U.S. patent number RE41,139 [Application Number 11/445,506] was granted by the patent office on 2010-02-16 for polyunsaturated fatty acids in plants.
This patent grant is currently assigned to Calgene LLC. Invention is credited to Debbie Knutzon.
United States Patent |
RE41,139 |
Knutzon |
February 16, 2010 |
Polyunsaturated fatty acids in plants
Abstract
The present invention relates to compositions and methods for
preparing polyunsaturated long chain fatty acids in plants, plant
parts and plant cells, such as leaves, roots, fruits and seeds.
Nucleic acid sequences and constructs encoding fatty acid
desaturases, including .DELTA.5-desaturases, .DELTA.6-desaturases
and .DELTA.12-desaturases, are used to generate transgenic plants,
plant parts and cells which contain and express one or more
transgenes encoding one or more desaturases. Expression of the
desaturases with different substrate specificities in the plant
system permit the large scale production of polyunsaturated long
chain fatty acids such as docosahexaenoic acid, eicosapentaenoic
acid, .alpha.-linolenic acid, gamma-linolenic acid, arachidonic
acid and the like for modification of the fatty acid profile of
plants, plant parts and tissues. Manipulation of the fatty acid
profiles allows for the production of commercial quantities of
novel plant oils and products.
Inventors: |
Knutzon; Debbie (Granite Bay,
CA) |
Assignee: |
Calgene LLC (Davis,
CA)
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Family
ID: |
22215271 |
Appl.
No.: |
11/445,506 |
Filed: |
May 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60089043 |
Jun 12, 1998 |
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Reissue of: |
09330235 |
Jun 10, 1999 |
06459018 |
Oct 1, 2002 |
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Current U.S.
Class: |
800/281; 800/298;
435/69.1; 435/468; 435/419 |
Current CPC
Class: |
C12N
15/8247 (20130101); C12P 7/6472 (20130101); C12P
7/6427 (20130101); C12N 9/0083 (20130101) |
Current International
Class: |
A01H
5/00 (20060101); C12N 15/82 (20060101); C12N
9/02 (20060101); C12P 7/64 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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550162 |
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Jul 1993 |
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EP |
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561569 |
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Sep 1993 |
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EP |
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644263 |
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Mar 1995 |
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EP |
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736598 |
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Jan 1996 |
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EP |
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WO 91/13972 |
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Sep 1991 |
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WO |
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WO 93/06712 |
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Apr 1993 |
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WO |
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WO 93/11245 |
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Jun 1993 |
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WO |
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WO 94/11516 |
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May 1994 |
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WO |
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WO 94/18337 |
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Aug 1994 |
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WO |
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WO 96/10086 |
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Apr 1996 |
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WO |
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WO 96/21022 |
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Jul 1996 |
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WO |
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WO 97/30582 |
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Aug 1997 |
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WO |
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Other References
Reddy and Thomas, "Expression of a cyanobacterial .DELTA..sup.6
desaturase gene results in gamma-linolenic acid production in
transgenic plants," Nature Biotechnology 14:639-642 (May 1996).
cited by other .
"Exciting prospects for stearidonic acid seed oils," Lipid
Technology (Nov. 1996). cited by other.
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Primary Examiner: McElwain; Elizabeth F
Attorney, Agent or Firm: Howrey LLP
Parent Case Text
This application claims the benefit of U.S. Provisional Application
No. 60/089,043 filed Jun. 12, 1998.
Claims
What is claimed is:
1. A method for producing stearidonic acid in a plant seed, said
method comprising: growing a plant having integrated into its
genome.Iadd.: .Iaddend. a first DNA construct comprising, in the 5'
to 3' direction of transcription, a promoter functional in a plant
seed cell, a DNA sequence encoding a delta-six desaturase
.Iadd.from the genus Mortierella.Iaddend., and a transcription
termination region functional in a plant cell, .Iadd.a second DNA
construct comprising, in the 5' to 3' direction of transcription, a
promoter function in a plant seed cell, and a DNA sequence encoding
a delta 15 desaturase, and a transcription termination region
functional in a plant cell, and a third DNA construct comprising,
in the 5' to 3' direction of transcription, a promoter functional
in a plant seed cell, and a DNA sequence encoding a delta 12
desaturase, and a transcription termination region functional in a
plant cell,.Iaddend. and growing said plant under conditions
whereby said delta-six desaturase.Iadd., delta 15 desaturase and
delta 12 desaturase .Iaddend..[.is.]. .Iadd.are
.Iaddend.expressed.
.[.2. The method according to claim 1 wherein said plant has a
second construct integrated into its genome, wherein said second
construct has in the 5' to 3' direction of transcription, a
promoter functional in a plant seed cell, and a DNA sequence
encoding a delta 12 desaturase..].
.[.3. The method according to claim 1 wherein said plant has a
second construct integrated into its genome, wherein said second
construct has in the 5' to 3' direction of transcription, a
promoter functional in a plant seed cell,and a DNA sequence
encoding a delta 15 desaturase..].
.[.4. The method of claim 1 wherein said desaturase encoding
sequence is from the genus Mortierella..].
5. The method according to claim 1, wherein said promoter .Iadd.for
the first DNA construct .Iaddend.is a napin promoter.
6. The method according to claim 1, wherein said promoter .Iadd.for
the first DNA construct .Iaddend.is from the soybean
.beta.-conglycinin 7S subunit transcription initiation region.
7. The method according to claim 1, wherein said method further
comprises extracting oil from said plant seed.
8. The method of claim 7, wherein said oil comprises about 5 weight
percent or greater stearidonic acid.
9. The method of claim 7, wherein said oil comprises about 10
weight percent or greater stearidonic acid.
10. The method of claim 7, wherein said oil comprises about 15
weight percent or greater stearidonic acid.
11. The method of claim 7, wherein said oil comprises about 20
weight percent or greater stearidonic acid.
12. The method of claim 7, wherein said oil comprises about 25
weight percent or greater stearidonic acid.
.Iadd.13. A method for producing stearidonic acid in a plant seed,
said method comprising: growing a plant having integrated into its
genome; a first DNA construct comprising, in the 5' to 3' direction
of transcription, a promoter functional in a plant seed cell, a DNA
sequence encoding a delta-six desaturase, and a transcription
termination region functional in a plant cell, a second DNA
construct comprising, in the 5' to 3' direction of transcription, a
promoter functional in a plant seed cell, and a DNA sequence
encoding a delta 15 desaturase, and a transcription termination
region functional in a plant cell, and a third DNA construct
comprising, in the 5' to 3' direction of transcription, a promoter
functional in a plant seed cell, and a DNA sequence encoding a
delta 12 desaturase, and a transcription termination region
functional in a plant cell, and growing said plant under conditions
whereby said delta-six desaturase, delta 15 desaturase, and
delta-12 desaturase are expressed..Iaddend.
.Iadd.14. The method according to claim 13, wherein said promoter
for the first DNA construct is a napin promoter..Iaddend.
.Iadd.15. The method according to claim 13, wherein said promoter
for the first DNA construct is from the soybean .beta.-conglycinin
7S subunit transcription initiation region..Iaddend.
.Iadd.16. The method according to claim 13, wherein said method
further comprises extracting oil from said plant seed..Iaddend.
.Iadd.17. The method of claim 16, wherein said oil comprises about
5 weight percent or greater stearidonic acid..Iaddend.
.Iadd.18. The method of claim 16, wherein said oil comprises about
10 weight percent or greater stearidonic acid..Iaddend.
.Iadd.19. The method of claim 16, wherein said oil comprises about
15 weight percent or greater stearidonic acid..Iaddend.
.Iadd.20. The method of claim 16, wherein said oil comprises about
20 weight percent or greater stearidonic acid..Iaddend.
.Iadd.21. The method of claim 16, wherein said oil comprises about
25 weight percent or greater stearidonic acid..Iaddend.
Description
TENHNICAL FIELD
This invention relates to modulating levels of enzymes and/or
enzyme components capable of altering the production of long chain
polyunsaturated fatty acids (PUFAS) in a host plant. The invention
is exemplified by the production of PUFAS in plants.
BACKGROUND
Three main families of polyunsaturated fatty acids (PUFAs) are the
3 fatty acids, exemplified by arachidonic acid, the .omega.9 fatty
acids exemplified by Mead acid, and the .omega.3 fatty acids,
exemplified by eicosapentaenoic acid. PUFAs are important
components of the plasma membrane of the cell, where they may be
found in such forms as phospholipids. PUFAs also serve as
precursors to other molecules of importance in human beings and
animals, including the prostacyclins, leukotrienes and
prostaglandins. PUFAs are necessary for proper development,
particularly in the developing infant brain, and for tissue
formation and repair.
Four major long chain PUFAs of importance include docosahexaenoic
acid (DHA) and eicosapentaenoic acid (EPA), which are primarily
found in different types of fish oil, gamma-linolenic acid (GLA),
which is found in the seeds of a number of plants, including
evening primrose (Oenothera biennis), borage (Borago officinalis)
and black currants (Ribes nigrum), and stearidonic acid (SDA),
which is found in marine oils and plant seeds. Both GLA and another
important long chain PUFA, arachidonic acid (ARA), are found in
filamentous fungi. ARA can be purified from animal tissues
including liver and adrenal gland. Mead acid accumulates in
essential fatty acid deficient animals.
For DHA, a number of sources exist for commercial production
including a variety of marine organisms, oils obtained from cold
water marine fish, and egg yolk fractions. For ARA, microorganisms,
including the genera Mortierella, Entomophthora, Phytium and
Porphyridium can be used for commercial production. Commercial
sources of SDA include the genera Trichodesma and Echium.
Commercial sources of GLA include evening primrose, black currants
and borage. However, there are several disadvantages associated
with commercial production of PUFAs from natural sources. Natural
sources of PUFAs, such as animals and plants, tend to have highly
heterogeneous oil compositions. The oils obtained from these
sources therefore can require extensive purification to separate
out one or more desired PUFAs or to produce an oil which is
enriched in one or more PUFA. Natural sources also are subject to
uncontrollable fluctuations in availability. Fish stocks may
undergo natural variation or may be depleted by overfishing. Fish
oils have unpleasant tastes and odors, which may be impossible to
economically separate from the desired product, and can render such
products unacceptable as food supplements. Animal oils, and
particularly fish oils, can accumulate environmental pollutants.
Weather and disease can cause fluctuation in yields from both fish
and plant sources. Cropland available for production of alternate
oil-producing crops is subject to competition from the steady
expansion of human populations and the associated increased need
for food production on the remaining arable land. Crops which do
produce PUFAs, such as borage, have not been adapted to commercial
growth and may not perform well in monoculture. Growth of such
crops is thus not economically competitive where more profitable
and better established crops can be grown. Large scale fermentation
of organisms such as Mortierella is also expensive. Natural animal
tissues contain low amounts of ARA and are difficult to process.
Microorganisms such as Porphyridium and Mortierella are difficult
to cultivate on a commercial scale.
Dietary supplements and pharmaceutical formulations containing
PUFAs can retain the disadvantages of the PUFA source. Supplements
such as fish oil capsules can contain low levels of the particular
desired component and thus require large dosages. High dosages
result in ingestion of high levels of undesired components,
including contaminants. Care must be taken in providing fatty acid
supplements, as overaddition may result in suppression of
endogenous biosynthetic pathways and lead to competition with other
necessary fatty acids in various lipid fractions in vivo, leading
to undesirable results. For example, Eskimos having a diet high in
.omega.3 fatty acids have an increased tendency to bleed (U.S. Pat.
No. 4,874,603). Unpleasant tastes and odors of the supplements can
make such regimens undesirable, and may inhibit compliance by the
patient.
A number of enzymes are involved in PUFA biosynthesis. Linoleic
acid (LA, 18:2 .DELTA.9, 12) is produced from oleic acid (18:1
.DELTA.9) by a .DELTA.12-desaturase. GLA (18:3 .DELTA.6, 9, 12) is
produced from linoleic acid (LA, 18:2 .DELTA.9, 12) by a
.DELTA.6-desaturase. ARA (20:4 .DELTA.5, 8, 11, 14) production from
DGLA (20:3 .DELTA.8, 11, 14) is catalyzed by a .DELTA.5-desaturase.
However, animals cannot desaturate beyond the .DELTA.9 position and
therefore cannot convert oleic acid (18:1 .DELTA.9) into linoleic
acid (18:2 .DELTA.9, 12). Likewise, .alpha.-linolenic acid (ALA,
18:3 .DELTA.9, 12, 15) cannot be synthesized by mammals. Other
eukaryotes, including fungi and plants, have enzymes which
desaturate at positions .DELTA.12 and .DELTA.15. The major
polyunsaturated fatty acids of animals therefore are either derived
from diet and/or from desaturation and elongation of linoleic acid
(18:2 .DELTA.9, 12) or -linolenic acid (18:3 .DELTA.9, 12, 15).
Poly-unsaturated fatty acids are considered to be useful for
nutritional, pharmaceutical, industrial, and other purposes. An
expansive supply of poly-unsaturated fatty acids from natural
sources and from chemical synthesis are not sufficient for
commercial needs. Therefore it is of interest to obtain genetic
material involved in PUFA biosynthesis from species that naturally
produce these fatty acids and to express the isolated material
alone or in combination in a heterologous system which can be
manipulated to allow production of commercial quantities of
PUFAS.
SUMMARY OF THE INVENTION
Novel compositions and methods are provided for preparation of
poly-unsaturated long chain fatty acids and desaturases in plants
and plant cells. The methods involve growing a host plant cell of
interest transformed with an expression cassette functional in a
host plant cell, the expression cassette comprising a
transcriptional and translational initiation regulatory region,
joined in reading frame 5' to a DNA sequence encoding a desaturase
polypeptide capable of modulating the production of PUFAs.
Expression of the desaturase polypeptide provides for an alteration
in the PUFA profile of host plant cells as a result of altered
concentrations of enzymes involved in PUFA biosynthesis. Of
particular interest is the selective control of PUFA production in
plant tissues and/or plant parts such as leaves, roots, fruits and
seeds. The invention finds use for example in the large scale
production of DHA, Mead Acid, EPA, ARA, DGLA, stearidonic acid GLA
and other fatty acids and for modification of the fatty acid
profile of edible plant tissues and/or plant parts.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows possible pathways for the synthesis of Mead acid (20:3
.DELTA.5, 8, 11), arachidonic acid (20:4 .DELTA.5, 8, 11, 14) and
stearidonic acid (18:4 .DELTA.6, 9, 12, 15) from palmitic acid
(C.sub.16) from a variety of organisms, including algae,
Mortierella and humans. These PUFAs can serve as precursors to
other molecules important for humans and other animals, including
prostacyclins, leukotrienes, and prostaglandins, some of which are
shown.
FIG. 2 shows possible pathways for production of PUFAs in addition
to ARA, including taxoleic acid and pinolenic, again compiled from
a variety of organisms.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In order to ensure a complete understanding of the invention, the
following definitions are provided: .DELTA.5-Desaturase: .DELTA.5
desaturase is an enzyme which introduces a double bond between
carbons 5 and 6 from the carboxyl end of a fatty acid molecule.
.DELTA.6-Desaturase: .DELTA.6-desaturase is an enzyme which
introduces a double bond between carbons 6 and 7 from the carboxyl
end of a fatty acid molecule. .DELTA.9-Desaturase:
.DELTA.9-desaturase is an enzyme which introduces a double bond
between carbons 9 and 10 from the carboxyl end of a fatty acid
molecule. .DELTA.12-Desaturase: .DELTA.12-desaturase is an enzyme
which introduces a double bond between carbons 12 and 13 from the
carboxyl end of a fatty acid molecule.
Fatty acids: Fatty acids are a class of compounds containing a
long-hydrocarbon chain and a terminal carboxylate group. Fatty
acids include the following:
TABLE-US-00001 Fatty Acid 12:0 Isuric acid 16:0 Palmitic acid 16:1
Palmitoleic acid 18:0 stearic acid 18:1 oleic acid .DELTA.9-18:1
18:2 .DELTA.5,9 Taxoleic acid .DELTA.5,9-18:2 18:2 .DELTA.6,9
6,9-octadecadienoic acid .DELTA.6,9-18:2 18:2 Linoleic acid
.DELTA.9,12-18:2 (LA) 18:3 .DELTA.6,9,12 Gasima-linoleic acid
.DELTA.6,9,12-18:3 (GLA) 18:3 .DELTA.5,9,12 Pinolenic acid
.DELTA.5,9,12-18:3 18:3 alpha-linolenic acid .DELTA.9,12,15-18:3
(ALA) 18:4 Stearidonic acid .DELTA.6,9,12,15-18:4 (SDA) 20:0
Arachidic acid 20:1 Eicosoenic Acid 20:2 .DELTA.8,11 .DELTA.8,11
20:3 .DELTA.5,8,11 Mend Acid .DELTA.5,8,11 22:0 Behenoic acid 22:1
erucic acid 22:2 Docanadienoic acid 20:4 .gamma.6 Arachidonic acid
.DELTA.5,8,11,14-20:4 (ARA) 20:3 .gamma.6 .gamma.6-eicosatrienoic
dihomo- .DELTA.8,11,14-20:3 (DGLA) gamma linolenic 20:5 .gamma.6
Eicosapentaenoic .DELTA.5,8,11,14,17-20:5 (EPA) (Timnodonic acid)
20:3 .gamma.6 .gamma.3-eicosatoanoic .DELTA.11,16,17-20:3 20:4
.gamma.6 .gamma.3-eicosatetraenoic .DELTA.8,11,14,17-20:4 22:5
.gamma.6 Docosapentanoic .DELTA.7,10,13,16,19-22:5 (.gamma.3DPA)
22:6 .gamma.6 Docosahexaenoic (cervonic .DELTA.7,10,13,16,19-22:6
(DHA) acid) 24:0 Lignoceric acid
Taking into account these definitions, the present invention is
directed to novel DNA sequences, and DNA constructs related to the
production of fatty acids in plants. Methods and compositions are
provided which permit modification of the poly-unsaturated long
chain fatty acid content of plant cells. Plant cells are
transformed with an expression cassette comprising a DNA encoding a
polypeptide capable of increasing the amount of one or more PUFA in
a plant cell. Desirably, integration constructs may be prepared
which provide for integration of the expression cassette into the
genome of a host cell. Host cells are manipulated to express a
sense or antisense DNA encoding a polypeptide(s) that has
desaturase activity. By "desaturase" is intended a polypeptide
which can desaturate one or more fatty acids to produce a mono- or
poly-unsaturated fatty acid or precursor thereof of interest. By
"polypeptide" is meant any chain of amino acids, regardless of
length or post-translational modification, for example,
glycosylation or phosphorylation. The substrate(s) for the
expressed enzyme may be produced by the host cell or may be
exogenously supplied.
To achieve expression in a host cell, the transformed DNA is
operably associated with transcriptional and translational
initiation and termination regulatory regions that are functional
in the host cell. Constructs comprising the gene to be expressed
can provide for integration into the genome of the host cell or can
autonomously replicate in the host cell. For production of taxoleic
acid, the expression cassettes generally used include a cassette
which provides for .DELTA.5 desaturase activity, particularly in a
host cell which produces or can take up oleic acid. For production
of .DELTA.6,9 18:2 or other .omega.9 unsaturated fatty acids, the
expression cassettes generally used include a cassette which
provides for .DELTA.6 desaturase activity, particularly in a host
cell which produces or can take up oleic acid. Production of a
oleic acid, taxoleic acid, or .omega.9 unsaturated fatty acids in a
plant having .DELTA.12 desaturase activity is favored by providing
an expression cassette. for an antisense .DELTA.12 transcript, or
by disrupting a .DELTA.12 desaturase gene. For production of
linoleic acid (LA), the expression cassettes generally used include
a cassette which provides for .DELTA.12 desaturase activity,
particularly in a host cell which produces or can take up oleic
acid. For production of ALA, the expression cassettes generally
used include a cassette which provides for .DELTA.15 to .omega.3
desaturase activity, particularly in a host cell which produces or
can take up LA. For production of GLA or SDA, the expression
cassettes generally used include a cassette which provides for
.DELTA.6 desaturase activity, particularly in a host cell which
produces or can take up LA or ALA, respectively. Production of
.omega.6-type unsaturated fatty acids, such as LA or GLA, in a
plant capable of producing ALA is favored by inhibiting the
activity of .DELTA.15 or .omega.3 type desaturase; this is
accomplished by providing and expression cassette for an antisense
.DELTA.15 or .omega.3 transcript, or by disrupting a .DELTA.15 or
.omega.3 desaturase gene. Similarly, production of LA or ALA in a
plant having .DELTA.6 desaturase activity is favored by providing
an expression cassette for an antisense A 6 transcript, or by
disrupting a .DELTA.6 desaturase gene. For production of ARA in a
host cell which produces or can take up DGLA, the expression
cassette generally used provides for D5 desaturase activity.
Production of .omega.6-type unsaturated fatty acids, such as ARA,
in a plant capable of producing ALA is favored by inhibiting the
activity of a .DELTA.15 or .omega.-3 desaturase; this is
accomplished by providing an expression cassette for an antisense
.DELTA.15 or .omega.3 transcript, or by disrupting a .DELTA.15 of
.omega.3 desaturase gene.
TRANSGENIC PLANT PRODUCTION OF FATTY ACIDS
Transgenic plant production of PUFAs offers several advantages over
purification from natural sources such as fish or plants.
Production of fatty acids from recombinant plants provides the
ability to alter the naturally occurring plant fatty acid profile
by providing new synthetic pathways in the host or by suppressing
undesired pathways, thereby increasing levels of desired PUFAs, or
conjugated forms thereof, and decreasing levels of undesired PUFAs.
Production of fatty acids in transgenic plants also offers the
advantage that expression of desaturase genes in particular tissues
and/or plant parts means that greatly increased levels of desired
PUFAs in those tissues and/or parts can be achieved, making
recovery from those tissues more economical. For example, the
desired PUFAs can be expressed in seed; methods of isolating seed
oils are well established. In addition to providing a source for
purification of desired PUFAs, seed oil components can be
manipulated through expression of desaturase genes, either alone or
in combination with other genes such as elongases, to provide seed
oils having a particular PUFA profile in concentrated form. The
concentrated seed oils then can be added to animal milks and/or
synthetic or semi-synthetic milks to serve as infant formulas where
human nursing is impossible or undesired, or in cases of
malnourishment or disease in both adults and infants.
For production of PUFAs, depending upon the host cell, the
availability of substrate, and the desired end product(s), several
polypeptides, particularly desaturases, are of interest including
those polypeptides which catalyze the conversion of stearic acid to
oleic acid, LA to GLA, of ALA to SDA, of oleic acid to LA, or of LA
to ALA, oleic acid to taxolic acid, LA to pinolenic acid, oleic
acid to 6,9-actadeca-dienoic acid which includes enzymes which
desaturate at the .DELTA.6, .DELTA.9, .DELTA.5, .DELTA.12,
.DELTA.15, .DELTA.5, or .omega.3 positions. Considerations for
choosing a specific polypeptide having desaturase activity include
the pH optimum of the polypeptide, whether the polypeptide is a
rate limiting enzyme or a component thereof, whether the desaturase
used is essential for synthesis of a desired polyunsaturated fatty
acid, and/or co-factors required by the polypeptide. The expressed
polypeptide preferably has parameters compatible with the
biochemical environment of its location in the host cell. For
example, the polypeptide may have to compete for substrate with
other enzymes in the host cell. Analyses of the K.sub.m and
specific activity of the polypeptide in question therefore are
considered in determining the suitability of a given polypeptide
for modifying PUFA production in a given host cell. The polypeptide
used in a particular situation therefore is one which can function
under the conditions present in the intended host cell but
otherwise can be any polypeptide having desaturase activity which
has the desired characteristic of being capable of modifying the
relative production of a desired PUFA. A scheme for the synthesis
of arachidonic acid (20:4 .DELTA.5, 8, 11, 14) from palmitic acid
(C.sub.16) is shown in FIG. 1. A key enzyme in this pathway is a
.DELTA.5-desaturase which converts DH-.gamma.-linolenic acid (DGLA,
eicosatrienoic acid) to ARA. Conversion of .alpha.-linolenic acid
(ALA) to stearidonic acid by a .DELTA.6-desaturase is also shown.
Production of PUFAs in addition to ARA, including EPA and DHA is
shown in FIG. 2. A key enzyme in the synthesis of arachidonic acid
(20:4 .DELTA.5, 8, 11, 14) from stearic acid (C.sub.18) is a
.DELTA.6-desaturase which converts the linoleic acid into
.gamma.-linolenic acid. Conversion of .alpha.-linolenic acid (ALA)
to stearidonic acid by a .DELTA.6-desaturase also is shown. For
production of ARA, the DNA sequence used encodes a polypeptide
having .DELTA.5 desaturase activity. In particular instances, this
can be coupled with an expression cassette which provides for
production of a polypeptide having .DELTA.6 desaturase activity
and, optionally, a transcription cassette providing for production
of antisense sequences to a .DELTA.15 transcription product. The
choice of combination of cassettes used depends in part on the PUFA
profile of the host cell. Where the host cell .DELTA.5-desaturase
activity is limiting, overexpression of .DELTA.5 desaturase alone
generally will be sufficient to provide for enhanced ARA
production.
SOURCES OF POLYPEPTIDES HAVING DESATURASE ACTIVITY
As sources of polypeptides having desaturase activity and
oligonucleotides encoding such polypeptides are organisms which
produce a desired poly-unsaturated fatty acid. As an example,
microorganisms having an ability to produce ARA can be used as a
source of .DELTA.5-desaturase genes; microorganisms which GLA or
SDA can be used as a source of .DELTA.6-desaturase and/or
.DELTA.12-desaturase genes. Such microorganisms include, for
example, those belonging to the genera Mortierella, Conidiobolus,
Pythium, Phytophathora, Penicillium, Porphyridium, Coidosporium,
Mucor, Fusarium, Aspergillus, Rhodotorula, and Entomophthora.
Within the genus Porphyridium, of particular interest is
Porphyridium cruentum. Within the genus Mortierella, of particular
interest are Mortierella elongata, Mortierella exigua, Mortierella
hygrophila, Mortierella ramanniana, var. angulispora, and
Mortierella alpina. Within the genus Mucor, of particular interest
are Mucro circinelloides and Mucor javanicus.
DNAs encoding desired desaturases can be identified in a variety of
ways. As an example, a source of the desired desaturase, for
example genomic or cDNA libraries from Mortierella, is screened
with detectable enzymatically- or chemically-synthesized probes,
which can be made from DNA, RNA, or non-naturally occurring
nucleotides, or mixtures thereof. Probes may be enzymatically
synthesized from DNAs of known desaturases for normal or
reduced-stringency hybridization methods. Oligonucleotide probes
also can be used to screen sources and can be based on sequences of
known desaturates, including sequences conserved among known
desaturases, or on peptide sequences obtained from the desired
purified protein. Oligonucleotide probes based on amino acid
sequences can be degenerate to encompass the degeneracy of the
genetic code, or can be biased in favor of the preferred codons of
the source organism. Oligonucleotides also can be used as primers
for PCR from reverse transcribed mRNA from a known or suspected
source; the PCR product can be the full length cDNA or can be used
to generate a probe to obtain the desired full length cDNA.
Alteratively, a desired protein can be entirely sequenced and total
synthesis of a DNA encoding that polypeptide performed.
Once the desired genomic or cDNA has been isolated, it can be
sequenced by known methods. It is recognized in the art that such
methods are subject to errors, such that multiple sequencing of the
same region is routine and is still expected to lead to measurable
rates of mistakes in the resulting deduced sequence, particularly
in regions having repeated domains, extensive secondary structure,
or unusual base compositions, such as regions with high GC base
content. When discrepancies arise, resequencing can be done and can
employ special methods. Special methods can include altering
sequencing conditions by using: different temperatures; different
enzymes; proteins which alter the ability of oligonucleotides to
form higher order structures; altered nucleotides such as ITP or
methylated dGTP; different gel compositions, for example adding
formamide; different primers or primers located at different
distances from the problem region; or different templates such as
single stranded DNAs. Sequencing of mRNA can also be employed.
For the most part, some or all of the coding sequence for the
polypeptide having desaturase activity is from a natural source. In
some situations, however, it is desirable to modify all or a
portion of the codons, for example, to enhance expression, by
employing host preferred codons. Host preferred codons can be
determined from the codons of highest frequency in the proteins
expressed in the largest amount in a particular host species of
interest. Thus, the coding sequence for a polypeptide having
desaturase activity can be synthesized in whole or in part. All or
portions of the DNA also can be synthesized to remove any
destabilizing sequences or regions of a secondary structure which
would be present in the transcribed mRNA. All or portions of the
DNA also can be synthesized to alter the base composition to one
more preferable in the desired host cell. Methods for synthesizing
sequences and bringing sequences together are well established in
the literature. In vitro mutagenesis and selection, site-directed
mutagenesis, or other means can be employed to obtain mutations of
naturally occurring desaturase genes to produce a polypeptide
having desaturase activity in vivo with more desirable physical and
kinetic parameters for function in the host cell, such as a longer
half-life or a higher rate of production of a desired
polyunsaturated fatty acid.
Desirable cDNAs have less than 60% A+T composition, preferably less
than 50% A+T composition. On a localized scale of a sliding window
of 20 base pairs, it is preferable that there are no localized
regions of the cDNA with greater than 75% A+T composition; with a
window of 60 base pairs, it is preferable that there are no
localized regions of the cDNA with greater than 60%, more
preferably no localized regions with greater than 55% A+T
composition.
Mortierella Alpina Desaturases
Of particular interest are the Mortierella alpina
.DELTA.5-desaturase, .DELTA.6-desaturase, .DELTA.12-desaturase and
.DELTA.15 desaturase. The gene encoding the Mortierella alpina
.DELTA.5-desaturase can be expressed in transgenic plants to effect
greater synthesis of ARA from DGLA, or pinolenic acid from LA,
taxoleic acid from oleic acid or Mead and from .DELTA.8, 11-20:2.
Other DNAs which are substantially identical in sequence to the
Mortierella alpina .DELTA.5-desaturase DNA, or which encode
polypeptides which are substantially identical in sequence to the
Mortierella alpina.DELTA.5-desaturase polypeptide, also can be
used. The gene encoding the Mortierella alpina .DELTA.6-desaturase
can be expressed in transgenic plants or animals to effect greater
synthesis of GLA from linoleic acid or stearidonic acid (SDA) from
ALA or of 6,9-octadecadienoic acid from oleic acid. Other DNAs
which are substantially identical in sequence to the Mortierella
alpina .DELTA.6-desaturase DNA, or which encode polypeptides which
are substantially identical in sequence to the Mortierella alpina
.DELTA.6-desaturase polypeptide, also can be used.
The gene encoding the Mortierella alpina .DELTA.12-desaturase can
be expressed in transgenic plants to effect greater synthesis of LA
from oleic acid. Other DNAs which are substantially identical to
the Mortierella alpina .DELTA.12-desaturase DNA, or which encode
polypeptides which are substantially identical to the Mortierella
alpina .DELTA.12-desaturase polypeptide, also can be used.
By substantially identical in sequence is intended an amino acid
sequence or nucleic acid sequence exhibiting in order of increasing
preference at least 60%, 80%, 90% or 95% homology to the
Mortierella alpina .DELTA.5-desaturase amino acid sequence or
nucleic acid sequence encoding the amino acid sequence. For
polypeptides, the length of comparison sequences generally is at
least 16 amino acids, preferably at least 20 amino acids, or most
preferably 35 amino acids. For nucleic acids, the length of
comparison sequences generally is at least 50 nucleotides,
preferably at least 60 nucleotides, and more preferably at least 75
nucleotides, and most preferably, 110 nucleotides. Homology
typically is measured using sequence analysis software, for
example, the Sequence Analysis software package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705, MEGAlign (DNAStar, Inc.,
1228 S. Park St., Madison, Wis. 53715), and MacVector (Oxford
Molecular Group, 2105 S. Bascom Avenue, Suite 200, Campbell, Calif.
95008). Such software matches similar sequences by assigning
degrees of homology to various substitutions, deletions, and other
modifications. Conservative substitutions typically include
substitutions within the following groups: glycine and alanine;
valine, isoleucine and leucine; aspartic acid, glutamic acid,
asparagine, and glutamine; serine and threonine; lysine and
arginine; and phenylalanine and tyrosine. Substitutions may also be
made on the basis of conserved hydrophobicity or hydrophilicity
(Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982), or on the
basis of the ability to assume similar polypeptide secondary
structure (Chou and Fasman, Adv. Enzymol. 47: 45-148, 1978).
EXPRESSION OF DESATURASE GENES
Once the DNA encoding a desaturase polypeptide has been obtained,
it is placed in a vector capable of replication in a host cell, or
is propagated in vitro by means of techniques such as PCR or long
PCR. Replicating vectors can include plasmids, phage, viruses,
cosmids and the like. Desirable vectors include those useful for
mutagenesis of the gene of interest or for expression of the gene
of interest in host cells. The technique of long PCR has made in
vitro propagation of large constructs possible, so that
modifications to the gene of interest, such as mutagenesis or
addition of expression signals, and propagation of the resulting
constructs can occur entirely in vitro without the use of a
replicating vector or a host cell.
For expression of a desaturase polypeptide, functional
transcriptional and translational initiation and termination
regions are operably linked to the DNA encoding the desaturase
polypeptide. Transcriptional and translational initiation and
termination regions are derived from a variety of nonexclusive
sources, including the DNA to be expressed, genes known or
suspected to be capable of expression in the desired system,
expression vectors, chemical synthesis, or from an endogenous locus
in a host cell. Expression in a plant tissue and/or plant part
presents certain efficiencies, particularly where the tissue or
part is one which is easily harvested, such as seed, leaves,
fruits, flowers, roots, etc. Expression can be targeted to that
location within the plant by using specific regulatory sequences,
such as those of U.S. Pat. No. 5,463,174, U.S. Pat. No. 4,943,674,
U.S. Pat. No. 5,106,739, U.S. Pat. No. 5,175,095, U.S. Pat. No.
5,420,034, U.S. Pat. No. 5,188,958, and U.S. Pat. No.
5,589,379.
Alternatively, the expressed protein can be an enzyme which
produces a product which may be incorporated, either directly or
upon further modifications, into a fluid fraction from the host
plant. In the present case, expression of desaturase genes, or
antisense desaturase transcripts, can alter the levels of specific
PUFAs, or derivatives thereof, found in plant parts and/or plant
tissues. The .DELTA.5-desaturase polypeptide coding region is
expressed either by itself or with other genes, in order to produce
tissues and/or plant parts containing higher proportions of desired
PUFAs or in which the PUFA composition more closely resembles that
of human breast milk (Prieto et al., PCT publication WO 95/24494).
The termination region can be derived from the 3' region of the
gene from which the initiation region was obtained or from a
different gene. A large number of termination regions are known to
and have been found to be satisfactory in a variety of hosts from
the same and different genera and species. The termination region
usually is selected more as a matter of convenience rather than
because of any particular property.
The choice of a host cell is influenced in part by the desired PUFA
profile of the transgenic cell, and the native profile of the host
cell. As an example, for production of linoleic acid from oleic
acid, the DNA sequence used encodes a polypeptide having .DELTA.12
desaturase activity, and for production of GLA from linoleic acid,
the DNA sequence used encodes a polypeptide having .DELTA.6
desaturase activity. Use of a host cell which expresses .DELTA.12
desaturase activity and lacks or is depleted in .DELTA.15
desaturase activity, can be used with an expression cassette which
provides for overexpression of .DELTA.6 desaturase alone generally
is sufficient to provide for enhanced GLA production in the
transgenic cell. Where the host cell expresses .DELTA.9 desaturase
activity, expression of both a .DELTA.12- and a .DELTA.6-desaturase
can provide for enhanced GLA production. In particular instances
where expression of .DELTA.6 desaturase activity is coupled with
expression of .DELTA.12 desaturase activity, it is desirable that
the host cell naturally have, or be mutated to have, low .DELTA.15
desaturase activity. Alternatively, a host cell for .DELTA.6
desaturase expression may have, or be mutated to have, high
.DELTA.12 desaturase activity.
Expression in a host cell can be accomplished in a transient or
stable fashion. Transient expression can occur from introduced
constructs which contain expression signals functional in the host
cell, but which constructs do not replicate and rarely integrate in
the host cell, or where the host cell is not proliferating.
Transient expression also can be accomplished by inducing the
activity of a regulatable promoter operably linked to the gene of
interest, although such inducible systems frequently exhibit a low
basal level of expression. Stable expression can be achieved by
introduction of a construct that can integrate into the host genome
or that autonomously replicates in the host cell. Stable expression
of the gene of interest can be selected for through the use of a
selectable marker located on or transfected with the expression
construct, followed by selection for cells expressing the marker.
When stable expression results from integration, integration of
constructs can occur randomly within the host genome or can be
targeted through the use of constructs containing regions of
homology with the host genome sufficient to target recombination
with the host locus. Where constructs are targeted to an endogenous
locus, all or some of the transcriptional and translation
regulatory regions can be provided by the endogenous locus.
When increased expression of the desaturase polypeptide in the
source plant is desired, several methods can be employed.
Additional genes encoding the desaturase polypeptide can be
introduced into the host organism. Expression from the native
desaturase locus also can be increased through homologous
recombination, for example by inserting a stronger promoter into
the host genome to cause increased expression, by removing
destabilizing sequences from either the mRNA or the encoded protein
by deleting that information from the host genome, or by adding
stabilizing sequences to the mRNA (see U.S. Pat. No. 4,910,141 and
U.S. Pat. No. 5,500,365.)
When it is desirable to express more than one different gene,
appropriate regulatory regions and expression methods, introduced
genes can be propagated in the host cell through use of replicating
vectors or by integration into the host genome. Where two or more
genes are expressed from separate replicating vectors, it is
desirable that each vector has a different means of replication.
Each introduced construct, whether integrated or not, should have a
different means of selection and should lack homology to the other
constructs to maintain stable expression and prevent reassortment
of elements among constructs. Judicious choices of regulatory
regions, selection means and method of propagation of the
introduced construct can be experimentally determined so that all
introduced genes are expressed at the necessary levels to provide
for synthesis of the desired products.
Constructs comprising the gene of interest may be introduced into a
host cell by standard techniques. These techniques include
transfection, infection, bolistic impact, electroporation,
microinjection, scraping, or any other method which introduces the
gene of interest into the host cell (see U.S. Pat. No. 4,743,548,
U.S. Pat. No. 4,795,855, U.S. Pat. No. 5,068,193, U.S. Pat. No.
5,188,958, U.S. Pat. No. 5,463,174, U.S. Pat. No. 5,565,346 and
U.S. Pat. No. 5,565,347). For convenience, a host cell which has
been manipulated by any method to take up a DNA sequence or
construct will be referred to as "transformed" or "recombinant"
herein. The subject host will have at least have one copy of the
expression construct and may have two or more, depending upon
whether the gene is integrated into the genome, amplified, or is
present on an extrachromosomal element having multiple copy
numbers.
The transformed host cell can be identified by selection for a
marker contained on the introduced construct. Alternatively, a
separate marker construct may be introduced with the desired
construct, as many transformation techniques introduce many DNA
molecules into host cells. Typically, transformed hosts are
selected for their ability to grow on selective media. Selective
media may incorporate an antibiotic or lack a factor necessary for
growth of the untransformed host, such as a nutrient or growth
factor. An introduced marker gene therefor may confer antibiotic
resistance, or encode an essential growth factor or enzyme, and
permit growth on selective media when expressed in the transformed
host cell. Desirably, resistance to kanamycin and the amino
glycoside G418 are of interest (see U.S. Pat. No. 5,034,322).
Selection of a transformed host can also occur when the expressed
marker protein can be detected, either directly or indirectly. The
marker protein may be expressed alone or as a fusion to another
protein. The marker protein can be detected by its enzymatic
activity; for example .beta. galactosidase can convert the
substrate X-gal to a colored product, and luciferase can convert
luciferin to a light-emitting product. The marker protein can be
detected by its light-producing or modifying characteristics; for
example, the green fluorescent protein of Aequorea victoria
fluoresces when illuminated with blue light. Antibodies can be used
to detect the marker protein or a molecular tag on, for example, a
protein of interest. Cells expressing the marker protein or tag can
be selected, for example, visually, or by techniques such as FACS
or panning using antibodies.
The PUFAs produced using the subject methods and compositions may
be found in the host plant tissue and/or plant part as free fatty
acids or in conjugated forms such as acylglycerols, phospholipids,
sulfolipids or glycolipids, and may be extracted from the host cell
through a variety of means well-known in the art. Such means may
include extraction with organic solvents, sonication, supercritical
fluid extraction using for example carbon dioxide, and physical
means such as presses, or combinations thereof. Of particular
interest is extraction with hexane or methanol and chloroform.
Where desirable, the aqueous layer can be acidified to protonate
negatively charged moieties and thereby increase partitioning of
desired products into the organic layer. After extraction, the
organic solvents can be removed by evaporation under a stream of
nitrogen. When isolated in conjugated forms, the products are
enzymatically or chemically cleaved to release the free fatty acid
or a less complex conjugate of interest, and are then subjected to
further manipulations to produce a desired end product. Desirably,
conjugated forms of fatty acids are cleaved with potassium
hydroxide.
Surprisingly, as demonstrated more fully in the examples below,
expression of the Mortierella .DELTA.6 desaturase leads to the
production of steariodonic acid in the oil extracted from seed
tissue of host plant cells. Furthermore, expression of the .DELTA.6
desaturase with additional desaturases provided for the enhanced
production of SDA in the seed oil.
Thus, the present invention provides methods for the production of
steariodonic acid (C18:4) in host plant cells. The methods allow
for the production of SDA in host plant cells ranging from about
0.3 wt % to at least about 30 wt %, preferably, from about 5 wt %
to at least about 25 wt %, more preferably from about 7 wt % to at
least about 25 wt %. The SDA is preferably produced in the seed oil
of host plants containing one or more expression constructs as
described herein.
Furthermore, the present invention provides a novel source of plant
oils containing steariodonic acid. The oils are preferably obtained
from the plant seed tissue. The seed oils contain amounts of SDA
ranging from about 0.3 wt % to at least about 30 wt %, preferably,
from about 5 wt % to at least about 25 wt %, more preferably from
about 7 wt % to at least about 25 wt %.
PURIFICATION OF FATTY ACIDS
If further purification is necessary, standard methods can be
employed. Such methods include extraction, treatment with urea,
fractional crystallization, HPLC, fractional distillation, silica
gel chromatography, high speed centrifugation or distillation, or
combinations of these techniques. Protection of reactive groups,
such as the acid or alkenyl groups, may be done at any step through
known techniques, for example alkylation or iodination. Methods
used include methylation of the fatty acids to produce methyl
esters. Similarly, protecting groups may be removed at any step.
Desirably, purification of fractions containing ARA, DHA and EPA is
accomplished by treatment with urea and/or fractional
distillation.
USES OF FATTY ACIDS
The uses of the fatty acids of subject invention are several.
Probes based on the DNAs of the present invention may find use in
methods for isolating related molecules or in methods to detect
organisms expressing desaturases. When used as probes, the DNAs or
oligonucleotides need to be detectable. This is usually
accomplished by attaching a label either at an internal site, for
example, via incorporation of a modified residue, or at the 5' or
3' terminus. Such labels can be directly detectable, can bind to a
secondary molecule that is detectably labeled, or can bind to an
unlabelled secondary molecule and a detectably labeled tertiary
molecule; this process can be extended as long as is practical to
achieve a satisfactorily detectable signal without unacceptable
levels of background signal. Secondary, tertiary, or bridging
systems can include use of antibodies directed against any other
molecule, including labels or other antibodies, or can involve any
molecules which bind to each other, for example a
biotin-streptavidin/avidin system. Detectable labels typically
include radioactive isotopes, molecules which chemically or
enzymatically produce or alter light, enzymes which produce
detectable reaction products, magnetic molecules, fluorescent
molecules or molecules whose fluorescence or light-emitting
characteristics change upon binding. Examples of labelling methods
can be found in U.S. Pat. No. 5,011,770. Alternatively, the binding
of target molecules can be directly detected by measuring the
change in heat of solution on binding of probe to target via
isothermal titration calorimetry, or by coating the probe or target
on a surface and detecting the change in scattering of light from
the surface produced by binding of target or probe, respectively,
as may be done with the BIAcore system.
The invention will be better understood by reference to the
following non-limiting examples.
EXAMPLES
Example 1
Expression of .omega.-3 Desaturase from C. Elegans in Transgenic
Plants
The .DELTA.15/.omega.-3 activity of Brassica napus can be increased
by the expression of an .omega.-3 desaturase from C. elegans. The
fat-1 cDNA clone (Genbank accession L41807; Spychalla, J. P.,
Kinney, A. J., and Browse, J. 1997 P.A.A.S. 94, 1142-1147, SEQ ID
NO:1 and SEQ ID NO:2) was obtained from john Browse at Washington
State University. The fat-1 cDNA was modified by PCR to introduce
cloning sites using the following primers: Fat-1forward (SEQ ID
NO:3): 5'-CUACUACUACUACTGCAGACAATGGTCGCTCATTCCTCAGA-3' Fat-1reverse
(SEQ ID NO:4): 5'-CAUCAUCAUCAUGCGGCCGCTTACTTGGCCTTTGCCTT-3'
These primers allowed the amplification of the entire coding region
and added PstI and NotI sites to the 5'- and 3'-ends, respectively.
The PCR product was subcloned into pAMP1 (GIBCOBRL) using the
CloneAmp system (GIBCOBRL) to create pCGN5562. The sequence was
verified by sequencing of both strands to be sure no changes were
introduced by PCR.
A once base pair difference was observed in the fat-1 coding region
from pCGN5562 vs. the fat-1 Genbank sequence. The C at position 705
of the fat-1 sequence was changed to an A in pCGN5562. This creates
a change of a GAC codon to GAA, changing the Asp residue at
position 231 of fat-1 to a Glu residue. This identical change was
observed in products of two independent PCR reactions using fat-1
template and most likely is not a result of PCR mis-incorporation
of a nucleotide. For seed specific expression, the Fat-1 coding
region was cut out of pCGN5562 as a PstI/NotI fragment and inserted
between the PstI/NotI sites of the binary vector, pCGN8623, to
create pCGN5563. PCGN5563 can be introduced into Brassica napus via
Agrobacterium-mediated transformation.
Construction of pCGN8623
The polylinker region of the napin promoter cassette, pCGN7770, was
replaced by ligating the following oligonucleotides:
5'-TCGACCTGCAGGAAGCTTGCGGCCGCGGATCC-3' (SEQ ID NO:5) and
5'-TCGAGGATCCGCGGCCGCAAGCTTCCTGCAGG-3' (SEQ ID NO:6). These
oligonucleotides were ligated into SalI/XhoI-digested pCGN7770 to
produce pCGN8619. These oligos encode BamHII, NotI, HindIII, and
PstI restriction sites. pCGN8619 contains the oligos oriented such
that the PstI site is closest to the napin 5' regulatory region. A
fragment containing the napin 5' regulatory region, polylinker, and
napin 3' region was removed from pCGN8619 by digestion with
Asp7181. The fragment was blunt-ended by filling in the 5'
overhangs with Klenow fragment then ligated into pCGN5139 that had
been digested with Asp7181 and HindIII and blunt-ended by filling
in the 5' overhangs with Klenow fragment. A plasmid containing the
insert oriented so that the napin promoter was closest to the
blunted Asp7181 site of pCGN5139 and the napin 3' was closest to
the blunted HindIII site was subjected to sequence analysis to
confirm both the insert orientation and the integrity of cloning
junctions. The resulting plasmid was designated pCGN8623.
To produce high levels of steariodonic acid in Brassica, the C.
elegans .omega.-3 desaturase can be combined with .DELTA.6- and
.DELTA.12-desaturases from Mortierella alpina. PCGN5563-transformed
plants may be crossed with pCGN5544-transformed plants expressing
the .DELTA.6-and .DELTA.12-desaturases, described below.
The resulting F1 seeds can be analyzed for steariodonic acid
content and selected F1 plants can be used for self-pollination to
produce F2 seed, or as donors for production of dihaploids, or
additional crosses.
An alternative method to combine the fat-1 cDNA with M. alpina
.DELTA.6 and .DELTA.12 desaturases is to combine them on one T-DNA
for transformation. The fat-1 coding region from pCGN5562 can be
cut out as a PstI/NotI fragment and inserted into PstI/NotI
digested pCGN8619. The transcriptional unit consisting of the napin
5' regulatory region, the fat-1 coding region, and the napin
3'-regulatory region can be cut out as a Sse83871 fragment and
inserted into pCGN5544 cut with Sse83871. The resulting plasmid
would contain three napin transcriptional units containing the C.
elegans .omega.-3 desaturase, M. alpina .DELTA.6 desaturase, and M.
alpina .DELTA.12 desaturase, all oriented in the same direction as
the 35S/nptII/tml transcriptional unit used for selection of
transformed tissue.
Example 2
Over-Expression of .DELTA.15-desaturase Activity in Transgenic
Canola
The .DELTA.15-desaturase activity of Brassica napus can be
increased by over-expression of the .DELTA.15-desaturase cDNA
clone.
A. B. napus .DELTA.15-desaturase cDNA clone was obtained by PCR
amplification of first-strand cDNA derived from B. napus cv.
212/86. The primers were based on published sequence: Genbank
#L01418 Arondel et al, 1992 Science 258:1353-1355 (SEQ ID NO:7 and
SEQ ID NO:8).
The following primers were used:
Bnd15-FORWARD (SEQ ID NO:9)
5'-CUACUACUACUAGAGCTCAGCGATGGTTGTTGCTATGGAC-3' Bnd15-REVERSE (SEQ
ID NO:10) 5'-CAUCAUCAUCAUGAATTCTTAATTGATTTTAGATTTG-3'
These primers allowed the amplification of the entire coding region
and added SacI and EcoRI sites to the 5'- and 3'-ends, respectively
The PCR product was subcloned into pAMP1 (GIBCOBRL) using the
CloneAmp system (GIBCOBRL) to create pCGN5520. The sequence was
verified by sequencing of both strands to be sure that the open
reading frame remained intact. For seed specific expression, the
.DELTA.15-desaturase coding region was cut out of pCGN5520 as a
BamHI/SalI fragment and inserted between the BglII and XhoI sites
of the pCGN7770, to create pCGN5557. The PstI fragment of pCGN5557
containing the napin 5'-regulatory region, B. napus
.DELTA.15-desaturase, and napin 3'-regulatory region was inserted
into the PstI site of the binary vector, pCGN5138 to produce
pCGN5558. pCGN5558 was introduced into Brassica napus via
Agrobacterium-mediated transformation.
To produce high levels of stearidonic acid in Brassica, the
.DELTA.15-desaturase can be combined with .DELTA.6- and
.DELTA.12-desaturases from Mortierella alpina. PCGN5558-transformed
plants may be crossed with pCGN5544-transformed plants expressing
the .DELTA.6 and .DELTA.12-desaturases. The resulting F1 seeds are
analyzed for stearidonic acid content. GC-FAME analysis of F1
half-seeds revealed a significant accumulation of SDA in the seed
oil of the Brassica lines. SDA levels (18:4) of greater than
approximately 25% were obtained in hemizygous lines and are
provided in Table 1. Selected F1 plants can be used for
self-pollination to produce F2 seed, or as donors for production of
dihaploids, or additional crosses.
TABLE-US-00002 TABLE 1 Strain ID* 16:0 18:0 18:1 18:2 18:2 18:3
18:3 18:3 18:4 20:0 20:1 20:2 22:- 0 22:1 22:2 C912 C91215 C6912
(5558-SP30021-26 X 6 1.34 2.97 9.58 9.58 52.79 34.76 18.03 25.21
0.7 0.56 0.16 0.41 0.03 0- 5544-LP30108-6-16-1-3) (5558-SP30021-26
X 4.45 0.86 10.42 9.06 9.06 49.08 25.68 23.4 23.45 0.5 0.84 0.55
0.49 0 0- .06 5544-LP30108-6-16-1-3) (5558-SP30021-26 X 5.8 2.36
12.5 11.13 11.13 47.47 18.86 28.61 17.55 1.01 0.86 0.3 0.85 0 -
0.07 5544-LP30108-6-16-1-3) (5558-SP30021-26 X 3.65 0.66 14.26
14.97 14.97 50.94 23.3 27.64 13.22 0.43 0.88 0.23 0.48 - 0.04 0
5544-LP30108-6-16-1-3) (5558-SP30021-26 X 4.86 2.42 18.74 14.23
14.23 46.22 23 23.22 10.67 0.89 0.92 0.18 0.7 0.0- 2 0
5544-LP30108-6-16-1-3) (5558-SP30021-26 X 6.57 1.07 16.79 14 14
48.98 32.88 16.09 10.24 0.52 0.94 0.22 0.39 0.02 - 0.01
5544-LP30108-6-16-1-3) (5558-SP30021-26 X 5.85 2.09 8.81 19.12
19.12 50.89 12.03 38.86 9.09 1.39 0.78 0.45 1.23 0- 0.04
5544-LP30108-6-16-1-3) (5558-SP30021-26 X 4.69 2.04 17.46 21.1 21.1
43.38 24.28 19.1 8.5 0.73 0.96 0.37 0.56 0 0 5544-LP30108-6-16-1-3)
(5558-SP30021-26 X 5.43 1.69 16.59 22.2 22.2 44.4 16.57 27.83 6.34
0.9 1.03 0.32 0.81 0.03- 0.05 5544-LP30108-6-16-1-3)
(5558-SP30021-26 X 4.28 1.34 18.83 27.24 27.24 40.54 20.91 19.63
5.03 0.73 0.88 0.27 0.7 0- 0 5544-LP30108-6-16-1-3)
(5558-SP30021-26 X 4.47 1.38 21.43 26.89 26.89 39.04 18.78 20.26
4.06 0.73 0.91 0.41 0.48 - 0 0 5544-LP30108-6-16-1-3)
(5558-SP30021-26 X 4.77 1.12 18.4 31.1 31.1 38.51 19.62 18.88 3.52
0.64 0.8 0.21 0.7 0 0 5544-LP30108-6-16-1-3) (5558-SP30021-26 X
4.34 1.86 24.73 35.49 35.49 28.79 10.79 18 1.91 0.67 1.07 0.45 0.48
0 0- .02 5544-LP30108-6-16-1-3) C91515- C6912- C912 ALA GLA
(5558-SP30021-26 X 4.71 1.75 20.72 34.68 34.68 34.01 4.65 29.36
1.62 0.71 0.89 0.1 0.63 0 - 0 5544-LP30108-6-16-1-3)
(5558-SP30021-26 X 4.3 0.8 40.79 14.34 14.36 37 36.88 0.12 0 0.43
1.47 0.29 0.29 0 0 5544-LP30108-6-16-1-3) (5558-SP30021-19 X 6.61
1.5 22.09 11.23 11.23 39.9 25.84 14.06 16.25 0.75 0.75 0.27 0.57 0-
0 5544-LP30108-6-16-1-1) (5558-SP30021-19 X 4.64 1.89 22.73 15.12
15.12 44.48 32.21 12.27 8.78 0.73 0.89 0.23 0.43 - 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 5.51 1.45 24.82 17.79
17.79 41.84 27.46 14.38 6.45 0.59 0.82 0.23 0.39 - 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 4.06 1.67 26.39 16.93
16.93 42.64 32.65 9.99 6 0.64 0.96 0.24 0.41 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 5.24 1.44 22.2 20.02
20.02 42.76 28.69 14.07 5.98 0.67 0.79 0.26 0.45 0- 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 5.34 2.2 22.68 18.6 18.6
43.14 31.45 11.69 5.5 0.82 0.87 0.25 0.53 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 3.98 2.9 25.23 21.21
21.21 38.78 24.6 14.18 4.98 1.02 1.04 0.24 0.57 0 - 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 3.94 1.77 28.92 20.89
20.89 37.02 21.71 15.32 4.96 0.64 1.09 0.3 0.43 0- 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 5.12 1.24 27.7 19.02
19.02 40.2 31.05 9.16 4.76 0.48 0.77 0.23 0.35 0 0-
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 4.16 1.52 28.59 21.99
21.99 36.85 23.33 13.53 4.55 0.6 0.98 0.27 0.41 0- 0
5544-LP30108-6-16-1-1) (5558-SP30021-19 X 4.91 1.32 30.46 18.01
18.01 38.59 30.23 8.36 4.34 0.58 0.93 0.25 0.4 0 - 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.66 1.52 29.52 20.52
20.52 36.61 20.09 16.52 5.63 0.67 1.12 0.14 0.52 - 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 5.09 1.81 25.81 21.54
21.54 38.2 22.52 15.68 4.92 0.75 0.96 0.12 0.57 0- .02 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.77 1.5 29.79 22.36
22.36 35.46 14.84 20.62 4.39 0.74 1.17 0.18 0.59 0- .02 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.71 1.45 32.18 23.86
23.86 32.32 17 15.32 3.92 0.63 1.12 0.15 0.5 0.02- 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.55 1.56 33.27 25.21
25.21 30.69 16.63 14.06 3.08 0.68 1.2 0.16 0.54 0- .03 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 4.04 1.52 33.63 24.47
24.47 30.72 18.19 12.53 3.07 0.63 1.17 0.14 0.46 - 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.67 1.58 31.98 26.13
26.13 30.89 15.92 14.97 3.05 0.69 1.21 0.16 0.51 - 0 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.58 1.8 30.2 27.22 27.22
31.42 15.48 15.94 2.85 0.79 1.21 0.17 0.61 0.- 02 0.01
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 4.68 1.41 28.32 28 28
32.22 14.92 17.3 2.74 0.65 1.1 0.18 0.53 0.01 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.5 1.46 34.13 25.92
25.92 29.7 16.77 12.93 2.65 0.67 1.26 0.15 0.51 0.- 01 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.9 1.68 33.44 26.18
26.18 29.43 16.11 13.31 2.6 0.72 1.23 0.18 0.5 0.0- 2 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.82 1.71 31.84 27.78
27.78 29.49 15.28 14.2 2.59 0.73 1.19 0.16 0.55 0- .02 0
5544-LP30108-6-16-1-1) (5558-SP30021-36 X 3.6 1.78 29.45 28.14
28.14 31.64 12.83 18.81 2.57 0.76 1.21 0.17 0.58 0- 0
5544-LP30108-6-16-1-1)
An alternative method to combine the B. napus .DELTA.15-desaturase
with M. alpina .DELTA.6 and .DELTA.12 desaturases is to combine
them on one T-DNA for transformation. The transcription cassette
consisting of the napin 5'-regulatory region, the
.DELTA.15-desaturase coding region, and the napin 3'-regulatory
region can be cut out of pCGN5557 as a SwaI fragment and inserted
into SwaI-digested pCGN5544. The resulting plasmid, pCGN5561,
contains three napin transcriptional units containing the M. alpina
.DELTA.6 desaturase, the B. napus .DELTA.15-desaturase, and the M.
alpina .DELTA.12 desaturase, all oriented in the same direction as
the 35S/nptII/tml transcriptional unit used for selection of
transformed tissue. In addition, the C. elegans .omega.-3
desaturase coding sequence was also cloned into pCGN5544 to create
the construct pCGN5565.
Pooled T2 seeds of plants containing 5561 contain significant
amounts of SDA (18:4), shown in Table 2. Levels of greater than
about 7% SDA are obtained in pooled 5561 segregating seed.
Furthermore, significant levels of SDA were obtained from seeds of
5565 Brassica lines, also shown in Table 2. As shown in Table 2,
with constructs 5561 and 5565, levels of SDA ranging from about 0.8
wt % to greater than about 7 wt % can be obtained.
TABLE-US-00003 TABLE 2 18:2 18:2 18:3 18:3 STRAIN ID 16:0 16:1 18:0
18:1 C6,9 C9,12 C6,9,12 C9,12,15 18:4 20:0 20:1 2- 0:2 22:0 22:1
22:2 5561-6 4.46 0.21 3.5 22.85 0 18.33 18.71 21.61 7.79 1.04 0.76
0.19 0.47 0 - 0 5561-4 4.14 0.15 2.62 33.07 0 21.07 17.61 14.56
4.39 0.87 0.92 0.14 0.39 0- 0.02 5561-2 4.26 0.15 2.21 30.42 0
22.02 21.06 12.88 4.25 0.89 0.98 0.2 0.51 0 - 0.02 5561-8 4.29 0.18
2 33.05 0 22.44 16.23 15.3 3.95 0.84 0.96 0.19 0.43 0 0.0- 4 5561-6
3.95 0.12 2.04 32.93 0 24.48 17.42 13.33 3.27 0.79 0.94 0.21 0.4
0.- 03 0.03 5561-7 4.26 0.17 2.02 38.4 0 23.3 13.35 13.3 2.73 0.75
1.06 0.16 0.41 0 0 5561-13 4.38 0.18 1.86 58.94 0.65 13.98 7.1 8.26
1.88 0.77 1.27 0.29 0.35 - 0.03 0 5561-15 4.29 0.15 2.3 40.96 0
26.63 8.58 12.98 1.51 0.83 1.07 0.19 0.45 0 - 0.02 5561-1 4.25 0.15
1.91 47.41 0 24.46 5.56 12.81 1 0.72 1.14 0.15 0.39 0 0 5561-5 4.07
0.16 1.96 52.29 0 20.88 5.02 12.17 0.97 0.72 1.16 0.21 0.28 0 -
0.06 CONTROL 3.89 0.21 1.65 58.48 0 22.44 0 11.03 0 0.6 1.15 0.16
0.27 0.01 0 STRAIN ID 16:0 18:0 18:1 18:2-C69 18:2-LA 18:3-GLA
18:3-ALA 18:4 20:0 5565-SP30021-7 4.03 1.93 41.24 0.43 14.46 21.39
6.62 7.38 0.68 5565-SP30021-12 3.95 2.46 40.19 0 30.35 7.3 10.92
2.57 0.7 5565-SP30021-9 4.03 1.82 35.76 0 33.49 8.63 11.58 2.54
0.51 5565-SP30021-3 3.86 1.8 32.3 0 35.57 11.3 10.05 2.37 0.61
5565-SP30021-1 3.98 1.92 59.99 1.84 11.24 8.07 7.46 2.32 0.76
5565-SP30021-8 4.67 1.72 38.95 0 30.38 8.99 10.83 2.25 0.52
5565-SP30021-10 4.03 1.43 47.04 0 26.96 5.97 11.1 1.35 0.52
5565-SP30021-6 3.87 1.77 46.73 0 28.79 5.31 10.4 0.79 0.56 CONTROL
3.89 1.65 58.48 0 22.44 0 11.03 0 0.6
Example 3
Expression of .DELTA.5 Desaturase in Plants Expression in
Leaves
Ma29 is a putative M. alpina .DELTA.5 desaturase as determined by
sequence homology (SEQ ID NO:11 and SEQ ID NO:12). This experiment
was designed to determine whether leaves expressing Ma29 (as
determined by Northern) were able to convert exogenously applied
DGLA (20:3) to ARA (20:4).
The Ma29 desaturase cDNA was modified by PCR to introduce
convenient restriction sites for cloning. The desaturase coding
region has been inserted into a d35 cassette under the control of
the double 35S promoter for expression in Brassica leaves
(pCGN5525) following standard protocols (see U.S. Pat. No.
5,424,200 and U.S. Pat. No. 5,106,739). Transgenic Brassica plants
containing pCGN5525 were generated following standard protocols
(see U.S. Pat. No. 5,188,958 and U.S. Pat. No. 5,463,174).
In the first experiment, three plants were used: a control,
LPO04-1, and two transgenics, 5525-23 and 5525-29. LP004 is a
low-linolenic Brassica variety. Leaves of each were selected for
one of three treatments: water, GLA or DGLA, GLA and DGLA were
purchased as sodium salts from NuChek Prep and dissolved in water
at 1 mg/ml. Aliquots were capped under N2 and stored at -70 degrees
C. Leaves were treated by applying a 50 .mu.l drop to the upper
surface and gently spreading with a gloved finger to cover the
entire surface. Applications were made approximately 30 minutes
before the end of the light cycle to minimize any photo-oxidation
of the applied fatty acids. After 6 days of treatment one leaf from
each treatment was harvested and cut in half through the mid rib.
One half was washed with water to attempt to remove unincorporated
fatty acid. Leaf samples were lyophilized overnight, and fatty acid
composition determined by gas chromatography (GC). The results are
shown in Table 3.
TABLE-US-00004 TABLE 3 Fatty Acid Analysis of Leaves from Ma29
Transgenic Brassica Plants SPL 16:00 16:01 18:00 18:01 18:02 18:3g
18:03 18:04 20:00 20:01 Treatment # % % % % % % % % % % Water 33
12.95 0.08 2.63 2.51 16.76 0 45.52 0 0.09 34 13.00 0.09 2.67 2.56
16.86 0 44.59 0 0.15 35 14.13 0.09 2.37 2.15 16.71 0 49.91 0 0.05
36 13.92 0.08 2.32 2.07 16.16 0 50.25 0 0.05 37 13.79 0.11 2.10
2.12 15.90 0.08 46.29 0 0.54 38 12.80 0.09 1.94 2.08 14.54 0.11
45.61 0 0.49 GLA 39 12.10 0.09 2.37 2.10 14.85 1.65 43.66 0 0 40
12.78 0.10 2.34 2.22 15.29 1.72 47.22 0 0.02 41 13.71 0.07 2.68
2.16 15.92 2.12 46.55 0 0 42 14.10 0.07 1.75 2.35 16.66 1.56 46.41
0 0.01 43 13.62 0.09 2.22 1.94 14.68 2.42 46.69 0 0.01 44 14.92
0.09 2.20 2.17 15.22 2.30 46.05 0 0.02 DGLA 45 12.45 0.14 2.30 2.28
15.65 0.07 44.62 0 0.01 46 12.67 0.15 2.69 2.50 15.96 0.09 42.77 0
0.01 47 12.56 0.23 3.40 1.98 13.57 0.03 45.52 0 0.01 48 13.07 0.24
3.60 2.51 13.54 0.04 45.13 0 0.01 49 13.26 0.07 2.81 2.34 16.05
0.04 43.89 0 0 50 13.53 0.07 2.84 2.41 16.07 0.02 44.90 0 0.01 SPL
20:02 20:03 20:04 20:05 22:00 22:01 22:02 22:03 22:06 24:0 24:1
Treatment # % % % % % % % % % % % Water 33 0 0 0.29 0 0.01 0.09
16.26 0 0 0.38 0.18 34 0.01 0 0 0 0.14 0.10 16.82 0.02 0.05 0.36
0.27 35 0.01 0 0.25 0 0.12 0.06 11.29 0.04 0.05 0.29 0.25 36 0 0.01
0.26 0 0.07 0.04 11.82 0.03 0.36 0.28 0.21 37 0.02 0 0.21 0 0.18
0.08 15.87 0.06 0.20 0.30 0.17 38 0.01 0 0.24 0 0.15 0.07 13.64
0.09 0.08 5.89 0.23 GLA 39 0.02 0.01 0.27 0 0.10 0.08 16.25 3.42
0.19 0.37 0.17 40 0.01 0 0.27 0 0.10 0.10 14.74 0.05 0.10 0.36 0.14
41 0 0 0.27 0 0.20 0.10 13.15 0.13 0.29 0.33 0.20 42 0 0 0.28 0
0.11 0.11 12.60 0.02 0.24 0.38 0.13 43 0.01 0 0.28 0 0.10 0.03
14.73 0.01 0.24 0.34 0.14 44 0.02 0 0.26 0 0.13 0.07 14.43 0.05
0.16 0.33 0.17 DGLA 45 0.06 1.21 0.26 0 0.07 0.07 18.67 0.02 0.21
0.36 0.13 46 0 1.94 0.27 0 0.11 0.09 17.97 0.09 0.39 0.41 0.11 47
0.01 0.69 0.96 0 0.11 0.07 17.96 0 0.22 0.49 0.20 48 0.01 0.70 0.74
0 0.14 0.09 17.14 0.05 0.32 0.52 0.10 49 0 0.35 1.11 0 0.10 0.07
17.26 0.07 0.23 0.39 0.18 50 0 0.20 0.87 0 0.21 0.07 15.73 0.04
0.15 0.37 0.18
Leaves treated with GLA contained from 1.56 to 2.4 wt % GLA. The
fatty acid analysis showed that the lipid composition of control
and transgenic leaves was essentially the same. Leaves of control
plants treated with DGLA contained 1.2-1.9 w % DGLA and background
amounts of ARA (0.26-0.27 wt %). Transgenic leaves contained only
0.2-0.7 wt % DGLA, but levels of ARA were increased (0.74-1.1 wt %)
indicating that the DGLA was converted to ARA in these leaves.
Expression in Seed
The purpose of this experiment was to determine whether a construct
with the seed specific napin promoter would enable expression in
seed.
The Ma29 cDNA was modified by PCR to introduce XhoI cloning sites
upstream and downstream of the start and stop codons, respectively,
using the following primers: Madxho-forward (SEQ ID NO:13):
5'-CUACUACUACUACTCGAGCAAGATGGGAACGGACCAAGG Madxho-reverse (SEQ ID
NO:14): 5'-CAUCAUCAUCAUCTCGAGCTACTCTTCCTTGGGACGGAG
The PCR product was subcloned into pAMP1 (GIBCOBRL) using the
CloneAmp system (GIBCOBRL) to create pCGN5522 and the .DELTA.5
desaturase sequence was verified by sequencing of both strands.
For seed-specific expression, the Ma29 coding region was cut out of
pCGN5522 as an XhoI fragment and inserted into the SalI site of the
napin expression cassette, pCGN3223, to create pCGN5528. The
HindIII fragment of pCGN5528 containing the napin 5' regulatory
region, the Ma29 coding region, and the napin 3' regulatory region
was inserted into the HindIII site of pCGN1557 to create pCGN5531.
Two copies of the napin transcriptional unit were inserted in
tandem. This tandem construct can permit higher expression of the
desaturases per genetic loci. pCGN5531 was introduced into Brassica
napus cv.LP004 via Agrobacterium mediated transformation.
The fatty acid composition of twenty-seed pools of mature T2 seeds
was analyzed by GC. Table 2 shows the results obtained with
independent transformed lines as compared to non-transformed LP004
seed. The transgenic seeds containing pCGN5531 contain two fatty
acids that are not present in the control seeds, identified as
taxoleic acid (5,9-18:2) and pinolenic acid (5,9,12-18:3), based on
their elution relative to oleic and linoleic acid. These would be
the expected products of .DELTA.5 desaturation of oleic and
linoleic acids. No other differences in fatty acid composition were
observed in the transgenic seeds.
Example 4
Production of D5-desaturated Fatty Acids in Transgenic Plants
The construction of pCGN5531 (.DELTA.5-desaturase) and fatty acid
composition of T2 seed pools is described in Example 3. This
example takes the seeds through one more generation and discusses
ways to maximize the .DELTA.5-desaturated fatty acids.
Example 3 describes the fatty acid composition of T2 seed pools of
pCGN5531-transformed B. napus cv. LP004 plants. To investigate the
segregation of .DELTA.5-desaturated fatty acids in the T2 seeds and
to identify individual plants to be taken on to subsequent
generations, half-seed analysis was done. Seeds were germinated
overnight in the dark at 30 degrees on water-soaked filter paper.
The outer cotyledon was excised for GC analysis and the rest of the
seedling was planted in soil. Results of some of these analyses are
shown in the accompanying Table 4. .DELTA.5,9-18:2 accumulated to
as high as 12% of the total fatty acids and A 5,9,12-18:3
accumulated to up to 0.77% of the fatty acids. These and other
individually selected T2 plants were grown in the greenhouse to
produce T3 seed.
TABLE-US-00005 TABLE 4 Composition of T2 Pooled Seed 16:0 16:1 18:0
18:1 .DELTA.5,9 18:2 18:2 .DELTA.5,9,12 18:3 18:3 20:0 20:1 20:2
22:0 22:1 24:0 % % % % .DELTA. % % .DELTA. % % % % % % % % LP004
control 3.86 0.15 3.05 69.1 0 18.51 0.01 1.65 1.09 1.40 0.03 0.63
0.- 05 0.42 5531-1 4.26 0.15 3.23 62.33 4.07 21.44 0.33 1.38 0.91
1.04 0.05 0.41 0.03 - 0.27 5531-2 3.78 0.14 3.37 66.18 4.57 17.31
0.27 1.30 1.03 1.18 0 0.47 0.01 0.3- 0 5531-6 3.78 0.13 3.47 63.61
6.21 17.97 0.38 1.34 1.04 1.14 0.05 0.49 0.02 - 0.26 5531-10 3.96
0.17 3.28 63.82 5.41 18.58 0.32 1.43 0.98 1.11 0.02 0.50 0 0.- 31
5531-16 3.91 0.17 3.33 64.31 5.03 18.98 0.33 1.39 0.96 1.11 0 0.44
0 0 5531-28 3.81 0.13 2.58 62.64 5.36 20.95 0.45 1.39 0.83 1.15
0.01 0.36 0.05- 0.21 Fatty acid analysis of selected T2 half-seeds
from pCGN5531-LP004 events CYCLE SPL ID NO STRAIN ID 12:0 14:0 16:0
16:1 18:0 18:1 18:2 .DELTA.5,9 18:2 .DELTA.9,12 18:3 .DELTA.5,9,12
18:3 .DELTA.9,12,15 97XX1539 93 5531-LP004-6 0.03 0.07 3.92 0.17
3.5 61.32 12.22 15.36 0.77 1.- 36 97XX1539 29 5531-LP004-6 0.01
0.04 3.6 0.09 3.23 63.77 10.63 14.47 0 1.22 97XX1539 38
5531-LP004-6 0.01 0.05 3.71 0.09 3.02 65.13 10.57 13.98 0 1.06-
97XX1539 41 5531-LP004-6 0.01 0.05 3.64 0.07 3.22 62.51 9.7 16.63 0
1.28 97XX1539 18 5531-LP004-6 0.02 0.06 3.69 0.09 3.36 63.79 9.63
15.29 0.63 1.- 15 97XX1539 85 5531-LP004-6 0.01 0.06 3.6 0.09 3.54
64.81 9.54 13.69 0.6 1.26- 98GC0023 98 5531-LP004-23 0.01 0.05 3.5
0.09 3.12 64.97 9.92 13.62 0.55 1.- 25 98GC0023 32 5531-LP004-23
0.01 0.05 3.43 0.08 2.62 65.21 9.83 14.28 0.59 1- .15 98GC0023 78
5531-LP004-23 0.01 0.05 3.45 0.07 2.78 64.97 9.34 14.69 0.58 1- .17
98GC0023 86 5531-LP004-23 0.01 0.05 3.32 0.08 2.7 64.18 9.08 15.99
0.68 1.- 18 98GC0023 67 5531-LP004-23 0.01 0.04 3.49 0.08 3.03
64.14 8.78 15.95 0.62 1- .08 98GC0023 52 5531-LP004-23 0.01 0.03
3.38 0.07 2.56 67.44 8.65 13.55 0.5 1.- 02
To maximize the accumulation of .DELTA.5,9 18:2 in seed oil, the
pCGN5531 construct could be introduced into a high oleic acid
variety of canola. A high-oleic variety could be obtained by
mutation, so-suppression, or antisense suppression of the .DELTA.12
and .DELTA.15 desaturases or other necessary co-factors.
To maximize accumulation of .DELTA.5,9,12 18:3 in canola, the
pCGN5531 construct could be introduced into a high linoleic strain
of canola. This could be achieved by crossing pCGN5531-transformed
plants with pCGN5542-(M. alpina .DELTA.12-desaturase) transformed
plants. Alternatively, the .DELTA.5 and .DELTA.12 desaturases could
be combined on one T-DNA for transformation. The transcriptional
unit consisting of the napin 5'regulatory region, the M. alpina
.DELTA.12-desaturase coding region, and the napin 3'-regulatory
region can be cut out of pCGN5541 as a NotI fragment. NotI/XbaI
linkers could be ligated and the resulting fragment inserted into
the XbaI site of pCGN5531. The resulting plasmid would contain
three napin transcriptional units containing the M. alpina
.DELTA.12 desaturase, and two copies of the napin/M. alpina
.DELTA.5 desaturase/napin unit, all oriented in the same direction
as the 35S/nptII/tml transcriptional unit used for selection of
transformed tissue.
Example 5
Expression of M. Alpina .DELTA.6 Desaturase in Brassica Napus
A nucleic acid sequence from a partial cDNA clone, Ma524, encoding
a .DELTA.6 fatty acid desaturase from Mortierella alpina was
obtained by random sequencing of clones from the M. alpina cDNA
library. The Ma524 cDNA was modified by PCR to introduce cloning
sites using the following primers: Ma524PCR-1 (SEQ ID NO:15)
5'-CUACUACUACUATCTAGACTCGAGACCATGGCTGCTGCT CCAGTGTG Ma524PCR-2 (SEQ
ID NO:16) 5'-CAUCAUCAUCAUAGGCCTCGAGTTACTGCGCCTTACCCAT
These primers allowed the amplification of the entire coding region
and added XbaI and XhoI sites to the 5'-end and XhoI and StuI sites
to the 3' end. The PCR product was subcloned into pAMP1 (GIBCOBRL)
using the CloneAmp system (GIBCOBRL) to create pCGN5535 and the
.DELTA.6 desaturase sequence was verified by sequencing of both
strands.
Construction of pCGN5544
Plant expression constructs were prepared to express the
Mortierella alpina .DELTA.6 desaturase and the Mortierella alpina
.DELTA.12 desaturase in a plant host cell. The constructs prepared
utilized transcriptional initiation regions derived from genes
preferentially expressed in a plant seed. Isolation of the cDNA
sequences encoding the M. alpina .DELTA.6 desaturase (SEQ ID NO:17
and SEQ ID NO:18) and M. alpina .DELTA.12 desaturase (SEQ ID NO:19
and SEQ ID NO:20) are described in PCT Publications WO 98/46763 and
WO 98/46764, the entireties of which are incorporated herein by
reference.
For seed-specific expression, the Ma524 coding region was cut out
of pCGN5535 as an XhoI fragment and inserted into the SalI site of
the napin expression cassette, pCGN3223, to create pCGN5536. The
NotI fragment of pCGN5536 containing the napin 5' regulatory
region, the Ma524 coding region, and the napin 3' regulatory region
was inserted into the NotI site of pCGN1557 to create pCGN5538.
The 5542 cDNA, encoding the M. alpina .DELTA.12 desaturase, was
modified by PCR to introduce cloning sites using the following
primers: Ma648PCR-for (SEQ ID NO:21)
5'-CUACUACUACUAGGATCCATGGCACCTCCCAACACT Ma648PCR-for (SEQ ID NO:22)
5'-CAUCAUCAUCAUGGTACCTCGAGTTACTTCTTGAAAAAGAC
These primers allowed the amplification of the entire coding region
and added a BamHI site to the 5' end and KpnI and XhoI sites to the
3' end. The PCR product was subcloned into pAMP1 (Gibco-BRL,
Gaithersburg, Md.) using the CloneAmp system (Gibco-BRL) to create
pCGN5540, and the .DELTA.12 desaturase sequence was verified by
sequencing of both strands.
A seed preferential expression construct was prepared for the
.DELTA.12 desaturase cDNA sequence. The Ma648 coding region was cut
out of pCGN5540 as a BamHI/XhoI fragment and inserted between the
BglII and XhoI sites of the napin expression cassette, pCGN3223
(described in U.S. Pat. No. 5,639,790), to create pCGN5542.
In order to express the M. alpina .DELTA.6 and .DELTA.12 desaturase
sequences from the same T-DNA, the following construct for
seed-preferential expression was prepared.
The NotI fragment of pCGN5536 containing the napin 5'
transcriptional initiation region, the Ma524 coding region, and the
napin 3' transcriptional termination region was inserted into the
NotI site of pCGN5542 to create pCGN5544. The expression cassettes
were oriented in such a way that the direction of transcription
from Ma524 and Ma648 and the nptII marker is the same.
For seed-specific expression, the Ma524 coding region was cut out
of pCGN5535 as an XhoI fragment and inserted into the SalI site of
the napin expression cassette, pCGN3223, to create pCGN5536. The
NotI fragment of pCGN5536 containing the napin 5 regulatory region,
the Ma524 coding region, and the napin 3' regulatory region was
inserted into the NotI site of pCGN1557 to create pCGN5538.
pCGN5538 was introduced into Brassica napus cv.LP004 via
Agrobacterium mediated transformation.
Maturing T2 seeds were collected from 6 independent transformation
events in the greenhouse. The fatty acid compositions of single
seeds was analyzed by GC. Table 5 shows the results of control
LP004 seeds and six 5538 lines. All of the 5538 lines except #8
produced seeds containing GLA. Presence of GLA segregated in these
seeds as is expected for the T2 selfed seed population. In addition
to GLA, the M. alpina .DELTA.6 desaturase is capable of producing
18:4 (stearidonic) and another fatty acid: .DELTA.6,9-18:2.
TABLE-US-00006 TABLE 5 Fatty Acid Analysis of Seeds from Ma524
Transgenic Brassica Plants SPL 16:00 16:01 18:0 18:1 6,9 18:2 18:2
18:3gs 18:3 18:4 20:1 22:0 22:1 24:0 24:1 # % % % % % % % % % % % %
% % LPOO4-1 4.33 0.21 3.78 72.49 0 13.97 0 1.7 0 1.34 0.71 0.02
0.58 0.27 .sup. -2 4.01 0.16 3.09 73.59 0 14.36 0.01 1.4 0 1.43
0.66 0.02 0.5 0.- 2 .sup. -3 4.12 0.19 3.56 70.25 0 17.28 0 1.57 0
1.28 0.5 0.02 0.39 0.2 .sup. -4 4.22 0.2 2.7 70.25 0 17.86 0 1.61 0
1.31 0.53 0.02 0.4 0.24 .sup. -5 4.02 0.16 3.41 72.91 0 14.45 0.01
1.45 0 1.37 0.7 0.02 0.51 0- .26 .sup. -6 4.22 0.18 3.23 71.47 0
15.92 0.01 1.52 0 1.32 0.69 0.02 0.51 - 0.27 .sup. -7 4.1 0.16 3.47
72.06 0 15.23 0 1.52 0 1.32 0.63 0.03 0.49 0.23- .sup. -9 4.01 0.17
3.71 72.98 0 13.97 0.01 1.41 0 1.45 0.74 0.03 0.58 - 0.23 .sup. -10
4.04 0.16 3.57 70.03 0 17.46 0 1.5 0 1.33 0.61 0.03 0.36 0.2- 4
5538-1-1 4.61 0.2 3.48 68.12 1.37 10.68 7.48 1.04 0.33 1.19 0.49
0.02 0.33- 0.13 .sup. -2 4.61 0.22 3.46 68.84 1.36 10.28 7.04 1.01
0.31 1.15 0.48 0.02- 0.39 0 .sup. -3 4.78 0.24 3.24 65.86 0 21.36 0
1.49 0 1.08 0.46 0.02 0.38 0.2- 2 .sup. -4 4.84 0.3 3.89 67.64 1.67
9.9 6.97 1.02 0.36 1.14 0.53 0.02 0.- 5 0.18 .sup. -5 4.64 0.2 3.58
64.5 3.61 8.85 10.14 0.95 0.48 1.19 0.47 0.01 0- .33 0.12 .sup. -6
4.91 0.27 3.44 66.51 1.48 11.14 7.74 1.15 0.33 1.08 0.49 0.02- 0.34
0.13 .sup. -7 4.87 0.22 3.24 65.78 1.27 11.92 8.38 1.2 0 1.12 0.47
0.02 0.3- 7 0.16 .sup. -8 4.59 0.22 3.4 70.77 0 16.71 0 1.35 0 1.14
0.48 0.02 0.39 0.15- .sup. -9 4.63 0.23 3.51 69.66 2.01 8.77 7.24
0.97 0 1.18 0.52 0.02 0.3- 0.11 .sup. -10 4.56 0.19 3.55 70.68 0
16.89 0 1.37 0 1.22 0.54 0.02 0.22 0.- 03 5538-3-1 4.74 0.21 3.43
67.52 1.29 10.91 7.77 1.03 0.28 1.11 0.5 0.02 0.35- 0.14 .sup. -2
4.72 0.21 3.24 67.42 1.63 10.37 8.4 0.99 0 1.12 0.49 0.02 0.3- 6
0.15 .sup. -3 4.24 0.21 3.52 71.31 0 16.53 0 1.33 0 1.12 0.45 0.02
0.4 0.14- .sup. -4 4.64 0.21 3.45 67.92 1.65 9.91 7.97 0.91 0.33
1.14 0.47 0.02 - 0.37 0.14 .sup. -5 4.91 0.25 3.31 67.19 0 19.92
0.01 1.39 0 1.05 0.48 0.02 0.37 - 0.14 .sup. -6 4.67 0.21 3.25
67.07 1.23 11.32 8.35 0.99 0 1.16 0.47 0.02 0.- 33 0.16 .sup. -7
4.53 0.19 2.94 64.8 4.94 8.45 9.95 0.93 0.44 1.13 0.37 0.01 0- .27
0.12 .sup. -8 4.66 0.22 3.68 67.33 0.71 12 6.99 1.1 0.24 1.18 0.48
0.03 0.3- 6 0.17 .sup. -9 4.65 0.24 3.11 67.42 0.64 12.71 6.93 1.16
0.25 1.08 0.45 0.02- 0.32 0.17 .sup. -10 4.88 0.27 3.33 65.75 0.86
12.89 7.7 1.1 0.24 1.08 0.46 0.01 - 0.34 0.16 5538-4-1 4.65 0.24
3.8 62.41 0 24.68 0 1.6 0.01 0.99 0.45 0.02 0.33 0.13 .sup. -2 5.37
0.31 3 57.98 0.38 18.04 10.5 1.41 0 0.99 0.48 0.02 0.3 0- .19 .sup.
-3 4.61 0.22 3.07 63.62 0.3 16.46 7.67 1.2 0 1.18 0.45 0.02 0.29-
0.14 .sup. -4 4.39 0.19 2.93 65.97 0 22.36 0 1.45 0 1.17 0.41 0.03
0.32 0.1- 5 .sup. -5 5.22 0.29 3.85 62.1 2.35 10.25 11.39 0.93 0.41
1.04 0.6 0.02 - 0.47 0.17 .sup. -6 4.66 0.18 2.85 66.79 0.5 13.03
7.66 0.97 0.22 1.28 0.42 0.02 - 0.31 0.14 .sup. -7 4.85 0.26 3.03
57.43 0.26 28.04 0.01 2.59 0.01 1.13 0.56 0.02- 0.4 0.23 .sup. -8
5.43 0.28 2.94 54.8 1.84 13.79 15.67 1.36 0.53 1.1 0.55 0.02 - 0.35
0.19 .sup. -9 4.88 0.24 3.32 62.3 0.58 14.86 9.04 1.34 0.29 1.13
0.52 0.02 - 0.37 0.19 .sup. -10 4.53 0.2 2.73 64.2 0.07 24.15 0
1.52 0 1.09 0.39 0.02 0.27 0- .17 5538-5-1 4.5 0.15 3.35 66.71 0.88
11.7 8.38 1.04 0.3 1.24 0.49 0.02 0.29 0- .17 .sup. -2 4.77 0.23
3.06 62.67 0.68 15.2 8.8 1.31 0.28 1.15 0.46 0.02 0- .3 0.19 .sup.
-3 4.59 0.22 3.61 64.33 2.29 9.95 10.57 1.01 0.45 1.21 0.48 0.02-
0.26 0.16 .sup. -4 4.86 0.26 3.4 67.69 0.65 12.24 6.61 1.09 0.23
1.07 0.45 0.02 - 0.32 0.15 .sup. -5 4.49 0.21 3.3 69.25 0.04 16.51
2.18 1.2 0 1.11 0.44 0.02 0.33- 0.16 .sup. -6 4.5 0.21 3.47 70.48
0.08 14.9 2.19 1.22 0 1.13 0.49 0.02 0.33- 0.16 .sup. -7 4.39 0.21
3.44 67.59 2.38 9.24 8.98 0.89 0 1.18 0.44 0.02 0.2- 8 0.14 .sup.
-8 4.52 0.22 3.17 68.33 0.01 18.91 0.73 1.32 0.01 1.08 0.45 0.02-
0.29 0.17 .sup. -9 4.68 0.2 3.05 64.03 1.93 11.03 11.41 1.02 0.01
1.15 0.39 0.02- 0.21 0.15 .sup. -10 4.57 0.2 3.1 67.21 0.61 12.62
7.68 1.07 0.25 1.14 0.43 0.02 - 0.25 0.15 5538-8-1 4.95 0.26 3.14
64.04 0 23.38 0 1.54 0 0.99 0.42 0.02 0.38 0.17 .sup. -2 4.91 0.26
3.71 62.33 0 23.97 0 1.77 0 0.95 0.53 0.02 0.42 0.1- 9 .sup. -3
4.73 0.25 4.04 63.83 0 22.36 0.01 1.73 0 1.05 0.55 0.02 0.45 - 0.16
.sup. -4 5.1 0.35 3.8 60.45 0 24.45 0.01 2.13 0 1.07 0.65 0.03 0.53
0.- 24 .sup. -5 4.98 0.3 3.91 62.48 0 23.44 0 1.77 0 1.01 0.51 0.01
0.43 0.21- .sup. -6 4.62 0.21 3.99 66.14 0 20.38 0 1.48 0 1.15 0.53
0.02 0.48 0.1- 9 .sup. -7 4.64 0.22 3.55 64.6 0 22.65 0 1.38 0 1.09
0.45 0.02 0.41 0.19- .sup. -8 5.65 0.38 3.18 56.6 0 30.83 0.02 0.02
0 0.98 0.55 0.03 0.39 0- .26 .sup. -9 8.53 0.63 6.9 51.76 0 26.01 0
0.01 0 1.41 1.21 0.07 0.96 0.33- .sup. -10 5.52 0.4 3.97 57.92 0
28.95 0 0.02 0 0.95 0.52 0.02 0.41 0.1- 6 5538-10-1 4.44 0.19 3.5
68.42 0 19.51 0 1.32 0 1.14 0.45 0.02 0.31 0.16 .sup. -2 4.57 0.21
3.07 66.08 0 21.99 0.01 1.36 0 1.12 0.41 0.02 0.31 - 0.16 .sup. -3
4.63 0.21 3.48 67.43 0 20.27 0.01 1.32 0 1.12 0.46 0.02 0.21 - 0.08
.sup. 4 4.69 0.19 3.22 64.62 0 23.16 0 1.35 0 1.08 0.46 0.02 0.33
0.2 .sup. -5 4.58 0.2 3.4 68.75 0 20.17 0.01 0.02 0 1.1 0.45 0.02
0.34 0.1- 7 .sup. -8 4.55 0.21 0 73.55 0.05 14.91 2.76 1.21 0.07
1.24 0.51 0.02 0.- 19 0 .sup. -9 4.58 0.21 3.28 66.19 0 21.55 0
1.36 0 1.12 0.43 0.02 0.33 0.1- 6 .sup. -10 4.52 0.2 3.4 68.37 0
19.33 0.01 1.3 0 1.13 0.46 0.02 0.35 0.- 18
Cross were made between transgenic Brassica 5544 lines producing
GLA and standard non-transformed canola varieties. Crosses between
5544 lines with Quantum, Eagle, and Ebony were conducted.
F1 half seeds were analyzed for SDA content and selected plants
were grown and allowed to self-pollinate to produce F2 seeds.
GC-FAME analysis of both single seed and half-seed samples from
such crosses revealed accumulation of significant levels of SDA
(Table 6). Half-seed analysis of 5544-LP108-6-16 with canola
variety Eagle yielded a level of approximately 6.3% SDA. Analysis
of F2 seed from a cross of 5544-LP108-12-1 with the canola variety
Ebony produced levels of SDA as high as about 7.4% SDA.
TABLE-US-00007 TABLE 6 18:2 18:2 18:3 18:3 STRAIN ID 16:0 16:1 18:0
18:1 C69 C912 C6912 C91215 18:4 20:0 20:1 20:2 22- :0 (SP30035-46 x
6.34 0.84 1.9 4.7 0 14.81 56.73 3.78 6.29 2.12 0.66 0.59 1.04
5544-LP30108-6-16) (SP30035-46 x 10.18 1.43 4.23 4.34 0 15.96 48.78
3.79 5.51 2.65 0.72 0.77 1.32 5544-LP30108-6-16) (SP30035-46 x 4.81
0.45 2.53 12.2 0 21.61 46.74 4.83 4.11 0.98 0.79 0.4 0.43
5544-LP30108-6-16) (SP30035-46 x 4.74 0.48 3.33 16.06 0 20.68 43.02
4.82 3.73 1.25 0.7 0.33 0.75 5544-LP30108-6-16) (SP30035-46 x 6.02
0.53 1.25 17.29 0 27.34 33.97 7.52 3.41 0.85 0.77 0.27 0.59
5544-LP30108-6-16) (SP30035-46 x 3.68 0.13 1.99 19.75 0.09 22.75
39.98 5.76 3.41 0.8 0.87 0.27 0.44 5544-LP30108-6-16) (SP30052-7 x
8.92 0.96 1.64 14.61 0 18.69 36.98 7.44 7.43 1.01 0.49 0.49 0- .95
5544-LP30108-12-1) (SP30052-7 x 9.02 0.89 1.88 10.69 0 16.73 43.39
6.8 6.76 1.05 0.57 0.75 1.- 07 5544-LP30108-12-1) (SP30052-7 x 7.76
0.59 1.86 8.15 0 16.04 52.24 4.65 5.3 1.04 0.59 0.69 0.8- 3
5544-LP30108-12-1) (SP30052-7 x 9.21 0.87 2.23 17.44 0 18.77 36.87
6.79 5.05 0.84 0.64 0.31 0- .71 5544-LP30108-12-1) (SP30052-7 x
5.76 0.31 1.6 20.38 0 24.36 29.94 10.9 4.41 0.69 0.78 0.23 0.- 48
5544-LP30108-12-1) (SP30052-7 x 4.03 0.22 1.3 16.87 0 19.3 46.67
5.33 4.17 0.53 0.75 0.35 0.3- 7 5544-LP30108-12-1) (SP30052-7 x
4.66 0.29 4.47 18.09 0.05 19.07 41.92 5.06 3.47 1.13 0.73 0.3- 5
0.57 5544-LP30108-12-1) (SP30052-7 x 4.91 0.26 3.13 18.16 0 18.53
43.99 4.64 3.43 1.01 0.79 0.37 0- .66 5544-LP30108-12-1)
Example 6
Production of .DELTA.6,9 18:2 in Canola Oil
Example 5 described construction of pCGN5538 designed to express
the M. alpina .DELTA.6 desaturase in seeds of transgenic canola.
Table 4 in that example showed examples of single seed analyses
from 6 independent transgenic events. Significant amounts of GLA
were produced, in addition to the .DELTA.-6,9 18:2 fatty acid.
A total of 29 independent pCGN5538-transformed transgenic plants of
the low-linolenic LP004 cultivar were regenerated and grown in the
greenhouse. Table 7 shows the fatty acid composition of 20-seed
pools of T2 seed from each event. Seven of the lines contained more
than 2% of the .DELTA.-6,9 18:2 in the seed pools. To identify and
select plants with high amounts of .DELTA.-6,9 18:2 to be taken on
to subsequent generations, half-seed analysis was done. Seeds were
germinated overnight in the dark at 30 degrees on water-soaked
filter paper. The outer cotyledon was excised for GC analysis and
the rest of the seedling was planted in soil. Based on results of
fatty acid analysis, selected T2 plants were grown in the
greenhouse to produce T3 seed. The-selection cycle was repeated;
pools of T3 seed were analyzed for .DELTA.-6,9 18:2, T3 half-seeds
were dissected and analyzed, and selected T3 plants were grown in
the greenhouse to produce T4 seed. Pools of T4 seed were analyzed
for fatty acid composition. Table 6 summarizes the results of this
process for lines derived from one of the original transgenic
events, 5538-LP004-25. Levels of .DELTA.-6,9 18:2 have thus been
maintained through 3 generations.
To maximize the amount of .DELTA.-6,9 18:2 that could be produced,
the pCGN5538 construct could be introduced into a high oleic acid
variety of canola either by transformation or crossing. A
high-oleic variety could be obtained by mutation, co-suppression,
or antisense suppression of the .DELTA.12 and .DELTA.15 desaturases
or other necessary co-factors.
TABLE-US-00008 TABLE 7 Fatty Acid Composition of 20-seed Pools of
pCON5538 T2 Seeds .DELTA.6,9 .DELTA.6,9,12 .DELTA.9,12,15 SPL
5538-LP004 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:2 18:3 18:3 18:4
20:0- 20:1 20:2 22:0 22:2 # event % % % % % % % % % % % % % % % %
31 0.02 0.06 4.07 0.07 0 59.4 5.4 10.07 15.93 1.2 0.6 0.98 1.16 0.0
0.44 - 0.03 29 0.01 0.05 3.81 0.14 0 60.7 4.53 10.9 14.77 1.03 0.55
1.09 1.26 0.0 0.4- 6 0.02 19 0.02 0.07 4.27 0.13 0 62.9 4.17 10.03
13.14 1.02 0.59 1.18 1.25 0.0 0.- 53 0.02 14 0 0 5.29 0.24 3.8 49.1
1.02 23.44 11.21 2.26 0.34 1.45 0.93 0.0 0.76 0- 22 0.02 0.05 3.87
0.09 0 64.1 2.59 12.57 11.18 1.27 0.6 1.18 1.08 0.1 0.5- 6 0 9 0.01
0.06 4.57 0.16 0 62.9 3.4 12.05 11.15 1.27 0.6 1.28 1.18 0.0 0.56 -
0.03 25 0.01 0.06 4.17 0.14 0 62.4 2.49 14.42 11.03 1.2 0.46 1.18
1.15 0.0 0.5- 3 0.01 15 0.01 0.05 3.94 0.11 0 65.2 2.08 12.77 10.9
1.04 0.43 1.1 1.24 0.0 0.48- 0.01 18 0 0.06 5.34 0.29 0 58.4 1.42
18.19 10.53 1.8 0.49 1.2 1 0.0 0.58 0.02 20 0.01 0.04 3.95 0.1 0
65.6 1.31 13.83 10.22 1.09 0.39 1.06 1.3 0.0 0.46- 0.01 7 0.02 0.07
4.04 0.11 0 62.1 0.92 18.12 8.72 1.77 0.35 1.26 1.19 0.0 0.58- 0 11
0.01 0.06 4.23 0.17 0 62.9 1.6 17.19 8.58 1.48 0.38 1.16 1.03 0.0
0.49- 0.01 27 0.02 0 3.99 0.15 0 65.3 0.64 17.85 7.89 1.36 0.31
1.08 1.21 0.0 0 0 2 0.01 0.05 4.02 0.14 0 66.4 1.2 15.74 7.58 1.22
0.32 10.6 1.19 0.0 0.45 - 0 28 0.01 0.04 3.77 0.11 0 67.5 0.79
15.56 7.58 1.12 0.28 0.97 1.23 0.0 0.4- 4 0 3 0.01 0.05 3.96 0.13 0
68.5 1.81 13.23 7.44 1.1 0.35 1.12 1.21 0.0 0.46 - 0.01 21 0.01
0.05 3.74 0.1 0 66.9 1.16 15.9 6.99 1.35 0.28 1.15 1.27 0.0 0.52 -
0 5 0.01 0.04 3.81 0.12 0 69.1 0.74 14.58 6.95 1.14 0.28 1.06 1.18
0.0 0.45- 0 6 0 0 2.84 0 3.06 62.5 1.55 18.44 6.94 1.21 0.39 1.04
1.33 0.1 0 0 4 0.01 0.05 3.88 0.11 0 66.9 0.64 16.21 6.89 1.52 0.31
1.09 1.21 0.0 0.5 - 0 30 0.01 0.04 3.89 0.12 0 68.6 0.72 15.58 6.47
1.17 0.23 1.03 1.07 0.0 0.4- 6 0 16 0.02 0.05 3.75 0.13 0 70.4 0.91
13.56 6.39 1.13 0.28 1.04 1.2 0.0 0.44- 0.01 26 0.01 0 3.77 0.12 0
67.6 0 21.08 3.61 1.37 0.13 0.96 1.16 0.0 0 0 23 0 0 4.92 0.22 0
65.2 0 22.23 3 1.79 0.11 1.28 1.11 0.0 0 0 24 0.01 0 3.84 0.13 0
68.4 0.36 21 2.09 1.53 0.08 1.06 1.27 0.0 0 0 10 0.01 0 3.74 0.11 0
70.4 0 20.82 0.65 1.3 0.03 1.05 1.18 0.0 0.46 0 12 0.01 0 3.83 0.12
0 69.9 0 21.61 0.34 1.34 0 1.06 1.12 0.0 0.47 0 17 0.01 0 0 0.13 0
72.6 0 23.03 0.24 1.51 0 1.18 1.21 0.0 0 0 8 0.01 0 4.54 0.2 0 64.9
0 25.65 0.22 1.94 0 1.38 1.01 0.0 0 0 13 0.01 0 3.99 0.16 0 65.8 0
25.9 0 1.58 0 1.17 1.16 0.0 0 0 LP004 0.01 0.04 3.46 0.09 0 69.9 0
21.95 0 1.37 0.01 0.9 1.25 0.0 0.42 0 control T2 Pool T3 Pool T3
selection T4 Pool STRAIN ID.DELTA. .DELTA.6,9 18:2 GLA .DELTA.6,9
18:2 GLA .DELTA.6,9 18:2 GLA .DELTA.6,9 18:2 .DELTA.6,9,12 18:3
5538-LP004-25 2.49 11.03 5538-LP004-25-3 9.1 11.92
5538-LP004-25-3-31 13.61 7.82 11.02 9.41 5538-LP004-25-3-30 6.51
7.93 10.27 8.7 5538-LP004-25-3-29 13.35 11.23 9.42 10.5
5538-LP004-25-3-28 9.92 24.1 9.37 10.19 5538-LP004-25-3-25 5.3
30.34 7.95 11.34 5538-LP004-25-2 3.87 11.08 5538-LP004-25-2-29
13.63 7.41 9.6 11.07 5538-LP004-25-2- 27 5.02 22.04 6.95 9.61
5538-LP004-25-2-26 1.21 26.84 4.31 7.45 5538-LP004-25-2-25 5.83
34.16 8.77 11.58 5538-LP004-25-13 10.53 11.19 5538-LP004-25-13-27
14.65 11.46 7.86 10.49 5538-LP004-25-13-26 11.18 13.04 9.33 10.01
5538-LP004-25-13-25 4.18 36.78 7.2 12.22 5538-LP004-25-1 3.05 11.16
5538-LP004-25-1-41 0 0 0.01 0.04 5538-LP004-25-1-28 3.43 19.98 4.63
6.53 5538-LP004-25-1-27 5.52 20.13 8.35 11.21 5538-LP004-25-1-26
0.1 25.16 5.52 8.59 5538-LP004-25-1-25 6.5 31.83 9.85 10.88
All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
SEQUENCE LISTINGS
1
2211391DNACaenorhabditis elegans 1 caagtttgag gtatggtcgc tcattcctca
gaagggttat ccgccacggc t ccggtcacc 60 ggcggagatg ttctggttga
tgctcgtgca tctcttgaag aaaaggaggc t ccacgtgat 120 gtgaatgcaa
acactaaaca ggccaccact gaagagccac gcatccaatt a ccaactgtg 180
gatgctttcc gtcgtgcaat tccagcacac tgtttcgaaa gagatctcgt t aaatcaatc
240 agatatttgg tgcaagactt tgcggcactc acaattctct actttgctct t
ccagctttt 300 gagtactttg gattgtttgg ttacttggtt tggaacattt
ttatgggagt t tttggattc 360 gcgttgttcg tcgttggaca cgattgtctt
catggatcat tctctgataa t cagaatctc 420 aatgatttca ttggacatat
cgccttctca ccactcttct ctccatactt c ccatggcag 480 aaaagtcaca
agcttcacca tgctttcacc aaccacattg acaaagatca t ggacacgtg 540
tggattcagg ataaggattg ggaagcaatg ccatcatgga aaagatggtt c aatccaatt
600 ccattctctg gatggcttaa atggttccca gtgtacactt tattcggttt c
tgtgatgga 660 tctcacttct ggccatactc ttcacttttt gttcgtaact
ctgaccgtgt t caatgtgta 720 atctctggaa tctgttgctg tgtgtgtgca
tatattgctc taacaattgc t ggatcatat 780 tccaattggt tctggtacta
ttgggttcca ctttctttct tcggattgat g ctcgtcatt 840 gttacctatt
tgcaacatgt cgatgatgtc gctgaggtgt acgaggctga t gaatggagc 900
ttcgtccgtg gacaaaccca aaccatcgat cgttactatg gactcggatt g gacacaacg
960 atgcaccata tcacagacgg acacgttgcc catcacttct tcaacaaaat c
ccacattac 1020 catctcatcg aagcaaccga aggtgtcaaa aaggtcttgg
agccgttgtc c gacacccaa 1080 tacgggtaca aatctcaagt gaactacgat
ttctttgccc gtttcctgtg g ttcaactac 1140 aagctcgact atctcgttca
caagaccgcc ggaatcatgc aattccgaac a actctcgag 1200 gagaaggcaa
aggccaagta aaagaatatc ccgtgccgtt ctagagtaca a caacaactt 1260
ctgcgttttc accggttttg ctctaattgc aatttttctt tgttctatat a tattttttt
1320 gctttttaat tttattctct ctaaaaaact tctacttttc agtgcgttga a
tgcataaag 1380 ccataactct t 1391 2402PRTCaenorhabditis elegans 2
Met Val Ala His Ser Ser Glu Gly Leu Ser A la Thr Ala Pro Val Thr 1
5 10 15 Gly Gly Asp Val Leu Val Asp Ala Arg Ala S er Leu Glu Glu
Lys Glu 20 25 30 Ala Pro Arg Asp Val Asn Ala Asn Thr Lys G ln Ala
Thr Thr Glu Glu 35 40 45 Pro Arg Ile Gln Leu Pro Thr Val Asp Ala P
he Arg Arg Ala Ile Pro 50 55 60 Ala His Cys Phe Glu Arg Asp Leu Val
Lys S er Ile Arg Tyr Leu Val 65 70 75 80 Gln Asp Phe Ala Ala Leu
Thr Ile Leu Tyr P he Ala Leu Pro Ala Phe 85 90 95 Glu Tyr Phe Gly
Leu Phe Gly Tyr Leu Val T rp Asn Ile Phe Met Gly 100 105 110 Val
Phe Gly Phe Ala Leu Phe Val Val Gly H is Asp Cys Leu His Gly 115
120 125 Ser Phe Ser Asp Asn Gln Asn Leu Asn Asp P he Ile Gly His
Ile Ala 130 135 140 Phe Ser Pro Leu Phe Ser Pro Tyr Phe Pro T rp
Gln Lys Ser His Lys 145 150 155 160 Leu His His Ala Phe Thr Asn His
Ile Asp L ys Asp His Gly His Val 165 170 175 Trp Ile Gln Asp Lys
Asp Trp Glu Ala Met P ro Ser Trp Lys Arg Trp 180 185 190 Phe Asn
Pro Ile Pro Phe Ser Gly Trp Leu L ys Trp Phe Pro Val Tyr 195 200
205 Thr Leu Phe Gly Phe Cys Asp Gly Ser His P he Trp Pro Tyr Ser
Ser 210 215 220 Leu Phe Val Arg Asn Ser Asp Arg Val Gln C ys Val
Ile Ser Gly Ile 225 230 235 240 Cys Cys Cys Val Cys Ala Tyr Ile Ala
Leu T hr Ile Ala Gly Ser Tyr 245 250 255 Ser Asn Trp Phe Trp Tyr
Tyr Trp Val Pro L eu Ser Phe Phe Gly Leu 260 265 270 Met Leu Val
Ile Val Thr Tyr Leu Gln His V al Asp Asp Val Ala Glu 275 280 285
Val Tyr Glu Ala Asp Glu Trp Ser Phe Val A rg Gly Gln Thr Gln Thr
290 295 300 Ile Asp Arg Tyr Tyr Gly Leu Gly Leu Asp T hr Thr Met
His His Ile 305 310 315 320 Thr Asp Gly His Val Ala His His Phe Phe
A sn Lys Ile Pro His Tyr 325 330 335 His Leu Ile Glu Ala Thr Glu
Gly Val Lys L ys Val Leu Glu Pro Leu 340 345 350 Ser Asp Thr Gln
Tyr Gly Tyr Lys Ser Gln V al Asn Tyr Asp Phe Phe 355 360 365 Ala
Arg Phe Leu Trp Phe Asn Tyr Lys Leu A sp Tyr Leu Val His Lys 370
375 380 Thr Ala Gly Ile Met Gln Phe Arg Thr Thr L eu Glu Glu Lys
Ala Lys 385 390 395 400 Ala Lys 341DNAsynthetic primer 3 cuacuacuac
uactgcagac aatggtcgct cattcctcag a 41 438DNAsynthetic primer 4
caucaucauc augcggccgc ttacttggcc tttgcctt 38 532DNAsynthetic
polylinker 5 tcgacctgca ggaagcttgc ggccgcggat cc 32 632DNAsynthetic
polylinker 6 tcgaggatcc gcggccgcaa gcttcctgca gg 32
71353DNABrassica napus 7 aatccatcaa acctttattc accacatttc
actgaaaggc cacacatcta g agagagaaa 60 cttcgtccaa atctctctct
ccagcgatgg ttgttgctat ggaccagcgc a gcaatgtta 120 acggagattc
cggtgcccgg aaggaagaag ggtttgatcc aagcgcacaa c caccgttta 180
agatcggaga tataagggcg gcgattccta agcattgctg ggtgaagagt c ctttgagat
240 ctatgagcta cgtcaccaga gacattttcg ccgtcgcggc tctggccatg g
ccgccgtgt 300 attttgatag ctggttcctc tggccactct actgggttgc
ccaaggaacc c ttttctggg 360 ccatcttcgt tcttggccac gactgtggac
atgggagttt ctcagacatt c ctctgctga 420 acagtgtggt tggtcacatt
cttcattcat tcatcctcgt tccttaccat g gttggagaa 480 taagccatcg
gacacaccac cagaaccatg gccatgttga aaacgacgag t cttgggttc 540
cgttgccaga aaagttgtac aagaacttgc cccatagtac tcggatgctc a gatacactg
600 tccctctgcc catgctcgct tacccgatct atctgtggta cagaagtcct g
gaaaagaag 660 ggtcacattt taacccatac agtagtttat ttgctccaag
cgagaggaag c ttattgcaa 720 cttcaactac ttgctggtcc ataatgttgg
ccactcttgt ttatctatcg t tcctcgttg 780 atccagtcac agttctcaaa
gtctatggcg ttccttacat tatctttgtg a tgtggttgg 840 acgctgtcac
gtacttgcat catcatggtc acgatgagaa gttgccttgg t acagaggca 900
aggaatggag ttatttacgt ggaggattaa caactattga tagagattac g gaatcttca
960 acaacatcca tcacgacatt ggaactcacg tgatccatca tcttttccca c
aaatccctc 1020 actatcactt ggtcgatgcc acgagagcag ctaaacatgt
gttaggaaga t actacagag 1080 agccgaagac gtcaggagca ataccgattc
acttggtgga gagtttggtc g caagtatta 1140 aaaaagatca ttacgtcagt
gacactggtg atattgtctt ctacgagaca g atccagatc 1200 tctacgttta
tgcttctgac aaatctaaaa tcaattaact tttcttccta g ctctattag 1260
gaataaacac tccttctctt ttacttattt gtttctgctt taagtttaaa a tgtactcgt
1320 gaaacctttt ttttattaat gtatttacgt tac 1353 8383PRTBrassica
napus 8 Met Val Val Ala Met Asp Gln Arg Ser Asn V al Asn Gly Asp
Ser Gly 1 5 10 15 Ala Arg Lys Glu Glu Gly Phe Asp Pro Ser A la Gln
Pro Pro Phe Lys 20 25 30 Ile Gly Asp Ile Arg Ala Ala Ile Pro Lys H
is Cys Trp Val Lys Ser 35 40 45 Pro Leu Arg Ser Met Ser Tyr Val Thr
Arg A sp Ile Phe Ala Val Ala 50 55 60 Ala Leu Ala Met Ala Ala Val
Tyr Phe Asp S er Trp Phe Leu Trp Pro 65 70 75 80 Leu Tyr Trp Val
Ala Gln Gly Thr Leu Phe T rp Ala Ile Phe Val Leu 85 90 95 Gly His
Asp Cys Gly His Gly Ser Phe Ser A sp Ile Pro Leu Leu Asn 100 105
110 Ser Val Val Gly His Ile Leu His Ser Phe I le Leu Val Pro Tyr
His 115 120 125 Gly Trp Arg Ile Ser His Arg Thr His His G ln Asn
His Gly His Val 130 135 140 Glu Asn Asp Glu Ser Trp Val Pro Leu Pro
G lu Lys Leu Tyr Lys Asn 145 150 155 160 Leu Pro His Ser Thr Arg
Met Leu Arg Tyr T hr Val Pro Leu Pro Met 165 170 175 Leu Ala Tyr
Pro Ile Tyr Leu Trp Tyr Arg S er Pro Gly Lys Glu Gly 180 185 190
Ser His Phe Asn Pro Tyr Ser Ser Leu Phe A la Pro Ser Glu Arg Lys
195 200 205 Leu Ile Ala Thr Ser Thr Thr Cys Trp Ser I le Met Leu
Ala Thr Leu 210 215 220 Val Tyr Leu Ser Phe Leu Val Asp Pro Val T
hr Val Leu Lys Val Tyr 225 230 235 240 Gly Val Pro Tyr Ile Ile Phe
Val Met Trp L eu Asp Ala Val Thr Tyr 245 250 255 Leu His His His
Gly His Asp Glu Lys Leu P ro Trp Tyr Arg Gly Lys 260 265 270 Glu
Trp Ser Tyr Leu Arg Gly Gly Leu Thr T hr Ile Asp Arg Asp Tyr 275
280 285 Gly Ile Phe Asn Asn Ile His His Asp Ile G ly Thr His Val
Ile His 290 295 300 His Leu Phe Pro Gln Ile Pro His Tyr His L eu
Val Asp Ala Thr Arg 305 310 315 320 Ala Ala Lys His Val Leu Gly Arg
Tyr Tyr A rg Glu Pro Lys Thr Ser 325 330 335 Gly Ala Ile Pro Ile
His Leu Val Glu Ser L eu Val Ala Ser Ile Lys 340 345 350 Lys Asp
His Tyr Val Ser Asp Thr Gly Asp I le Val Phe Tyr Glu Thr 355 360
365 Asp Pro Asp Leu Tyr Val Tyr Ala Ser Asp L ys Ser Lys Ile Asn
370 375 380 940DNAsynthetic primer 9 cuacuacuac uagagctcag
cgatggttgt tgctatggac 40 1037DNAsynthetic primer 10 caucaucauc
augaattctt aattgatttt agatttg 37 111482DNAMortierella alpina 11
gcttcctcca gttcatcctc catttcgcca cctgcattct ttacgaccgt t aagcaagat
60 gggaacggac caaggaaaaa ccttcacctg ggaagagctg gcggcccata a
caccaagga 120 cgacctactc ttggccatcc gcggcagggt gtacgatgtc
acaaagttct t gagccgcca 180 tcctggtgga gtggacactc tcctgctcgg
agctggccga gatgttactc c ggtctttga 240 gatgtatcac gcgtttgggg
ctgcagatgc cattatgaag aagtactatg t cggtacact 300 ggtctcgaat
gagctgccca tcttcccgga gccaacggtg ttccacaaaa c catcaagac 360
gagagtcgag ggctacttta cggatcggaa cattgatccc aagaatagac c agagatctg
420 gggacgatac gctcttatct ttggatcctt gatcgcttcc tactacgcgc a
gctctttgt 480 gcctttcgtt gtcgaacgca catggcttca ggtggtgttt
gcaatcatca t gggatttgc 540 gtgcgcacaa gtcggactca accctcttca
tgatgcgtct cacttttcag t gacccacaa 600 ccccactgtc tggaagattc
tgggagccac gcacgacttt ttcaacggag c atcgtacct 660 ggtgtggatg
taccaacata tgctcggcca tcacccctac accaacattg c tggagcaga 720
tcccgacgtg tcgacgtctg agcccgatgt tcgtcgtatc aagcccaacc a aaagtggtt
780 tgtcaaccac atcaaccagc acatgtttgt tcctttcctg tacggactgc t
ggcgttcaa 840 ggtgcgcatt caggacatca acattttgta ctttgtcaag
accaatgacg c tattcgtgt 900 caatcccatc tcgacatggc acactgtgat
gttctggggc ggcaaggctt t ctttgtctg 960 gtatcgcctg attgttcccc
tgcagtatct gcccctgggc aaggtgctgc t cttgttcac 1020 ggtcgcggac
atggtgtcgt cttactggct ggcgctgacc ttccaggcga a ccacgttgt 1080
tgaggaagtt cagtggccgt tgcctgacga gaacgggatc atccaaaagg a ctgggcagc
1140 tatgcaggtc gagactacgc aggattacgc acacgattcg cacctctgga c
cagcatcac 1200 tggcagcttg aactaccagg ctgtgcacca tctgttcccc
aacgtgtcgc a gcaccatta 1260 tcccgatatt ctggccatca tcaagaacac
ctgcagcgag tacaaggttc c ataccttgt 1320 caaggatacg ttttggcaag
catttgcttc acatttggag cacttgcgtg t tcttggact 1380 ccgtcccaag
gaagagtaga agaaaaaaag cgccgaatga agtattgccc c ctttttctc 1440
caagaatggc aaaaggagat caagtggaca ttctctatga ag 1482
12446PRTMortierella alpina 12 Met Gly Thr Asp Gln Gly Lys Thr Phe
Thr T rp Glu Glu Leu Ala Ala 1 5 10 15 His Asn Thr Lys Asp Asp Leu
Leu Leu Ala I le Arg Gly Arg Val Tyr 20 25 30 Asp Val Thr Lys Phe
Leu Ser Arg His Pro G ly Gly Val Asp Thr Leu 35 40 45 Leu Leu Gly
Ala Gly Arg Asp Val Thr Pro V al Phe Glu Met Tyr His 50 55 60 Ala
Phe Gly Ala Ala Asp Ala Ile Met Lys L ys Tyr Tyr Val Gly Thr 65 70
75 80 Leu Val Ser Asn Glu Leu Pro Ile Phe Pro G lu Pro Thr Val Phe
His 85 90 95 Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr P he Thr Asp
Arg Asn Ile 100 105 110 Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly A
rg Tyr Ala Leu Ile Phe 115 120 125 Gly Ser Leu Ile Ala Ser Tyr Tyr
Ala Gln L eu Phe Val Pro Phe Val 130 135 140 Val Glu Arg Thr Trp
Leu Gln Val Val Phe A la Ile Ile Met Gly Phe 145 150 155 160 Ala
Cys Ala Gln Val Gly Leu Asn Pro Leu H is Asp Ala Ser His Phe 165
170 175 Ser Val Thr His Asn Pro Thr Val Trp Lys I le Leu Gly Ala
Thr His 180 185 190 Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val T rp
Met Tyr Gln His Met 195 200 205 Leu Gly His His Pro Tyr Thr Asn Ile
Ala G ly Ala Asp Pro Asp Val 210 215 220 Ser Thr Ser Glu Pro Asp
Val Arg Arg Ile L ys Pro Asn Gln Lys Trp 225 230 235 240 Phe Val
Asn His Ile Asn Gln His Met Phe V al Pro Phe Leu Tyr Gly 245 250
255 Leu Leu Ala Phe Lys Val Arg Ile Gln Asp I le Asn Ile Leu Tyr
Phe 260 265 270 Val Lys Thr Asn Asp Ala Ile Arg Val Asn P ro Ile
Ser Thr Trp His 275 280 285 Thr Val Met Phe Trp Gly Gly Lys Ala Phe
P he Val Trp Tyr Arg Leu 290 295 300 Ile Val Pro Leu Gln Tyr Leu
Pro Leu Gly L ys Val Leu Leu Leu Phe 305 310 315 320 Thr Val Ala
Asp Met Val Ser Ser Tyr Trp L eu Ala Leu Thr Phe Gln 325 330 335
Ala Asn His Val Val Glu Glu Val Gln Trp P ro Leu Pro Asp Glu Asn
340 345 350 Gly Ile Ile Gln Lys Asp Trp Ala Ala Met G ln Val Glu
Thr Thr Gln 355 360 365 Asp Tyr Ala His Asp Ser His Leu Trp Thr S
er Ile Thr Gly Ser Leu 370 375 380 Asn Tyr Gln Ala Val His His Leu
Phe Pro A sn Val Ser Gln His His 385 390 395 400 Tyr Pro Asp Ile
Leu Ala Ile Ile Lys Asn T hr Cys Ser Glu Tyr Lys 405 410 415 Val
Pro Tyr Leu Val Lys Asp Thr Phe Trp G ln Ala Phe Ala Ser His 420
425 430 Leu Glu His Leu Arg Val Leu Gly Leu Arg P ro Lys Glu Glu
435 440 445 1339DNAsynthetic primer 13 cuacuacuac uactcgagca
agatgggaac ggaccaagg 39 1439DNAsynthetic primer 14 caucaucauc
auctcgagct actcttcctt gggacggag 39 1547DNAsynthetic primer 15
cuacuacuac uatctagact cgagaccatg gctgctgctc cagtgtg 47
1640DNAsynthetic primer 16 caucaucauc auaggcctcg agttactgcg
ccttacccat 40 171617DNAMortierella alpina 17 cgacactcct tccttcttct
cacccgtcct agtccccttc aacccccctc t ttgacaaag 60 acaacaaacc
atggctgctg ctcccagtgt gaggacgttt actcgggccg a ggttttgaa 120
tgccgaggct ctgaatgagg gcaagaagga tgccgaggca cccttcttga t gatcatcga
180 caacaaggtg tacgatgtcc gcgagttcgt ccctgatcat cccggtggaa g
tgtgattct 240 cacgcacgtt ggcaaggacg gcactgacgt ctttgacact
tttcaccccg a ggctgcttg 300 ggagactctt gccaactttt acgttggtga
tattgacgag agcgaccgcg a tatcaagaa 360 tgatgacttt gcggccgagg
tccgcaagct gcgtaccttg ttccagtctc t tggttacta 420 cgattcttcc
aaggcatact acgccttcaa ggtctcgttc aacctctgca t ctggggttt 480
gtcgacggtc attgtggcca agtggggcca gacctcgacc ctcgccaacg t gctctcggc
540 tgcgcttttg ggtctgttct ggcagcagtg cggatggttg gctcacgact t
tttgcatca 600 ccaggtcttc caggaccgtt tctggggtga tcttttcggc
gccttcttgg g aggtgtctg 660 ccagggcttc tcgtcctcgt ggtggaagga
caagcacaac actcaccacg c cgcccccaa 720 cgtccacggc gaggatcccg
acattgacac ccaccctctg ttgacctgga g tgagcatgc 780 gttggagatg
ttctcggatg tcccagatga ggagctgacc cgcatgtggt c gcgtttcat 840
ggtcctgaac cagacctggt tttacttccc cattctctcg tttgcccgtc t ctcctggtg
900 cctccagtcc attctctttg tgctgcctaa cggtcaggcc cacaagccct c
gggcgcgcg 960 tgtgcccatc tcgttggtcg agcagctgtc gcttgcgatg
cactggacct g gtacctcgc 1020 caccatgttc ctgttcatca aggatcccgt
caacatgctg gtgtactttt t ggtgtcgca 1080 ggcggtgtgc ggaaacttgt
tggcgatcgt gttctcgctc aaccacaacg g tatgcctgt 1140 gatctcgaag
gaggaggcgg tcgatatgga tttcttcacg aagcagatca t cacgggtcg 1200
tgatgtccac ccgggtctat ttgccaactg gttcacgggt ggattgaact a tcagatcga
1260 gcaccacttg ttcccttcga tgcctcgcca caacttttca
aagatccagc c tgctgtcga 1320 gaccctgtgc aaaaagtaca atgtccgata
ccacaccacc ggtatgatcg a gggaactgc 1380 agaggtcttt agccgtctga
acgaggtctc caaggctgcc tccaagatgg g taaggcgca 1440 gtaaaaaaaa
aaacaaggac gttttttttc gccagtgcct gtgcctgtgc c tgcttccct 1500
tgtcaagtcg agcgtttctg gaaaggatcg ttcagtgcag tatcatcatt c tccttttac
1560 cccccgctca tatctcattc atttctctta ttaaacaact tgttcccccc t
tcaccg 1617 18457PRTMortierella alpina 18 Met Ala Ala Ala Pro Ser
Val Arg Thr Phe T hr Arg Ala Glu Val Leu 1 5 10 15 Asn Ala Glu Ala
Leu Asn Glu Gly Lys Lys A sp Ala Glu Ala Pro Phe 20 25 30 Leu Met
Ile Ile Asp Asn Lys Val Tyr Asp V al Arg Glu Phe Val Pro 35 40 45
Asp His Pro Gly Gly Ser Val Ile Leu Thr H is Val Gly Lys Asp Gly 50
55 60 Thr Asp Val Phe Asp Thr Phe His Pro Glu A la Ala Trp Glu Thr
Leu 65 70 75 80 Ala Asn Phe Tyr Val Gly Asp Ile Asp Glu S er Asp
Arg Asp Ile Lys 85 90 95 Asn Asp Asp Phe Ala Ala Glu Val Arg Lys L
eu Arg Thr Leu Phe Gln 100 105 110 Ser Leu Gly Tyr Tyr Asp Ser Ser
Lys Ala T yr Tyr Ala Phe Lys Val 115 120 125 Ser Phe Asn Leu Cys
Ile Trp Gly Leu Ser T hr Val Ile Val Ala Lys 130 135 140 Trp Gly
Gln Thr Ser Thr Leu Ala Asn Val L eu Ser Ala Ala Leu Leu 145 150
155 160 Gly Leu Phe Trp Gln Gln Cys Gly Trp Leu A la His Asp Phe
Leu His 165 170 175 His Gln Val Phe Gln Asp Arg Phe Trp Gly A sp
Leu Phe Gly Ala Phe 180 185 190 Leu Gly Gly Val Cys Gln Gly Phe Ser
Ser S er Trp Trp Lys Asp Lys 195 200 205 His Asn Thr His His Ala
Ala Pro Asn Val H is Gly Glu Asp Pro Asp 210 215 220 Ile Asp Thr
His Pro Leu Leu Thr Trp Ser G lu His Ala Leu Glu Met 225 230 235
240 Phe Ser Asp Val Pro Asp Glu Glu Leu Thr A rg Met Trp Ser Arg
Phe 245 250 255 Met Val Leu Asn Gln Thr Trp Phe Tyr Phe P ro Ile
Leu Ser Phe Ala 260 265 270 Arg Leu Ser Trp Cys Leu Gln Ser Ile Leu
P he Val Leu Pro Asn Gly 275 280 285 Gln Ala His Lys Pro Ser Gly
Ala Arg Val P ro Ile Ser Leu Val Glu 290 295 300 Gln Leu Ser Leu
Ala Met His Trp Thr Trp T yr Leu Ala Thr Met Phe 305 310 315 320
Leu Phe Ile Lys Asp Pro Val Asn Met Leu V al Tyr Phe Leu Val Ser
325 330 335 Gln Ala Val Cys Gly Asn Leu Leu Ala Ile V al Phe Ser
Leu Asn His 340 345 350 Asn Gly Met Pro Val Ile Ser Lys Glu Glu A
la Val Asp Met Asp Phe 355 360 365 Phe Thr Lys Gln Ile Ile Thr Gly
Arg Asp V al His Pro Gly Leu Phe 370 375 380 Ala Asn Trp Phe Thr
Gly Gly Leu Asn Tyr G ln Ile Glu His His Leu 385 390 395 400 Phe
Pro Ser Met Pro Arg His Asn Phe Ser L ys Ile Gln Pro Ala Val 405
410 415 Glu Thr Leu Cys Lys Lys Tyr Asn Val Arg T yr His Thr Thr
Gly Met 420 425 430 Ile Glu Gly Thr Ala Glu Val Phe Ser Arg L eu
Asn Glu Val Ser Lys 435 440 445 Ala Ala Ser Lys Met Gly Lys Ala Gln
450 455 191488DNAMortierella alpina 19 gtcccctgtc gctgtcggca
caccccatcc tccctcgctc cctctgcgtt t gtccttggc 60 ccaccgtctc
tcctccaccc tccgagacga ctgcaactgt aatcaggaac c gacaaatac 120
acgatttctt tttactcagc accaactcaa aatcctcaac cgcaaccctt t ttcaggatg
180 gcacctccca acactatcga tgccggtttg acccagcgtc atatcagcac c
tcggcccca 240 aactcggcca agcctgcctt cgagcgcaac taccagctcc
ccgagttcac c atcaaggag 300 atccgagagt gcatccctgc ccactgcttt
gagcgctccg gtctccgtgg t ctctgccac 360 gttgccatcg atctgacttg
ggcgtcgctc ttgttcctgg ctgcgaccca g atcgacaag 420 tttgagaatc
ccttgatccg ctatttggcc tggcctgttt actggatcat g cagggtatt 480
gtctgcaccg gtgtctgggt gctggctcac gagtgtggtc atcagtcctt c tcgacctcc
540 aagaccctca acaacacagt tggttggatc ttgcactcga tgctcttggt c
ccctaccac 600 tcctggagaa tctcgcactc gaagcaccac aaggccactg
gccatatgac c aaggaccag 660 gtctttgtgc ccaagacccg ctcccaggtt
ggcttgcctc ccaaggagaa c gctgctgct 720 gccgttcagg aggaggacat
gtccgtgcac ctggatgagg aggctcccat t gtgactttg 780 ttctggatgg
tgatccagtt cttgttcgga tggcccgcgt acctgattat g aacgcctct 840
ggccaagact acggccgctg gacctcgcac ttccacacgt actcgcccat c tttgagccc
900 cgcaactttt tcgacattat tatctcggac ctcggtgtgt tggctgccct c
ggtgccctg 960 atctatgcct ccatgcagtt gtcgctcttg accgtcacca
agtactatat t gtcccctac 1020 ctctttgtca acttttggtt ggtcctgatc
accttcttgc agcacaccga t cccaagctg 1080 ccccattacc gcgagggtgc
ctggaatttc cagcgtggag ctctttgcac c gttgaccgc 1140 tcgtttggca
agttcttgga ccatatgttc cacggcattg tccacaccca t gtggcccat 1200
cacttgttct cgcaaatgcc gttctaccat gctgaggaag ctacctatca t ctcaagaaa
1260 ctgctgggag agtactatgt gtacgaccca tccccgatcg tcgttgcggt c
tggaggtcg 1320 ttccgtgagt gccgattcgt ggaggatcag ggagacgtgg
tctttttcaa g aagtaaaaa 1380 aaaagacaat ggaccacaca caaccttgtc
tctacagacc tacgtatcat g tagccatac 1440 cacttcataa aagaacatga
gctctagagg cgtgtcattc gcgcctcc 1488 20399PRTMortierella alpina 20
Met Ala Pro Pro Asn Thr Ile Asp Ala Gly L eu Thr Gln Arg His Ile 1
5 10 15 Ser Thr Ser Ala Pro Asn Ser Ala Lys Pro A la Phe Glu Arg
Asn Tyr 20 25 30 Gln Leu Pro Glu Phe Thr Ile Lys Glu Ile A rg Glu
Cys Ile Pro Ala 35 40 45 His Cys Phe Glu Arg Ser Gly Leu Arg Gly L
eu Cys His Val Ala Ile 50 55 60 Asp Leu Thr Trp Ala Ser Leu Leu Phe
Leu A la Ala Thr Gln Ile Asp 65 70 75 80 Lys Phe Glu Asn Pro Leu
Ile Arg Tyr Leu A la Trp Pro Val Tyr Trp 85 90 95 Ile Met Gln Gly
Ile Val Cys Thr Gly Val T rp Val Leu Ala His Glu 100 105 110 Cys
Gly His Gln Ser Phe Ser Thr Ser Lys T hr Leu Asn Asn Thr Val 115
120 125 Gly Trp Ile Leu His Ser Met Leu Leu Val P ro Tyr His Ser
Trp Arg 130 135 140 Ile Ser His Ser Lys His His Lys Ala Thr G ly
His Met Thr Lys Asp 145 150 155 160 Gln Val Phe Val Pro Lys Thr Arg
Ser Gln V al Gly Leu Pro Pro Lys 165 170 175 Glu Asn Ala Ala Ala
Ala Val Gln Glu Glu A sp Met Ser Val His Leu 180 185 190 Asp Glu
Glu Ala Pro Ile Val Thr Leu Phe T rp Met Val Ile Gln Phe 195 200
205 Leu Phe Gly Trp Pro Ala Tyr Leu Ile Met A sn Ala Ser Gly Gln
Asp 210 215 220 Tyr Gly Arg Trp Thr Ser His Phe His Thr T yr Ser
Pro Ile Phe Glu 225 230 235 240 Pro Arg Asn Phe Phe Asp Ile Ile Ile
Ser A sp Leu Gly Val Leu Ala 245 250 255 Ala Leu Gly Ala Leu Ile
Tyr Ala Ser Met G ln Leu Ser Leu Leu Thr 260 265 270 Val Thr Lys
Tyr Tyr Ile Val Pro Tyr Leu P he Val Asn Phe Trp Leu 275 280 285
Val Leu Ile Thr Phe Leu Gln His Thr Asp P ro Lys Leu Pro His Tyr
290 295 300 Arg Glu Gly Ala Trp Asn Phe Gln Arg Gly A la Leu Cys
Thr Val Asp 305 310 315 320 Arg Ser Phe Gly Lys Phe Leu Asp His Met
P he His Gly Ile Val His 325 330 335 Thr His Val Ala His His Leu
Phe Ser Gln M et Pro Phe Tyr His Ala 340 345 350 Glu Glu Ala Thr
Tyr His Leu Lys Lys Leu L eu Gly Glu Tyr Tyr Val 355 360 365 Tyr
Asp Pro Ser Pro Ile Val Val Ala Val T rp Arg Ser Phe Arg Glu 370
375 380 Cys Arg Phe Val Glu Asp Gln Gly Asp Val V al Phe Phe Lys
Lys 385 390 395 2136DNAsynthetic primer 21 cuacuacuac uaggatccat
ggcacctccc aacact 36 2241DNAsynthetic primer 22 caucaucauc
auggtacctc gagttacttc ttgaaaaaga c 41
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