Note: Descriptions are shown in the official language in which they were submitted.
CA 02547678 1996-07-03
PATENT
ATTORNEY DOCKET N0: 07148/042CA1
CANOLA OIL HAVING INCREASED OLEIC ACID AND
DECREASED LINOLENIC ACID CONTENT
Technical Field
This invention relates to a Brassica canola oil
having an elevated oleic acid content and a decreased
linolenic .acid profile in the seed oil. The invention also
relates to methods by which such an oil may be produced.
Background of the Invention
DiEas high in saturated fats increase low density
lipoproteins (LDL) which mediate the deposition of
cholesterol on blood vessels. High plasma levels of serum
cholesterol are closely correlated with atherosclerosis and
coronary heart disease (Conner et al., Coronary Heart
Disease: Prevention, Complications, and Treatment, pp. 43-
64, 1985). By producing oilseed Brassica varieties with
reduced levels of individual and total saturated fats in the
seed oil, oil-based food products which contain less
saturated fats can be produced. Such products will benefit
public health by reducing the incidence of atherosclerosis
and coronary heart disease.
The dietary effects of monounsaturated fats have
also been shown to have dramatic effects on health. Oleic '
acid, the only monounsaturated fat in most edible vegetable
oils, lowers LDL as effectively as linoleic acid, but does
not affect high density lipoproteins (HDL) levels (Mattson,
F.H., J. Am. Diet. Assoc., 89:387-391, 1989; Mensink et al.,
New England J. Med., 321:436-441, 1989). Oleic acid is at
least as effective in lowering plasma cholesterfll as a diet
low in fat and high in carbohydrates (Grundy, S.M., New
England J. Med., 314:745-748, 1986; Mensink et al.,~~Nev~i ' ' ' ' '
England J. Med., 321:436-441, 1989). In fact, a high oleic
acid diet is preferable to low fat, high carbohydrate diets
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for diabetics (Garg et al., New England J. Med., 319:829-
834, 1988). Diets high in monounsaturated fats are also
correlated with reduced systolic blood pressure (Williams et
al., J. Am. Med. Assoc., 257:3251-3256, 1987)
Epidemiological studies have demonstrated that the
"Mediterranean" diet, which is high in fat and
monounsaturates, is not associated with coronary heart
disease.
Intensive breeding has produced Brassica plants
whose seed oil contains less than 2% erucic acid. The same
varieties have also been bred so that the defatted meal
contains less than 30 ~mol glucosinolates/gram. Brassica
seeds, or oils extracted from Brassica seeds, that contain
less than 2% erucic acid (Cz2:1), and produce a meal with
less than,30 ~,mol glucosinolates/gram are referred to as
canola seeds or canola oils. Plant lines producing such
seeds are also referred to as canola lines or varieties.
Many breeding studies have been directed to
alteration of the fatty acid composition in seeds of
Brassica varieties. For example, Pleines and Freidt, Fat
Sci. Technol., 90(5), 167-171 (1988) describe plant lines
with reduced C18,3 levels (2.5-5.8%) combined with high oleic
content ('73-79%) . Roy and Tarr, Z. Pflanzenzuchtg, 95 (3) ,
201-209 (1985) teaches .transfer of genes through an
interspec_ific cross from Brassica juncea into Brassica napus
resulting in a reconstituted line combining high linoleic
with low linolenic acid content. Roy and Tarr, Plant
Breeding, 98, 89-96 (1987) discuss prospects for development
of B. napus L. having improved linolenic and linolenic acid
content. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses
the effect of temperature on the fatty acid composition of
oils from several seed crops including rapeseed.
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Mutations can be induced with extremely high doses
of radiation and/or chemical mutagens (Gaul, H. Radiation
Botany (1964) 4:155-232). High dose levels which exceed
LD50, and typically reach LD90, led to maximum achievable
mutation rates. In mutation breeding of Brassica varieties,
high levels of chemical mutagens alone or combined with
radiation have induced a limited number of fatty acid
mutations (Rakow, G.Z. Pflanzenzuchtg~(1973) 69:62-82).
Ral~;ow and McGregor, J. Amer. Oil Chem. Soc., 50,
400-403 (Oct. 1973) discuss problems associated with
selecting mutants affecting seed linoleic and linolenic acid
levels: T'he low a-linolenic acid mutation derived from the
Rakow mutation breeding program did not have direct
commercial application because of low seed yield. The first
commercial. cultivar using the low a-linolenic acid mutation
derived in 1973 was released in 1988 as the variety Stellar
(Scarth, R. et al., Can. J. Plant Sci. (1988) 68:509-511).
The a-linolenic acid content of Stellar seeds was greater
than 3% and the linoleic acid content was about 28%.
Chemical and/or radiation mutagenesis has been used
in an attempt to develop an endogenous canola oil having an
oleic acid content of greater than 79% and an a-linolenic
acid content of less than 5%. along, et .al., EP 0 323 753
B1. However, the lowest a-linolenic acid level achieved was
about 2.7%. PCT publication WO 91/05910 discloses
mutagenesis of a starting Brassica napus line in order to
increase the oleic acid content in the seed oil. However,
the oleic acid content in canola oil extracted from seeds of
such mutant lines did not exceed 80%.
The quality of canola oil and its suitability for
different end uses is in large measure determined by the
Irelative proportion of the various fatty acids present in
the seed triacylglycerides. As an example, the oxidative
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stability of canola oil, especially at high temperatures, decreases as the
proportion of
tn-unsaturated acids increases. Oxidative stability decreases to a lesser
extent as
the proportion of di-unsaturated acids increases. However, it has not been
possible
to alter the fatty acid composition in Brassica seeds beyond certain limits.
Thus, an
endogenous canola oil having altered fatty acid compositions in seeds is not
available fur certain specialty uses. Instead, such specialty oils typically
are prepared
from canola oil by further processing, such as hydrogenation and/or
fractionation.
Summar)i of the Invention
In accordance with an aspect of the invention, An endogenous 8rassica seed
oil, said oil has an oleic acid content of greater than about 80%, a linoleic
acid
content of about 2% to about 6%, an a-linolenic acid content of less than 2.5%
and
an erucic acid content of less than about 2%, said oleic acid content,
linoleic acid
content a-linolenic acid content and erucic acid content determined after
hydrolysis
15 of said oil.
In accordance with an aspect of the present invention there is provided a
Brassica plant cell containing at least one recombinant nucleic acid
construct, said
construct comprising at least one seed-specific regulatory sequence operably
linked
in sense orientation to a mutant delta-12 fatty acid desaturase gene coding
2o sequence, said plant cell exhibiting a seed-specific reduction in native
delta-12
desaturase gene expression.
In accordance with another aspect of the invention, crushed seeds, said
seeds containing at least one recombinant nucleic acid construct, said at
least one
construct comprises:
25 a) a regulatory sequence fragment operably linked to a wild-type
nticrosomal delta-12 fatty acid desaturase coding sequence fragment; and
b) a regulatory sequence fragment operably linked to a wild-type
microsomal delta-15 fatty acid desaturase coding sequence fragment, said seeds
yielding an oil having an oleic acid content of about 86% or greater and an
erucic
3o acid content of less than about 2%, said oleic acid content and erucic acid
content
determined after hydrolysis of said oil.
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In accordance with a further aspect of the invention, crushed seeds, said
seeds coni:aining at least one recombinant nucleic acid construct, said at
least
one construct comprises:
a) a regulatory sequence fragment operably linked to a wild-type
s microsomal delta-12 fatty acid desaturase coding sequence fragment; and
b) a regulatory sequence fragment operably linked to a wild-type
microsomal delta-15 fatty acid desaturase coding sequence fragment.
said seeds yielding an. oil having an oleic acid content of 80% or greater, an
a-
linolenic acid content of about 2.5% or less and an erucic acid content of
less
than about 2%, said, oleic acid content, linolenic acid content and erucic
acid
content determined after hydrolysis of said oil.
In accordance with another aspect of the invention, a method of producing
an endogenous oil from 8rassica seeds, comprises the steps of:
a) creating at least one transgenic Brassica plant having a seed-
specific reduction in microsomai delta-12 fatty acid desaturase gene
expression
and a seed-specific reduction in microsomal delta-15 fatty acid desaturase
gene
expression, wherein said transgenic plant contains at least one recombinant
nucleic acid construct, said at least one construct compnslng a regulatory
sequence fragment operably linked to a wild-type microsomal delta-12 fatty
acid
2o desaturase coding sequence fragment and a regulatory sequence fragment
operably linked to a wild-type microsomal delta-15 fatty acid desaturase
coding
sequence fragment;
b) crushing seeds produced from said plant; and
c) extracting said oil from said seeds, said oil having an oleic acid
25 content of about 86% or greater and an erucic acid content of less than
about
2%, said oleic acid content and erucic acid content determined after
hydrolysis of
said oil.
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In accordance with a further aspect of the invention, a method of
producing an endogenous oil from Brassica seeds, comprises the steps of:
a) creating at least one tmansgenic Brassica plant having a seed-
specific reduction in microsomal delta-12 fatty acid desaturase gene
expression
and a seed-specific reduction in rnicrosomal delta-15 fatty acid desaturase
gene
expression, wherein said transgenic plant contains at least one recombinant
nucleic acid construct, said at least one construct comprising a regulatory
sequence fragment operably lirdced to a wild-type microsomal delta-12 fatty
acid
desaturase coding sequence fragment and a regulatory
sequence fragment operably linked to a wild-type microsomal delta-15 fatty
acid
desaturase coding sequence fragment;
b) crushing seeds produced from said plant; and
c) extracting said oil from said seeds, said oil having an oleic acid
content of about 80% or greater, an a-linolenic acid content of 2.5% or less
and
an erucic acid content of less than about 2%. said oleic acid content and
crude
acid content determined after hydrolysis of said oil.
Description of the Preferred Embodiments
The term "fatty acid desaturase" refers to an enzyme which catalyzes the
2o breakage of a carbon-hydrogen bond and the introduction of a carbon-carbon
double bond into a fatty acid molecule. The fatty acid may be free or
esterified to
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another molecule including, but not limited to, acyl-carrier
protein, coenzyme A, sterols and the glycerol moiety of
glycerolip:ids. The term "glycerolipid desaturases" refers
to a subset of the fatty acid desaturases that act on fatty
acyl moieties esterified to a glycerol backbone. "Delta-12
desaturase" refers to a fatty acid desaturase that catalyzes
the formation of a double bond between carbon positions 6
and 7 (numbered from the methyl end), (i.e., those that
correspond to carbon positions 12 and 13 (numbered from the
carbonyl carbon) of an 18 carbon-long fatty acyl chain.
"Delta-15 desaturase" refers to a fatty acid desaturase that
catalyzes the formation of a double bond between carbon
positions 3 and 4 (numbered from the methyl end), (i.e.,
those that correspond to carbon positions 15 and 16
(numbered from the carbonyl carbon) of an 18 carbon-long
fatty acyl chain. "Microsomal desaturase" refers to the
cytoplasmic location of the enzyme, while "chloroplast
desaturase" and "plastid desaturase" refer to the plastid
location of the enzyme. It should be noted that these fatty
acid desat:ur.ases have never been isolated and characterized
as proteins. Accordingly, the terms such as "delta-12
desaturase" and "delta-15 desaturase" are used as a
convenience to describe the proteins encoded by nucleic acid
fragments that have been isolated based on the phenotypic '
effects caused by their disruption. They do not imply any
catalytic mechanism. For example, delta-12 desaturase
refers to the enzyme that catalyzes the formation of a
double bond between carbons 12 and 13 of an 18 carbon fatty
acid irrespective of whether it "counts" the carbons from
the methyl, carboxyl end, or the first double bond.
Microsomal delta-12 fatty acid desaturase (also
known as omega-6 fatty acid desaturase, cytoplasmic~ole'ic '
desaturase or oleate desaturase) is involved in the
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enzymatic conversion of oleic acid to linoleic acid. A
microsomal delta-12 desaturase has been cloned and
characterized using T-DNA tagging. Okuley, et al., Plant
Cell 6:147-158 (1994). The nucleotide sequences of higher
plant genes encoding microsomal delta-12 fatty acid
desaturase are described in Lightner et al., W094/11516.
Microsomal delta-15 fatty acid desaturase (also
known as omega-3 fatty acid desaturase, cytoplasmic linoleic
acid desaturase or linoleate desaturase) is involved in the
enzymatic conversion of linoleic acid to a-linolenic acid.
Sequences of higher plant genes encoding microsomal and
plastid delta-15 fatty acid desaturases are disclosed in
Yadav, N., et al., Plant Physiol., 103:467-476 (1993), WO
93/11245 and Arondel, V. et al., Science, 258:1353-1355
(1992) .
Brassica species have more than one gene for
endogenous microsomal delta-12 desaturase and more than one
gene for endogenous microsomal delta-15 desaturase. The
genes for microsomal delta-12 desaturase are designated Fad2
while the genes for microsomal delta-15 desaturase are
designated Fad3. In amphidiploids, each gene is derived
from one of the ancestral genomes making up the species
under consideration. The full-length coding sequences for
the wild-type Fad2 genes from Brassica napus (termed the D
form and the F form) are shown in SEQ ID NO:1 and SEQ ID
N0:5, respectively. The full-length coding sequence for a
wild-type Fad3 gene is disclosed in WO 93/11245.
The inventors have discovered canola oils that have
novel fatty acid compositions, e.g., very high oleic acid
levels and very low a-linolenic acid levels. Such oils may
be obtained by crushing seeds of transgenic Brassica plants
1exhibiting a seed-specific reduction'in delta-12 desaturase
and delta-15 desaturase activity; oil of the invention is
_ g _
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extracted therefrom. Expression of Fad2 and Fad3 in seeds
is reduced such that the resulting seed oil possesses very
high levels of oleic acid and very low levels of a-linolenic
acid. The fatty acid composition of the endogenous seed
oil, as determined after hydrolysis of fatty acid esters
reflects the novel fatty acid composition of such seeds.
The fatty acid composition of oils disclosed herein
is determined by techniques known to the skilled artisan,
e.g., hydrolysis of esterified fatty acids
(triacylglycerides and the like) in a bulk seed sample
followed by gas-liquid chromatography (GLC) analysis of
fatty acid methyl esters.
In one embodiment, an oil of the invention has an
oleic acid content of about 80% or greater, as well as a
surprisingly low a-linolenic acid content of about 2.5% or
less. The oleic acid content is preferably from about 84%
to about 89%, more preferably from about 86% to about 89%.
The a-linolenic acid preferably is from about 1% to less
than about 2.5%, more preferably from about 1% to about 2%.
The linoleic acid content of an oil of this
embodiment typically is less than about 10%, preferably less
than about 7%, more preferably from about 2% to about 6%.
Ca.nola seed is crushed by techniques known in the
art. The seed typicaliy is tempered by spraying the seed
with water to raise the moisture to, for example, 8.5%. The
tempered seed is flaked using smooth roller with, for
example, a gap setting of 0.23 to 0.27 mm. Heat may be
applied to the flakes to deactivate enzymes, facilitate
further cell rupturing, coalesce the oil droplets and
agglomerate protein particles in order to ease the
extraction process.
,. . . ,.
Typically, oil is removed from the heated cariola
flakes by a screw press to press out a major fraction of the
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oil from the flakes. The resulting press cake contains some
residual o:il.
Crude oil produced from the pressing operation
typically is passed through a settling tank with a slotted
wire drainage top to remove the solids expressed out with
the oil in the screw pressing operation. The clarified oil
can be passed through a plate and frame filter to remove the
remaining fine solid particles.
Canola press cake produced from the screw pressing
operation can be extracted with commercial n-Hexane. The
canola oil recovered from the extraction process is combined
with the clarified oil from the screw pressing operation,
resulting in a blended crude oil.
Free fatty acids and gums typically are removed from
the crude oil by heating in a batch refining tank to which
food grade phosphoric acid has been added. The acid serves
to convert: the non-hydratable phosphatides to a hydratable
form, and to chelate minor metals that are present in the
crude oil. The phosphatides and the metal salts are removed
from the oil along with the soapstock. The oil-acid mixture
is treated with sodium hydroxide solution to neutralize the
free fatty acids and the phosphoric acid in the acid-oil
mixture. The neutralized free fatty acids, phosphatides,
etc. (soapstock) are drained off from the neutralized oil. '
A water wash may be done to further reduce the soap content
of the oil. The oil may be bleached and deodorized before
use, if desired, by techniques known in the art.
A transgenic plant disclosed herein contains at
least one recombinant nucleic acid construct. The construct
or constructs comprise an oleate desaturase coding sequence
fragment and a linoleate desaturase coding sequence
fragment, both of which are expressed preferentially iri
developing seeds. Seed-specific expression of the
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recombinant desaturases results in a seed-specific reduction
in native desaturase gene expression. The seed-specific
defect in delta-12 and delta-15 desaturase gene expression
alters the fatty acid composition in mature seeds produced
on the plant, so that the oil obtained from such seeds has
the novel fatty acid compositions disclosed herein.
Typically, the oleate and linoleate desaturase
sequence fragments are present on separate constructs and
are introduced into the non-transgenic parent on separate
plasmids. The desaturase fragments may be isolated or
derived from, e.g., Brassica spp., soybean (Glycine max),
sunflower and Arabidopsis. Preferred host or recipient
organisms for introduction of a nucleic acid construct are
oil-producing species, such as Brassica napus, B. rapa and
B. juncea.
A transgenic plant disclosed herein preferably is
homozygous for the transgene containing construct. Such a
plant may be used as a parent to develop plant lines or may
itself be a member of a plant line, i.e., be one of a group
of plants that display little or no genetic variation
between individuals for the novel oil composition trait.
Such lines may be created by several generations of self-
pollination and selection, or vegetative propagation from a
single parent using tissue or cell culture techniques. '
Other means of breeding plant lines from a parent plant are
known in the art.
Progeny of a transgenic plant are included within
the scope of the invention, provided that such progeny
exhibit the novel seed oil characteristics disclosed herein.
Progeny of an instant plant include, for example, seeds
formed on Fl, Fz, F3, and subsequent generation plants, or
seeds formed on BCl, BC2, BC3 and subsequent generatio'ii ~ '
plants.
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A seed-specific reduction in Fad2 and Fad3 gene
expression may be achieved by techniques including, but not
limited to, antisense and cosuppression. These phenomena
significantly reduce expression of the gene product by the
native genes (wild-type or mutated). The reduction in gene
expression can be inferred from the decreased level of
reaction product and the increased level of substrate in
seeds (e.g., decreased 18:2 and increased 18:1), compared to
the corresponding levels in plant tissues expressing the
native genes.
The preparation of antisense and cosuppression
constructs for inhibition of fatty acid desaturases may
utilize fragments containing the transcribed sequence for
the Fad2 and Fad3 fatty acid desaturase genes in canola.
These genes have been cloned and sequenced as discussed
hereinabove.
Antisense RNA has been used to inhibit plant target
genes in a tissue-specific manner. van der Krol et al.,
Biotechniques 6:958-976 (1988). Antisense inhibition has
been shown using the entire cDNA sequence as well as a
partial cDNA sequence. Sheehy et al., Proc. Natl. Acad.
Sci. USA 85:8805-8809 (1988); Cannon et al., Plant Mol.
Biol. 15:39-47 (1990). There is also evidence that 3' non-
coding sequence fragment and 5' coding sequence fragments,
containing as few as 41 base-pairs of a 1.87 kb cDNA, can
play important roles in antisense inhibition. (Ch'ng et
al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989);
Cannon et al., supra.
The phenomenon of cosuppression has also been used
to inhibit plant target genes in a tissue-specific manner.
Cosuppression of an endogenous gene using a full-length cDNA
sequence as well as a partial cDNA sequence'(730 by of a
1770 by cDNA) are known. Napoli et al., The Plant Cell
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2:279-289 {1990); van der Krol et~al., The Plant Cell 2:291-
299 (1990); Smith et al., Mol. Gen. Genetics 224:477-481
(1990) .
Nucleic acid fragments comprising a partial or a
full-length delta-12 or delta-15 fatty acid desaturase
coding sequence are operably linked to at least one suitable
regulatory sequence in antisense orientation (for antisense
constructs) or in sense orientation (for cosuppression
constructs). Molecular biology techniques for preparing
such chimeric genes are known in the art. The chimeric gene
is introduced into a Brassica plant and transgenic progeny
displaying a fatty acid composition disclosed herein due to
antisense or cosuppression are identified. Transgenic
plants that produce a seed oil having a fatty acid
composition disclosed herein are selected for use in the
invention. Experimental procedures to develop and identify
cosuppressed plants involve breeding techniques and fatty
acid analytical techniques known in the art.
One may use a partial cDNA sequence for
cosuppression as well as for antisense inhibition. For
example, cosuppression of delta-12 desaturase and delta-15
desaturase in Brassica napus may be achieved by expressing,
in the sense orientation, the entire or partial seed delta-
12 desaturase cDNA found in pCF2-165D. See WO 04/11516. '
Seed-specific expression of native Fad2 and Fad3
genes can also be inhibited by non-coding regions of an
introduced copy of the gene. See, e.g., Brusslan, J.A. et
al. (1993) Plant Cell 5:667-677; Matzke, M.A. et al., Plant
Molecular Biology 16:821-830). One skilled in the art can
readily isolate genomic DNA containing sequences that flank
desaturase coding sequences and use the non-coding regions
for antisense or cosuppression inhibition. ~ '""~ ' ' ' ' '
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Regulatory sequences typically do not themselves
code for a gene product. Instead, regulatory sequences
affect the expression level of the mutant coding sequence.
Examples of regulatory sequences are known in the art and
include, without limitation, promoters of genes expressed
during embryogenesis, e.g., a napin promoter, a phaseolin
promoter, a oleosin promoter and a cruciferin promoter.
Native regulatory sequences, including the native promoters,
of delta-12 and delta-15 fatty acid desaturase genes can be
readily isolated by those skilled in the art and used in
constructs of the invention. Other examples of suitable
regulatory sequences include enhancers or enhancer-like
elements, introns and 3' non-coding regions such as poly A
sequences. Further examples of suitable regulatory
sequences for the proper expression of mutant or wild-type
delta-l2~or mutant delta-15 coding sequences are known in
the art.
In preferred embodiments, regulatory sequences are
seed-specific, i.e., the chimeric desaturase gene product is
preferentially expressed in developing seeds and expressed
at low levels or not at all in the remaining tissues of the
plant. Seed-specific regulatory sequences preferably
stimulate or induce expression of the recombinant desaturase
coding sequence fragment at a time that coincides with or
slightly precedes expression of the native desaturase gene.
Murphy et al., J. Plant Physiol. 135:63-69 (1989').
Transgenic plants for use in the invention are
created by transforming plant cells of Erassica species,
Such techniques include, without limitation, Agrobacterium-
mediated transformation, electroporation and particle gun
transformation. Illustrative examples of transformation
"" ' " "techniques are described in U.S. Patent 5,2.04,253, (.particle
gun) and U.S. Patent 5,188,958 (Agrobacterium).
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Transformation methods utilizing the Ti and Ri plasmids of Agrobacferium spp.
typically use binary type vectors. Walkerpeach, C. et al., in Plant Molecular
Biology Manual, S. Gelvin and R. Schilperoort, eds., Kluwer Dordrecht, CI:I-19
(1994). If cell or tissue cultures are used as the recipient tissue for
transformation, plants can be regenerated from transformed cultures by
techniques known to those skilled in the art.
One or more recombinant nucleic acid constructs, suitable for antisense or
cosuppression of native Fad2 and Fad3 genes are introduced, and at least one
transgenic Brassica plant is obtained. Seeds produced by the transgenic
plants)
are grown and either selfed or outcrossed to obtain plants homozygous for the
recombinant construct. Seeds are analyzed as discussed above in order to
identify those homozygotes having native fatty acid desaturase activities
inhibited
by the mechanisms discussed above. Homozygotes may be entered into a
breeding program, e.g., to increase seed, to introgress the novel oil
composition
trait into other lines or species, or for further selection of other desirable
traits
(disease resistance, yield and the like)
Fatty acid composition is followed during the breeding program by
analysis of a bulked seed sample or of a single half-seed. Half-seed analysis
useful because the viability of the embryo is maintained and thus those seeds
2o having a desired fatty acid profile may be advanced to the next generation.
However, half-seed analysis is also known to be an inaccurate representation
of
the genotype of the seed being analyzed. Bulk seed analysis typically yields a
more accurate representation of the fatty acid profile in seeds of a given
genotype.
Procedures for analysis of tatty acid composition are known in the art.
These procedures can be used to
CA 02547678 1996-07-03
identify individuals to be retained in a breeding program;
the procedures can also be used to determine the product
specifications of commercial or pilot plant oils.
The relative content of each fatty acid in canola
seeds can be determined either by direct trans-
esterification of individual seeds in methanolic HZS04
(2.5%) or by hexane extraction of bulk seed samples followed
by trans-esterification of an aliquot~in 1% sodium methoxide
in methanol. Fatty acid methyl esters can be extracted from
the methanolic solutions into hexane after the addition of
an equal volume of water.
For example, a seed sample from each transformant in
a breeding program is crushed with a mortar and pestle and
extracted 4 times with 8 mL hexane at about 50°C. The
extracts from each sample are reduced in volume and two
aliquots~are taken for esterification. Separation of the
fatty acid methyl esters can be carried out by gas-liquid
chromatography using an Omegawaxk320 column (Supelco Inc.,
0.32 mm ID X 30M) run isothermally at 220° and cycled to
260° between each injection.
Alternatively, seed samples from a breeding program
are ground and extracted in methanol/KOH, extracted with
iso-octane, and fatty acids separated by gas chromatography.
A method to produce an oil of the invention
comprises the creation of at least one Brassica plant having
a seed-specific reduction in Fad2 and Fad3 gene expression,
as discussed above. Seeds produced by such a plant, or its
progeny, are crushed and the oil is extracted from the
crushed seeds. Such lines produce seeds yielding an oil of
the invention, e.g., an oiI having from about 80% to about
88% oleic acid, from about 1% to about 2% a-linolenic acid
' " '''and less than about 2% erucic acid. , , ,
* Trade-mark
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Alternatively, such a plant can be created by
crossing two parent plants, one exhibiting a reduction in
Fad2 gene expression and the other exhibiting a reduction in
Fad3 gene expression. Progeny of the cross are outcrossed
or selfed in order to obtain progeny seeds homozygous for
both traits.
Transgenic plants having a substantial reduction in
Fad2 and Fad3 gene expression in seeds have novel fatty acid
profiles in oil extracted from such seeds, compared to known
canola plants, e.g., the reduction in both desaturase
activities results in a novel combination of high oleic and
lower a-linolenic acid in seed oils. By combining seed-
specific inhibition of microsomal delta-12 desaturase with
seed-specific inhibition of microsomal delta-15 desaturase,
one obtains very low levels of seed a-linolenic acid,
without adversely affecting agronomic properties.
It is noteworthy that Fad2 and Fad3 cosuppression
constructs provide a novel means for producing canola oil
having 86% oleic acid or greater. A method of producing a
canola oil having greater than 86% oleic acid comprises the
creation of a transgenic Brassica plant containing at least
one recombinant nucleic acid construct, which constructs)
comprises an oleate desaturase coding sequence expressed
preferentially in developing seeds and a linoleate '
desaturase coding sequence expressed preferentially in
developing seeds. A pz-oportion of the plants that are
homozygous for the transgenes have seed-specific
cosuppression of the native linoleate desaturase. Seeds
produced by such transgenic cosuppressed plants are crushed
and the oil is extracted therefrom. The oil has about 86%
or greater oleic acid and less than about 2% erucic acid.
The oleic acid content can be as high as 89%. ' ''"' ~ ~ ~ ' ,
- 17 -
CA 02547678 1996-07-03
Transgenic plants exhibiting cosuppression of Fad2
and Fad3 produce seeds having a very high oleic acid
content. This result was unexpected because it was not
known if one could obtain plants in which inhibition of Fad2
and Fad3 via cosuppression was sufficient to achieve an
oleic acid level of 86x or greater in seeds. Indeed, it was
not known if two cosuppressed'genes in fatty acid metabolism
could be introduced in canola without the first
cosuppression gene interfering with the second cosuppression
gene, or without adversely affecting other agronomic traits.
Marker-assisted breeding techniques may be used to
identify and follow a desired fatty acid composition during
the breeding process. Such markers may include RFLP, RAPD,
or PCR markers, for example. Marker-assisted breeding
techniques may be used in addition to, or as an alternative
to, other sorts of identification techniques. An example of
marker-assisted breeding is the use of PCR primers that
specifically amplify the junction between a promoter
fragment and the coding sequence of a Fad2 gene.
While the invention is susceptible to various
modifications and alternative forms, certain specific
embodiments thereof are described in the general methods and
examples set forth below. For example the invention may be
applied to all Brassica species, including B. rapa, B.
juncea, and B. hirta, to produce substantially similar
results. It should be understood, however, that these
examples are not intended to limit the invention to the
particular forms disclosed. Instead, the disclosure is to
cover all modifications, equivalents and alternatives
falling within the scope of the invention.
EXAMPLE 1 ~ '
CONSTRUCTS FOR COSUPPRESSION OF DELTA-12 FATTY
- 18 -
CA 02547678 1996-07-03
ACID DESATURASE AND DELTA-15 FATTY ACID DESATURASE
The wild-type Brassica cDNA coding sequence for the delta-12
desaturase D form was cloned as described in WO 94/11516. Briefly, rapeseed
s cDNAs encoding cytoplasmic oleate (18:1 ) desaturase were obtained by
screening a cDNA library made from developing rapeseed using a heterologous
probe derived from an Arabidopsis cDNA fragment encoding the same enzyme.
(Okuley et al 1994) . The full-length coding sequence of Fad2 is found as SEQ
ID
N0:1. Rapeseed cDNAs encoding the cytoplasmic linoleate (18:2) desaturase
(Fad3) were obtained as described in WO 93/11245. See also (Yadav et. al
1993) . Seed specific expression of these cDNAs in transgenic rapeseed was
driven by ane of four different seed storage protein promoters, napin, oleosin
and
cruciferin promoters from 8. napus and a phaseolin promoter from Phaseolus
vulgaris.
~5 Detailed procedures for manipulation of DNA fragments by restriction
endonuclease digestion, size separation by agarose gel electrophoresis,
isolation
of DNA fragments from agarose gels, ligation of DNA fragments, modification of
cut ends of DNA fragments and transformation of E. coli cells with plasmids
have been described. Sambrook et al., (Molecular Cloning, A Laboratory Manual,
20 2nd ed (1989) Cold Spring Harbor Laboratory Press); Ausubel et al., Current
Protocols in Molecular Biology (1989) John Wiley & Sons) Plant molecular
biology procedures are described in Plant Molecular Biology Manual, Gelvin S.
and Schilperoort, R. eds. Kiuwer, Dordrecht (1994).
The plasmid pZS212 was used to construct binary vectors for these
25 experiments. pZS212 contains a chimeric CaMV35S/NPT gene for use in
selecting kanamycin resistant
19
CA 02547678 1996-07-03
transformed plant cells, the left and right border of an
Agrobacterium Ti plasmid T-DNA, the E. coli lacZ a-
complementing segment with unique restriction endonuclease
sites for EcoRI, KpnI, BamHI and SalI, the bacterial
replication origin from the Pseudomonas plasmid pVSl and a
bacterial Tn5 NPT gene for selection of transformed
Agrobacterium. See WO 94/11516, p. 100.
The first construct was prepared by inserting a
full-length mutant Brassica Fad2 D gene coding sequence
fragment in sense orientation between the phaseolin promoter
and phaseolin 3' poly A region of plasmid pCW108. The full-
length coding sequence of the mutant gene is found in SEQ ID
N0:3.
The pCW108 vector contains the bean phaseolin
promoter and 3' untranslated region and was derived from the
commercially available pUClB plasmid (Gibco-BRL) via
plasmids AS3 and pCW104. Plasmid AS3 contains 495 base
pairs of the Phaseolus vulgaris phaseolin promoter starting
with 5'-TGGTCTTTTGGT-3' followed by the entire 1175 base
pairs of the 3' untranslated region of the same gene.
Sequence descriptions of the 7S seed storage protein
promoter are found in Doyle et al., J. Biol. Chem. 261:9228-
9238 (1986) and Slightom et al., Proc. Natl. Acad. Sci. USA,
80:1897-1901 (1983). Further sequence description may be '
found in WO 91/13993. The fragment was cloned into the Hind
III site of pUCl8. The additional cloning sites of the
pUCl8 multiple cloning region (Eco RI, Sph I, Pst I and Sal
I) were removed by digesting with Eco RI and Sal I, filling
in the ends with Klenow and relegating to yield the plasmid
pCW104. A new multiple cloning site was created between the
495bp of the 5' phaseolin and the 1175bp of the 3' phaseolin
by inserting a dimer of complementary synthetic °"' ' ' '
oligonucleotides to create the plasmid pCW108. See WO
- 20 -
CA 02547678 1996-07-03
94/11516. This plasmid contains unique Nco I, Sma I, Kpn I
and Xba I sites directly behind the phaseolin promoter.
The phaseolin promoter:mutantFad2:phaseolin poly A
construct in pCw108 was excised and cloned between the
SalI/EcoRI sites of pZS212. The resulting plasmid was
designated pIMC201.
A second plasmid was constructed by inserting the
full-length wild type Brassica Fad2 D gene coding sequence
into the NotI site of plasmid pIMC401, which contains a 2.2
kb napin expression cassette. See, e.g., W094/11516, page
102. The 5'-napin:Fad2:napin poly A-3' construct was
inserted into the SalI site of pZS212 and the resulting 17.2
Kb plasmid was termed pIMC127. Napin promoter sequences are
also disclosed in U.S. Patent 5,420,034.
A third plasmid, pIMC135, was constructed in a
manner similar to that described above for pIMC127. Plasmid
pIMC135 contains a 5' cruciferin promoter fragment operably
linked in sense orientation to the full-length wild-type
Brassica Fad2 D gene coding sequence, followed by a
cruciferin 3' poly A fragment. The 5'-cruciferin:Fad2
D:cruciferin polyA cassette was inserted into pZS212; the
resulting plasmid was termed pIMC135. Suitable cruciferin
regulatory sequences are disclosed in Rodin, J. et al., J.
Biol. Chem. 265:2720 (1990); Ryan, A. et al., Nucl. Acids ,
Res. 17:3584 (1989) and Simon, A. et al., Plant Mol. Biol.
5:191 (1985). Suitable sequences are also disclosed in the
Genbank computer database, e.g., Accession No. M93103.
A fourth plasmid, pIMC133 was constructed in a
manner similar to that described above. Plasmid pIMC133
contains a 5' oleosin promoter fragment operably linked in
sense orientation to the full-length Brassica Fad2 D gene
coding sequence, followed by a 3' oleosin poly A fragment. . , , ,
See, e.g., WO 93/20216,
- 21 -
CA 02547678 1996-07-03
A napin-Fad3 construct was made by first isolating a delta-15
desaturase coding sequence fragment from pBNSF3-f 2. The fragment contained
the full-length coding sequence of the desaturase, disclosed as SEQ ID NO: 6
in
WO 93/11245. The 1.2 kb fragment was fitted with linkers and ligated into
pIMC401. The 5'napin:Fad3:3'napin cassette was inserted into the Sal I site of
pZS212; the resulting plasmid was designated pIMC110.
EXAMPLE 2
~o CREATION OF TRANSGENIC COSUPPRESSED PLANTS
The plasmids pIMC2O1, pIMC127, pIMC135, pIMC133 and pIMCllO were
introduced into Agrobacferium strain LBA4404/pAL44O4 by a freeze-thaw
method. The plasmids were introduced into Brassica napus cultivar Westar by
the method of Agrobacterium-mediated transformation as described in
W094/11516. Transgenic progeny plants containing pIMC201 were designated
as the WS2O1 series. Plants transformed with pIMC127 were designated as the
WS687 series. Plants transformed with pIMC135 were designated as the
WS691 series. Plants transformed with pIMC133 were designated as the
WS692 series. Plants transformed with pIMCllO were designated as the WS663
2o series.
Unless indicated otherwise, fatty acid percentages described herein are
percent by weight of the oil in the indicated seeds as determined after
extraction
and hydrolysis.
From about 50 to 350 transformed plants (Ti generation) were produced
2s for each cDNA and promoter combination. Ti plants were selfed to obtain T2
seed. T2 samples in which cosuppression events occurred were
22
CA 02547678 1996-07-03
identified from the fatty acid profile and from the presence
of the tra.nsgene by molecular analysis. The transformed
plants were screened for phenotype by analysis of the
relative fatty acid contents of bulk seed from the first
transformed generation by GC separation of fatty acid methyl
esters.
T2 seed was sown in 4-inch pots containing Pro-Mix
soil. The plants, along with Westar controls, were grown at
25~3°C/18~3°C, 14/10 hr day/night conditions in the
greenhouse. At flowering, the terminal raceme was self-
pollinated by bagging. At maturity, seed was individually
harvested from each plant, labelled, and stored to ensure
that the source of the seed was known.
Fatty acid profiles were determined as described in
WO 91/05910. For chemical analysis, 10-seed bulk samples
were hand ground with a glass rod in a 15-mL polypropylene
tube and extracted in 1.2 mL 0.25 N KOH in 2:1
ether/methanol. The sample was vortexed for 30 sec. and
heated for 60 sec. in a 60°C water bath. Four mL of
saturated NaCl and 2.4 mL of iso-octane were added, and the
mixture was vortexed again. After phase separation, 600 ~L
of the upper organic phase were pipetted into individual
vials and stored under nitrogen at -5°C. One uL samples
were injected into a Supelco SP-2330*fused silica capillary ,
column (0.25 mm ID, 30 M length, 0.20 ~m df).
The gas chromatograph was set at 180°C for 5.5
minutes, then programmed for a 2°C/minute increase to 212°C,
and held at this temperature for 1.5 minutes. Total run
time was 23 minutes. Chromatography settings were: Column
head pressure - 15 psi., Column flow (He) - 0.7 mL/min.,
Auxiliary and Column flow - 33 mL/min., Hydrogen flow - 33
mL/min., Air flow - 400 mL/min., Injector temperature T , ,
250°C, Detector temperature - 300°C, Split vent - 1/15.
*Traae~mark - 23 -
CA 02547678 1996-07-03
Table 1 shows the content of the seven major fatty
acids in mature seeds from transgenic cosuppressed plants
homozygous for the napin:Fad3 construct or the napin:Fad2
construct (T4 or later generation). Over expression
phenotypes and cosuppression phenotypes were observed for
both chimeric genes (oleate desaturase and linoleate
desaturase); data for plants exhibiting the cosuppression
phenotype are shown in the Table. '
As shown in Table 1, the homozygous Fad2-
cosuppressed seed had,a a-linolenic acid content of about
2.9%, which was less than half that of the Westar control;
the oleic acid content increased to about 84.1%. The
homozygous Fad3-cosuppressed seed had an a-linolenic acid of
about 1.2%; the oleic acid and linoleic acid contents in
Fad3-cosuppressed plants increased slightly compared to
Westar. The results demonstrate that inhibiting gene
expression of either enzyme by cosuppression resulted in a
change in fatty acid composition of the seed oil.
TABLE 1
Fattv Acid Profiles in Oil From Cosupt~ression Canola Seed
TRANSGENE FATTY ACID (% OF TOTAL FATTY ACIDS
CONSTRUCTION 16:0 18:0 18:1 18:2 18:3 20:0 20:1 22:0 24:0
non-transformed
Westar 3.9 1.8 67.0 19.0 7.5 0.6 0.8 0.6 0.1
napin:Fad2
(co-suppression) 4.3 1.4 84.1 5.2 2.9 0.6 0.9 0.5 0.2
napin:Fad3
(co-suppression) 3.8 1.5 68.5 22.1 1.2 0.6 1.1 0.4 0.1
,. ,. , . , , ~, , , , ..
- 24 -
CA 02547678 1996-07-03
TABLE 2
Fatty Acid Oil From pression Seeds
Profiles in Cosun Canola
Construct Fatty Ac id ion
Composit
Line (p romoter/coding tuence) 18:0 18:1 18:218:3
sec 16:0
663-40 napin/Fad3 3.9 1.4 71.2 20.11.2
687-193 napin/Fad2 4.0 1.5 82.8 5.9 3.7
691-215 cruciferin/Fad23.3 1.3 86.5 3.0 3.7
692-090-3 oleosin/Fad2 3.4 1.3 86.5 2.6 3.9
692-105-11 oleosin/Fad2 ' 3.4 1.3 86.2 2.7 4.2
201-389 phaseolin/MFad24.2 2,.7 84.6 4.7 3.7
A23
TABLE 3
Range of Fatty Acid Profiles for Fad2 and Fad3
Cosuppression Lines Tested in the Field
Fatty Acid Composition
Line No. Vector Min/Max C16:0 C18:0 C18;1 C18:2 C1_ 8-3
663-40 pIMC110 Min 3.5 2.3 73.5 16.3 0.8
Max 4.7 2.2 64.0 24.2 1.5
687-193 pIMCl27 Min 3.4 3.1 83.3 3.8 2.3
Max 3.4 2.1 85.5 3.2 2.5
2 0 692-105 pIMC133 Min 3.7 2.7 84.6 2.8 2.4
Max 3.3 2.3 86.3 2.1 2.7
691-215 pIMC135 Min 3.2 2.4 84.6 3.0 2.5
Max 3.0 2.0 86.3 2.6 2.5
Table 2 shows the fatty acid.profile in T4 or later
homozygous seeds produced by six individual plants having
various promoter-desaturase gene combinations. The seeds
were obtained from greenhouse-grown plants. The results
indicate that the oleic acid content ranged from about 82.8%
to about 86.5% among the lines carrying the Fad2 constructs.
The phaseolin:mutated Fad2 construct was as successful as
the wild-type Fad2 constructs in achieving seed-specific
Fad2 cosuppression.
The napin:Fad3 cosuppressed plant line had an
" . " . .unusually low a-linolenic acid contend of 1.,2% . However,
the oleic acid content was only 71.2% and the linoleic acid
- 25 -
CA 02547678 1996-07-03
content was similar to that of the non-transformed control
Westar in Table 1.
Homozygous seeds from four of the lines in Table 2
were planted in a field nursery in Colorado and self-
pollinated. Seed samples from several plants of each line
were collected and separately analyzed for fatty acid
composition. The results for~the 663-40 plant having the
minimum and the 663-40 plant having the maximum linolenic
acid content observed in the field are shown in Table 3.
The results for the 687-193, 692-105 and 691-215 plants
having the minimum and maximum oleic acid content in the
field are also shown in Table 3.
The results in Table 3 demonstrate that the fatty
acid profile in field-grown seeds of cosuppressed transgenic
plants was similar to that in the greenhouse-grown seeds
(Table 2)I, indicating that the cosuppression trait confers a
stable fatty acid composition on the oil. The results also
indicate that an oil having the combination of an oleic acid
content of 86% or greater and an a-linolenic acid content of
2.5% or less could not :be obtained from plants cosuppressed
for either Fad2 or Fad3 alone.
EXAMPLE 3
OIL CONTENT IN SEEDS OF
PLANTS EXHIBITING Fad2 and Fad3 COSUPPRESSION
Crosses were made between the napin:Fad3
cosuppressed line 663-40 and three Fad2 cosuppressed lines,
691-215, 692-090-3 and 692-105-11. F1 plants were selfed
for 2 generations in the greenhouse to obtain F3 generation
seed that was homozygous for both recombinant constructs.
.,.. , .. ",. , ~ ,
- 26 -
CA 02547678 1996-07-03
TABLE 4
Fatty Acid Profile in F3 Seedsof
Lines Exhibiting Fad2 and Cosuppression
Fad3
Line # Construct 16:019:0 18:1 18:2 18:3
663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2
691-215 cruciferin/Fad2 3.9 1.3 86.5 3.0 3.7
663-40X691-215 napin/Fad3 & crUCiferin/Fad23 I 86 5 1 . 5 i
. . , .
2 4 2 2
16:018:0 18:1 18:2 18:3
663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2
692-090-3 oleosin/Fad2 3.4 1.3 86.5 2.6 3.9
663-40X692-090-3napin/Fad3 & oleosin/Fad2 3.4 1.5 85.5 5.0 1.7
16:018:0 18:1 18:2 18:3
663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2
692-105-11 oleosin/Fad2 3.4 1.3 86.2 2.7 4.2
663-40X692-105-ilnapin/Fad3 & oleosin/Fad2 3.4 1.4 86.8 4.6 1.4
The seed fatty acid profiles of the parent lines and
a representative F3 cosuppressed line are shown in Table 4.
Plants expressing both cosuppression constructs exhibited an
oleic acid level of about 86% or greater. Moreover, this
high level of oleic acid was present in combination with an
unusually low level of a-linolenic acid, less than 2.0%.
However, the linoleic acid content in the F3 seeds increased
from about 2.6-3.0% to about 4.6-5.2%.
These results demonstrate that a canola oil can be
extracted from rapeseeds that contains greater than 80%
oleic acid and less than 2.5% a-linolenic acid. Results
similar to those obtained using cosuppression constructs are
achieved when antisense constructs are used.
The canola oil extracted from Fad2 and Fad3
cosuppressed F3 seed, or progeny thereof, is found to have
superior oxidative stability compared to the oil extracted
from westar seed. The improved oxidative stability of such
an oil is measured after refining, bleaching and
deodorizing, using the Accelerated Oxygen Method (AOM),
- 27 -
CA 02547678 1996-07-03
American Oil Chemists' Society Official Method Cd 12-57 for
fat stability, Active Oxygen Method (revised 1989). The
improved oxidative stability is also demonstrated when using
the Oxidative Stability Index method. The improved
oxidative stability is measured in the absence of added
antioxidants.
EXAMPLE 4 '
OIL CONTENT IN SEEDS OF PLANTS HAVING Fad3 COSUPPRESSION
AND CHEMICALLY-INDUCED Fad2 MUTATIONS
Q4275 is a doubly mutagenized B. napes line having
defects in the Fad2 gene. Q4275 was derived by chemical
mutagenesis of B. napes line IMC129, which carries a
mutation in the Fad2 D gene; the coding sequence of the
mutated gene is shown in SEQ ID N0:3. Line IMC129 was
itself derived by chemical mutagenesis of the cultivar
Westar, as disclosed in WO 91/05910. Genetic segregation
analysis of crosses between Q4275 and other tatty acid
mutant lines indicated that Q4275 carried a mutation in the
B. napes Fad2 F gene in. addition to the IMC129 Fad2 D gene
mutation. Q4275 thus carries chemically induced mutations
in both Fad2 genes.
A cross was made between Q4275 and the napin:Fad3
cosuppressed line 663-40. F1 plants were selfed in the
greenhouse and F2 plants that were homozygous for the
recombinant construct and the Fad2 D and Fad2 F mutated
genes were identified by fatty acid profile analysis of the
F3 generation seed. After selfing to homozygosity, the
fatty acid profiles in seeds of a representative homozygous
plant was analyzed and compared to the profile of the parent
plants, as shown in Table 5.
' The~results show that an oil'h~ving greater than 87%
oleic acid and less than 1.5% a-linolenic acid can be
- 2a -
CA 02547678 1996-07-03
obtained from a transgenic Brassica plant containing a seed-
specific reduction in Fad3 gene expression as well as
chemically-induced mutations in Fad2 genes.
TABLE 5
Fatty Acid Profile of Fad3 Cosuppression, Fad2 Mutated Seeds
16:0 18:0 18:1 18:2 18:3
663-40 3.9 1.4 71.2 20.1 1.2
Q4275 3.3 1.5 86.7 2.2 3.1
Q4275 X 663-40 3.2 1.6 87.6 4.2 1.3
TABLE 6
Range of Fatty Acid Profiles for Fad3 Cosuppression,
Fad2 Mutated Lines Tested in the Field
16:0 18:0 18:1 18:2 18:3
663-40 Min 3.5 2.3 73.5 16.3 0.8
Max 4.7 2.2 64.0 24.2 1.5
Q4275 Min 3.2 3.3 85.0 1.8 2.0
Max 3.0 2.3 86.6 1.7 2.6
Q4275 X 663-40 Min 3.2 2.0 85.1 5.3 0.9
Max 3.2 2.9 84.0 6.0 1.5
Additional seed from the homozygous plant described
above was planted in the field and self-pollinated. Mature
seeds from several progeny plants were separately analyzed
for their fatty acid profile. The fatty acid profile for
the progeny plant having the minimum linolenic acid content
and the plant having the maximum linolenic acid content are
shown in Table 6. The results show that the homozygous
plant having Fad2 mutations and Fad3 cosuppression had a
,.", ~ , ,,
fatty acid profile in the field that was similar tolthat of
- 29 -
CA 02547678 1996-07-03
the greenhouse-grown seed (Table 5), indicating that the
Fad3 cosuppression trait and the chemically-induced Fad2
mutants conferred a stable fatty acid composition on seeds
of this plant. Thus, an oil of the invention can be
obtained from either field-grown seeds or greenhouse-grown
seeds.
Because of the decreased a-linolenic acid content
and increased oleic acid content, an oil of the invention is
useful in food and industrial applications. Oils which are
low in a-linolenic acid have increased oxidative stability.
The rate of oxidation of lipid fatty acids increases with
higher levels of linolenic acid leading to off-flavors and
off-odors in foods. The present invention provides novel
canola oils that are low in a-linolenic acid.
To the extent not already indicated, it will be
understood by those of ordinary skill in the art that any
one of the various specific embodiments herein described and
illustrated may be further modified to incorporate features
shown in other of the specific embodiments.
The foregoing detailed description has been provided
for a better understanding of the invention only and no
unnecessary limitation should be understood therefrom as
some modifications will be apparent to those skilled in the
art without deviating from the spirit and scope of the
appended claims.
,..,
- 30 -
CA 02547678 1996-07-03
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: DeBonte, L. et al.
(ii) TITLE OF INVENTION: CANOLA OIL HAVING INCREASED OLEIC ACID AND
DECREASED LINOLENIC ACID CONTENT
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sim & McBurney
(B) STREET: 330 University Avenue, 6th Floor
(C) CITY: Toronto
(D) STATE: ON
(E) COUNTRY: Canada
(F) ZIP: M5G 1R7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 03-JUL-1996
(C) CLASSIFICATION:
(vii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BARTOSZEWICZ, Lola, A.
(B) REGISTRATION NUMBER: 10037
(C) REFERENCE/DOCKET NUMBER: 8722-53 LAB
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 416/849-8420
(B) TELEFAX: 416/595-1163
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1155 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi ) ORIGINAL SOURCE
(A) ORGANISM: Brassica napus
(ix) FEATURE:
(D) OTHER INFORMATION: Wild type D form.
31
CA 02547678 1996-07-03
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT 48
Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96
Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
GTC GGA GAA CTC AAG AAA GCA ATC (:CA CCG CAC TGT TTC AAA CGC TCG 144
Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser
35 40 45
ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192
Ile Pro Arg Ser Phe Ser Tyr Leu :Cle Trp Asp Ile Ile Ile Ala Ser
50 55 60
TGC TTC TAC TAC NTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT 240
Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro
65 70 75 80
CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAA GGG TGC GTC 288
Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
CTA ACC GGC GTC TGG GTC ATA GCC CAC GAA TGC GGC CAC CAC GCC TTC 336
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
100 105 110
AGC GAC TAC CAG TGG CTT GAC GAC ACC GTC GGT CTC ATC TTC CAC TCC 384
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
115 120 125
TTC CTC CTC GTC CCT TAC TTC TCC 'rGG AAG TAC AGT CAT CGC AGC CAC 432
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His
130 135 140
CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCG TTG 576
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTC CGT 624
Tyr Leu Ala Phe Asn Val Ser Gly .Arg Pro Tyr Asp Gly Gly Phe Arg
195 200 205
TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672
Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu
210 215 220
CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720
Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu
32
CA 02547678 1996-07-03
225 230 235 240
TTC CGT TAC GCC GCC GGC CAG GGA GTG GCC TCG ATG GTC TGC TTC TAC 768
Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
GGA GTC CCG CTT CTG ATT GTC AAT (JGT TTC CTC GTG TTG ATC ACT TAC 816
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCC GAG TGG 864
Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp
275 280 285
GAT TGG TTC AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TAC GGA ATC 912
Asp Trp Phe Arg Gly Ala Leu Ala 'rhr Val Asp Arg Asp Tyr Gly Ile
290 295 300
TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCC CAT CAT 960
Leu Asn Lys Val Phe His Asn Ile 'rhr Asp Thr His Val Ala His His
305 310 315 320
CCG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCT ACC AAG GCG 1008
Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala
325 330 335
ATA AAG CCG ATA (.TG GGA GAG TAT 'rAT CAG TTC GAT GGG ACG CCG GTG 1056
Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val
340 345 350
GTT AAG GCG ATG TGG AGG GAG GCG i~AG GAG TGT ATC TAT GTG GAA CCG 1104
Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro
355 360 365
GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153
Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu
370 375 380
GA 1155
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Gly Ala Gly Gly Arg Met Gln 'Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser
35 40 45
33
CA 02547678 1996-07-03
Ile Pro Arg Ser Phe Ser Tyr Leu Tle Trp Asp Ile Ile Ile Ala Ser
50 55 60
Cys Phe Tyr Tyr Xaa Ala Thr Thr 'Tyr Phe Pro Leu Leu Pro His Pro
65 70 75 80
Leu Ser Tyr Phe Ala Trp Pro Leu 'Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
100 105 110
Ser Asp Tyr Gln 'Trp Leu Asp Asp 'Thr Val Gly Leu Ile Phe His Ser
115 120 125
Phe Leu Leu Val Pro Tyr Phe Ser 'Trp Lys Tyr Ser His Arg Ser His
130 135 140
His Ser Asn Thr Gly Ser Leu Glu .Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
Tyr Leu Ala Phe Asn Val Ser Gly .Arg Pro Tyr Asp Gly Gly Phe Arg
195 200 205
Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu
210 215 220
Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu
225 230 235 240
Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
Leu Gln His Thr :His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp
275 280 285
Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile
290 295 300
Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His
305 310 315 320
Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala
325 330 335
Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val
340 345 350
Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro
355 360 365
34
CA 02547678 1996-07-03
Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu
370 375 380
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE: CHARACTERISTICS:
(A) LENGTH: 1155 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Brassica napus
(vii) IMMEDIATE SOURCE:
(B) CLONE: IMC129
(ix) FEATURE-
(D) OTHER INFORMATION: G to A transversion
mutation at nucleotide 316 of l.he D form.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAG AAG TCT 48
Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96
Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144
Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser
35 40 45
ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192
Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser
50 55 60
TGC TTC TAC TAC NTC GCC ACC ACT 'TAC TTC CCT CTC CTC CCT CAC CCT 240
Cys Phe Tyr Tyr Xaa Ala Thr Thr 'Tyr Phe Pro Leu Leu Pro His Pro
65 70 75 80
CTC TCC TAC TTC GCC TGG CCT CTC 'TAC TGG GCC TGC CAA GGG TGC GTC 288
Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
CTA ACC GGC GTC TGG GTC ATA GCC CAC AAG TGC GGC CAC CAC GCC TTC 336
Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe
100 105 110
AGC GAC TAC CAG TGG CTT GAC GAC ACC GTC GGT CTC ATC TTC CAC TCC 384
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
115 120 125
CA 02547678 1996-07-03
TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGC AGC CAC 432
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His
130 135 140
CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCG TTG 576
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTC CGT 624
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg
195 200 205
TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672
Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu
210 215 220
CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720
Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu
225 230 235 240
TTC CGT TAC GCC GCC GGC CAG GGA GTG GCC TCG ATG GTC TGC TTC TAC 768
Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
GGA GTC CCG CTT CTG ATT GTC AAT GGT TTC CTC GTG TTG ATC ACT TAC 816
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCC GAG TGG 864
Leu Gln His Thr His Pro Ser Leu :Pro His Tyr Asp Ser Ser Glu Trp
275 280 285
GAT TGG TTC AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TAC GGA ATC 912
Asp Trp Phe Arg Gly Ala Leu Ala 'Thr Val Asp Arg Asp Tyr Gly Ile
290 295 300
TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCC CAT CAT 960
Leu Asn Lys Val Phe His Asn Ile 'Thr Asp Thr His Val Ala His His
305 310 315 320
CCG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCT ACC AAG GCG 1008
Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala
325 330 335
ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTC GAT GGG ACG CCG GTG 1056
Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val
340 345 350
GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104
Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro
355 360 365
36
CA 02547678 1996-07-03
GAC AGG CAA GGT GAG AAG AAA GGT CiTG TTC TGG TAC AAC AAT AAG TTA T 1153
Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu
370 375 380
GA 1155
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser
35 40 45
Ile Pro Arg Ser Phe Ser Tyr Leu :Lle Trp Asp Ile Ile Ile Ala Ser
50 55 60
Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro
65 70 75 80
Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His Ala Phe
100 105 110
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
115 120 125
Phe Leu Leu Val Pro Tyr Phe Ser 'rrp Lys Tyr Ser His Arg Ser His
130 135 140
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg
195 200 205
Cys His Phe His Pro Asn Ala Pro Tle Tyr Asn Asp Arg Glu Arg Leu
210 215 220
Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu
225 230 235 240
37
CA 02547678 1996-07-03
Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp
275 280 285
Asp Trp Phe Arg C;ly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile
290 295 300
Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His
305 310 315 320
Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala
325 330 335
Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly Thr Pro Val
340 345 350
Val Lys Ala Met Trp Arg Glu Ala hys Glu Cys Ile Tyr Val Glu Pro
355 360 365
Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu
370 375 380
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1155 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sing:Le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Brassica napes
(ix) FEATURE:
(D) OTHER INFORMATION: Wild type F form.
(xi) SEQUENCE DESCRIPTION: S1EQ ID N0:5:
ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT 48
Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
GAA ACC GAC AAC ATC AAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96
Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144
38
CA 02547678 1996-07-03
Val Gly Glu Leu Lys Lys Ala Ile :Pro Pro His Cys Phe Lys Arg Ser
35 40 45
ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCC TCC 192
Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser
50 55 60
TGC TTC TAC TAC GTC GCC ACC ACT 'rAC TTC CCT CTC CTC CCT CAC CCT 240
Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro
65 70 75 80
CTC TCC TAC TTC GCC TGG CCT CTC 'TAC TGG GCC TGC CAG GGC TGC GTC 288
Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
CTA ACC GGC GTC TGG GTC ATA GCC CAC GAG TGC GGC CAC CAC GCC TTC 336
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
100 105 110
AGC GAC TAC CAG TGG CTG GAC GAC ACC GTC GGC CTC ATC TTC CAC TCC 384
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
115 120 125
TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGA CGC CAC 432
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His
130 135 140
CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG 480
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCT TTG 528
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCT TTG 576
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
TAC TTA GCC TTC AAC GTC TCG GGG AGA CCT TAC GAC GGC GGC TTC GCT 624
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala
195 200 205
TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGT CTC 672
Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu
210 215 220
CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGT CTC 720
Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu
225 230 235 240
TAC CGC TAC GCT GCT GTC CAA GGA GTT GCC TCG ATG GTC TGC TTC TAC 768
Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
GGA GTT CCG CTT CTG ATT GTC AAT GGG TTC TTA GTT TTG ATC ACT TAC 816
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAT GAC TCG TCT GAG TGG 864
39
CA 02547678 1996-07-03
Leu Gln His Thr His Pro Ser Leu F'ro His Tyr Asp Ser Ser Glu Trp
275 280 285
GATTGG TTGAGGGGA GCTTTGGCC ACCGTTGAC AGAGACTAC GGAATC 912
AspTrp LeuArgGly AlaLeuAla ThrValAsp ArgAspTyr GlyIle
290 295 300
TTGAAC AAGGTCTTC CACAATATC ACGGACACG CACGTGGCG CATCAC 960
LeuAsn LysValPhe HisAsnIle ThrAspThr HisValAla HisHis
305 310 315 320
CTGTTC TCGACCATG CCGCATTAT C:ATGCGATG GAAGCTACG AAGGCG 1008
LeuPhe SerThrMet ProHisTyr HisAlaMet GluAlaThr LysAla
325 330 335
ATAAAG CCGATACTG GGAGAGTAT TATCAGTTG CATGGGACG CCGGTG 1056
IleLys ProIleLeu GlyGluTyr TyrGlnLeu HisGlyThr ProVal
340 345 350
GTTAAG GCGATGTGG AGGGAGGCG AAGGAGTGT ATCTATGTG GAACCG 1104
ValLys AlaMetTrp ArgGluAla LysGluCys IleTyrVal GluPro
355 360 365
GACAGG CAAGGTGAG AAGAAAGGT GTGTTCTGG TACAACAAT AAGTTA 1153
T
AspArg GlnGlyGlu LysLysGly ValPheTrp TyrAsnAsn LysLeu
370 375 380
GA 1155
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys Lys Ser
1 5 10 15
Glu Thr Asp Asn Tle Lys Arg Val Pro Cys Glu Thr Pro Pro Phe Thr
20 25 30
val Gly Glu Leu Lys Lys Ala Ile 1?ro Pro His Cys Phe Lys Arg Ser
35 40 45
Ile Pro Arg Ser Phe Ser Tyr Leu :Lle Trp Asp Ile Ile Ile Ala Ser
50 55 60
Cys Phe Tyr Tyr Val Ala Thr Thr 'ryr Phe Pro Leu Leu Pro His Pro
65 70 75 80
Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val
85 90 95
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe
CA 02547678 1996-07-03
100 105 110
Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser
115 120 125
Phe Leu Leu Val Pro Tyr Phe Ser '.Crp Lys Tyr Ser His Arg Arg His
130 135 140
His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160
Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu
165 170 175
Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu
180 185 190
Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala
195 200 205
Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu
210 215 220
Gln Ile Tyr Ile Ser Asp Ala Gly :Ile Leu Ala Val Cys Tyr Gly Leu
225 230 235 240
Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met Val Cys Phe Tyr
245 250 255
Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr
260 265 270
Leu Gln His Thr His Pro Ser Leu :Pro His Tyr Asp Ser Ser Glu Trp
275 280 285
Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile
290 295 300
Leu Asn Lys Val Phe His Asn Ile 'rhr Asp Thr His Val Ala His His
305 310 315 320
Leu Phe Ser Thr Met Pro His Tyr :fiis Ala Met Glu Ala Thr Lys Ala
325 330 335
Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Leu His Gly Thr Pro Val
340 345 350
Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro
355 360 365
Asp Arg Gln Gly Glu Lys Lys Gly 'Val Phe Trp Tyr Asn Asn Lys Leu
370 375 380
0
41
