
or vectors may also contain a promoterless gene that may utilize an
endogenous promoter upon insertion.
[0108] Nucleic acid molecules that may be used in plant
transformation or transfection may be any of the nucleic acid
molecules of the present invention. It is not intended that the
present invention be limited to the illustrated embodiments.
Exemplary nucleic acid molecules have been described in Part A of
the Detailed Description, and further non-limiting exemplary
nucleic acid molecules are described below and illustrated in FIGS.
1-4, and in the Examples.
[0109] Referring now to the drawings, embodiments of the nucleic
acid molecule of the present invention are shown in FIGS. 1-4. As
described above, the nucleic acid molecule comprises (a) a first
set of DNA sequences and (b) a second set of DNA sequences, which
are located on one or more T-DNA regions, each of which is flanked
by a right border and a left border. Within the T-DNA regions the
direction of transcription is shown by arrows. The nucleic acid
molecules described may have their DNA sequences arranged in
monocistronic or polycistronic configurations. Preferred
configurations include a configuration in which the first set of
DNA sequences and the second set of DNA sequences are located on a
single T-DNA region.
[0110] In each of the illustrated embodiments, the first set of DNA
sequences comprises one or more sequences which when expressed are
capable of selectively reducing one or both of the protein and
transcript encoded by a gene selected from the group consisting of
FAD2, FAD3, and FATB. Preferably each sequence in the first set of
DNA sequences is capable, when expressed, of suppressing the
expression of a different gene, including without limitation
different gene family members. The sequences may include coding
sequences, intron sequences, 3'UTR sequences, 5'UTR sequences,
other non-coding sequences, or any combination of the foregoing.
The first set of DNA sequences may be expressed in any suitable
form, including as a dsRNA construct, a sense cosuppression
construct, or as an antisense construct. The first set of DNA
sequences is operably linked to at least one promoter which drives
expression of the sequences, which can be any promoter functional
in a plant, or any plant promoter. Preferred promoters include, but
are not limited to, a napin promoter, a 7S.alpha. promoter, a
7s.alpha.' promoter, an arcelin promoter, or a FAD2-1A
promoter.
[0111] The second set of DNA sequences comprises coding sequences,
each of which is a DNA sequence that encodes a sequence that when
expressed is capable of increasing one or both of the protein and
transcript encoded by a gene selected from the group consisting of
KAS I, KAS IV, delta-9 desaturase, and CP4 EPSPS. Each coding
sequence is associated with a promoter, which can be any promoter
functional in a plant, or any plant promoter. Preferred promoters
for use in the second set of DNA sequences are an FMV promoter
and/or seed-specific promoters. Particularly preferred
seed-specific promoters include, but are not limited to, a napin
promoter, a 7S.alpha. promoter, a 7s.alpha.' promoter, an arcelin
promoter, a delta-9 desaturase promoter, or a FAD2-1A promoter.
[0112] In the embodiments depicted in FIGS. 1 and 2, the first set
of DNA sequences, when expressed, is capable of forming a dsRNA
molecule that is capable of suppressing the expression of one or
both of the protein and transcript encoded by, or transcribed from,
a gene selected from the group consisting of FAD2, FAD3, and FATB.
The first set of DNA sequences depicted in FIG. 1 comprises three
non-coding sequences, each of which expresses an RNA sequence (not
shown) that exhibits identity to a non-coding region of a gene
selected from the group consisting of FAD2, FAD3, and FATB genes.
The non-coding sequences each express an RNA sequence that exhibits
at least 90% identity to a non-coding region of a gene selected
from the group consisting of FAD2, FAD3, and FATB genes. The first
set of DNA sequences also comprises three antisense sequences, each
of which expresses an antisense RNA sequence (not shown) that is
capable of forming a double-stranded RNA molecule with its
respective corresponding RNA sequence (as expressed by the
non-coding sequences).
[0113] The non-coding sequences may be separated from the antisense
sequences by a spacer sequence, preferably one that promotes the
formation of a dsRNA molecule. Examples of such spacer sequences
include those set forth in Wesley et al., supra, and Hamilton et
al., Plant J., 15:737-746 (1988). In a preferred aspect, the spacer
sequence is capable of forming a hairpin structure as illustrated
in Wesley et al., supra. Particularly preferred spacer sequences in
this context are plant introns or parts thereof. A particularly
preferred plant intron is a spliceable intron. Spliceable introns
include, but are not limited to, an intron selected from the group
consisting of PDK intron, FAD3-1A or FAD3-1B intron #5, FAD3 intron
#1, FAD3 intron #3A, FAD3 intron #3B, FAD3 intron #3C, FAD3 intron
#4, FAD3 intron #5, FAD2 intron #1, and FAD2-2 intron. Preferred
spliceable introns include, but are not limited to, an intron
selected from the group consisting of FAD3 intron #1, FAD3 intron
#3A, FAD3 intron #3B, FAD3 intron #3C, and FAD3 intron #5. Other
preferred spliceable introns include, but are not limited to, a
spliceable intron that is about 0.75 kb to about 1.1 kb in length
and is capable of facilitating an RNA hairpin structure. One
non-limiting example of a particularly preferred spliceable intron
is FAD3 intron #5.
[0114] Referring now to FIG. 1, the nucleic acid molecule comprises
two T-DNA regions, each of which is flanked by a right border and a
left border. The first T-DNA region comprises the first set of DNA
sequences that is operably linked to a promoter, and the first
T-DNA region further comprises a first part of the second set of
DNA sequences that comprises a first promoter operably linked to a
first coding sequence, and a second promoter operably linked to a
second coding sequence. The second T-DNA region comprises a second
part of the second set of DNA sequences that comprises a third
promoter operably linked to a third coding sequence. In a preferred
embodiment depicted in FIG. 2, the nucleic acid molecule comprises
a single T-DNA region, which is flanked by a right border and a
left border. The first and second sets of DNA sequences are all
located on the single T-DNA region.
[0115] In the dsRNA-expressing embodiments shown in FIGS. 1 and 2,
the order of the sequences may be altered from that illustrated and
described, however the non-coding sequences and the antisense
sequences preferably are arranged around the spacer sequence such
that, when expressed, the first non-coding sequence can hybridize
to the first antisense sequence, the second non-coding sequence can
hybridize to the second antisense sequence, and the third
non-coding sequence can hybridize to the third antisense sequence
such that a single dsRNA molecule can be formed. Preferably the
non-coding sequences are in a sense orientation, and the antisense
sequences are in an antisense orientation relative to the promoter.
The numbers of non-coding, antisense, and coding sequences, and the
various relative positions thereof on the T-DNA region(s) may also
be altered in any manner suitable for achieving the goals of the
present invention.
[0116] Referring now to FIGS. 3 and 4, the illustrated nucleic acid
molecule comprises a T-DNA region flanked by a right border and a
left border, on which are located the first and second sets of DNA
sequences. The first set of DNA sequences is operably linked to a
promoter and a transcriptional termination signal. The second set
of DNA sequences that comprises a first promoter operably linked to
a first coding sequence, a second promoter operably linked to a
second coding sequence, and a third promoter operably linked to a
third coding sequence. The transcriptional termination signal can
be any transcriptional termination signal functional in a plant, or
any plant transcriptional termination signal. Preferred
transcriptional termination signals include, but are not limited
to, a pea Rubisco E9 3' sequence, a Brassica napin 3' sequence, a
tml 3' sequence, and a nos 3' sequence.
[0117] In the embodiment depicted in FIG. 3, the first set of DNA
sequences, when expressed, is capable of forming a sense
cosuppression construct that is capable of suppressing the
expression of one or more proteins or transcripts encoded by, or
derived from, a gene selected from the group consisting of FAD2,
FAD3, and FATB. The first set of DNA sequences comprises three
non-coding sequences, each of which expresses an RNA sequence (not
shown) that exhibits identity to one or more non-coding region(s)
of a gene selected from the group consisting of FAD2, FAD3, and
FATB genes. The non-coding sequences each express an RNA sequence
that exhibits at least 90% identity to one or more non-coding
region(s) of a gene selected from the group consisting of FAD2,
FAD3, and FATB genes. The order of the non-coding sequences within
the first set of DNA sequences may be altered from that illustrated
and described herein, but the non-coding sequences are arranged in
a sense orientation relative to the promoter.
[0118] FIG. 4 depicts an embodiment in which the first set of DNA
sequences, when expressed, is capable of forming an antisense
construct that is capable of suppressing the expression of one or
more proteins or transcripts encoded by, or derived from, a gene
selected from the group consisting of FAD2, FAD3, and FATB. The
first set of DNA sequences comprises three antisense sequences,
each of which expresses an antisense RNA sequence (not shown) that
exhibits identity to one or more non-coding region(s) of a gene
selected from the group consisting of FAD2, FAD3, and FATB genes.
The antisense sequences each express an antisense RNA sequence that
exhibits at least 90% identity to one or more non-coding region(s)
of a gene selected from the group consisting of FAD2, FAD3, and
FATB genes. The order of the antisense sequences within the first
set of DNA sequences may be altered from that illustrated and
described herein, but the antisense sequences are arranged in an
antisense orientation relative to the promoter.
[0119] The above-described nucleic acid molecules are preferred
embodiments which achieve the objects, features and advantages of
the present invention. It is not intended that the present
invention be limited to the illustrated embodiments. The
arrangement of the sequences in the first and second sets of DNA
sequences within the nucleic acid molecule is not limited to the
illustrated and described arrangements, and may be altered in any
manner suitable for achieving the objects, features and advantages
of the present invention as described herein and illustrated in the
accompanying drawings.
E. Transgenic Organisms, and Methods for Producing Same
[0120] Any of the nucleic acid molecules and constructs of the
invention may be introduced into a plant or plant cell in a
permanent or transient manner. Preferred nucleic acid molecules and
constructs of the present invention are described above in Parts A
through D of the Detailed Description, and in the Examples. Another
embodiment of the invention is directed to a method of producing
transgenic plants which generally comprises the steps of selecting
a suitable plant or plant cell, transforming the plant or plant
cell with a recombinant vector, and obtaining a transformed host
cell.
[0121] In a preferred embodiment the plant or cell is, or is
derived from, a plant involved in the production of vegetable oils
for edible and industrial uses. Especially preferred are temperate
oilseed crops. Plants of interest include, but are not limited to,
rapeseed (canola and High Erucic Acid varieties), maize, soybean,
crambe, mustard, castor bean, peanut, sesame, cotton, linseed,
safflower, oil palm, flax, sunflower, and coconut. The invention is
applicable to monocotyledonous or dicotyledonous species alike, and
will be readily applicable to new and/or improved transformation
and regulatory techniques.
[0122] Methods and technology for introduction of DNA into plant
cells are well known to those of skill in the art, and virtually
any method by which nucleic acid molecules may be introduced into a
cell is suitable for use in the present invention. Non-limiting
examples of suitable methods include: chemical methods; physical
methods such as microinjection, electroporation, the gene gun,
microprojectile bombardment, and vacuum infiltration; viral
vectors; and receptor-mediated mechanisms. Other methods of cell
transformation can also be used and include but are not limited to
introduction of DNA into plants by direct DNA transfer into pollen,
by direct injection of DNA into reproductive organs of a plant, or
by direct injection of DNA into the cells of immature embryos
followed by the rehydration of desiccated embryos.
[0123] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells. See, e.g., Fraley et
al., Bio/Technology 3:629-635 (1985); Rogers et al., Methods
Enzynol. 153:253-277 (1987). The region of DNA to be transferred is
defined by the border sequences and intervening DNA is usually
inserted into the plant genome. Spiehnann et al., Mol. Gen. Genet.
205:34 (1986). Modern Agrobacterium transformation vectors are
capable of replication in E. coli as well as Agrobacterium,
allowing for convenient manipulations. Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New
York, pp. 179-203 (1985).
[0124] The regeneration, development and cultivation of plants from
single plant protoplast transformants or from various transformed
explants is well known in the art. See generally, Maliga et al.,
Methods in Plant Molecular Biology, Cold Spring Harbor Press
(1995); Weissbach and Weissbach, In: Methods for Plant Molecular
Biology, Academic Press, San Diego, Calif. (1988). Plants of the
present invention can be part of or generated from a breeding
program, and may also be reproduced using apomixis. Methods for the
production of apomictic plants are known in the art. See, e.g.,
U.S. Pat. No. 5,811,636.
[0125] In a preferred embodiment, a plant of the present invention
that includes nucleic acid sequences which when expressed are
capable of selectively reducing the level of a FAD2, FAD3, and/or
FATB protein, and/or a FAD2, FAD3, and/or FATB transcript is mated
with another plant of the present invention that includes nucleic
acid sequences which when expressed are capable of overexpressing
another enzyme. Preferably the other enzyme is selected from the
group consisting of beta-ketoacyl-ACP synthase I, beta-ketoacyl-ACP
synthase IV, delta-9 desaturase, and CP4 EPSPS.
F. Products of the Present Invention
[0126] The plants of the present invention may be used in whole or
in part. Preferred plant parts include reproductive or storage
parts. The term "plant parts" as used herein includes, without
limitation, seed, endosperm, ovule, pollen, roots, tubers, stems,
leaves, stalks, fruit, berries, nuts, bark, pods, seeds and
flowers. In a particularly preferred embodiment of the present
invention, the plant part is a seed.
[0127] Any of the plants or parts thereof of the present invention
may be processed to produce a feed, meal, protein, or oil
preparation. A particularly preferred plant part for this purpose
is a seed. In a preferred embodiment the feed, meal, protein or oil
preparation is designed for livestock animals, fish or humans, or
any combination. Methods to produce feed, meal, protein and oil
preparations are known in the art. See, e.g. U.S. Pat. Nos.
4,957,748, 5,100,679, 5,219,596, 5,936,069, 6,005,076, 6,146,669,
and 6,156,227. In a preferred embodiment, the protein preparation
is a high protein preparation. Such a high protein preparation
preferably has a protein content of greater than 5% w/v, more
preferably 10% w/v, and even more preferably 15% w/v.
[0128] In a preferred oil preparation, the oil preparation is a
high oil preparation with an oil content derived from a plant or
part thereof of the present invention of greater than 5% w/v, more
preferably 10% w/v, and even more preferably 15% w/v. In a
preferred embodiment the oil preparation is a liquid and of a
volume greater than 1, 5, 10 or 50 liters. The present invention
provides for oil produced from plants of the present invention or
generated by a method of the present invention. Such an oil may
exhibit enhanced oxidative stability. Also, such oil may be a minor
or major component of any resultant product.
[0129] Moreover, such oil may be blended with other oils. In a
preferred embodiment, the oil produced from plants of the present
invention or generated by a method of the present invention
constitutes greater than 0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by
volume or weight of the oil component of any product. In another
embodiment, the oil preparation may be blended and can constitute
greater than 10%, 25%, 35%, 50% or 75% of the blend by volume. Oil
produced from a plant of the present invention can be admixed with
one or more organic solvents or petroleum distillates.
[0130] Seeds of the plants may be placed in a container. As used
herein, a container is any object capable of holding such seeds. A
container preferably contains greater than about 500, 1,000, 5,000,
or 25,000 seeds where at least about 10%, 25%, 50%, 75% or 100% of
the seeds are derived from a plant of the present invention. The
present invention also provides a container of over about 10,000,
more preferably about 20,000, and even more preferably about 40,000
seeds where over about 10%, more preferably about 25%, more
preferably 50% and even more preferably about 75% or 90% of the
seeds are seeds derived from a plant of the present invention. The
present invention also provides a container of over about 10 kg,
more preferably about 25 kg, and even more preferably about 50 kg
seeds where over about 10%, more preferably about 25%, more
preferably about 50% and even more preferably about 75% or 90% of
the seeds are seeds derived from a plant of the present
invention.
G. Oil Compositions
[0131] For many oil applications, saturated fatty acid levels are
preferably less than 8% by weight, and more preferably about 2-3%
by weight. Saturated fatty acids have high melting points which are
undesirable in many applications. When used as a feedstock for
fuel, saturated fatty acids cause clouding at low temperatures, and
confer poor cold flow properties such as pour points and cold
filter plugging points to the fuel. Oil products containing low
saturated fatty acid levels may be preferred by consumers and the
food industry because they are perceived as healthier and/or may be
labeled as "saturated fat free" in accordance with FDA guidelines.
In addition, low saturate oils reduce or eliminate the need to
winterize the oil for food applications such as salad oils. In
biodiesel and lubricant applications oils with low saturated fatty
acid levels confer improved cold flow properties and do not cloud
at low temperatures.
[0132] The factors governing the physical properties of a
particular oil are complex. Palmitic, stearic and other saturated
fatty acids are typically solid at room temperature, in contrast to
the unsaturated fatty acids, which remain liquid. Because saturated
fatty acids have no double bonds in the acyl chain, they remain
stable to oxidation at elevated temperatures. Saturated fatty acids
are important components in margarines and chocolate formulations,
but for many food applications, reduced levels of saturated fatty
acids are desired.
[0133] Oleic acid has one double bond, but is still relatively
stable at high temperatures, and oils with high levels of oleic
acid are suitable for cooking and other processes where heating is
required. Recently, increased consumption of high oleic oils has
been recommended, because oleic acid appears to lower blood levels
of low density lipoproteins ("LDLs") without affecting levels of
high density lipoproteins ("HDLs"). However, some limitation of
oleic acid levels is desirable, because when oleic acid is degraded
at high temperatures, it creates negative flavor compounds and
diminishes the positive flavors created by the oxidation of
linoleic acid. Neff et al., JAOCS, 77:1303-1313 (2000); Warner et
al., J. Agric. Food Chem. 49:899-905 (2001). Preferred oils have
oleic acid levels that are 65-85% or less by weight, in order to
limit off-flavors in food applications such as frying oil and fried
food. Other preferred oils have oleic acid levels that are greater
than 55% by weight in order to improve oxidative stability.
[0134] Linoleic acid is a major polyunsaturated fatty acid in foods
and is an essential nutrient for humans. It is a desirable
component for many food applications because it is a major
precursor of fried food flavor substances such as 2,4 decadienal,
which make fried foods taste good. However, linoleic acid has
limited stability when heated. Preferred food oils have linoleic
acid levels that are 10% or greater by weight, to enhance the
formation of desirable fried food flavor substances, and also are
25% or less by weight, so that the formation of off-flavors is
reduced. Linoleic acid also has cholesterol-lowering properties,
although dietary excess can reduce the ability of human cells to
protect themselves from oxidative damage, thereby increasing the
risk of cardiovascular disease. Toborek et al., Am J. Clin. J.
75:119-125 (2002). See generally Flavor Chemistry of Lipid Foods,
editors D. B. Min & T. H. Smouse, Am Oil Chem. Soc., Champaign,
Ill. (1989).
[0135] Linoleic acid, having a lower melting point than oleic acid,
further contributes to improved cold flow properties desirable in
biodiesel and biolubricant applications. Preferred oils for most
applications have linoleic acid levels of 30% or less by weight,
because the oxidation of linoleic acid limits the useful storage or
use-time of frying oil, food, feed, fuel and lubricant products.
See generally, Physical Properties of Fats, Oils, and Emulsifiers,
ed. N. Widlal, AOCS Press (1999); Erhan & Asadauskas, Lubricant
Basestocks from Vegetable Oils, Industrial Crops and Products,
11:277-282 (2000). In addition, high linoleic acid levels in cattle
feed can lead to undesirably high levels of linoleic acid in the
milk of dairy cattle, and therefore poor oxidative stability and
flavor. Timmons et al., J. Dairy Sci. 84:2440-2449 (2001). A
broadly useful oil composition has linoleic acid levels of 10-25%
by weight.
[0136] Linolenic acid is also an important component of the human
diet. It is used to synthesize the .omega.-3 family of long-chain
fatty acids and the prostaglandins derived therefrom. However, its
double bonds are highly susceptible to oxidation, so that oils with
high levels of linolenic acid deteriorate rapidly on exposure to
air, especially at high temperatures. Partial hydrogenation of such
oils is often necessary before they can be used in food products to
retard the formation of off-flavors and rancidity when the oil is
heated, but hydrogenation creates unhealthy trans fatty acids which
can contribute to cardiovascular disease. To achieve improved
oxidative stability, and reduce the need to hydrogenate oil,
preferred oils have linolenic acid levels that are 8% or less by
weight, 6% or less, 4% or less, and more preferably 0.5-2% by
weight of the total fatty acids in the oil of the present
invention.
[0137] The oil of the present invention can be a blended oil,
synthesized oil or in a preferred embodiment an oil generated from
an oilseed having an appropriate oil composition. In a preferred
embodiment, the oil is a soybean oil. The oil can be a crude oil
such as crude soybean oil, or can be a processed oil, for example
the oil can be refined, bleached, deodorized, and/or winterized. As
used herein, "refining" refers to a process of treating natural or
processed fat or oil to remove impurities, and may be accomplished
by treating fat or oil with caustic soda, followed by
centrifugation, washing with water, and heating under vacuum.
"Bleaching" refers to a process of treating a fat or oil to remove
or reduce the levels of coloring materials in the fat or oil.
Bleaching may be accomplished by treating fat or oil with activated
charcoal or Fullers (diatomaceous) earth. "Deodorizing" refers to a
process of removing components from a fat or oil that contribute
objectionable flavors or odors to the end product, and may be
accomplished by use of high vacuum and superheated steam washing.
"Winterizing" refers to a process of removing saturated glycerides
from an oil, and may be accomplished by chilling and removal of
solidified portions of fat from an oil.
[0138] A preferred oil of the present invention has a low saturate
oil composition, or a zero saturate oil composition. In other
preferred embodiments, oils of the present invention have increased
oleic acid levels, reduced saturated fatty levels, and reduced
polyunsaturated fatty acid levels. In a preferred embodiment, the
oil is a soybean oil. The percentages of fatty acid content, or
fatty acid levels, used herein refer to percentages by weight.
[0139] In a first embodiment, an oil of the present invention
preferably has an oil composition that is 55 to 80% oleic acid, 10
to 40% linoleic acid, 6% or less linolenic acid, and 2 to 8%
saturated fatty acids; more preferably has an oil composition that
is 55 to 80% oleic acid, 10 to 39% linoleic acid, 4.5% or less
linolenic acid, and 3 to 6% saturated fatty acids; and even more
preferably has an oil composition that is 55 to 80% oleic acid, 10
to 39% linoleic acid, 3.0% or less linolenic acid, and 2 to 3.6%
saturated fatty acids.
[0140] In a second embodiment, an oil of the present invention
preferably has an oil composition that is 65 to 80% oleic acid, 10
to 30% linoleic acid, 6% or less linolenic acid, and 2 to 8%
saturated fatty acids; more preferably has an oil composition that
is 65 to 80% oleic acid, 10 to 29% linoleic acid, 4.5% or less
linolenic acid, and 3 to 6% saturated fatty acids; and even more
preferably has an oil composition that is 65 to 80% oleic acid, 10
to 29% linoleic acid, 3.0% or less linolenic acid, and 2 to 3.6%
saturated fatty acids.
[0141] In other embodiments, the percentage of oleic acid is 50% or
greater, 55% or greater; 60% or greater, 65% or greater, 70% or
greater; 75% or greater, or 80% or greater; or is a range from 50
to 80%; 55 to 80%; 55 to 75%; 55 to 65%; 65 to 80%; 65 to 75%; 65
to 70%; or 69 to 73%. Suitable percentage ranges for oleic acid
content in oils of the present invention also include ranges in
which the lower limit is selected from the following percentages:
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, or 80 percent;
and the upper limit is selected from the following percentages: 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, or 90 percent.
[0142] In these other embodiments, the percentage of linoleic acid
in an oil of the present invention is a range from 10 to 40%; 10 to
39%; 10 to 30%; 10 to 29%; 10 to 28%; 10 to 25%; 10 to 21%; 10 to
20%; 11 to 30%; 12 to 30%; 15 to 25%; 20 to 25%; 20 to 30%; or 21
to 24%. Suitable percentage ranges for linoleic acid content in
oils of the present invention also include ranges in which the
lower limit is selected from the following percentages: 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30 percent; and the upper limit is selected from the following
percentages: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, or 40 percent.
[0143] In these other embodiments, the percentage of linolenic acid
in an oil of the present invention is 10% or less; 9% or less; 8%
or less; 7% or less; 6% or less; 5% or less; 4.5% or less; 4% or
less; 3.5% or less; 3% or less; 3.0% or less; 2.5% or less; or 2%
or less; or is a range from 0.5 to 2%; 0.5 to 3%; 0.5 to 4.5%; 0.5%
to 6%; 3 to 5%; 3 to 6%; 3 to 8%; 1 to 2%; 1 to 3%; or 1 to 4%. In
these other embodiments, the percentage of saturated fatty acids in
an oil composition of the present invention is 15% or less; 14% or
less; 13% or less; 12% or less, 11% or less; 10% or less; 9% or
less; 8% or less; 7% or less; 6% or less; 5% or less; 4% or less;
or 3.6% or less; or is a range from 2 to 3%; 2 to 3.6%; 2 to 4%; 2
to 8%; 3 to 15%; 3 to 10%; 3 to 8%; 3 to 6%; 3.6 to 7%; 5 to 8%; 7
to 10%; or 10 to 15%.
[0144] An oil of the present invention is particularly suited to
use as a cooking or frying oil. Because of its reduced
polyunsaturated fatty acid content, the oil of the present
invention does not require the extensive processing of typical oils
because fewer objectionable odorous and colorant compounds are
present. In addition, the low saturated fatty acid content of the
present oil improves the cold flow properties of the oil, and
obviates the need to heat stored oil to prevent it from
crystallizing or solidifying. Improved cold flow also enhances
drainage of oil from fried food material once it has been removed
from frying oil, thereby resulting in a lower fat product. See
Bouchon et al., J. Food Science 66: 918-923 (2001). The low levels
of linolenic acid in the present oil are particularly advantageous
in frying to reduce off-flavors.
[0145] The present oil is also well-suited for use as a salad oil
(an oil that maintains clarity at refrigerator temperatures of
40-50 degrees Fahrenheit). Its improved clarity at refrigerator
temperatures, due to its low saturated fatty acid and moderate
linoleic acid content, reduces or eliminates the need to winterize
the oil before use as a salad oil.
[0146] In addition, the moderate linoleic and low linolenic acid
content of the present oil make it well-suited for the production
of shortening, margarine and other semi-solid vegetable fats used
in foodstuffs. Production of these fats typically involves
hydrogenation of unsaturated oils such as soybean oil, corn oil, or
canola oil. The increased oxidative and flavor stability of the
present oil mean that it need not be hydrogenated to the extent
that typical vegetable oil is for uses such as margarine and
shortening, thereby reducing processing costs and the production of
unhealthy trans isomers.
[0147] An oil of the present invention is also suitable for use as
a feedstock to produce biodiesel, particularly because biodiesel
made from such an oil has improved cold flow, improved ignition
quality (cetane number), improved oxidative stability, and reduced
nitric oxide emissions. Biodiesel is an alternative diesel fuel
typically comprised of methyl esters of saturated, monounsaturated,
and polyunsaturated C.sub.16-C.sub.22 fatty acids. Cetane number is
a measure of ignition quality--the shorter the ignition delay time
of fuel in the engine, the higher the cetane number. The ASTM
standard specification for biodiesel fuel (D 6751-02) requires a
minimum cetane number of 47.
[0148] The use of biodiesel in conventional diesel engines results
in substantial reductions of pollutants such as sulfates, carbon
monoxide, and particulates compared to petroleum diesel fuel, and
use in school buses can greatly reduce children's exposure to toxic
diesel exhaust. A limitation to the use of 100% conventional
biodiesel as fuel is the high cloud point of conventional soy
biodiesel (2 degrees C.) compared to number 2 diesel (-16 degrees
C.). Dunn et al., Recent. Res. Devel. in Oil Chem., 1:31-56 (1997).
Biodiesel made from oil of the present invention has an improved
(reduced) cloud point and cold filter plugging point, and may also
be used in blends to improve the cold-temperature properties of
biodiesel made from inexpensive but highly saturated sources of fat
such as animal fats (tallow, lard, chicken fat) and palm oil.
Biodiesel can also be blended with petroleum diesel at any
level.
[0149] Biodiesel is typically obtained by extracting, filtering and
refining soybean oil to remove free fats and phospholipids, and
then transesterifying the oil with methanol to form methyl esters
of the fatty acids. See, e.g., U.S. Pat. No. 5,891,203. The
resultant soy methyl esters are commonly referred to as
"biodiesel." The oil of the present invention may also be used as a
diesel fuel without the formation of methyl esters, such as, for
example, by mixing acetals with the oil. See, e.g., U.S. Pat. No.
6,013,114. Due to its improved cold flow and oxidative stability
properties, the oil of the present invention is also useful as a
lubricant, and as a diesel fuel additive. See, e.g., U.S. Pat. Nos.
5,888,947, 5,454,842 and 4,557,734.
[0150] Soybeans, and oils of the present invention are also
suitable for use in a variety of soyfoods made from whole soybeans,
such as soymilk, soy nut butter, natto, and tempeh, and soyfoods
made from processed soybeans and soybean oil, including soybean
meal, soy flour, soy protein concentrate, soy protein isolates,
texturized soy protein concentrate, hydrolyzed soy protein, whipped
topping, cooking oil, salad oil, shortening, and lecithin. Whole
soybeans are also edible, and are typically sold to consumers raw,
roasted, or as edamame. Soymilk, which is typically produced by
soaking and grinding whole soybeans, may be consumed as is,
spray-dried, or processed to form soy yoghurt, soy cheese, tofu, or
yuba. The present soybean or oil may be advantageously used in
these and other soyfoods because of its improved oxidative
stability, the reduction of off-flavor precursors, and its low
saturated fatty acid level.
[0151] The following examples are illustrative and not intended to
be limiting in any way.
EXAMPLES
Example 1
Suppression Constructs
1A. FAD2-1 Constructs
[0152] The FAD2-1A intron (SEQ ID NO: 1) is cloned into the
expression cassette, pCGN3892, in sense and antisense orientations.
The vector pCGN3892 contains the soybean 7S promoter and a pea rbcS
3'. Both gene fusions are then separately ligated into pCGN9372, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter. The resulting expression constructs (pCGN5469 sense and
pCGN5471 antisense) are used for transformation of soybean.
[0153] The FAD2-1B intron (SEQ ID NO: 2) is fused to the 3' end of
the FAD2-1A intron in plasmid pCGN5468 (contains the soybean 7S
promoter fused to the FAD2-1A intron (sense) and a pea rbcS 3') or
pCGN5470 (contains the soybean 7S promoter fused to the FAD2-1A
intron (antisense) and a pea rbcS 3') in sense or antisense
orientation respectively. The resulting intron combination fusions
are then ligated separately into pCGN9372, a vector that contains
the CP4 EPSPS gene regulated by the FMV promoter. The resulting
expression constructs (pCGN5485, FAD2-1A & FAD2-1B intron sense
and pCGN5486, FAD2-1A & FAD2-1B intron antisense) are used for
transformation of soybean.
1B. FAD3-1 Constructs
[0154] FAD3-1A introns #1, #2, #4 and #5 (SEQ ID NOs: 7, 8, 10 and
11, respectively), FAD3-1B introns #3C (SEQ ID NO: 23) and #4 (SEQ
ID NO: 24), are all ligated separately into pCGN3892, in sense or
antisense orientations. pCGN3892 contains the soybean 7S promoter
and a pea rbcS 3'. These fusions are ligated into pCGN9372, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter for transformation into soybean. The resulting expression
constructs (pCGN5455, FAD3-1A intron #4 sense; pCGN5459, FAD3-1A
intron #4 antisense; pCGN5456, FAD3 intron #5 sense; pCGN5460,
FAD3-1A intron #5 antisense; pCGN5466, FAD3-1A intron #2 antisense;
pCGN5473, FAD3-1A intron #1 antisense) are used for transformation
of soybean.
1C. FatB Constructs
[0155] The soybean FATB intron II sequence (SEQ ID NO: 30) is
amplified via PCR using a FATB partial genomic clone as a template.
PCR amplification is carried out as follows: 1 cycle, 95.degree. C.
for 10 min; 25 cycles, 95.degree. C. for 30 sec, 62.degree. C. for
30 sec, 72.degree. C. for 30 sec; 1 cycle, 72.degree. C. for 7 min.
PCR amplification results in a product that is 854 bp long,
including reengineered restriction sites at both ends. The PCR
product is cloned directly into the expression cassette pCGN3892 in
sense orientation, by way of XhoI sites engineered onto the 5' ends
of the PCR primers, to form pMON70674. Vector pCGN3892 contains the
soybean 7S promoter and a pea rbcS 3'. pMON70674 is then cut with
NotI and ligated into pMON41164, a vector that contains the CP4
EPSPS gene regulated by the FMV promoter. The resulting gene
expression construct, pMON70678, is used for transformation of
soybean using Agrobacterium methods.
[0156] Two other expression constructs containing the soybean FATB
intron II sequence (SEQ ID NO: 30) are created. pMON70674 is cut
with NotI and ligated into pMON70675 which contains the CP4 EPSPS
gene regulated by the FMV promoter and the KAS IV gene regulated by
the napin promoter, resulting in pMON70680. The expression vector
pMON70680 is then cut with SnaBI and ligated with a gene fusion of
the jojoba delta-9 desaturase gene (SEQ ID NO: 41) in sense
orientation regulated by the 7S promoter. The expression constructs
pMON70680 and pMON70681 are used for transformation of soybean
using Agrobacterium methods.
1D. Combination Constructs
[0157] Expression constructs are made containing various
permutations of a first set of DNA sequences. The first set of DNA
sequences are any of those described, or illustrated in FIGS. 5 and
6, or any other set of DNA sequences that contain either various
combinations of sense and antisense FAD2, FAD3, and FATB non-coding
regions so that they are capable of forming dsRNA constructs, sense
cosuppression constructs, antisense constructs, or various
combinations of the foregoing.
[0158] FIGS. 5(a)-(c) depict several first sets of DNA sequences
which are capable of expressing sense cosuppression or antisense
constructs according to the present invention, the non-coding
sequences of which are described in Tables 1 and 2 below. The
non-coding sequences may be single sequences, combinations of
sequences (e.g. the 5'UTR linked to the 3'UTR), or any combination
of the foregoing. To express a sense cosuppression construct, all
of the non-coding sequences are sense sequences, and to express an
antisense construct, all of the non-coding sequences are antisense
sequences. FIG. 5(d) depicts a first set of DNA sequences which is
capable of expressing sense and antisense constructs according to
the present invention.
[0159] FIGS. 6(a)-c) depict several first sets of DNA sequences
which are capable of expressing dsRNA constructs according to the
present invention, the noncoding sequences of which are described
in Tables 1 and 2 below. The first set of DNA sequences depicted in
FIG. 6 comprises pairs of related sense and antisense sequences,
arranged such that, e.g. the RNA expressed by the first sense
sequence is capable of forming a double-stranded RNA with the
antisense RNA expressed by the first antisense sequence. For
example, referring to FIG. 6(a) and illustrative combination No. 1
(of Table 1), the first set of DNA sequences comprises a sense
FAD2-1 sequence, a sense FAD3-1 sequence, an antisense FAD2-1
sequence and an antisense FAD3-1 sequence. Both antisense sequences
correspond to the sense sequences so that the expression products
of the first set of DNA sequences are capable of forming a
double-stranded RNA with each other. The sense sequences may be
separated from the antisense sequences by a spacer sequence,
preferably one that promotes the formation of a dsRNA molecule.
Examples of such spacer sequences include those set forth in Wesley
et al., supra, and Hamilton et al., Plant J, 15:737-746 (1988). The
promoter is any promoter functional in a plant, or any plant
promoter. Non-limiting examples of suitable promoters are described
in Part D of the Detailed Description.
[0160] The first set of DNA sequences is inserted in an expression
construct in either the sense or anti-sense orientation using a
variety of DNA manipulation techniques. If convenient restriction
sites are present in the DNA sequences, they are inserted into the
expression construct by digesting with the restriction
endonucleases and ligation into the construct that has been
digested at one or more of the available cloning sites. If
convenient restriction sites are not available in the DNA
sequences, the DNA of either the construct or the DNA sequences is
modified in a variety of ways to facilitate cloning of the DNA
sequences into the construct. Examples of methods to modify the DNA
include by PCR, synthetic linker or adapter ligation, in vitro
site-directed mutagenesis, filling in or cutting back of
overhanging 5' or 3' ends, and the like. These and other methods of
manipulating DNA are well known to those of ordinary skill in the
art.
TABLE-US-00001 TABLE 1 Illustrative Non-Coding Sequences (sense or
antisense) Combinations First Second Third Fourth 1 FAD2-1A or B
FAD3-1A or B or C 2 FAD3-1A or B or C FAD2-1A or B 3 FAD2-1A or B
FAD3-1A or B or C different FAD3-1A or B or C sequence 4 FAD2-1A or
B FAD3-1A or B or C FATB 5 FAD2-1A or B FATB FAD3-1A or B or C 6
FAD3-1A or B or C FAD2-1A or B FATB 7 FAD3-1A or B or C FATB
FAD2-1A or B 8 FATB FAD3-1A or B or C FAD2-1A or B 9 FATB FAD2-1A
or B FAD3-1A or B or C 10 FAD2-1A or B FAD3-1A or B or C different
FAD3-1A FATB or B or C sequence 11 FAD3-1A or B or C FAD2-1A or B
different FAD3-1A FATB or B or C sequence 12 FAD3-1A or B or C
different FAD3-1A FAD2-1A or B FATB or B or C sequence 13 FAD2-1A
or B FAD3-1A or B or C FATB different FAD3-1A or B or C sequence 14
FAD3-1A or B or C FAD2-1A or B FATB different FAD3-1A or B or C
sequence 15 FAD3-1A or B or C different FAD3-1A FATB FAD2-1A or B
or B or C sequence 16 FAD2-1A or B FATB FAD3-1A or B or C different
FAD3-1A or B or C sequence 17 FAD3-1A or B or C FATB FAD2-1A or B
different FAD3-1A or B or C sequence 18 FAD3-1A or B or C FATB
different FAD3-1A FAD2-1A or B or B or C sequence 19 FATB FAD2-1A
or B FAD3-1A or B or C different FAD3-1A or B or C sequence 20 FATB
FAD3-1A or B or C FAD2-1A or B different FAD3-1A or B or C sequence
21 FATB FAD3-1A or B or C different FAD3-1A FAD2-1A or B or B or C
sequence
TABLE-US-00002 TABLE 2 Correlation of SEQ ID NOs with Sequences in
Table 1 FAD2-1A FAD2-1B FAD3-1A FAD3-1B FAD3-1C FATB 3'UTR SEQ NO:
5 n/a SEQ NO: 16 SEQ NO: 26 n/a SEQ NO: 36 5'UTR SEQ NO: 6 n/a SEQ
NO: 17 SEQ NO: 27 n/a SEQ NO: 37 5' + 3' UTR (or Linked SEQ n/a
Linked SEQ Linked SEQ n/a Linked SEQ 3' + 5' UTR) NOs: 5 and 6 NOs:
16 and 17 NOs: 26 and 27 NOs: 36 and 37 Intron #1 SEQ NO: 1 SEQ NO:
2 SEQ NO: 7 SEQ NO: 19 n/a SEQ NO: 29 Intron #2 n/a n/a SEQ NO: 8
SEQ NO: 20 n/a SEQ NO: 30 Intron #3 n/a n/a n/a n/a n/a SEQ NO: 31
Intron #3A n/a n/a SEQ NO: 9 SEQ NO: 21 n/a n/a Intron #3B n/a n/a
SEQ NO: 12 SEQ NO: 22 n/a n/a Intron #3C n/a n/a SEQ NO: 13 SEQ NO:
23 n/a n/a Intron #4 n/a n/a SEQ NO: 10 SEQ NO: 24 SEQ NO: 14 SEQ
NO: 32 Intron #5 n/a n/a SEQ NO: 11 SEQ NO: 25 n/a SEQ NO: 33
Intron #6 n/a n/a n/a n/a n/a SEQ NO: 34 Intron #7 n/a n/a n/a n/a
n/a SEQ NO: 35
Example 2
Combination Constructs
[0161] In FIGS. 7-15, promoters are indicated by arrows, encoding
sequences (both coding and non-coding) are indicated by pentagons
which point in the direction of transcription, sense sequences are
labeled in normal text and antisense sequences are labeled in
upside-down text. The abbreviations used in these Figures include:
7Sa=7Sa promoter, 7Sa'=7S.alpha.' promoter, Br napin=Brassica napin
promoter; FMV=an FMV promoter; ARC=arcelin promoter; RBC E9
3'=Rubisco E9 termination signal; Nos 3'=nos termination signal;
TML 3'=tml termination signal; napin 3'=napin termination signal;
`3 (in the same box as FAD or FAT)=3` UTR; 5' (in the same box as
FAD or FAT)=5'UTR; Cr=Cuphea pulcherrima; Gm=Glycine max;
Rc=Ricinus conmunis; FAB2=a FAB2 allele of a stearoyl-desaturase
gene; and Intr or Int=intron.
2A. dsRNA Constructs
[0162] FIGS. 7-9 depict nucleic acid molecules of the present
invention in which the first sets of DNA sequences are capable of
expressing dsRNA constructs. The first set of DNA sequences
depicted in FIGS. 7-9 comprise pairs of related sense and antisense
sequences, arranged such that, e.g., the RNA expressed by the first
sense sequence is capable of forming a double-stranded RNA with the
antisense RNA expressed by the first antisense sequence. The sense
sequences may be adjacent to the antisense sequences, or separated
from the antisense sequences by a spacer sequence, as shown in FIG.
9.
[0163] The second set of DNA sequences comprises coding sequences,
each of which is a DNA sequence that encodes a sequence that when
expressed is capable of increasing one or both of the protein and
transcript encoded by a gene selected from the group consisting of
KAS I, KAS IV, delta-9 desaturase, and CP4 EPSPS. Each coding
sequence is associated with a promoter, which can be any promoter
functional in a plant, or any plant promoter, and may be an FMV
promoter, a napin promoter, a 7S (either 7S.alpha. or 7S.alpha.')
promoter, an arcelin promoter, a delta-9 desaturase promoter, or a
FAD2-1A promoter.
[0164] Referring now to FIG. 7, soybean FAD2-1 intron 1 (SEQ ID NO:
1 or 2), FAD3-1A 3'UTR (SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO:
36) sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in sense and antisense orientations,
separated by a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11),
into a vector containing the soybean 7S.alpha.' promoter and a tml
3' termination sequence, by way of XhoI sites engineered onto the
5' ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. Vectors containing a C. pulcherima KAS IV gene (SEQ ID
NO: 39) regulated by a Brassica napin promoter and a Brassica napin
3' termination sequence, and a R. communis delta-9 desaturase
(FAB2) gene (SEQ ID NO: 40) regulated by a soybean FAD2 promoter
and a nos 3' termination sequence, are cut with appropriate
restriction enzymes, and ligated into pMON41164. The resulting gene
expression construct, pMON68539, is depicted in FIG. 7 and is used
for transformation using methods as described herein.
[0165] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A intron
4 (SEQ ID NO: 10), and FATB intron II (SEQ ID NO: 30) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense and antisense orientations, separated by
a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11), into a
vector containing the soybean 7S.alpha.' promoter and a tml 3'
termination sequence, by way of XhoI sites engineered onto the 5'
ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. The resulting gene expression construct, pMON68540, is
depicted in FIG. 7 and is used for transformation using methods as
described herein.
[0166] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A intron
4 (SEQ ID NO: 10), and FATB intron II (SEQ ID NO: 30) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense and antisense orientations, separated by
a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11), into a
vector containing the soybean 7S.alpha.' promoter and a tml 3'
termination sequence, by way of XhoI sites engineered onto the 5'
ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. A vector containing a C. pulcherrima KAS Iv gene (SEQ ID
NO: 39) regulated by a Brassica napin promoter and a Brassica napin
3' termination sequence is cut with appropriate restriction
enzymes, and ligated into pMON41164. The resulting gene expression
construct, pMON68544, is depicted in FIG. 7 and is used for
transformation using methods as described herein.
[0167] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A intron
4 (SEQ ID NO: 10), FATB intron II (SEQ ID NO: 30), and FAD3-1B
intron 4 (SEQ ID NO: 24) sequences are amplified via PCR to result
in PCR products that include reengineered restriction sites at both
ends. The PCR products are cloned directly, in sense and antisense
orientations, separated by a spliceable soybean FAD3-1A intron 5
(SEQ ID NO: 11), into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. The vector is then
cut with NotI and ligated into pMON41164, a vector that contains
the CP4 EPSPS gene regulated by the FMV promoter and a pea Rubisco
E9 3' termination sequence. The resulting gene expression
construct, pMON68546, is depicted in FIG. 7 and is used for
transformation using methods as described herein.
[0168] Referring now to FIG. 8, soybean FAD2-1 intron 1 (SEQ ID NO:
1 or 2), FAD3-1A 3'UTR (SEQ ID NO: 16), and FATB 3'UTR(SEQ ID NO:
36) sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in sense and antisense orientations,
separated by a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11),
into a vector containing the soybean 7S.alpha.' promoter and a tml
3' termination sequence, by way of XhoI sites engineered onto the
5' ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. The resulting gene expression construct, pMON68536, is
depicted in FIG. 8 and is used for transformation using methods as
described herein.
[0169] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A 3'UTR
(SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense and antisense orientations, separated by
a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11), into a
vector containing the soybean 7S.alpha.' promoter and a tinl 3'
termination sequence, by way of XhoI sites engineered onto the 5'
ends of the PCR primers. A vector containing a R. communis delta-9
desaturse (FAB2) gene (SEQ ID NO: 40) regulated by a soybean FAD2
promoter and a nos 3' termination sequence, is cut with appropriate
restriction enzymes, and ligated just upstream of the tml 3'
termination sequence. The vector is then cut with NotI and ligated
into pMON41164, a vector that contains the CP4 EPSPS gene regulated
by the FMV promoter and a pea Rubisco E9 3' termination sequence.
The resulting gene expression construct, pMON68537, is depicted in
FIG. 8 and is used for transformation using methods as described
herein.
[0170] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A 3'UTR
(SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense and antisense orientations, separated by
a spliceable soybean FAD3-1A intron 5 (SEQ ID NO: 11), into a
vector containing the soybean 7S.alpha.' promoter and a tml 3'
termination sequence, by way of XhoI sites engineered onto the 5'
ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. A vector containing a C. pulcherrima KAS IV gene (SEQ ID
NO: 39) regulated by a Brassica napin promoter and a Brassica napin
3' termination sequence is cut with appropriate restriction
enzymes, and ligated into pMON41164. The resulting gene expression
construct, pMON68538, is depicted in FIG. 8 and is used for
transformation using methods as described herein.
[0171] Referring now to FIG. 9, soybean FAD2-1 3'UTR (SEQ ID NO:
5), FATB 3'UTR (SEQ ID NO: 36), FAD3-1A 3'UTR (SEQ ID NO: 16), and
FAD3-1B 3'UTR (SEQ ID NO: 26) sequences are amplified via PCR to
result in PCR products that include reengineered restriction sites
at both ends. The PCR products are cloned directly, in sense and
antisense orientations, separated by a spliceable soybean FAD3-1A
intron 5 (SEQ ID NO: 11), into a vector containing the soybean
7S.alpha.' promoter and a tml 3' termination sequence, by way of
XhoI sites engineered onto the 5' ends of the PCR primers. The
vector is then cut with XhoI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. The resulting gene
expression construct, pMON80622, is depicted in FIG. 9 and is used
for transformation using methods as described herein.
[0172] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FATB 3'UTR (SEQ ID NO:
36), and FAD3-1A 3'UTR (SEQ ID NO: 16) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in sense
and antisense orientations, separated by a spliceable soybean
FAD3-1A intron 5 (SEQ ID NO: 11), into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
The vector is then cut with NotI and ligated into pMON41164, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, pMON80623, is depicted in FIG.
9 and is used for transformation using methods as described
herein.
[0173] Soybean FAD2-1 5'UTR-3'UTR (SEQ ID NOs: 6 and 5, ligated
together), FATB 5'UTR-3'UTR (SEQ ID NOs: 37 and 36, ligated
together), FAD3-1A 3'UTR (SEQ ID NO: 16) and FAD3-1B 5'UTR-3'UTR
(SEQ ID NOs: 27 and 26, ligated together) sequences are amplified
via PCR to result in PCR products that include reengineered
restriction sites at both ends. The PCR products are cloned
directly, in sense and antisense orientations, into a vector
containing the soybean 7S.alpha.' promoter and a tml 3' termination
sequence, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vector is then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, O5, is depicted in FIG. 9 and
is used for transformation using methods as described herein.
[0174] Soybean FAD2-1 5'UTR-3'UTR (SEQ D) NOs: 6 and 5, ligated
together), FATB 5'UTR-3'UTR (SEQ ID NOs: 37 and 36, ligated
together) and FAD3-1A 3'UTR (SEQ ID NO: 16) sequences are amplified
via PCR to result in PCR products that include reengineered
restriction sites at both ends. The PCR products are cloned
directly, in sense and antisense orientations, into a vector
containing the soybean 7S.alpha.' promoter and a tml 3' termination
sequence, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vector is then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. A
vector containing a C. pulcherrima KAS IV gene (SEQ ID NO: 39)
regulated by a Brassica napin promoter and a Brassica napin 3'
termination sequence is cut with appropriate restriction enzymes,
and ligated into pMON41164. The resulting gene expression
construct, O6, is depicted in FIG. 9 and is used for transformation
using methods as described herein.
2B. Sense Cosuppression Constructs
[0175] FIGS. 10-13 depict nucleic acid molecules of the present
invention in which the first sets of DNA sequences are capable of
expressing sense cosuppression constructs. The second set of DNA
sequences comprises coding sequences, each of which is a DNA
sequence that encodes a sequence that when expressed is capable of
increasing one or both of the protein and transcript encoded by a
gene selected from the group consisting of KASI, KAS IV, delta-9
desaturase, and CP4 EPSPS. Each coding sequence is associated with
a promoter, which is any promoter functional in a plant, or any
plant promoter, and may be an FMV promoter, a napin promoter, a 7S
promoter (either 7S.alpha. or 7S.alpha.'), an arcelin promoter, a
delta-9 desaturase promoter, or a FAD2-1A promoter.
[0176] Referring now to FIG. 10, soybean FAD2-1 intron 1 (SEQ ID
NO: 1 or 2), FAD3-1C intron 4 (SEQ ID NO: 14), FATB intron II (SEQ
ID NO: 30), FAD3-1A intron 4 (SEQ ID NO: 10), and FAD3-1B intron 4
(SEQ ID NO: 24) sequences are amplified via PCR to result in PCR
products that include reengineered restriction sites at both ends.
The PCR products are cloned directly, in sense orientation, into a
vector containing the soybean 7S.alpha.' promoter and a pea Rubisco
E9 3' termination sequence, by way of XhoI sites engineered onto
the 5' ends of the PCR primers. The vector is then cut with NotI
and ligated into pMON41164, a vector that contains the CP4 EPSPS
gene regulated by the FMV promoter and a pea Rubisco E9 3'
termination sequence. The resulting gene expression construct,
pMON68522, is depicted in FIG. 10 and is used for transformation
using methods as described herein.
[0177] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A intron
4 (SEQ ID NO: 10), FAD3-1B intron 4 (SEQ ID NO: 24), and FATB
intron II (SEQ ID NO: 30) sequences are amplified via PCR to result
in PCR products that include reengineered restriction sites at both
ends. The PCR products are cloned directly, in sense orientation,
into a vector containing the soybean 7S.alpha.' promoter and a tml
3' termination sequence, by way of XhoI sites engineered onto the
5' ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. Vectors containing a C. pulcherrima KAS IV gene (SEQ ID
NO: 39) regulated by a Brassica napin promoter and a Brassica napin
3' termination sequence, and a R. communis delta-9 desaturase
(FAB2) gene (SEQ ID NO: 40) regulated by a soybean FAD2 promoter
and a nos 3' termination sequence, are cut with appropriate
restriction enzymes, and ligated into pMON41164. The resulting gene
expression construct, pMON80614, is depicted in FIG. 10 and is used
for transformation using methods as described herein.
[0178] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A 3'UTR
(SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
The vector is then cut with NotI and ligated into pMON41164, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, pMON68531, is depicted in FIG.
10 and is used for transformation using methods as described
herein.
[0179] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A 3'UTR
(SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
The vector is then cut with NotI and ligated into pMON41164, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter and a pea Rubisco E9 3' termination sequence. Vectors
containing a C. pulcherrima KAS IV gene (SEQ ID NO: 39) regulated
by a Brassica napin promoter and a Brassica napin 3' termination
sequence, and a R. communis delta-9 desaturase (FAB2) gene (SEQ ID
NO: 40) regulated by a soybean FAD2 promoter and a nos 3'
termination sequence, are cut with appropriate restriction enzymes,
and ligated into pMON41164. The resulting gene expression
construct, pMON68534, is depicted in FIG. 10 and is used for
transformation using methods as described herein.
[0180] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A 3'UTR
(SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are
amplified via PCR to result in PCR products that include
reengineered restriction sites at both ends. The PCR products are
cloned directly, in sense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
The vector is then cut with NotI and ligated into pMON41164, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter and a pea Rubisco E9 3' termination sequence. A vector
containing a R. communis delta-9 desaturase (FAB2) gene (SEQ ID NO:
40) regulated by a soybean FAD2 promoter and a nos 3' termination
sequence, is cut with appropriate restriction enzymes, and ligated
into pMON41164. The resulting gene expression construct, pMON68535,
is depicted in FIG. 10 and is used for transformation using methods
as described herein.
[0181] Referring now to FIG. 11, soybean FAD2-1 3'UTR (SEQ D NO:
5), FAD3-1A 3'UTR (SEQ ID NO: 16), and FATB 3'UTR (SEQ ID NO: 36)
sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in sense orientation, into a vector
containing the soybean 7S.alpha.' promoter and a tml 3' termination
sequence, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vector is then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, pMON80605, is depicted in FIG.
11 and is used for transformation using methods as described
herein.
[0182] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FAD3-1A 3'UTR (SEQ ID
NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. The vector is then
cut with NotI and ligated into pMON41164, a vector that contains
the CP4 EPSPS gene regulated by the FMV promoter and a pea Rubisco
E9 3' termination sequence. A vector containing a C. pulcherrima
KAS IV gene (SEQ ID NO: 39) regulated by a Brassica napin promoter
and a Brassica napin 3' termination sequence is cut with
appropriate restriction enzymes, and ligated into pMON4164. The
resulting gene expression construct, pMON80606, is depicted in FIG.
11 and is used for transformation using methods as described
herein.
[0183] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FAD3-1A 3'UTR (SEQ ID
NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. The vector is then
cut with NotI and ligated into pMON41164, a vector that contains
the CP4 EPSPS gene regulated by the FMV promoter and a pea Rubisco
E9 3' termination sequence. A vector containing a R. communis
delta-9 desaturase (FAB2) gene (SEQ ID NO: 40) regulated by a
soybean FAD2 promoter and a nos 3' termination sequence is cut with
appropriate restriction enzymes, and ligated into pMON41164. The
resulting gene expression construct, pMON80607, is depicted in FIG.
11 and is used for transformation using methods as described
herein.
[0184] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FAD3-1A 3'UTR (SEQ ID
NO: 16), and FATB 3'UTR (SEQ ID NO: 36) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. The vector is then
cut with NotI and ligated into pMON41164, a vector that contains
the CP4 EPSPS gene regulated by the FMV promoter and a pea Rubisco
E9 3' termination sequence. Vectors containing a C. pulcherrima KAS
IV gene (SEQ ID NO: 39) regulated by a Brassica napin promoter and
a Brassica napin 3' termination sequence, and a R. communis delta-9
desaturase (FAB2) gene (SEQ ID NO: 40) regulated by a soybean FAD2
promoter and a nos 3' termination sequence, are cut with
appropriate restriction enzymes, and ligated into pMON41164. The
resulting gene expression construct, pMON80614, is depicted in FIG.
11 and is used for transformation using methods as described
herein.
[0185] Referring now to FIG. 12, soybean FAD2-1 3'UTR (SEQ ID NO:
5), FATB 3'UTR (SEQ ID NO: 36), and FAD3-1A 3'UTR (SEQ ID NO: 16)
sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in sense orientation, into a vector
containing the soybean 7S.alpha. promoter and a tml 3' termination
sequence, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vector is then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, pMON80629, is depicted in FIG.
12 and is used for transformation using methods as described
herein.
[0186] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2), FAD3-1A intron
4 (SEQ ID NO: 10), FATB intron II (SEQ ID NO: 30), and FAD3-1A
intron 4 (SEQ ID NO: 10) sequences are amplified via PCR to result
in PCR products that include reengineered restriction sites at both
ends. The PCR products are cloned directly, in sense orientation,
into a vector containing the soybean 7S.alpha. promoter and a tml
3' termination sequence, by way of XhoI sites engineered onto the
5' ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. The resulting gene expression construct, pMON81902, is
depicted in FIG. 12 and is used for transformation using methods as
described herein.
[0187] Soybean FAD2-1 5'UTR-3'UTR (SEQ ID NOs: 6 and 5, ligated
together), FAD3-1 5'UTR-3'UTR (SEQ ID NOs: 17 and 16, ligated
together, or 27 and 26, ligated together), and FATB 5'UTR-3'UTR
(SEQ ID NOs: 37 and 36, ligated together) sequences are amplified
via PCR to result in PCR products that include reengineered
restriction sites at both ends. The FAD2-1 PCR product is cloned
directly, in sense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
Similarly, the FAD3-1 PCR product is cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. The FATB PCR
product is cloned directly, in sense orientation, into a vector
containing the arcelin promoter and a tml 3' termination sequence,
by way of XhoI sites engineered onto the 5' ends of the PCR
primers. These vectors are then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, O1, is depicted in FIG. 12 and
is used for transformation using methods as described herein.
[0188] Soybean FAD2-1 5'UTR-3'UTR (SEQ ID NOs: 6 and 5, ligated
together), FAD3-1 5'UTR-3'UTR (SEQ ID NOs: 17 and 16, ligated
together, or 27 and 26, ligated together), and FATB 5'UTR-3'UTR
(SEQ ID NOs: 37 and 36, ligated together) sequences are amplified
via PCR to result in PCR products that include reengineered
restriction sites at both ends. The FAD2-1 PCR product is cloned
directly, in sense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
Similarly, the FAD3-1 PCR product is cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5'ends of the PCR primers. The FATB PCR product
is cloned directly, in sense orientation, into a vector containing
the arcelin promoter and a tml 3' termination sequence, by way of
XhoI sites engineered onto the 5' ends of the PCR primers. These
vectors are then cut with NotI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. A vector containing a C.
pulcherrima KAS IV gene (SEQ ID NO: 39) regulated by a Brassica
napin promoter and a Brassica napin 3' termination sequence is cut
with appropriate restriction enzymes, and ligated into pMON41164.
The resulting gene expression construct, O2, is depicted in FIG. 12
and is used for transformation using methods as described
herein.
[0189] Referring now to FIG. 13, soybean FAD2-1 5'UTR-3'UTR (SEQ ID
NOs: 6 and 5, ligated together), FATB 5'UTR-3'UTR (SEQ ID NOs: 37
and 36, ligated together), FAD3-1A 3'UTR (SEQ ID NO: 16), and
FAD3-1B 5'UTR-3'UTR (SEQ ID NOs: 27 and 26, ligated together)
sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in sense orientation, into a vector
containing the soybean 7S.alpha.' promoter and a tml 3' termination
sequence, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vectors are then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. A
vector containing a C. pulcherrima KAS IV gene (SEQ ID NO: 39)
regulated by a Brassica napin promoter and a Brassica napin 3'
termination sequence is cut with appropriate restriction enzymes,
and ligated into pMON41164. The resulting gene expression
construct, O7, is depicted in FIG. 13 and is used for
transformation using methods as described herein.
[0190] Soybean FAD2-1 intron 1 (SEQ ID NO: 1 or 2) is amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in sense
orientation, into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, by way of XhoI sites
engineered onto the 5' ends of the PCR primers. Soybean FATB
5'UTR-3'UTR (SEQ ID NOs: 37 and 36, ligated together), FAD3-1A
3'UTR (SEQ ID NO: 16), and FAD3-1B 5'UTR-3'UTR (SEQ ID NOs: 27 and
26, ligated together) sequences are amplified via PCR to result in
PCR products that include reengineered restriction sites at both
ends. The PCR products are cloned directly, in sense orientation,
into a vector containing the soybean 7S.alpha. promoter and a nos
3' termination sequence, by way of XhoI sites engineered onto the
5' ends of the PCR primers. The vectors are then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. A vector containing a C. pulcherrima KAS IV gene (SEQ ID
NO: 39) regulated by a Brassica napin promoter and a Brassica napin
3' termination sequence is cut with appropriate restriction
enzymes, and ligated into pMON41164. The resulting gene expression
construct, O9, is depicted in FIG. 13 and is used for
transformation using methods as described herein.
2C. Antisense Constructs
[0191] FIG. 14 depicts nucleic acid molecules of the present
invention in which the first sets of DNA sequences are capable of
expressing antisense constructs, and FIG. 15 depicts nucleic acid
molecules of the present invention in which the first sets of DNA
sequences are capable of expressing combinations of sense and
antisense constructs. The second set of DNA sequences comprises
coding sequences, each of which is a DNA sequence that encodes a
sequence that when expressed is capable of increasing one or both
of the protein and transcript encoded by a gene selected from the
group consisting of KAS I, KAS IV, delta-9 desaturase, and CP4
EPSPS. Each coding sequence is associated with a promoter, which is
any promoter functional in a plant, or any plant promoter, and may
be an FMV promoter, a napin promoter, a 7S (either 7S.alpha. or
7S.alpha.') promoter, an arcelin promoter, a delta-9 desaturase
promoter, or a FAD2-1A promoter.
[0192] Referring now to FIG. 14, soybean FAD2-1 3'UTR (SEQ ID NO:
5), FATB 3'UTR (SEQ D NO: 36), and FAD3-1A 3'UTR (SEQ ID NO: 16)
sequences are amplified via PCR to result in PCR products that
include reengineered restriction sites at both ends. The PCR
products are cloned directly, in antisense orientation, into a
vector containing the soybean 7S.alpha.' promoter and a tml 3'
termination sequence, by way of XhoI sites engineered onto the 5'
ends of the PCR primers. The vector is then cut with NotI and
ligated into pMON41164, a vector that contains the CP4 EPSPS gene
regulated by the FMV promoter and a pea Rubisco E9 3' termination
sequence. The resulting gene expression construct, pMON80615, is
depicted in FIG. 14 and is used for transformation using methods as
described herein.
[0193] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FATB 3'UTR (SEQ ID NO:
36), and FAD3-1A 3'UTR (SEQ ID NO: 16) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in
antisense orientation, into a vector containing the soybean
7S.alpha.' promoter and a tml 3' termination sequence, by way of
XhoI sites engineered onto the 5' ends of the PCR primers. The
vector is then cut with NotI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. A vector containing a C.
pulcherrima KAS IV gene (SEQ ID NO: 39) regulated by a Brassica
napin promoter and a Brassica napin 3' termination sequence is cut
with appropriate restriction enzymes, and ligated into pMON41164.
The resulting gene expression construct, pMON80616, is depicted in
FIG. 14 and is used for transformation using methods as described
herein.
[0194] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FATB 3'UTR (SEQ ID NO:
36), and FAD3-1A 3'UTR (SEQ ID NO: 16) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in
antisense orientation, into a vector containing the soybean
7S.alpha.' promoter and a tml 3' termination sequence, by way of
XhoI sites engineered onto the 5' ends of the PCR primers. The
vector is then cut with NotI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. A vector containing a R.
communis delta-9 desaturase (FAB2) gene (SEQ ID NO: 40) regulated
by a soybean FAD2 promoter and a nos 3' termination sequence, is
cut with appropriate restriction enzymes, and ligated into
pMON41164. The resulting gene expression construct, pMON80617, is
depicted in FIG. 14 and is used for transformation using methods as
described herein.
[0195] Soybean FAD2-1 3'UTR (SEQ ID NO: 5), FATB 3'UTR (SEQ ID NO:
36), and FAD3-1A 3'UTR (SEQ ID NO: 16) sequences are amplified via
PCR to result in PCR products that include reengineered restriction
sites at both ends. The PCR products are cloned directly, in
antisense orientation, into a vector containing the soybean
7S.alpha. promoter and a tml 3' termination sequence, by way of
XhoI sites engineered onto the 5' ends of the PCR primers. The
vector is then cut with NotI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. The resulting gene
expression construct, pMON80630, is depicted in FIG. 14 and is used
for transformation using methods as described herein.
[0196] Soybean FAD2-1 5'UTR-3'UTR (SEQ ID NOs: 6 and 5, ligated
together), FATB 5'UTR-3'UTR (SEQ ID NOs: 37 and 36, ligated
together), FAD3-1A 3'UTR (SEQ ID NO: 16), and FAD3-1B 5'UTR-3'UTR
(SEQ ID NOs: 27 and 26, ligated together) sequences are amplified
via PCR to result in PCR products that include reengineered
restriction sites at both ends. The PCR products are cloned
directly, in antisense orientation, into a vector containing the
soybean 7S.alpha.' promoter and a tml 3' termination sequence, by
way of XhoI sites engineered onto the 5' ends of the PCR primers.
The vector is then cut with NotI and ligated into pMON41164, a
vector that contains the CP4 EPSPS gene regulated by the FMV
promoter and a pea Rubisco E9 3' termination sequence. A vector
containing a C. pulcherrima KAS IV gene (SEQ ID NO: 39) regulated
by a Brassica napin promoter and a Brassica napin 3' termination
sequence is cut with appropriate restriction enzymes, and ligated
into pMON41164. The resulting gene expression construct, O8, is
depicted in FIG. 14 and is used for transformation using methods as
described herein.
[0197] Referring now to FIG. 15, soybean FAD2-1 5'UTR-3'UTR (SEQ ID
NOs: 6 and 5, ligated together), FAD3-1A 5'UTR-3'UTR (SEQ ID NOs:
17 and 16, ligated together), and FATB 5'UTR-3'UTR (SEQ ID NOs: 37
and 36, ligated together) sequences are amplified via PCR to result
in PCR products that include reengineered restriction sites at both
ends. The PCR products are cloned directly in sense and antisense
orientation into a vector containing the soybean 7S.alpha.'
promoter and a tml 3' termination sequence, with an additional
soybean 7S.alpha. promoter located between the sense and antisense
sequences, by way of XhoI sites engineered onto the 5' ends of the
PCR primers. The vector is then cut with NotI and ligated into
pMON41164, a vector that contains the CP4 EPSPS gene regulated by
the FMV promoter and a pea Rubisco E9 3' termination sequence. The
resulting gene expression construct, O3, is depicted in FIG. 15 and
is used for transformation using methods as described herein.
[0198] Soybean FAD2-1 5'UTR-3'UTR (SEQ ID NOs: 6 and 5, ligated
together), FAD3-1A 5'UTR-3'UTR (SEQ ID NOs: 27 and 26, ligated
together), and FATB 5'UTR-3'UTR (SEQ ID NOs: 37 and 36, ligated
together) sequences are amplified via PCR to result in PCR products
that include reengineered restriction sites at both ends. The PCR
products are cloned directly in sense and antisense orientation
into a vector containing the soybean 7S.alpha.' promoter and a tml
3' termination sequence, with an additional soybean 7S.alpha.
promoter located between the sense and antisense sequences, by way
of XhoI sites engineered onto the 5' ends of the PCR primers. The
vector is then cut with NotI and ligated into pMON41164, a vector
that contains the CP4 EPSPS gene regulated by the FMV promoter and
a pea Rubisco E9 3' termination sequence. A vector containing a C.
pulcherrima KAS IV gene (SEQ ID NO: 39) regulated by a Brassica
napin promoter and a Brassica napin 3' termination sequence is cut
with appropriate restriction enzymes, and ligated into pMON41164.
The resulting gene expression construct, O4, is depicted in FIG. 15
and is used for transformation using methods as described
herein.
[0199] The above-described nucleic acid molecules are preferred
embodiments which achieve the objects, features and advantages of
the present invention. It is not intended that the present
invention be limited to the illustrated embodiments. The
arrangement of the sequences in the first and second sets of DNA
sequences within the nucleic acid molecule is not limited to the
illustrated and described arrangements, and may be altered in any
manner suitable for achieving the objects, features and advantages
of the present invention as described herein, illustrated in the
accompanying drawings, and encompassed within the claims.
Example 3
Plant Transformation and Analysis
[0200] The constructs of Examples 1 and 2 are stably introduced
into soybean (for example, Asgrow variety A4922 or Asgrow variety
A3244 or Asgrow variety A3525) by the methods described earlier,
including the methods of McCabe et al., Bio/Technology, 6:923-926
(1988), or Agrobacterium-mediated transformation. Transformed
soybean plants are identified by selection on media containing
glyphosate. Fatty acid compositions are analyzed from seed of
soybean lines transformed with the constructs using gas
chromatography. In addition, any of the constructs may contain
other sequences of interest, as well as different combinations of
promoters.
[0201] For some applications, modified fatty acid compositions are
detected in developing seeds, whereas in other instances, such as
for analysis of oil profile, detection of fatty acid modifications
occurring later in the FAS pathway, or for detection of minor
modifications to the fatty acid composition, analysis of fatty acid
or oil from mature seeds is preferred. Furthermore, analysis of oil
and/or fatty acid content of individual seeds may be desirable,
especially in detection of oil modification in the segregating R1
seed populations. As used herein, R0 indicates the plant and seed
arising from transformation/regeneration protocols described
herein, and R1 indicates plants and seeds generated from the
transgenic R0 seed.
[0202] Fatty acid compositions are determined for the seed of
soybean lines transformed with the constructs of Example 2. One to
ten seeds of each of the transgenic and control soybean lines are
ground individually using a tissue homogenizer (Pro Scientific) for
oil extraction. Oil from ground soybean seed is extracted overnight
in 1.5 ml heptane containing triheptadecanoin (0.50 mg/ml).
Aliquots of 200 .mu.l of the extracted oil are derivatized to
methyl esters with the addition of 500 .mu.l sodium methoxide in
absolute methanol. The derivatization reaction is allowed to
progress for 20 minutes at 50.degree. C. The reaction is 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 are resolved by gas chromatography (GC) on a
Hewlett-Packard model 6890 GC (Palo Alto, Calif.). 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 is
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 are 250.degree. C. and
270.degree. C., respectively.
Example 4
Synthesized Fuel Oil with Improved Biodiesel Properties
[0203] A synthesized fuel oil fatty acid composition is prepared
having the following mixtures of fatty acid methyl esters: 73.3%
oleic acid, 21.4% linoleic acid, 2.2% palmitic acid, 2.1% linolenic
acid and 1.0% stearic acid (all by weight). Purified fatty acid
methyl esters are obtained from Nu-Chek Prep, Inc., Elysian, Minn.,
USA. The cetane number and ignition delay of this composition is
determined by the Southwest Research Institute using an Ignition
Quality Tester ("IQT") 613 (Southwest Research Institute, San
Antonio, Tex., USA).
[0204] An IQT consists of a constant volume combustion chamber that
is electrically heated, a fuel injection system, and a computer
that is used to control the experiment, record the data and provide
interpretation of the data. The fuel injection system includes a
fuel injector nozzle that forms an entrance to the combustion
chamber. A needle lift sensor in the fuel injector nozzle detects
fuel flow into the combustion chamber. A pressure transducer
attached to the combustion chamber measures cylinder pressure, the
pressure within the combustion chamber. The basic concept of an IQT
is measurement of the time from the start of fuel injection into
the combustion chamber to the start of combustion. The
thermodynamic conditions in the combustion chamber are precisely
controlled to provide consistent measurement of the ignition delay
time.
[0205] For a cetane number and ignition delay test, the test fuel
is filtered using a 5-micron filter. The fuel reservoir, injection
line, and nozzle are purged with pressurized nitrogen. The fuel
reservoir is then cleaned with a lint free cloth. A portion of the
test fuel is used to flush the fuel reservoir, injection line, and
nozzle. The reservoir is filled with the test fuel and all air is
bled from the system. The reservoir is pressurized to 50 psig. The
method basically consists of injecting at high pressure a precisely
metered quantity of the test fuel into the combustion chamber that
is charged with air to the desired pressure and temperature. The
measurement consists of determining the time from the start of
injection to the onset of combustion, often referred to as the
ignition delay time. This determination is based on the measured
needle lift and combustion chamber pressures. The normal cetane
rating procedure calls for setting the skin temperature at
567.5.degree. C. and the air pressure at 2.1 MPa.
[0206] A fuel with a known injection delay is run in the IQT
combustion bomb at the beginning of the day to make sure the unit
is operating within normal parameters. The test synthetic is then
run. The known fuel is run again to verify that the system has not
changed. Once the fuel reservoir is reconnected to the fuel
injection pump, the test procedure is initiated on the PC
controller. The computer controls all the procedure, including the
air charging, fuel injection, and exhaust events. 32 repeat
combustion events are undertaken.
[0207] The ignition delay is the time from the start of injection
to the start of ignition. It is determined from the needle lift and
cylinder pressure data. The rise of the injection needle signals
start of injection. The cylinder pressure drops slightly due to the
cooling effect of the vaporization of the fuel. Start of combustion
is defined as the recovery time of the cylinder pressure--increases
due to combustion to the pressure it was just prior to fuel
injection.
[0208] The measured ignition delay times are then used to determine
the cetane number based on a calibration curve that is incorporated
into the data acquisition and reduction software. The calibration
curve, consisting of cetane number as a function of ignition delay
time, is generated using blends of the primary reference fuels and
NEG check fuels. In the case of test fuels that are liquid at
ambient conditions, the calibration curve is checked on a daily
basis using at least one check fuel of known cetane number (Ryan,
"Correlation of Physical and Chemical Ignition Delay to Cetane
Number", SAE Paper 852103 (1985); Ryan, "Diesel Fuel Ignition
Quality as Determined in a Constant Volume Combustion Bomb", SAE
Paper 870586 (1986); Ryan, "Development of a Portable Fuel Cetane
Quality Monitor", Belvoir Fuels and Lubricants Research Facility
Report No. 277, May (1992); Ryan, "Engine and Constant Volume Bomb
Studies of Diesel Ignition and Combustion", SAE Paper 881616
(1988); and Allard et al., "Diesel Fuel Ignition Quality as
Determined in the Ignition Quality Tester ("IQT")", SAE Paper
961182 (1996)). As shown in Table 3, the synthesized oil
composition exhibits cetane numbers and ignition delays that are
suitable for use for example, without limitation, as a biodiesel
oil.
TABLE-US-00003 TABLE 3 Ignition Fuel Test Cetane Std. Dev. Delay
Std. Dev. Name Number Number Cetane No. (ms) Ign. Delay
Check-High.sup.1 1777 49.55 0.534 4.009 0.044 Check-High 1778 49.33
0.611 4.028 0.051 Average 49.4 4.02 Synthesized Oil 1779 55.02
1.897 3.622 0.116 Synthesized Oil 1780 55.65 1.807 3.583 0.109
Synthesized Oil 1781 55.63 1.649 3.583 0.098 Average 55.4 3.60
Check-High 1786 49.2 0.727 4.04 0.061 .sup.1The fuel called
"Check-High" is a calibration fuel. It should have a cetane number
of 49.3 .+-. 0.5. The unit is checked with the calibration before
and after running the synthetic test fuel.
[0209] The density (ASTM D-4052) cloud point (ASTM D-2500), pour
point (ASTM D-97), and cold filter plugging point (IP 309/ASTM
D-6371) are determined for the synthesized oil using ASTM D
protocols. ASTM D protocols are obtained from ASTM, 100 Barr Harbor
Drive, West Conshohocken, Pa., USA. The results of these tests are
set forth in Table 4. As shown in Table 4, the synthesized oil
composition exhibits numbers that are suitable for use as, for
example without limitation, as a biodiesel oil.
TABLE-US-00004 TABLE 4 TEST METHOD RESULTS Density ASTM D-4052
0.8791 g/mL Cloud Point ASTM D-2500 -18 deg. C. Pour Point ASTM
D-97 -21 deg. C. Cold Filter Plugging Point IP 309 (same -21 deg.
C. as ASTM D-6371)
[0210] Levels of nitric oxide emissions are estimated by evaluating
the unsaturation levels of a biologically-based fuel, by measuring
the fuel density and indirectly calculating the estimated emissions
levels, or by directly measuring. There are also standard protocols
available for directly measuring levels of nitric oxide emissions.
The synthesized oil is estimated to have lower nitric oxide
emissions levels than methyl esters of fatty acids made from
conventional soybean oil based on an evaluation of the overall
level of unsaturation in the synthesized oil. Oils containing
larger numbers of double bonds, i.e., having a higher degree of
unsaturation, tend to produce higher nitric oxide emissions. The
oil has a total of 123 double bonds, as compared to conventional
soybean oil's total of 153 double bonds, as shown in Table 5.
TABLE-US-00005 TABLE 5 SYNTHETIC OIL 73% oleic acid (18:1) .times.
1 double bond = 73 22% linoleic acid (18:2) .times. 2 double bonds
= 44 2% linolenic acid (18:3) .times. 3 double bonds = 6 TOTAL
double bonds 123 CONVENTIONAL SOYBEAN OIL 23% oleic acid (18:1)
.times. 1 double bond = 23 53% linoleic acid (18:2) .times. 2
double bonds = 106 8% linolenic acid (18:3) .times. 3 double bonds
= 24 TOTAL double bonds 153
[0211] As reported by the National Renewable Energy Laboratory,
Contract No. ACG-8-17106-02 Final Report, The Effect Of Biodiesel
Composition On Engine Emissions From A DDC Series 60 Diesel Engine,
(June 2000), nitric acid emissions of biodiesel compositions are
predicted by the formula y=46.959.times.-36.388 where y is the
oxide emissions in grams/brake horse power hours; and x is the
density of biodiesel. The formula is based on a regression analysis
of nitric acid emission data in a test involving 16 biodiesel
fuels. The test makes use of a 1991 calibration, production series
60 model Detroit Diesel Corporation engine.
[0212] The density of the synthesized oil is determined by
Southwest Research Institute using the method ASTM D4052. The
result shown in Table 4 is used in the above equation to predict a
nitric oxide emission value of 4.89 g/bhp-h. This result is
compared to a control soybean product. The National Renewable
Energy Laboratory report gives the density and nitric oxide
emissions of a control soy based biodiesel (methyl soy ester IGT).
The density of the control biodiesel is 0.8877 g/mL, giving a
calculated nitric oxide emission of 5.30 g/bhp-h. This calculated
emission value is similar to the experimental value for nitric
oxide emission of 5.32 g/bhp-h. The synthesized oil composition
exhibits improved numbers compared to the control and is suitable
for use, for example without limitation, as a biodiesel oil.
Example 5
Optimum Fatty Acid Composition for Healthy Serum Lipid Levels
[0213] The cholesterol lowering properties of vegetable
compositions are determined to identify fatty acid compositions
that have a more favorable effect on serum lipid levels than
conventional soybean oil (i.e., lower LDL-cholesterol and higher
HDL-cholesterol). Published equations based on 27 clinical trials
(Mensink, R. P. and Katan, M. B. Arteriosclerosis and Thrombosis,
12:911-919 (1992)) are used to compare the effects on serum lipid
levels in humans of new oilseed compositions with that of normal
soybean oil.
[0214] Table 6 below presents the results of the change in serum
lipid levels where 30% of dietary energy from carbohydrate is
substituted by lipids. The results show that soybean oil already
has favorable effects on serum lipids when it replaces
carbohydrates in the diet. Improvements on this composition are
possible by lowering saturated fat levels and by obtaining a
linoleic acid level between 10-30% of the total fatty acids,
preferably about 15-25% of the total fatty acids. When the
proportion of linoleic acid is less than 10% of the total fatty
acids, the new composition raises LDL-cholesterol compared to
control soybean oil, even though the saturated fat content is
lowered to 5% of the total fatty acids. When the proportion of
linoleic acid is increased, the ability of the composition to raise
serum HDL levels is reduced. Therefore, the preferred linoleic acid
composition is determined to be about 15-25% of the total fatty
acids.
TABLE-US-00006 TABLE 6 Fatty acids Other Serum C16:0 C18:0 C18:1
C18:2 C18:3 (C20:1) Liplds Soy control (%) 11.000 4.000 23.400
53.200 7.800 0.600 Proportion of 30% fat E (%) 3.300 1.200 7.020
15.960 2.340 0.180 LDL Calculation (mg/dl) 4.224 1.536 1.685 8.778
1.287 0.043 -6.033 HDL Calc (mg/dl) 1.551 0.564 2.387 4.469 0.655
0.061 9.687 3% 18:2, <6% sat (%) 3.000 2.000 85.000 3.000 3.000
4.000 Proportion of 30% fat E (%) 0.900 0.600 25.500 0.900 0.900
1.200 LDL Calculation (mg/dl) 1.152 0.768 6.120 0.495 0.495 0.288
-5.478 vs. control (mg/dl) 0.555 HDL calculation (mg/dl) 0.423
0.282 8.670 0.252 0.252 0.408 10.287 vs. control (mg/dl) 0.600 10%
18:2, <6% sat (%) 3.000 2.000 72.000 10.000 3.000 10.000
Proportion of 30% fat E (%) 0.900 0.600 21.600 3.000 0.900 3.000
LDL Calculation (mg/dl) 1.152 0.768 5.184 1.650 0.495 0.720 -6.129
vs. control (mg/dl) -0.096 HDL calculation (mg/dl) 0.423 0.282
7.344 0.840 0.252 1.020 10.161 vs. control (mg/dl) 0.474 20% 18:2,
<6% sat (%) 3.000 2.000 65.000 20.000 3.000 7.000 Proportion of
30% fat E (%) 0.900 0.600 19.500 6.000 0.900 2.100 LDL Calculation
(mg/dl) 1.152 0.768 4.680 3.300 0.495 0.504 -7.059 vs. control
(mg/dl) -1.026 HDL calculation (mg/dl) 0.423 0.282 6.630 1.680
0.252 0.714 9.981 vs. control (mg/dl) 0.294 21% 18:2, <3.2% sat
(%) 2.000 1.000 72.000 21.000 1.000 3.000 Proportion of 30% fat E
(%) 0.600 0.300 21.600 6.300 0.300 0.900 LDL Calculation (mg/dl)
0.768 0.384 5.184 3.465 0.165 0.216 -7.878 vs. control (mg/dl)
-1.845 HDL calculation (mg/dl) 0.282 0.141 7.344 1.764 0.084 0.306
9.921 vs. control (mg/dl) 0.234 30% 18:2, <6% sat (%) 3.000
2.000 57.000 30.000 3.000 5.000 Proportion of 30% fat E (%) 0.900
0.600 17.100 9.000 0.900 1.500 LDL Calculation (mg/dl) 1.152 0.768
4.104 4.950 0.495 0.360 -7.989 vs. control (mg/dl) -1.956 HDL
calculation (mg/dl) 0.423 0.282 5.814 2.520 0.252 0.510 9.801 vs.
control (mg/dl) 0.114
Sequence CWU 1
1
411420DNAGlycine maxFAD2-1A intron 1 1gtaaattaaa ttgtgcctgc
acctcgggat atttcatgtg gggttcatca tatttgttga 60ggaaaagaaa ctcccgaaat
tgaattatgc atttatatat cctttttcat ttctagattt 120cctgaaggct
taggtgtagg cacctagcta gtagctacaa tatcagcact tctctctatt
180gataaacaat tggctgtaat gccgcagtag aggacgatca caacatttcg
tgctggttac 240tttttgtttt atggtcatga tttcactctc tctaatctct
ccattcattt tgtagttgtc 300attatcttta gatttttcac tacctggttt
aaaattgagg gattgtagtt ctgttggtac 360atattacaca ttcagcaaaa
caactgaaac tcaactgaac ttgtttatac tttgacacag 4202405DNAGlycine
maxFAD2-1B intron 1 2gtatgatgct aaattaaatt gtgcctgcac cccaggatat
ttcatgtggg attcatcatt 60tattgaggaa aactctccaa attgaatcgt gcatttatat
tttttttcca tttctagatt 120tcttgaaggc ttatggtata ggcacctaca
attatcagca cttctctcta ttgataaaca 180attggctgta ataccacagt
agagaacgat cacaacattt tgtgctggtt accttttgtt 240ttatggtcat
gatttcactc tctctaatct gtcacttccc tccattcatt ttgtacttct
300catatttttc acttcctggt tgaaaattgt agttctcttg gtacatacta
gtattagaca 360ttcagcaaca acaactgaac tgaacttctt tatactttga cacag
40531704DNAGlycine maxFAD2-1B promoter 3actatagggc acgcgtggtc
gacggcccgg gctggtcctc ggtgtgactc agccccaagt 60gacgccaacc aaacgcgtcc
taactaaggt gtagaagaaa cagatagtat ataagtatac 120catataagag
gagagtgagt ggagaagcac ttctcctttt tttttctctg ttgaaattga
180aagtgttttc cgggaaataa ataaaataaa ttaaaatctt acacactcta
ggtaggtact 240tctaatttaa tccacacttt gactctatat atgttttaaa
aataattata atgcgtactt 300acttcctcat tatactaaat ttaacatcga
tgattttatt ttctgtttct cttctttcca 360cctacataca tcccaaaatt
tagggtgcaa ttttaagttt attaacacat gtttttagct 420gcatgctgcc
tttgtgtgtg ctcaccaaat tgcattcttc tctttatatg ttgtatttga
480attttcacac catatgtaaa caagattacg tacgtgtcca tgatcaaata
caaatgctgt 540cttatactgg caatttgata aacagccgtc cattttttct
ttttctcttt aactatatat 600gctctagaat ctctgaagat tcctctgcca
tcgaatttct ttcttggtaa caacgtcgtc 660gttatgttat tattttattc
tatttttatt ttatcatata tatttcttat tttgttcgaa 720gtatgtcata
ttttgatcgt gacaattaga ttgtcatgta ggagtaggaa tatcacttta
780aaacattgat tagtctgtag gcaatattgt cttctttttc ctcctttatt
aatatatttt 840gtcgaagttt taccacaagg ttgattcgct ttttttgtcc
ctttctcttg ttctttttac 900ctcaggtatt ttagtctttc atggattata
agatcactga gaagtgtatg catgtaatac 960taagcaccat agctgttctg
cttgaattta tttgtgtgta aattgtaatg tttcagcgtt 1020ggctttccct
gtagctgcta caatggtact gtatatctat tttttgcatt gttttcattt
1080tttcttttac ttaatcttca ttgctttgaa attaataaaa caatataata
tagtttgaac 1140tttgaactat tgcctattca tgtaattaac ttattcactg
actcttattg tttttctggt 1200agaattcatt ttaaattgaa ggataaatta
agaggcaata cttgtaaatt gacctgtcat 1260aattacacag gaccctgttt
tgtgcctttt tgtctctgtc tttggttttg catgttagcc 1320tcacacagat
atttagtagt tgttctgcat acaagcctca cacgtatact aaaccagtgg
1380acctcaaagt catggcctta cacctattgc atgcgagtct gtgacacaac
ccctggtttc 1440catattgcaa tgtgctacgc cgtcgtcctt gtttgtttcc
atatgtatat tgataccatc 1500aaattattat atcatttata tggtctggac
cattacgtgt actctttatg acatgtaatt 1560gagtttttta attaaaaaaa
tcaatgaaat ttaactacgt agcatcatat agagataatt 1620gactagaaat
ttgatgactt attctttcct aatcatattt tcttgtattg atagccccgc
1680tgtccctttt aaactcccga gaga 170444497DNAGlycine maxFAD2-1A
genomic clone 4cttgcttggt aacaacgtcg tcaagttatt attttgttct
tttttttttt atcatatttc 60ttattttgtt ccaagtatgt catattttga tccatcttga
caagtagatt gtcatgtagg 120aataggaata tcactttaaa ttttaaagca
ttgattagtc tgtaggcaat attgtcttct 180tcttcctcct tattaatatt
ttttattctg ccttcaatca ccagttatgg gagatggatg 240taatactaaa
taccatagtt gttctgcttg aagtttagtt gtatagttgt tctgcttgaa
300gtttagttgt gtgtaatgtt tcagcgttgg cttcccctgt aactgctaca
atggtactga 360atatatattt tttgcattgt tcattttttt cttttactta
atcttcattg ctttgaaatt 420aataaaacaa aaagaaggac cgaatagttt
gaagtttgaa ctattgccta ttcatgtaac 480ttattcaccc aatcttatat
agtttttctg gtagagatca ttttaaattg aaggatataa 540attaagagga
aatacttgta tgtgatgtgt ggcaatttgg aagatcatgc gtagagagtt
600taatggcagg ttttgcaaat tgacctgtag tcataattac actgggccct
ctcggagttt 660tgtgcctttt tgttgtcgct gtgtttggtt ctgcatgtta
gcctcacaca gatatttagt 720agttgttgtt ctgcatataa gcctcacacg
tatactaaac gagtgaacct caaaatcatg 780gccttacacc tattgagtga
aattaatgaa cagtgcatgt gagtatgtga ctgtgacaca 840acccccggtt
ttcatattgc aatgtgctac tgtggtgatt aaccttgcta cactgtcgtc
900cttgtttgtt tccttatgta tattgatacc ataaattatt actagtatat
cattttatat 960tgtccatacc attacgtgtt tatagtctct ttatgacatg
taattgaatt ttttaattat 1020aaaaaataat aaaacttaat tacgtactat
aaagagatgc tcttgactag aattgtgatc 1080tcctagtttc ctaaccatat
actaatattt gcttgtattg atagcccctc cgttcccaag 1140agtataaaac
tgcatcgaat aatacaagcc actaggcatg gtaaattaaa ttgtgcctgc
1200acctcgggat atttcatgtg gggttcatca tatttgttga ggaaaagaaa
ctcccgaaat 1260tgaattatgc atttatatat cctttttcat ttctagattt
cctgaaggct taggtgtagg 1320cacctagcta gtagctacaa tatcagcact
tctctctatt gataaacaat tggctgtaat 1380gccgcagtag aggacgatca
caacatttcg tgctggttac tttttgtttt atggtcatga 1440tttcactctc
tctaatctct ccattcattt tgtagttgtc attatcttta gatttttcac
1500tacctggttt aaaattgagg gattgtagtt ctgttggtac atattacaca
ttcagcaaaa 1560caactgaaac tcaactgaac ttgtttatac tttgacacag
ggtctagcaa aggaaacaac 1620aatgggaggt agaggtcgtg tggcaaagtg
gaagttcaag ggaagaagcc tctctcaagg 1680gttccaaaca caaagccacc
attcactgtt ggccaactca agaaagcaat tccaccacac 1740tgctttcagc
gctccctcct cacttcattc tcctatgttg tttatgacct ttcatttgcc
1800ttcattttct acattgccac cacctacttc cacctccttc ctcaaccctt
ttccctcatt 1860gcatggccaa tctattgggt tctccaaggt tgccttctca
ctggtgtgtg ggtgattgct 1920cacgagtgtg gtcaccatgc cttcagcaag
taccaatggg ttgatgatgt tgtgggtttg 1980acccttcact caacactttt
agtcccttat ttctcatgga aaataagcca tcgccgccat 2040cactccaaca
caggttccct tgaccgtgat gaagtgtttg tcccaaaacc aaaatccaaa
2100gttgcatggt tttccaagta cttaaacaac cctctaggaa gggctgtttc
tcttctcgtc 2160acactcacaa tagggtggcc tatgtattta gccttcaatg
tctctggtag accctatgat 2220agttttgcaa gccactacca cccttatgct
cccatatatt ctaaccgtga gaggcttctg 2280atctatgtct ctgatgttgc
tttgttttct gtgacttact ctctctaccg tgttgcaacc 2340ctgaaagggt
tggtttggct gctatgtgtt tatggggtgc ctttgctcat tgtgaacggt
2400tttcttgtga ctatcacata tttgcagcac acacactttg ccttgcctca
ttacgattca 2460tcagaatggg actggctgaa gggagctttg gcaactatgg
acagagatta tgggattctg 2520aacaaggtgt ttcatcacat aactgatact
catgtggctc accatctctt ctctacaatg 2580ccacattacc atgcaatgga
ggcaaccaat gcaatcaagc caatattggg tgagtactac 2640caatttgatg
acacaccatt ttacaaggca ctgtggagag aagcgagaga gtgcctctat
2700gtggagccag atgaaggaac atccgagaag ggcgtgtatt ggtacaggaa
caagtattga 2760tggagcaacc aatgggccat agtgggagtt atggaagttt
tgtcatgtat tagtacataa 2820ttagtagaat gttataaata agtggatttg
ccgcgtaatg actttgtgtg tattgtgaaa 2880cagcttgttg cgatcatggt
tataatgtaa aaataattct ggtattaatt acatgtggaa 2940agtgttctgc
ttatagcttt ctgcctaaaa tgcacgctgc acgggacaat atcattggta
3000atttttttaa aatctgaatt gaggctactc ataatactat ccataggaca
tcaaagacat 3060gttgcattga ctttaagcag aggttcatct agaggattac
tgcataggct tgaactacaa 3120gtaatttaag ggacgagagc aactttagct
ctaccacgtc gttttacaag gttattaaaa 3180tcaaattgat cttattaaaa
ctgaaaattt gtaataaaat gctattgaaa aattaaaata 3240tagcaaacac
ctaaattgga ctgattttta gattcaaatt taataattaa tctaaattaa
3300acttaaattt tataatatat gtcttgtaat atatcaagtt ttttttttta
ttattgagtt 3360tggaaacata taataaggaa cattagttaa tattgataat
ccactaagat cgacttagta 3420ttacagtatt tggatgattt gtatgagata
ttcaaacttc actcttatca taatagagac 3480aaaagttaat actgatggtg
gagaaaaaaa aatgttattg ggagcatatg gtaagataag 3540acggataaaa
atatgctgca gcctggagag ctaatgtatt ttttggtgaa gttttcaagt
3600gacaactatt catgatgaga acacaataat attttctact tacctatccc
acataaaata 3660ctgattttaa taatgatgat aaataatgat taaaatattt
gattctttgt taagagaaat 3720aaggaaaaca taaatattct catggaaaaa
tcagcttgta ggagtagaaa ctttctgatt 3780ataattttaa tcaagtttaa
ttcattcttt taattttatt attagtacaa aatcattctc 3840ttgaatttag
agatgtatgt tgtagcttaa tagtaatttt ttatttttat aataaaattc
3900aagcagtcaa atttcatcca aataatcgtg ttcgtgggtg taagtcagtt
attccttctt 3960atcttaatat acacgcaaag gaaaaaataa aaataaaatt
cgaggaagcg cagcagcagc 4020tgataccacg ttggttgacg aaactgataa
aaagcgctgt cattgtgtct ttgtttgatc 4080atcttcacaa tcacatctcc
agaacacaaa gaagagtgac ccttcttctt gttattccac 4140ttgcgttagg
tttctacttt cttctctctc tctctctctc tcttcattcc tcatttttcc
4200ctcaaacaat caatcaattt tcattcagat tcgtaaattt ctcgattaga
tcacggggtt 4260aggtctccca ctttatcttt tcccaagcct ttctctttcc
ccctttccct gtctgcccca 4320taaaattcag gatcggaaac gaactgggtt
cttgaatttc actctagatt ttgacaaatt 4380cgaagtgtgc atgcactgat
gcgacccact cccccttttt tgcattaaac aattatgaat 4440tgaggttttt
cttgcgatca tcattgcttg aattgaatca tattaggttt agattct
44975206DNAGlycine maxFAD2-1A 3'UTR 5tggagcaacc aatgggccat
agtgggagtt atggaagttt tgtcatgtat tagtacataa 60ttagtagaat gttataaata
agtggatttg ccgcgtaatg actttgtgtg tattgtgaaa 120cagcttgttg
cgatcatggt tataatgtaa aaataattct ggtattaatt acatgtggaa
180agtgttctgc ttatagcttt ctgcct 2066125DNAGlycine maxFAD2-1A 5'UTR
6ccatatacta atatttgctt gtattgatag cccctccgtt cccaagagta taaaactgca
60tcgaataata caagccacta ggcatgggtc tagcaaagga aacaacaatg ggaggtagag
120gtcgt 1257191DNAGlycine maxFAD3-1A intron 1 7gtaataattt
ttgtgtttct tactcttttt tttttttttt tgtttatgat atgaatctca 60cacattgttc
tgttatgtca tttcttcttc atttggcttt agacaactta aatttgagat
120ctttattatg tttttgctta tatggtaaag tgattcttca ttatttcatt
cttcattgat 180tgaattgaac a 1918346DNAGlycine maxFAD3-1A intron 2
8ttagttcata ctggcttttt tgtttgttca tttgtcattg aaaaaaaatc ttttgttgat
60tcaattattt ttatagtgtg tttggaagcc cgtttgagaa aataagaaat cgcatctgga
120atgtgaaagt tataactatt tagcttcatc tgtcgttgca agttctttta
ttggttaaat 180ttttatagcg tgctaggaaa cccattcgag aaaataagaa
atcacatctg gaatgtgaaa 240gttataactg ttagcttctg agtaaacgtg
gaaaaaccac attttggatt tggaaccaaa 300ttttatttga taaatgacaa
ccaaattgat tttgatggat tttgca 3469142DNAGlycine maxFAD3-1A intron 3A
9gtatgtgatt aattgcttct cctatagttg ttcttgattc aattacattt tatttatttg
60gtaggtccaa gaaaaaaggg aatctttatg cttcctgagg ctgttcttga acatggctct
120tttttatgtg tcattatctt ag 142101228DNAGlycine maxFAD3-1A intron 4
10taacaaaaat aaatagaaaa tagtgggtga acacttaaat gcgagatagt aatacctaaa
60aaaagaaaaa aatataggta taataaataa tataactttc aaaataaaaa gaaatcatag
120agtctagcgt agtgtttgga gtgaaatgat gttcacctac cattactcaa
agattttgtt 180gtgtccctta gttcattctt attattttac atatcttact
tgaaaagact ttttaattat 240tcattgagat cttaaagtga ctgttaaatt
aaaataaaaa acaagtttgt taaaacttca 300aataaataag agtgaaggga
gtgtcatttg tcttctttct tttattgcgt tattaatcac 360gtttctcttc
tctttttttt ttttcttctc tgctttccac ccattatcaa gttcatgtga
420agcagtggcg gatctatgta aatgagtggg gggcaattgc acccacaaga
ttttattttt 480tatttgtaca ggaataataa aataaaactt tgcccccata
aaaaataaat attttttctt 540aaaataatgc aaaataaata taagaaataa
aaagagaata aattattatt aattttatta 600ttttgtactt tttatttagt
ttttttagcg gttagatttt tttttcatga cattatgtaa 660tcttttaaaa
gcatgtaata tttttatttt gtgaaaataa atataaatga tcatattagt
720ctcagaatgt ataaactaat aataatttta tcactaaaag aaattctaat
ttagtccata 780aataagtaaa acaagtgaca attatatttt atatttactt
aatgtgaaat aatacttgaa 840cattataata aaacttaatg acaggagata
ttacatagtg ccataaagat attttaaaaa 900ataaaatcat taatacactg
tactactata taatattcga tatatatttt taacatgatt 960ctcaatagaa
aaattgtatt gattatattt tattagacat gaatttacaa gccccgtttt
1020tcatttatag ctcttacctg tgatctattg ttttgcttcg ctgtttttgt
tggtcaaggg 1080acttagatgt cacaatatta atactagaag taaatattta
tgaaaacatg taccttacct 1140caacaaagaa agtgtggtaa gtggcaacac
acgtgttgca tttttggccc agcaataaca 1200cgtgtttttg tggtgtacta aaatggac
122811625DNAGlycine maxFAD3-1A intron 5 11gtacatttta ttgcttattc
acctaaaaac aatacaatta gtacatttgt tttatctctt 60ggaagttagt cattttcagt
tgcatgattc taatgctctc tccattctta aatcatgttt 120tcacacccac
ttcatttaaa ataagaacgt gggtgttatt ttaatttcta ttcactaaca
180tgagaaatta acttatttca agtaataatt ttaaaatatt tttatgctat
tattttatta 240caaataatta tgtatattaa gtttattgat tttataataa
ttatattaaa attatatcga 300tattaatttt tgattcactg atagtgtttt
atattgttag tactgtgcat ttattttaaa 360attggcataa ataatatatg
taaccagctc actatactat actgggagct tggtggtgaa 420aggggttccc
aaccctcctt tctaggtgta catgctttga tacttctggt accttcttat
480atcaatataa attatatttt gctgataaaa aaacatggtt aaccattaaa
ttcttttttt 540aaaaaaaaaa ctgtatctaa actttgtatt attaaaaaga
agtctgagat taacaataaa 600ctaacactca tttggattca ctgca
6251298DNAGlycine maxFAD3-1A intron 3B 12ggtgagtgat tttttgactt
ggaagacaac aacacattat tattataata tggttcaaaa 60caatgacttt ttctttatga
tgtgaactcc atttttta 9813115DNAGlycine maxFAD3-1A intron 3C
13ggtaactaaa ttactcctac attgttactt tttcctcctt ttttttatta tttcaattct
60ccaattggaa atttgaaata gttaccataa ttatgtaatt gtttgatcat gtgca
115141037DNAGlycine maxFad3-1C intron 4 14gtaacaaaaa taaatagaaa
atagtgagtg aacacttaaa tgttagatac taccttcttc 60ttcttttttt tttttttttt
gaggttaatg ctagataata gctagaaaga gaaagaaaga 120caaatatagg
taaaaataaa taatataacc tgggaagaag aaaacataaa aaaagaaata
180atagagtcta cgtaatgttt ggatttttga gtgaaatggt gttcacctac
cattactcaa 240agattctgtt gtctacgtag tgtttggact ttggagtgaa
atggtgttca cctaccatta 300ctcagattct gttgtgtccc ttagttactg
tcttatattc ttagggtata ttctttattt 360tacatccttt tcacatctta
cttgaaaaga ttttaattat tcattgaaat attaacgtga 420cagttaaatt
aaaataataa aaaattcgtt aaaacttcaa ataaataaga gtgaaaggat
480catcattttt cttctttctt ttattgcgtt attaatcatg cttctcttct
tttttttctt 540cgctttccac ccatatcaaa ttcatgtgaa gtatgagaaa
atcacgattc aatggaaagc 600tacaggaacy ttttttgttt tgtttttata
atcggaatta atttatactc cattttttca 660caataaatgt tacttagtgc
cttaaagata atatttgaaa aattaaaaaa attattaata 720cactgtacta
ctatataata tttgacatat atttaacatg attttctatt gaaaatttgt
780atttattatt ttttaatcaa aacccataag gcattaattt acaagaccca
tttttcattt 840atagctttac ctgtgatcat ttatagcttt aagggactta
gatgttacaa tcttaattac 900aagtaaatat ttatgaaaaa catgtgtctt
accccttaac cttacctcaa caaagaaagt 960gtgataagtg gcaacacacg
tgttgctttt ttggcccagc aataacacgt gtttttgtgg 1020tgtacaaaaa tggacag
1037154010DNAGlycine maxpartial FAD3-1A genomic clone 15acaaagcctt
tagcctatgc tgccaataat ggataccaac aaaagggttc ttcttttgat 60tttgatccta
gcgctcctcc accgtttaag attgcagaaa tcagagcttc aataccaaaa
120cattgctggg tcaagaatcc atggagatcc ctcagttatg ttctcaggga
tgtgcttgta 180attgctgcat tggtggctgc agcaattcac ttcgacaact
ggcttctctg gctaatctat 240tgccccattc aaggcacaat gttctgggct
ctctttgttc ttggacatga ttggtaataa 300tttttgtgtt tcttactctt
tttttttttt ttttgtttat gatatgaatc tcacacattg 360ttctgttatg
tcatttcttc ttcatttggc tttagacaac ttaaatttga gatctttatt
420atgtttttgc ttatatggta aagtgattct tcattatttc attcttcatt
gattgaattg 480aacagtggcc atggaagctt ttcagatagc cctttgctga
atagcctggt gggacacatc 540ttgcattcct caattcttgt gccataccat
ggatggttag ttcatactgg cttttttgtt 600tgttcatttg tcattgaaaa
aaaatctttt gttgattcaa ttatttttat agtgtgtttg 660gaagcccgtt
tgagaaaata agaaatcgca tctggaatgt gaaagttata actatttagc
720ttcatctgtc gttgcaagtt cttttattgg ttaaattttt atagcgtgct
aggaaaccca 780ttcgagaaaa taagaaatca catctggaat gtgaaagtta
taactgttag cttctgagta 840aacgtggaaa aaccacattt tggatttgga
accaaatttt atttgataaa tgacaaccaa 900attgattttg atggattttg
caggagaatt agccacagaa ctcaccatga aaaccatgga 960cacattgaga
aggatgagtc atgggttcca gtatgtgatt aattgcttct cctatagttg
1020ttcttgattc aattacattt tatttatttg gtaggtccaa gaaaaaaggg
aatctttatg 1080cttcctgagg ctgttcttga acatggctct tttttatgtg
tcattatctt agttaacaga 1140gaagatttac aagaatctag acagcatgac
aagactcatt agattcactg tgccatttcc 1200atgtttgtgt atccaattta
tttggtgagt gattttttga cttggaagac aacaacacat 1260tattattata
atatggttca aaacaatgac tttttcttta tgatgtgaac tccatttttt
1320agttttcaag aagccccgga aaggaaggct ctcacttcaa tccctacagc
aatctgtttc 1380cacccagtga gagaaaagga atagcaatat caacactgtg
ttgggctacc atgttttctc 1440tgcttatcta tctctcattc attaactagt
ccacttctag tgctcaagct ctatggaatt 1500ccatattggg taactaaatt
actcctacat tgttactttt tcctcctttt ttttattatt 1560tcaattctcc
aattggaaat ttgaaatagt taccataatt atgtaattgt ttgatcatgt
1620gcagatgttt gttatgtggc tggactttgt cacatacttg catcaccatg
gtcaccacca 1680gaaactgcct tggtaccgcg gcaaggtaac aaaaataaat
agaaaatagt gggtgaacac 1740ttaaatgcga gatagtaata cctaaaaaaa
gaaaaaaata taggtataat aaataatata 1800actttcaaaa taaaaagaaa
tcatagagtc tagcgtagtg tttggagtga aatgatgttc 1860acctaccatt
actcaaagat tttgttgtgt cccttagttc attcttatta ttttacatat
1920cttacttgaa aagacttttt aattattcat tgagatctta aagtgactgt
taaattaaaa 1980taaaaaacaa gtttgttaaa acttcaaata aataagagtg
aagggagtgt catttgtctt 2040ctttctttta ttgcgttatt aatcacgttt
ctcttctctt tttttttttt cttctctgct 2100ttccacccat tatcaagttc
atgtgaagca gtggcggatc tatgtaaatg agtggggggc 2160aattgcaccc
acaagatttt attttttatt tgtacaggaa taataaaata aaactttgcc
2220cccataaaaa ataaatattt tttcttaaaa taatgcaaaa taaatataag
aaataaaaag 2280agaataaatt attattaatt ttattatttt gtacttttta
tttagttttt ttagcggtta 2340gatttttttt tcatgacatt atgtaatctt
ttaaaagcat gtaatatttt tattttgtga 2400aaataaatat aaatgatcat
attagtctca gaatgtataa actaataata attttatcac 2460taaaagaaat
tctaatttag tccataaata agtaaaacaa gtgacaatta tattttatat
2520ttacttaatg tgaaataata cttgaacatt ataataaaac ttaatgacag
gagatattac 2580atagtgccat aaagatattt taaaaaataa aatcattaat
acactgtact actatataat 2640attcgatata tatttttaac atgattctca
atagaaaaat tgtattgatt atattttatt 2700agacatgaat ttacaagccc
cgtttttcat ttatagctct tacctgtgat ctattgtttt 2760gcttcgctgt
ttttgttggt caagggactt agatgtcaca atattaatac tagaagtaaa
2820tatttatgaa aacatgtacc ttacctcaac aaagaaagtg tggtaagtgg
caacacacgt 2880gttgcatttt tggcccagca ataacacgtg tttttgtggt
gtactaaaat ggacaggaat 2940ggagttattt aagaggtggc ctcaccactg
tggatcgtga ctatggttgg atcaataaca 3000ttcaccatga cattggcacc
catgttatcc
accatctttt cccccaaatt cctcattatc 3060acctcgttga agcggtacat
tttattgctt attcacctaa aaacaataca attagtacat 3120ttgttttatc
tcttggaagt tagtcatttt cagttgcatg attctaatgc tctctccatt
3180cttaaatcat gttttcacac ccacttcatt taaaataaga acgtgggtgt
tattttaatt 3240tctattcact aacatgagaa attaacttat ttcaagtaat
aattttaaaa tatttttatg 3300ctattatttt attacaaata attatgtata
ttaagtttat tgattttata ataattatat 3360taaaattata tcgatattaa
tttttgattc actgatagtg ttttatattg ttagtactgt 3420gcatttattt
taaaattggc ataaataata tatgtaacca gctcactata ctatactggg
3480agcttggtgg tgaaaggggt tcccaaccct cctttctagg tgtacatgct
ttgatacttc 3540tggtaccttc ttatatcaat ataaattata ttttgctgat
aaaaaaacat ggttaaccat 3600taaattcttt ttttaaaaaa aaaactgtat
ctaaactttg tattattaaa aagaagtctg 3660agattaacaa taaactaaca
ctcatttgga ttcactgcag acacaagcag caaaaccagt 3720tcttggagat
tactaccgtg agccagaaag atctgcgcca ttaccatttc atctaataaa
3780gtatttaatt cagagtatga gacaagacca cttcgtaagt gacactggag
atgttgttta 3840ttatcagact gattctctgc tcctccactc gcaacgagac
tgagtttcaa actttttggg 3900ttattattta ttgattctag ctactcaaat
tacttttttt ttaatgttat gttttttgga 3960gtttaacgtt ttctgaacaa
cttgcaaatt acttgcatag agagacatgg 401016184DNAGlycine maxFAD3-1A
3'UTR 16gtttcaaact ttttgggtta ttatttattg gattctagct actcaaatta
cttttttttt 60aatgttatgt tttttggagt ttaacgtttt ctgaacaact tgcaaattac
ttgcatagag 120agacatggaa tatttatttg aaattagtaa ggtagtaata
ataaattttg aattgtcagt 180ttca 18417143DNAGlycine maxFAD3-1A 5'UTR
17tgcggttata taaatgcact atcccataag agtatttttc gaagatttcc ttcttcctat
60tctaggtttt tacgcaccac gtatccctga gaaaagagag gaaccacact ctctaagcca
120aagcaaaagc agcagcagca gca 143182683DNAGlycine maxpartial FAD3-1B
genomic clone 18gttcaagcac agcctctaca acatgttggt aatggtgcag
ggaaagaaga tcaagcttat 60tttgatccaa gtgctccacc acccttcaag attgcaaata
tcagagcagc aattccaaaa 120cattgctggg agaagaacac attgagatct
ctgagttatg ttctgaggga tgtgttggta 180gtgactgcat tggtagctgc
agcaatcggc ttcaatagct ggttcttctg gccactctat 240tggcctgcac
aaggcacaat gttttgggca ctttttgttc ttggacatga ttggtaacta
300attattatta caaattgtta tgttatgtta tgttatgttg ttgtgccttt
ttctcagtga 360tgctttagtc atttcatttc acttggttat gcatgattgt
tcgttcatat gttctgtcat 420ggtgagttct aatttgattg atgcatggaa
cagtggtcat ggaagttttt caaacagtcc 480tttgttgaac agcattgtgg
gccacatctt gcactcttca attcttgtac cataccatgg 540atggtcggtt
ccttttagca acttttcatg ttcactttgt ccttaaattt ttttttatgt
600ttgttaaaaa atctttggtc tgatttaaca acctaaccat ttttacaact
catggatttt 660ttgcaggaga attagccaca ggactcacca tcagaaccat
ggccatgttg agaaggatga 720atcatgggtt ccggtattac tatgagtttg
cttgattaat ttccacattt tttctttctt 780cttaatttta atcagtggtt
agatttggtt gtgttccgat agaagaaaag ggggtatcta 840gagagatgtg
aatttcatga agtggttcat gattatgtgt ctttatgcct ttatgtcagc
900ttacagagaa agtttacaag aatctagaca acatgacaag aatgatgaga
ttcactcttc 960ctttccccat ctttgcatac cccttttatt tggtgagacc
ctctttttcc agaatgacag 1020cattatttta ctatatagta cctcaatttt
tatatttcta aaattttgaa ttcttgaaat 1080tgaaaggaaa ggactttatt
gggtctagca tctcactctc tctttgtgat atgaaccata 1140tatttcagtg
gagcagaagc cctggaaaag aaggctctca tttcaaccct tacagcaact
1200tgttctctcc tggtgagaga agagatgtgc taacttcaac tctatgttgg
ggcatcatgc 1260tttctgtgct tctctatctt tccctcacaa tgggtccact
ttttatgctc aagctctatg 1320gggttcccta tttggtaatc tcactctcac
actttcttta tacatcgcac gccagtgtgg 1380gttatttgca acctacaccg
aagtaatgcc ctataattaa tgaggttaac acatgtccaa 1440gtccaatatt
ttgttcactt atttgaactt gaacatgtgt agatcttcgt catgtggctg
1500gatttcgtca cgtacttgca tcatcatggt tacaagcaga aactgccttg
gtaccgtggc 1560caggtatccc atttaacaca atttgtttca ttaacatttt
aagagaattt ttttttcaaa 1620atagttttcg aaattaagca aataccaagc
aaattgttag atctacgctt gtacttgttt 1680taaagtcaaa ttcatgacca
aattgtcctc acaagtccaa accgtccact attttatttt 1740cacctacttt
atagcccaat ttgccatttg gttacttcag aaaagagaac cccatttgta
1800gtaaatatat tatttatgaa ttatggtagt ttcaacataa aacatactta
tgtgcagttt 1860tgccatcctt caaaagaagg tagaaactta ctccatgtta
ctctgtctat atgtaatttc 1920acaggaatgg agttatctaa ggggtggtct
tacaacagta gatcgcgact atggttggat 1980caacaacatt caccatgaca
ttggcaccca tgttatccat caccttttcc ctcaaattcc 2040acattatcat
ttaatcgaag cggtattaat tctctatttc acaagaaatt attgtatgtc
2100tgcctatgtg atctaagtca attttcacat aacacatgat caaactttct
taattctttc 2160ttctaaattg aaaaagtgga ttatatgtca attgaaaatt
ggtcaagacc acaaacatgt 2220gatgatctcc caccttacat ataataattt
ctcctattct acaatcaata atccttctat 2280ggtcctgaat tgttcctttc
ttttttcatt ttcttattct ttttgttgtc ccacaataga 2340ctaaagcagc
aaaggcagtg ctaggaaagt attatcgtga gcctcagaaa tctgggccat
2400tgccacttca tctaataaag tacttgctcc acagcataag tcaggatcac
ttcgttagcg 2460actctggcga cattgtgtac taccagactg attcccagct
ccacaaagat tcttggaccc 2520agtccaacta aagtttttga tgctacattt
acctatttca ctcttaaata ctatttccta 2580tgtaatatgt aatttagaat
atgttaccta ctcaaatcaa ttaggtgaca tgtataagct 2640ttcataaatt
atgctagaaa tgcacttact tttcaaagca tgc 268319160DNAGlycine maxFAD3-1B
intron 1 19gtaactaatt attattacaa attgttatgt tatgttatgt tatgttgttg
tgcctttttc 60tcagtgatgc tttagtcatt tcatttcact tggttatgca tgattgttcg
ttcatatgtt 120ctgtcatggt gagttctaat ttgattgatg catggaacag
16020119DNAGlycine maxFAD3-1B intron 2 20gttcctttta gcaacttttc
atgttcactt tgtccttaaa ttttttttta tgtttgttaa 60aaaatctttg gtctgattta
acaacctaac catttttaca actcatggat tttttgcag 11921166DNAGlycine
maxFAD3-1B intron 3a 21gtattactat gagtttgctt gattaatttc cacatttttt
ctttcttctt aattttaatc 60agtggttaga tttggttgtg ttccgataga agaaaagggg
gtatctagag agatgtgaat 120ttcatgaagt ggttcatgat tatgtgtctt
tatgccttta tgtcag 16622156DNAGlycine maxFAD3-1B intron 3b
22gtgagaccct ctttttccag aatgacagca ttattttact atatagtacc tcaattttta
60tatttctaaa attttgaatt cttgaaattg aaaggaaagg actttattgg gtctagcatc
120tcactctctc tttgtgatat gaaccatata tttcag 15623148DNAGlycine
maxFAD3-1B intron 3c 23gtaatctcac tctcacactt tctttataca tcgcacgcca
gtgtgggtta tttgcaacct 60acaccgaagt aatgccctat aattaatgag gttaacacat
gtccaagtcc aatattttgt 120tcacttattt gaacttgaac atgtgtag
14824351DNAGlycine maxFAD3-1B intron 4 24taacacaatt tgtttcatta
acattttaag agaatttttt tttcaaaata gttttcgaaa 60ttaagcaaat accaagcaaa
ttgttagatc tacgcttgta cttgttttaa agtcaaattc 120atgaccaaat
tgtcctcaca agtccaaacc gtccactatt ttattttcac ctactttata
180gcccaatttg ccatttggtt acttcagaaa agagaacccc atttgtagta
aatatattat 240ttatgaatta tggtagtttc aacataaaac atacttatgt
gcagttttgc catccttcaa 300aagaaggtag aaacttactc catgttactc
tgtctatatg taatttcaca g 35125277DNAGlycine maxFAD3-1B intron 5
25gtattaattc tctatttcac aagaaattat tgtatgtctg cctatgtgat ctaagtcaat
60tttcacataa cacatgatca aactttctta attctttctt ctaaattgaa aaagtggatt
120atatgtcaat tgaaaattgg tcaagaccac aaacatgtga tgatctccca
ccttacatat 180aataatttct cctattctac aatcaataat ccttctatgg
tcctgaattg ttcctttctt 240ttttcatttt cttattcttt ttgttgtccc acaatag
27726158DNAGlycine maxFAD3-1B 3'UTR 26agtttttgat gctacattta
cctatttcac tcttaaatac tatttcctat gtaatatgta 60atttagaata tgttacctac
tcaaatcaat taggtgacat gtataagctt tcataaatta 120tgctagaaat
gcacttactt ttcaaagcat gctatgtc 1582783DNAGlycine maxFAD3-1B 5'UTR
27tctaatacga ctcactatag ggcaagcagt ggtatcaacg cagagtacgc gggggtaaca
60gagaaagaaa catttgagca aaa 83284083DNAGlycine maxFATB genomic
clone 28gggaaacaac aaggacgcaa aatgacacaa tagcccttct tccctgtttc
cagcttttct 60ccttctctct ctccatcttc ttcttcttct tcactcagtc aggtacgcaa
acaaatctgc 120tattcattca ttcattcctc tttctctctg atcgcaaact
gcacctctac gctccactct 180tctcattttc tcttcctttc tcgcttctca
gatccaactc ctcagataac acaagaccaa 240acccgctttt tctgcatttc
tagactagac gttctaccgg agaaggttct cgattctttt 300ctcttttaac
tttattttta aaataataat aatgagagct ggatgcgtct gttcgttgtg
360aatttcgagg caatggggtt ctcattttcg ttacagttac agattgcatt
gtctgctttc 420ctcttctccc ttgtttcttt gccttgtctg atttttcgtt
tttatttctt acttttaatt 480tttggggatg gatatttttt ctgcattttt
tcggtttgcg atgttttcag gattccgatt 540ccgagtcaga tctgcgccgg
cttatacgac gaatttgttc ttattcgcaa cttttcgctt 600gattggcttg
ttttacctct ggaatctcac acgtgatcaa ataagcctgc tattttagtt
660gaagtagaat ttgttcttta tcggaaagaa ttctatggat ctgttctgaa
attggagcta 720ctgtttcgag ttgctatttt ttttagtagt attaagaaca
agtttgcctt ttattttaca 780tttttttcct ttgcttttgc caaaagtttt
tatgatcact ctcttctgtt tgtgatataa 840ctgatgtgct gtgctgttat
tatttgttat ttggggtgaa gtataatttt ttgggtgaac 900ttggagcatt
tttagtccga ttgatttctc gatatcattt aaggctaagg ttgacctcta
960ccacgcgttt gcgtttgatg ttttttccat ttttttttta tctcatatct
tttacagtgt 1020ttgcctattt gcatttctct tctttatccc ctttctgtgg
aaaggtggga gggaaaatgt 1080attttttttt tctcttctaa cttgcgtata
ttttgcatgc agcgacctta gaaattcatt 1140atggtggcaa cagctgctac
ttcatcattt ttccctgtta cttcaccctc gccggactct 1200ggtggagcag
gcagcaaact tggtggtggg cctgcaaacc ttggaggact aaaatccaaa
1260tctgcgtctt ctggtggctt gaaggcaaag gcgcaagccc cttcgaaaat
taatggaacc 1320acagttgtta catctaaaga aggcttcaag catgatgatg
atctaccttc gcctcccccc 1380agaactttta tcaaccagtt gcctgattgg
agcatgcttc ttgctgctat cacaacaatt 1440ttcttggccg ctgaaaagca
gtggatgatg cttgattgga agccacggcg acctgacatg 1500cttattgacc
cctttgggat aggaaaaatt gttcaggatg gtcttgtgtt ccgtgaaaac
1560ttttctatta gatcatatga gattggtgct gatcgtaccg catctataga
aacagtaatg 1620aaccatttgc aagtaagtcc gtcctcatac aagtgaatct
ttatgatctt cagagatgag 1680tatgctttga ctaagatagg gctgtttatt
tagacactgt aattcaattt catatataga 1740taatatcatt ctgttgttac
ttttcatact atatttatat caactatttg cttaacaaca 1800ggaaactgca
cttaatcatg ttaaaagtgc tgggcttctt ggtgatggct ttggttccac
1860gccagaaatg tgcaaaaaga acttgatatg ggtggttact cggatgcagg
ttgtggtgga 1920acgctatcct acatggttag tcatctagat tcaaccatta
catgtgattt gcaatgtatc 1980catgttaagc tgctatttct ctgtctattt
tagtaatctt tatgaggaat gatcactcct 2040aaatatattc atggtaatta
ttgagactta attatgagaa ccaaaatgct ttggaaattt 2100gtctgggatg
aaaattgatt agatacacaa gctttataca tgatgaacta tgggaaacct
2160tgtgcaacag agctattgat ctgtacaaga gatgtagtat agcattaatt
acatgttatt 2220agataaggtg acttatcctt gtttaattat tgtaaaaata
gaagctgata ctatgtattc 2280tttgcatttg ttttcttacc agttatatat
accctctgtt ctgtttgagt actactagat 2340gtataaagaa tgcaattatt
ctgacttctt ggtgttgggt tgaagttaga taagctatta 2400gtattattat
ggttattcta aatctaatta tctgaaattg tgtgtctata tttgcttcag
2460gggtgacata gttcaagtgg acacttgggt ttctggatca gggaagaatg
gtatgcgtcg 2520tgattggctt ttacgtgact gcaaaactgg tgaaatcttg
acaagagctt ccaggtagaa 2580atcattctct gtaattttcc ttcccctttc
cttctgcttc aagcaaattt taagatgtgt 2640atcttaatgt gcacgatgct
gattggacac aattttaaat ctttcaaaca tttacaaaag 2700ttatggaacc
ctttcttttc tctcttgaag atgcaaattt gtcacgactg aagtttgagg
2760aaatcatttg aattttgcaa tgttaaaaaa gataatgaac tacatatttt
gcaggcaaaa 2820acctctaatt gaacaaactg aacattgtat cttagtttat
ttatcagact ttatcatgtg 2880tactgatgca tcaccttgga gcttgtaatg
aattacatat tagcattttc tgaactgtat 2940gttatggttt tggtgatcta
cagtgtttgg gtcatgatga ataagctgac acggaggctg 3000tctaaaattc
cagaagaagt cagacaggag ataggatctt attttgtgga ttctgatcca
3060attctagaag aggataacag aaaactgact aaacttgacg acaacacagc
ggattatatt 3120cgtaccggtt taagtgtatg tcaactagtt tttttgtaat
tgttgtcatt aatttctttt 3180cttaaattat ttcagatgtt gctttctaat
tagtttacat tatgtatctt cattcttcca 3240gtctaggtgg agtgatctag
atatcaatca gcatgtcaac aatgtgaagt acattgactg 3300gattctggag
gtatttttct gttcttgtat tctaatccac tgcagtcctt gttttgttgt
3360taaccaaagg actgtccttt gattgtttgc agagtgctcc acagccaatc
ttggagagtc 3420atgagctttc ttccgtgact ttagagtata ggagggagtg
tggtagggac agtgtgctgg 3480attccctgac tgctgtatct ggggccgaca
tgggcaatct agctcacagt ggacatgttg 3540agtgcaagca tttgcttcga
ctcgaaaatg gtgctgagat tgtgaggggc aggactgagt 3600ggaggcccaa
acctatgaac aacattggtg ttgtgaacca ggttccagca gaaagcacct
3660aagattttga aatggttaac ggttggagtt gcatcagtct ccttgctatg
tttagactta 3720ttctggcctc tggggagagt tttgcttgtg tctgtccaat
caatctacat atctttatat 3780ccttctaatt tgtgttactt tggtgggtaa
gggggaaaag ctgcagtaaa cctcattctc 3840tctttctgct gctccatatt
tcatttcatc tctgattgcg ctactgctag gctgtcttca 3900atatttaatt
gcttgatcaa aatagctagg catgtatatt attattcttt tctcttggct
3960caattaaaga tgcaattttc attgtgaaca cagcataact attattctta
ttatttttgt 4020atagcctgta tgcacgaatg acttgtccat ccaatacaac
cgtgattgta tgctccagct 4080cag 408329109DNAGlycine maxFATB intron I
29gtacgcaaac aaatctgcta ttcattcatt cattcctctt tctctctgat cgcaaactgc
60acctctacgc tccactcttc tcattttctc ttcctttctc gcttctcag
10930836DNAGlycine maxFATB intron II 30gttctcgatt cttttctctt
ttaactttat ttttaaaata ataataatga gagctggatg 60cgtctgttcg ttgtgaattt
cgaggcaatg gggttctcat tttcgttaca gttacagatt 120gcattgtctg
ctttcctctt ctcccttgtt tctttgcctt gtctgatttt tcgtttttat
180ttcttacttt taatttttgg ggatggatat tttttctgca ttttttcggt
ttgcgatgtt 240ttcaggattc cgattccgag tcagatctgc gccggcttat
acgacgaatt tgttcttatt 300cgcaactttt cgcttgattg gcttgtttta
cctctggaat ctcacacgtg atcaaataag 360cctgctattt tagttgaagt
agaatttgtt ctttatcgga aagaattcta tggatctgtt 420ctgaaattgg
agctactgtt tcgagttgct atttttttta gtagtattaa gaacaagttt
480gccttttatt ttacattttt ttcctttgct tttgccaaaa gtttttatga
tcactctctt 540ctgtttgtga tataactgat gtgctgtgct gttattattt
gttatttggg gtgaagtata 600attttttggg tgaacttgga gcatttttag
tccgattgat ttctcgatat catttaaggc 660taaggttgac ctctaccacg
cgtttgcgtt tgatgttttt tccatttttt ttttatctca 720tatcttttac
agtgtttgcc tatttgcatt tctcttcttt atcccctttc tgtggaaggt
780gggagggaaa atgtattttt tttttctctt ctaacttgcg tatattttgc atgcag
83631169DNAGlycine maxFATB intron III 31gtaagtccgt cctcatacaa
gtgaatcttt atgatcttca gagatgagta tgctttgact 60aagatagggc tgtttattta
gacactgtaa ttcaatttca tatatagata atatcattct 120gttgttactt
ttcatactat atttatatca actatttgct taacaacag 16932525DNAGlycine
maxFATB intron IV 32gttagtcatc tagattcaac cattacatgt gatttgcaat
gtatccatgt taagctgcta 60tttctctgtc tattttagta atctttatga ggaatgatca
ctcctaaata tattcatggt 120aattattgag acttaattat gagaaccaaa
atgctttgga aatttgtctg ggatgaaaat 180tgattagata cacaagcttt
atacatgatg aactatggga aaccttgtgc aacagagcta 240ttgatctgta
caagagatgt agtatagcat taattacatg ttattagata aggtgactta
300tccttgttta attattgtaa aaatagaagc tgatactatg tattctttgc
atttgttttc 360ttaccagtta tatataccct ctgttctgtt tgagtactac
tagatgtata aagaatgcaa 420ttattctgac ttcttggtgt tgggttgaag
ttagataagc tattagtatt attatggtta 480ttctaaatct aattatctga
aattgtgtgt ctatatttgc ttcag 52533389DNAGlycine maxFATB intron V
33gtagaaatca ttctctgtaa ttttccttcc cctttccttc tgcttcaagc aaattttaag
60atgtgtatct taatgtgcac gatgctgatt ggacacaatt ttaaatcttt caaacattta
120caaaagttat ggaacccttt cttttctctc ttgaagatgc aaatttgtca
cgactgaagt 180ttgaggaaat catttgaatt ttgcaatgtt aaaaaagata
atgaactaca tattttgcag 240gcaaaaacct ctaattgaac aaactgaaca
ttgtatctta gtttatttat cagactttat 300catgtgtact gatgcatcac
cttggagctt gtaatgaatt acatattagc attttctgaa 360ctgtatgtta
tggttttggt gatctacag 38934106DNAGlycine maxFATB intron VI
34tatgtcaact agtttttttg taattgttgt cattaatttc ttttcttaaa ttatttcaga
60tgttgctttc taattagttt acattatgta tcttcattct tccagt
1063582DNAGlycine maxFATB intron VII 35gtatttttct gttcttgtat
tctaatccac tgcagtcctt gttttgttgt taaccaaagg 60actgtccttt gattgtttgc
ag 8236208DNAGlycine maxFATB 3'UTR 36gatttgaaat ggttaacgat
tggagttgca tcagtctcct tgctatgttt agacttattc 60tggttccctg gggagagttt
tgcttgtgtc tatccaatca atctacatgt ctttaaatat 120atacaccttc
taatttgtga tactttggtg ggtaaggggg aaaagcagca gtaaatctca
180ttctcattgt aattaaaaaa aaaaaaaa 20837229DNAGlycine maxFATB 5'UTR
37acaattacac tgtctctctc ttttccaaaa ttagggaaac aacaaggacg caaaatgaca
60caatagccct tcttccctgt ttccagcttt tctccttctc tctctctcca tcttcttctt
120cttcttcact cagtcagatc caactcctca gataacacaa gaccaaaccc
gctttttctg 180catttctaga ctagacgttc taccggagaa gcgaccttag aaattcatt
229381398DNACuphea pulcherrimaKAS I gene 38atgcattccc tccagtcacc
ctcccttcgg gcctccccgc tcgacccctt ccgccccaaa 60tcatccaccg tccgccccct
ccaccgagca tcaattccca acgtccgggc cgcttccccc 120accgtctccg
ctcccaagcg cgagaccgac cccaagaagc gcgtcgtgat caccggaatg
180ggccttgtct ccgttttcgg ctccgacgtc gatgcgtact acgacaagct
cctgtcaggc 240gagagcggga tcggcccaat cgaccgcttc gacgcctcca
agttccccac caggttcggc 300ggccagattc gtggcttcaa ctccatggga
tacattgacg gcaaaaacga caggcggctt 360gatgattgcc ttcgctactg
cattgtcgcc gggaagaagt ctcttgagga cgccgatctc 420ggtgccgacc
gcctctccaa gatcgacaag gagagagccg gagtgctggt tgggacagga
480atgggtggtc tgactgtctt ctctgacggg gttcaatctc ttatcgagaa
gggtcaccgg 540aaaatcaccc ctttcttcat cccctatgcc attacaaaca
tggggtctgc cctgctcgct 600attgaactcg gtctgatggg cccaaactat
tcaatttcca ctgcatgtgc cacttccaac 660tactgcttcc atgctgctgc
taatcatatc cgccgtggtg aggctgatct tatgattgct 720ggaggcactg
aggccgcaat cattccaatt gggttgggag gctttgtggc ttgcagggct
780ctgtctcaaa ggaacgatga ccctcagact gcctctaggc cctgggataa
agaccgtgat 840ggttttgtga tgggtgaagg tgctggagtg ttggtgctgg
agagcttgga acatgcaatg 900aaacgaggag cacctattat tgcagagtat
ttgggaggtg caatcaactg tgatgcttat 960cacatgactg acccaagggc
tgatggtctc ggtgtctcct cttgcattga gagtagcctt 1020gaagatgctg
gcgtctcacc tgaagaggtc aattacataa atgctcatgc gacttctact
1080ctagctgggg atctcgccga gataaatgcc atcaagaagg ttttcaagaa
cacaaaggat 1140atcaaaatta atgcaactaa gtcaatgatc ggacactgtc
ttggagcctc tggaggtctt 1200gaagctatag cgactattaa gggaataaac
accggctggc ttcatcccag cattaatcaa 1260ttcaatcctg agccatccgt
ggagttcgac actgttgcca acaagaagca gcaacacgaa 1320gttaatgttg
cgatctcgaa ttcatttgga ttcggaggcc acaactcagt cgtggctttc
1380tcggctttca agccatga
1398391218DNACuphea pulcherrima 39atgggtgtgg tgactcctct aggccatgac
cctgatgttt tctacaataa tctgcttgat 60ggaacgagtg gcataagcga gatagagacc
tttgattgtg ctcaatttcc tacgagaatt 120gctggagaga tcaagtcttt
ctccacagat ggttgggtgg ccccgaagct ctctaagagg 180atggacaagt
tcatgctata catgctgacc gctggcaaga aagcattaac agatggtgga
240atcaccgaag atgtgatgaa agagctagat aaaagaaaat gcggagttct
cattggctca 300gcaatgggtg gaatgaaggt attcaatgat gccattgaag
ccctaaggat ttcatataag 360aagatgaatc ccttttgtgt acctttcgct
accacaaata tgggatcagc tatgcttgca 420atggacttgg gatggatggg
gcccaactac tcgatatcta ctgcttgtgc aacgagtaac 480ttttgtataa
tgaatgctgc gaaccatata atcagaggcg aagcagatgt gatgctttgc
540gggggctcag atgcggtaat catacctatt ggtatgggag gttttgttgc
atgccgagct 600ttgtcccaga gaaattccga ccctactaaa gcttcaagac
catgggacag taatcgtgat 660ggatttgtta tgggggaagg agctggagtg
ctactactag aggagttgga gcatgcaaag 720aaaagaggtg cgactattta
cgcagaattt ctaggtggga gtttcacttg cgatgcctac 780cacatgaccg
agcctcaccc tgatggagct ggagtgattc tctgcataga gaaggctttg
840gctcagtcag gagtctctag ggaagacgta aattacataa atgcccatgc
cacatccact 900ccggctggag atatcaaaga gtaccaagct cttatccact
gtttcggcca aaacagagag 960ttaaaagtta attcaaccaa atcaatgatt
ggtcaccttc tcggagcagc cggtggtgtg 1020gaagcagttt cagtagttca
ggcaataagg actgggtgga tccatccgaa tattaatttg 1080gaaaacccag
atgaaggcgt ggatacaaaa ttgctcgtgg gtcctaagaa ggagagactg
1140aacgttaagg tcggtttgtc taattcattt gggtttggtg ggcacaactc
gtccatactc 1200ttcgcccctt acatctag 1218401191DNARicinus
communisdelta-9 desaturase 40atggctctca agctcaatcc tttcctttct
caaacccaaa agttaccttc tttcgctctt 60ccaccaatgg ccagtaccag atctcctaag
ttctacatgg cctctaccct caagtctggt 120tctaaggaag ttgagaatct
caagaagcct ttcatgcctc ctcgggaggt acatgttcag 180gttacccatt
ctatgccacc ccaaaagatt gagatcttta aatccctaga caattgggct
240gaggagaaca ttctggttca tctgaagcca gttgagaaat gttggcaacc
gcaggatttt 300ttgccagatc ccgcctctga tggatttgat gagcaagtca
gggaactcag ggagagagca 360aaggagattc ctgatgatta ttttgttgtt
ttggttggag acatgataac ggaagaagcc 420cttcccactt atcaaacaat
gctgaatacc ttggatggag ttcgggatga aacaggtgca 480agtcctactt
cttgggcaat ttggacaagg gcatggactg cggaagagaa tagacatggt
540gacctcctca ataagtatct ctacctatct ggacgagtgg acatgaggca
aattgagaag 600acaattcaat atttgattgg ttcaggaatg gatccacgga
cagaaaacag tccatacctt 660gggttcatct atacatcatt ccaggaaagg
gcaaccttca tttctcatgg gaacactgcc 720cgacaagcca aagagcatgg
agacataaag ttggctcaaa tatgtggtac aattgctgca 780gatgagaagc
gccatgagac agcctacaca aagatagtgg aaaaactctt tgagattgat
840cctgatggaa ctgttttggc ttttgctgat atgatgagaa agaaaatttc
tatgcctgca 900cacttgatgt atgatggccg agatgataat ctttttgacc
acttttcagc tgttgcgcag 960cgtcttggag tctacacagc aaaggattat
gcagatatat tggagttctt ggtgggcaga 1020tggaaggtgg ataaactaac
gggcctttca gctgagggac aaaaggctca ggactatgtt 1080tgtcggttac
ctccaagaat tagaaggctg gaagagagag ctcaaggaag ggcaaaggaa
1140gcacccacca tgcctttcag ctggattttc gataggcaag tgaagctgta g
1191411194DNASimmondsia chinensisdelta-9 desaturase 41atggcgttga
agcttcacca cacggccttc aatccttcca tggcggttac ctcttcggga 60cttcctcgat
cgtatcacct cagatctcac cgcgttttca tggcttcttc tacaattgga
120attacttcta aggagatacc caatgccaaa aagcctcaca tgcctcctag
agaagctcat 180gtgcaaaaga cccattcaat gccgcctcaa aagattgaga
ttttcaaatc cttggagggt 240tgggctgagg agaatgtctt ggtgcatctt
aaacctgtgg agaagtgttg gcaaccacaa 300gattttctac ccgacccggc
ctccgaggga tttatggatc aagtcaagga gttgagggaa 360agaaccaaag
aaatcccgga tgagtacctt gtggtgttgg ttggcgatat gatcactgaa
420gaagctcttc cgacctacca gacgatgcta aacacgctcg atggagtacg
tgatgagacg 480ggtgccagcc ttacttcttg ggctatctgg acccgggcat
ggaccgctga agagaatagg 540cacggtgatc ttttgaacaa gtatctttac
cttactggtc gagttgacat gaagcagata 600gagaagacaa tccagtatct
aatcggatct ggaatggacc ctcgaagtga aaacaacccc 660tatctaggct
tcatctacac ttccttccaa gagagagcaa ccttcatctc ccatggaaac
720accgctaggc tcgccaaaga ccacggcgac tttcaactag cacaagtatg
tggcatcatc 780gctgcagatg agaagcgcca cgaaactgcc tacacaaaaa
ttgtcgaaaa gctctttgaa 840atcgacccag acggcgctgt tctagcacta
gctgacatga tgagaaagaa ggtttccatg 900ccagcccact taatgtatga
tggcaaagat gacaatctct ttgagaacta ctcagccgtc 960gctcaacaaa
ttggagttta caccgcgaag gactacgctg acatcctcga acacctcgtt
1020aatcgctgga aagtcgagaa tttaatgggt ctgtctggcg agggacataa
ggctcaagat 1080ttcgtatgtg ggttggcccc gaggatcagg aaactcgggg
agagagctca gtcgctaagc 1140aaaccggtat ctcttgtccc cttcagctgg
attttcaaca aggaattgaa ggtt 1194
* * * * *