U.S. patent application number 12/354018 was filed with the patent office on 2009-05-28 for limnanthes oil genes.
This patent application is currently assigned to E. I. DU PONT DE NEMOURS AND COMPANY. Invention is credited to Edgar B. Cahoon, William D. Hitz, Anthony J. Kinney, Steven J. Vollmer.
Application Number | 20090138992 12/354018 |
Document ID | / |
Family ID | 40670906 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090138992 |
Kind Code |
A1 |
Cahoon; Edgar B. ; et
al. |
May 28, 2009 |
LIMNANTHES OIL GENES
Abstract
This invention relates to an isolated nucleic acid fragment
encoding an enzyme involved in lipid biosynthesis. The invention
also relates to the construction of a chimeric gene encoding all or
a portion of the enzyme involved in lipid biosynthesis, in sense or
antisense orientation, wherein expression of the chimeric gene
results in production of altered levels of the enzyme involved in
lipid biosynthesis in a transformed host cell.
Inventors: |
Cahoon; Edgar B.; (Lincoln,
NE) ; Hitz; William D.; (Wilmington, DE) ;
Kinney; Anthony J.; (Wilmington, DE) ; Vollmer;
Steven J.; (Wilmington, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1122B, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Assignee: |
E. I. DU PONT DE NEMOURS AND
COMPANY
|
Family ID: |
40670906 |
Appl. No.: |
12/354018 |
Filed: |
January 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10855854 |
May 27, 2004 |
7495149 |
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12354018 |
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09664840 |
Sep 19, 2000 |
6838594 |
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10855854 |
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Current U.S.
Class: |
800/312 ;
800/298 |
Current CPC
Class: |
C12N 9/1029 20130101;
C07H 21/04 20130101; C12N 9/0083 20130101; C12N 15/8247
20130101 |
Class at
Publication: |
800/312 ;
800/298 |
International
Class: |
A01H 5/00 20060101
A01H005/00 |
Claims
1-34. (canceled)
35. A seed obtained from a transgenic plant wherein the seed
comprises a desaturated fatty acid wherein the fatty acid comprises
a double bond in the delta-5 position.
36. The seed of claim 35 wherein the transgenic plant is
soybean.
37. An oil obtained from the seed of a transgenic plant wherein the
oil comprises a desaturated fatty acid wherein the fatty acid
comprises a double bond in the delta-5 position.
38. The oil of claim 37 wherein the transgenic plant is
soybean.
39. Seed oil produced by a method comprising a desaturated fatty
acid wherein the fatty acid comprises a double bond in the delta-5
position, the method comprising: (a) transforming a plant cell with
a chimeric gene operably linked to a regulatory sequence, said
chimeric gene comprising: (i) a polynucleotide encoding a
polypeptide having delta-5 acyl-CoA desaturase activity, wherein
the polypeptide has an amino acid sequence of at least 95% sequence
identity, based on the Clustal alignment method, when compared to
SEQ ID NO:2, or (ii) the full complement of the nucleotide
sequence. (b) growing a fertile plant from the transformed plant
cell of step (a); (c) obtaining a seed from the plant of step (b);
and (d) processing the seed of step (c) to obtain oil wherein the
oil comprises a desaturated fatty acid wherein the fatty acid
comprises a double bond in the delta-5 position.
40. Seed oil produced by a method comprising a desaturated fatty
acid wherein the fatty acid comprises a double bond in the delta-5
position, the method comprising: (a) transforming a plant cell with
a chimeric gene operably linked to a regulatory sequence, said
chimeric gene comprising: (i) a first polynucleotide encoding a
first polypeptide having delta-5 acyl-CoA desaturase activity,
wherein the first polypeptide has an amino acid sequence of at
least 95% sequence identity, based on the Clustal alignment method,
when compared to SEQ ID NO:2, or (ii) the full complement of the
first nucleotide sequence. (iii) a second polynucleotide encoding a
second polypeptide having fatty acyl-CoA elongase activity, wherein
the second polypeptide has an amino acid sequence of at least 95%
sequence identity, based on the Clustal alignment method, when
compared to SEQ ID NO:5, or (iv) the full complement of the first
nucleotide sequence. (b) growing a fertile plant from the
transformed plant cell of step (a); (c) obtaining a seed from the
plant of step (b); and (d) processing the seed of step (c) to
obtain oil wherein the oil comprises a desaturated fatty acid
wherein the fatty acid comprises a double bond in the delta-5
position.
41. Seed oil with reduced levels of 16 carbon fatty acids produced
by a method comprising: (a) transforming a plant cell with a
chimeric gene operably linked to a regulatory sequence, said
chimeric gene comprising: (i) a polynucleotide encoding a
polypeptide having fatty acyl-CoA elongase activity, wherein the
polypeptide has an amino acid sequence of at least 95% sequence
identity, based on the Clustal alignment method, when compared to
SEQ ID NO:5, or (ii) the full complement of the nucleotide
sequence. (b) growing a fertile plant from the transformed plant
cell of step (a); (c) obtaining a seed from the plant of step (b);
and (d) processing the seed of step (c) to obtain oil wherein the
oil comprises a lower level of 16 carbon fatty acids than oil
obtained from a seed obtained from a plant that was grown from a
plant cell that was not transformed with the chimeric gene of step
(a).
42. Seed oil with increased levels of 20 carbon fatty acids
produced by a method comprising: (a) transforming a plant cell with
a chimeric gene operably linked to a regulatory sequence, said
chimeric gene comprising: (i) a polynucleotide encoding a
polypeptide having fatty acyl-CoA elongase activity, wherein the
polypeptide has an amino acid sequence of at least 95% sequence
identity, based on the Clustal alignment method, when compared to
SEQ ID NO:5, or (ii) the full complement of the nucleotide
sequence. (b) growing a fertile plant from the transformed plant
cell of step (a); (c) obtaining a seed from the plant of step (b);
and (d) processing the seed of step (c) to obtain oil wherein the
oil comprises a higher level of 20 carbon fatty acids than oil
obtained from a seed obtained from a plant that was grown from a
plant cell that was not transformed with the chimeric gene of step
(a).
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/664,840, filed Sep. 19, 2000, which claims the benefit of
U.S. Provisional Application No. 60/078,736 filed Mar. 20,
1998.
FIELD OF THE INVENTION
[0002] This invention is in the field of plant molecular biology.
More specifically, this invention pertains to nucleic acid
fragments encoding enzymes involved in lipid biosynthesis in plants
and seeds.
BACKGROUND OF THE INVENTION
[0003] Improved means to manipulate fatty acid compositions, from
biosynthetic or natural plant sources, are of paramount importance.
For example, edible oil sources containing the minimum possible
amounts of saturated fatty acids are desired for dietary reasons
and alternatives to current sources of highly saturated oil
products, such as tropical oils are needed.
[0004] Fatty acids are used in plant membranes and in neutral
lipids that are formed for energy storage in developing seed
tissues. The fatty acid composition (polarity, chain-length and
degree of unsaturation) of a membrane determines its physical
properties. The most common fatty acids contain 16 or 18 carbons
(C16 or C18) with one or more double bonds. Fatty acids with longer
(C20 or C22) or shorter (C12 or C14) carbon chains are unusual as
are hydroxylated fatty acids and fatty acids with different
positions of the double bonds (delta-5 or delta-6). Higher plants
appear to synthesize common fatty acids via a metabolic pathway in
plant plastid organelles (i.e., chloroplasts, proplastids, or other
related organelles) with intermediates bound to acyl carrier
proteins as part of the Fatty Acid Synthesis (FAS) complex. The
pathways involved in the synthesis of common fatty acids in
developing oilseeds are now well understood and are relatively easy
to manipulate. In fatty acid biosynthesis, delta-9 acyl-lipid
desaturase/delta-9 acyl-CoA desaturase most commonly introduces a
double bond at the delta-9 position of a C18 saturated fatty acid
(i.e., the desaturation of stearoyl-ACP(C18:0-ACP) to
oleoyl-ACP(C18:1-ACP)) to produce mono-unsaturated fatty acids.
Several other fatty-acid desaturase enzymes are known in higher
plants such as delta-6 and delta-5 desaturases that further
desaturate mono-unsaturated fatty acids to make polyunsaturated
fatty acids. There are a number of naturally occurring
mono-unsaturated fatty acids with double bonds in positions other
than the ninth carbon from the fatty acid carboxyl group. For
example, the triacylglycerols of Limnanthes alba and a number of
other gymnosperms all contain mono-unsaturated fatty acids with a
double bond at the delta-5 position. This activity may be catalyzed
by a delta-5 desaturase that, unlike the delta-9 desaturase which
uses 18:1-CoA as a substrate for the desaturation reaction, may
instead use 20:0-CoA (Pollard, M. R. and Stumpf, P. K. (1980) Plant
Physiol 66:649-655; Moreau, R. A. et al. (1981) Arch Biochem
Biophys 209:376-384).
[0005] Meadowfoam (Limnanthes alba) is a plant native to the higher
elevations of northern California and southern Oregon. The
triacylglycerol fraction of the mature seed is composed principally
of fatty acids containing 20 or 22 carbons and one or two double
bonds (20:1, 22:1 and 22:2). This double bond is unusual in that it
is in a position not normally found in the fatty acids of common
plant oils: the delta-5 position. The Limnanthes elongase appears
to prefer palmitoyl-CoA (16:0-CoA) as its substrate instead of
oleoyl-CoA (18:1 delta-9-CoA), the common substrate for the known
plant fatty acid elongases. In Limnanthes the 16:0-CoA is elongated
to 20:0-CoA and is desaturated to 20:1 delta-5. This is in contrast
to the formation of 20:1 delta-11 as in Arabidopsis or Canola where
the 18:1 delta-9 is elongated to 20:1 delta-11 (Pollard, M. R. and
Stumpf, P. K. (1980) Plant Physiol 66:649-655). The genes encoding
the Limnanthes delta-5 desaturase and the fatty acyl elongase
functions have not been isolated to date and are the subject of the
present application.
[0006] Although most plants contain at least trace amounts of very
long chain fatty acids, the FAS is not involved in the de novo
production of these very long chain fatty acids. Instead the
products of FAS are exported from the plastid and converted to
acyl-CoA derivatives which then serve as the substrates for the
fatty acid elongation system (FAE). The gene involved in the
Arabidopsis FAE has been localized to the FAE1 locus. The jojoba
oil consists mainly of waxes which are esters of monounsaturated
fatty acids and alcohols most of which contain fatty acid chains
with more than 18 carbons. Elongation to form very long chain fatty
acids in Arabidopsis, jojoba and rapeseed uses malonyl-CoA and
acyl-CoA as substrates (Lassner, M. W. et al. (1996) Plant Cell
8:281-292). In Limnanthes biosynthesis of 20:0 fatty acids occurs
predominantly by a chain elongation of palmitate as the initial
substrate; thus the enzyme catalyzing this reaction should be
similar but yet distinct from the enzyme involved in the production
of very long chain fatty acids through the elongation of
malonyl-CoA.
[0007] The ability to manipulate fatty acid biosynthetic pathways
by genetic engineering will allow changes to be made in the fatty
acid composition of plant oils and/or to introduce completely new
pathways into oilseeds in order to produce novel biopolymers from
acetyl-CoA. Limnanthes oils and fatty acids have potential use as
industrial agents. Estolides are oligomeric fatty acids containing
a secondary ester linkage on the alkyl backbone of the fatty acids.
The 20:1 delta-5 fatty acids present in Limnanthes oil are useful
for the production of polyestolides where the unique delta-5 bond
stabilizes the compound (Isbell, T. A. and Kleiman, R. (1996) J Am
Oil Chem Soc 73:1097-1107). Biodegradation of polyestiolides
derived from the Limnanthes monounsaturated fatty acids appears to
be slower than the biodegradation of polyestolides derived from
soybean oils or oleic oils but biodegradation continues with time
so that all estolides are probably ultimately degraded in nature
(Ehran, S. M. and Kleiman, R. (1997) J Am Oil Chem Soc 74:605-606).
This resistance to bacterial degradation suggests that
polyestolides derived from 20:1 delta-5 fatty acids will produce
lubricants, greases, plastics, inks, cosmetics and surfactants with
a long shelf life.
SUMMARY OF THE INVENTION
[0008] The instant invention relates to isolated nucleic acid
fragments encoding Limnanthes oil biosynthetic enzymes.
Specifically, this invention concerns an isolated nucleic acid
fragment encoding a delta-5 acyl-CoA desaturase or a fatty acyl-CoA
elongase. In addition, this invention relates to a nucleic acid
fragment that is complementary to the nucleic acid fragment
encoding a delta-5 acyl-CoA desaturase or fatty acyl-CoA elongase.
Also disclosed is the extension of 16:0-CoA to 20:0 by the
Limnanthes fatty acyl-CoA elongase. We also show that the delta-5
desaturase, in the absence of 20:0-CoA, will insert a double bond
at the delta-5 position of 16:0 and 18:0-CoA.
[0009] An additional embodiment of the instant invention pertains
to a polypeptide encoding all or a substantial portion of an enzyme
involved in lipid biosynthesis selected from the group consisting
of a delta-5 acyl-CoA desaturase and fatty acyl-CoA elongase.
[0010] In another embodiment, the instant invention relates to a
chimeric gene encoding a delta-5 acyl-CoA desaturase or a fatty
acyl-CoA elongase, or to a chimeric gene that comprises a nucleic
acid fragment that is complementary to a nucleic acid fragment
encoding a delta-5 acyl-CoA desaturase or a fatty acyl-CoA
elongase, operably linked to suitable regulatory sequences, wherein
expression of the chimeric gene results in production of the
encoded protein in a transformed host cell.
[0011] In a further embodiment, the instant invention concerns a
transformed host cell comprising in its genome a chimeric gene
encoding a delta-5 acyl-CoA desaturase or a fatty acyl-CoA
elongase, operably linked to suitable regulatory sequences.
Expression of the chimeric gene results in production of the
encoded protein in the transformed host cell. The transformed host
cell can be of eukaryotic or prokaryotic origin, and include cells
derived from higher plants and microorganisms. The invention also
includes transformed embryos and plants that arise from transformed
host cells of higher plants, and seeds derived from such
transformed plants.
[0012] An additional embodiment of the instant invention concerns a
method of altering the level of a delta-5 acyl-CoA desaturase or a
fatty acyl-CoA elongase in a transformed host cell comprising: a)
transforming a host cell with a chimeric gene comprising a nucleic
acid fragment encoding a delta-5 acyl-CoA desaturase or a fatty
acy-CoAl elongase; and b) growing the transformed host cell under
conditions that are suitable for expression of the chimeric gene
wherein expression of the chimeric gene results in production of
altered levels of a delta-5 acyl-CoA desaturase or a fatty acyl-CoA
elongase in the transformed host cell.
[0013] An addition embodiment of the instant invention concerns a
method for obtaining a nucleic acid fragment encoding all or a
substantial portion of an amino acid sequence encoding a delta-5
acyl-CoA desaturase or a fatty acyl-CoA elongase.
[0014] In a further embodiment, the instant invention concerns a
method for producing a desaturated fatty acid comprising a double
bond in the delta-5 position in a host cell, and seeds, oils and
methods of producing seed oils wherein the seeds and oils comprise
a desaturated fatty acid wherein the fatty acid comprises a double
bond in the delta-5 position.
[0015] An additional embodiment of the instant invention is a
method of reducing the level of 16 carbon fatty acids in a host
cell and a method of increasing the level of 20 carbon fatty acids
in a host cell, and seeds, oils and methods of producing seed oils
with reduced levels of 16 carbon fatty acids or increased levels or
20 carbon fatty acids.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0016] The invention can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application.
[0017] FIG. 1 shows the pathways for the formation of long-chain
fatty acids found in Limnanthes seeds. Biosynthesis of palmitate
(16:0), stearate (18:0) and oleate (18:1) occurs in the plastid
while elongation of palmitate to arachidonate and delta-5
desaturation occurs in the endoplasmic reticulum (adapted from
Pollard, M. R. and Stumpf, P. K. (1980) Plant Physiol
66:649-655).
[0018] FIG. 2 shows an alignment of the amino acid sequences from
Arabidopsis thaliana delta-9 desaturase (SEQ ID NO:3) and the
instant Limnanthes delta-5 acyl-CoA desaturase (lde.pk0008.b9; SEQ
ID NO:2). Amino acids which are identical among both sequences are
indicated with an asterisk (*) above the alignment. Dashes are used
by the program to maximize alignment of the sequences.
[0019] FIG. 3 shows the tracings from gas chromatograms obtained
for the oils of wild type soybean embryos (FIG. 3(A)) and of
soybean embryos expressing the Limnanthes fatty acyl-CoA elongase
(FIG. 3(B), demonstrating the production of C20 fatty acids in the
transformed soybean embryos. The fatty acids corresponding to the
various peaks of the chromatogram are indicated.
[0020] FIG. 4 shows the decrease in 16:0 fatty acid accumulation
concomitant with the increase in 20:0 fatty acids in individual
transgenic soybean embryos expressing the Limnanthes fatty acyl-CoA
elongase.
[0021] FIG. 5 shows the tracings from gas chromatograms obtained
for the oils of wild type soybean embryos (FIG. 5(A)) and of
soybean embryos expressing the Limnanthes delta-5-acyl-CoA
desaturase (FIG. 5(B)). The relevant fatty acids are indicated by
their retention time: 2.209 is 16:0; 2.271 is 16:1 .DELTA.5; 3.477
is 18:0; 3.530 is 18:1 .DELTA.5; and 3.567 is 18:1 .DELTA.9.
[0022] FIG. 6 shows the GC-MS analysis of fatty acid methyl esters
prepared from soybean embryos expressing the Limnanthes delta-5
acyl-CoA desaturase demonstrating the formation of 16:1 delta-5 and
18:1 delta-5 fatty acids. FIG. 6(A) presents the gas chromatogram
wherein DMDS derivatives of methyl hexadecenoic acid were
identified using a selected ion scan for 362 m/z. FIG. 6(B) is the
mass spectrum of the largest of the two peaks apparent in FIG.
6(A). FIG. 6(C) presents the gas chromatogram wherein DMDS
derivatives of methyl octadecenoic acid were identified using a
selected ion scan for 390 m/z. FIG. 6(D) is the mass spectrum of
the front shoulder of the largest peak that is apparent in FIG.
6(C).
[0023] The following sequence descriptions and Sequence Listing
attached hereto comply with the rules governing nucleotide and/or
amino acid sequence disclosures in patent applications as set forth
in 37 C.F.R. .sctn. 1.821-1.825.
[0024] SEQ ID NO: 1 is the nucleotide sequence comprising the
entire cDNA insert in clone lde.pk0008.b9 encoding an entire
Limnanthes delta-5 acyl-CoA desaturase.
[0025] SEQ ID NO:2 is the deduced amino acid sequence of an entire
Limnanthes delta-5 acyl-CoA desaturase derived from the nucleotide
sequence of SEQ ID NO: 1.
[0026] SEQ ID NO:3 is the amino acid sequence of an Arabidopsis
thaliana delta-9 desaturase having an NCBI General Identifier No:
2970034.
[0027] SEQ ID NO:4 is the nucleotide sequence comprising the contig
assembled from cDNA insert in clones lde.pk0008.d5 and
lde.pk0015.d10 encoding an entire Limnanthes fatty acyl-CoA
elongase.
[0028] SEQ ID NO:5 is the deduced amino acid sequence of an entire
Limnanthes fatty acyl-CoA elongase derived from the nucleotide
sequence of SEQ ID NO:4.
[0029] SEQ ID NO:6 is the nucleotide sequence comprising a portion
of the cDNA insert in clone lde.pk0010.e4 encoding a portion of a
Limnanthes fatty acyl-CoA elongase.
[0030] SEQ ID NO:7 is the deduced amino acid sequence of a portion
of a Limnanthes fatty acyl-CoA elongase derived from the nucleotide
sequence of SEQ ID NO:6.
[0031] The Sequence Listing contains the one letter code for
nucleotide sequence characters and the three letter codes for amino
acids as defined in conformity with the IUPAC-IUBMB standards
described in Nucleic Acids Research 13:3021-3030 (1985) and in the
Biochemical Journal 219 (No. 2):345-373 (1984) which are herein
incorporated by reference. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn. 1.822.
DETAILED DESCRIPTION OF THE INVENTION
[0032] In the context of this disclosure, a number of terms shall
be utilized. As used herein, an "isolated nucleic acid fragment" is
a polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA. As used herein, "contig" refers to an
assemblage of overlapping nucleic acid sequences to form one
contiguous nucleotide sequence. For example, several DNA sequences
can be compared and aligned to identify common or overlapping
regions. The individual sequences can then be assembled into a
single contiguous nucleotide sequence.
[0033] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the protein encoded by the DNA
sequence. "Substantially similar" also refers to nucleic acid
fragments wherein changes in one or more nucleotide bases does not
affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by antisense or co-suppression
technology. "Substantially similar" also refers to modifications of
the nucleic acid fragments of the instant invention such as
deletion or insertion of one or more nucleotides that do not
substantially affect the functional properties of the resulting
transcript vis-a-vis the ability to mediate alteration of gene
expression by antisense or co-suppression technology or alteration
of the functional properties of the resulting protein molecule. It
is therefore understood that the invention encompasses more than
the specific exemplary sequences.
[0034] For example, it is well known in the art that antisense
suppression and co-suppression of gene expression may be
accomplished using nucleic acid fragments representing less than
the entire coding region of a gene, and by nucleic acid fragments
that do not share 100% sequence identity with the gene to be
suppressed. Moreover, alterations in a gene which result in the
production of a chemically equivalent amino acid at a given site,
but do not effect the functional properties of the encoded protein,
are well known in the art. Thus, a codon for the amino acid
alanine, a hydrophobic amino acid, may be substituted by a codon
encoding another less hydrophobic residue, such as glycine, or a
more hydrophobic residue, such as valine, leucine, or isoleucine.
Similarly, changes which result in substitution of one negatively
charged residue for another, such as aspartic acid for glutamic
acid, or one positively charged residue for another, such as lysine
for arginine, can also be expected to produce a functionally
equivalent product. Nucleotide changes which result in alteration
of the N-terminal and C-terminal portions of the protein molecule
would also not be expected to alter the activity of the protein.
Each of the proposed modifications is well within the routine skill
in the art, as is determination of retention of biological activity
of the encoded products. Moreover, substantially similar nucleic
acid fragments may also be characterized by their ability to
hybridize, under stringent conditions (0.1.times.SSC, 0.1% SDS,
65.degree. C.), with the nucleic acid fragments disclosed
herein.
[0035] Substantially similar nucleic acid fragments of the instant
invention may also be characterized by the percent similarity of
the amino acid sequences that they encode to the amino acid
sequences disclosed herein, as determined by algorithms commonly
employed by those skilled in this art. Preferred are those nucleic
acid fragments whose nucleotide sequences encode amino acid
sequences that are 80% similar to the amino acid sequences reported
herein. More preferred nucleic acid fragments encode amino acid
sequences that are 90% similar to the amino acid sequences reported
herein. Most preferred are nucleic acid fragments that encode amino
acid sequences that are 95% similar to the amino acid sequences
reported herein. Sequence alignments and percent similarity
calculations were performed using the Megalign program of the
LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences was performed using the
Clustal method of alignment (Higgins, D. G. and Sharp, P. M. (1989)
CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP
LENGTH PENALTY=10). Default parameters for pairwise alignments
using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and
DIAGONALS SAVED=5.
[0036] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to afford putative
identification of that polypeptide or gene, either by manual
evaluation of the sequence by one skilled in the art, or by
computer-automated sequence comparison and identification using
algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more
contiguous amino acids or thirty or more nucleotides is necessary
in order to putatively identify a polypeptide or nucleic acid
sequence as homologous to a known protein or gene. Moreover, with
respect to nucleotide sequences, gene specific oligonucleotide
probes comprising 20-30 contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12-15 bases may be used as amplification
primers in PCR in order to obtain a particular nucleic acid
fragment comprising the primers. Accordingly, a "substantial
portion" of a nucleotide sequence comprises enough of the sequence
to afford specific identification and/or isolation of a nucleic
acid fragment comprising the sequence. The instant specification
teaches partial or complete amino acid and nucleotide sequences
encoding one or more particular plant proteins. The skilled
artisan, having the benefit of the sequences as reported herein,
may now use all or a substantial portion of the disclosed sequences
for purposes known to those skilled in this art. Accordingly, the
instant invention comprises the complete sequences as reported in
the accompanying Sequence Listing, as well as substantial portions
of those sequences as defined above.
[0037] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without effecting
the amino acid sequence of an encoded polypeptide. Accordingly, the
instant invention relates to any nucleic acid fragment that encodes
all or a substantial portion of the amino acid sequence encoding
the delta-5 acyl-CoA desaturase or the fatty acyl-CoA elongase
proteins as set forth in SEQ ID NOs:2, 5 and 7. The skilled artisan
is well aware of the "codon-bias" exhibited by a specific host cell
in usage of nucleotide codons to specify a given amino acid.
Therefore, when synthesizing a gene for improved expression in a
host cell, it is desirable to design the gene such that its
frequency of codon usage approaches the frequency of preferred
codon usage of the host cell.
[0038] "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments which are then
enzymatically assembled to construct the entire gene. "Chemically
synthesized", as related to a sequence of DNA, means that the
component nucleotides were assembled in vitro. Manual chemical
synthesis of DNA may be accomplished using well established
procedures, or automated chemical synthesis can be performed using
one of a number of commercially available machines. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codon bias of
the host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where
sequence information is available.
[0039] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers any gene
that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0040] "Coding sequence" refers to a DNA sequence that codes for a
specific amino acid sequence. "Regulatory sequences" refer to
nucleotide sequences located upstream (5' non-coding sequences),
within, or downstream (3' non-coding sequences) of a coding
sequence, and which influence the transcription, RNA processing or
stability, or translation of the associated coding sequence.
Regulatory sequences may include promoters, translation leader
sequences, introns, and polyadenylation recognition sequences.
[0041] "Promoter" refers to a DNA sequence capable of controlling
the expression of a coding sequence or functional RNA. In general,
a coding sequence is located 3' to a promoter sequence. The
promoter sequence consists of proximal and more distal upstream
elements, the latter elements often referred to as enhancers.
Accordingly, an "enhancer" is a DNA sequence which can stimulate
promoter activity and may be an innate element of the promoter or a
heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic DNA segments. It is understood by those skilled in the
art that different promoters may direct the expression of a gene in
different tissues or cell types, or at different stages of
development, or in response to different environmental conditions.
Promoters which cause a gene to be expressed in most cell types at
most times are commonly referred to as "constitutive promoters".
New promoters of various types useful in plant cells are constantly
being discovered; numerous examples may be found in the compilation
by Okamuro and Goldberg, (1989) Biochemistry of Plants 15: 1-82. It
is further recognized that since in most cases the exact boundaries
of regulatory sequences have not been completely defined, DNA
fragments of different lengths may have identical promoter
activity.
[0042] The "translation leader sequence" refers to a DNA sequence
located between the promoter sequence of a gene and the coding
sequence. The translation leader sequence is present in the fully
processed mRNA upstream of the translation start sequence. The
translation leader sequence may affect processing of the primary
transcript to mRNA, mRNA stability or translation efficiency.
Examples of translation leader sequences have been described
(Turner, R. and Foster, G. D. (1995) Molecular Biotechnology
3:225).
[0043] The "3'non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include polyadenylation
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor. The use of different 3' non-coding sequences is
exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.
[0044] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from posttranscriptional processing of the primary
transcript and is referred to as the mature RNA. "Messenger RNA
(mRNA)" refers to the RNA that is without introns and that can be
translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene (U.S. Pat. No. 5,107,065, incorporated herein by
reference). The complementarity of an antisense RNA may be with any
part of the specific gene transcript, i.e., at the 5' non-coding
sequence, 3' non-coding sequence, introns, or the coding sequence.
"Functional RNA" refers to sense RNA, antisense RNA, ribozyme RNA,
or other RNA that may not be translated but yet has an effect on
cellular processes.
[0045] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0046] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide. "Antisense inhibition" refers to the production of
antisense RNA transcripts capable of suppressing the expression of
the target protein. "Overexpression" refers to the production of a
gene product in transgenic organisms that exceeds levels of
production in normal or non-transformed organisms. "Co-suppression"
refers to the production of sense RNA transcripts capable of
suppressing the expression of identical or substantially similar
foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated
herein by reference).
[0047] "Altered levels" refers to the production of gene product(s)
in transgenic organisms in amounts or proportions that differ from
that of normal or non-transformed organisms.
[0048] "Mature" protein refers to a post-translationally processed
polypeptide; i.e., one from which any pre- or propeptides present
in the primary translation product have been removed. "Precursor"
protein refers to the primary product of translation of mRNA; i.e.,
with pre- and propeptides still present. Pre- and propeptides may
be but are not limited to intracellular localization signals.
[0049] A "chloroplast transit peptide" is an amino acid sequence
which is translated in conjunction with a protein and directs the
protein to the chloroplast or other plastid types present in the
cell in which the protein is made. "Chloroplast transit sequence"
refers to a nucleotide sequence that encodes a chloroplast transit
peptide. A "signal peptide" is an amino acid sequence which is
translated in conjunction with a protein and directs the protein to
the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant
Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed
to a vacuole, a vacuolar targeting signal (supra) can further be
added, or if to the endoplasmic reticulum, an endoplasmic reticulum
retention signal (supra) may be added. If the protein is to be
directed to the nucleus, any signal peptide present should be
removed and instead a nuclear localization signal included (Raikhel
(1992) Plant Phys. 100:1627-1632).
[0050] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
organisms. Examples of methods of plant transformation include
Agrobacterium-mediated transformation (De Blaere et al. (1987)
Meth. Enzymol. 143:277) and particle-accelerated or "gene gun"
transformation technology (Klein T M et al. (1987) Nature (London)
327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by
reference).
[0051] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, 1989 (hereinafter "Maniatis").
[0052] Nucleic acid fragments encoding at least a portion of two
enzymes involved in lipid biosynthesis have been isolated and
identified by comparison of random plant cDNA sequences to public
databases containing nucleotide and protein sequences using the
BLAST algorithms well known to those skilled in the art. The
identity of these enzymes has been confirmed by functional analysis
as set forth in Example 6. Table 1 lists the proteins that are
described herein, and the designation of the cDNA clones that
comprise the nucleic acid fragments encoding these proteins.
TABLE-US-00001 TABLE 1 Limnanthes Oil Biosynthetic Enzymes Enzyme
Clone Plant Delta-5 Acyl-CoA Desaturase lde.pk0008.b9 Limnanthes
douglasii Fatty Acyl-CoA Elongase Contig of: Limnanthes douglasii
lde.pk0008.d5 Xlde.pk0015.d10 lde.pk0010.e4 Limnanthes
douglasii
[0053] The nucleic acid fragments of the instant invention may be
used to isolate cDNAs and genes encoding homologous proteins from
the same or other plant species. Isolation of homologous genes
using sequence-dependent protocols is well known in the art.
Examples of sequence-dependent protocols include, but are not
limited to, methods of nucleic acid hybridization, and methods of
DNA and RNA amplification as exemplified by various uses of nucleic
acid amplification technologies (e.g., polymerase chain reaction,
ligase chain reaction).
[0054] For example, genes encoding other delta-5 acyl-CoA
desaturase or fatty acyl-CoA elongase homologs, either as cDNAs or
genomic DNAs, could be isolated directly by using all or a portion
of the instant nucleic acid fragments as DNA hybridization probes
to screen libraries from any desired plant employing methodology
well known to those skilled in the art. Specific oligonucleotide
probes based upon the instant nucleic acid sequences can be
designed and synthesized by methods known in the art (Maniatis).
Moreover, the entire sequences can be used directly to synthesize
DNA probes by methods known to the skilled artisan such as random
primer DNA labeling, nick translation, or end-labeling techniques,
or RNA probes using available in vitro transcription systems. In
addition, specific primers can be designed and used to amplify a
part or all of the instant sequences. The resulting amplification
products can be labeled directly during amplification reactions or
labeled after amplification reactions, and used as probes to
isolate full length cDNA or genomic fragments under conditions of
appropriate stringency.
[0055] In addition, two short segments of the instant nucleic acid
fragments may be used in polymerase chain reaction protocols to
amplify longer nucleic acid fragments encoding homologous genes
from DNA or RNA. The polymerase chain reaction may also be
performed on a library of cloned nucleic acid fragments wherein the
sequence of one primer is derived from the instant nucleic acid
fragments, and the sequence of the other primer takes advantage of
the presence of the polyadenylic acid tracts to the 3' end of the
mRNA precursor encoding plant genes. Alternatively, the second
primer sequence may be based upon sequences derived from the
cloning vector. For example, the skilled artisan can follow the
RACE protocol (Frohman et al., (1988) Proc. Natl. Acad. Sci. USA
85:8998) to generate cDNAs by using PCR to amplify copies of the
region between a single point in the transcript and the 3' or 5'
end. Primers oriented in the 3' and 5' directions can be designed
from the instant sequences. Using commercially available 3' RACE or
5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be
isolated (Ohara et al., (1989) Proc. Natl. Acad. Sci. USA 86:5673;
Loh et al., (1989) Science 243:217). Products generated by the 3'
and 5' RACE procedures can be combined to generate full-length
cDNAs (Frohman, M. A. and Martin, G. R., (1989) Techniques
1:165).
[0056] Availability of the instant nucleotide and deduced amino
acid sequences facilitates immunological screening of cDNA
expression libraries. Synthetic peptides representing portions of
the instant amino acid sequences may be synthesized. These peptides
can be used to immunize animals to produce polyclonal or monoclonal
antibodies with specificity for peptides or proteins comprising the
amino acid sequences. These antibodies can be then be used to
screen cDNA expression libraries to isolate full-length cDNA clones
of interest (Lerner, R. A. (1984) Adv. Immunol. 36:1;
Maniatis).
[0057] Oil biosynthesis in plants has been fairly well-studied (see
Harwood (1989) in Critical Reviews in Plant Sciences, Vol. 8:1-43).
As used herein, "Oilseed crops" refers to plant species which
produce and store triacylglycerol in specific organs, primarily in
seeds. In particular, for purposes of this disclosure, "oilseed
crops" refers to soybean, corn, sunflower, peanut, safflower,
sesame, niger, cotton, cocoa, linseed (flax), low linoleic flax,
castor, oil palm, coconut, canola and other Brassica oilseed
species such as B. napus, B. campestris, B. oleracea, B. carinata,
B. juncea, B. nigra, B. adpressa, B. tournefortii, B.
fruticulosas.
[0058] The nucleic acid fragments of the instant invention may be
used to create transgenic plants in which the disclosed delta-5
acyl-CoA desaturase or fatty acyl-CoA elongase are present at
higher or lower levels than normal or in cell types or
developmental stages in which they are not normally found. This
would have the effect of altering the level of fatty acid
saturation and chain length in those cells. As demonstrated in
Example 6 below, overexpression of the Limnanthes fatty acyl-CoA
elongase in an oilseed crop results in the elongation of palmitic
acid (16:0) to arachidonic acid (20:0). Overexpression of the
Limnanthes delta-5 acyl-CoA desaturase in an oilseed crop results
in the introduction of a double bond at the delta-5 position of a
fatty acid chain, resulting in the production of 16:1 and 18:1
delta-5 fatty acids. Overexpression of both of these genes in an
oilseed crop will enable the production of 20:1 delta-5 fatty
acids. There are at least two positive effects emanating from this:
the reduction of the saturated fatty acids (especially 16:0) in
food oils and the production of fatty acids (20:1 delta-5) with a
myriad of industrial uses.
[0059] Overexpression of the delta-5 acyl-CoA desaturase or the
fatty acyl-CoA elongase proteins of the instant invention may be
accomplished by first constructing a chimeric gene in which the
coding region is operably linked to a promoter capable of directing
expression of a gene in the desired tissues at the desired stage of
development. For reasons of convenience, the chimeric gene may
comprise promoter sequences and translation leader sequences
derived from the same genes. 3' Non-coding sequences encoding
transcription termination signals may also be provided. The instant
chimeric gene may also comprise one or more introns in order to
facilitate gene expression.
[0060] Plasmid vectors comprising the instant chimeric gene can
then constructed. The choice of plasmid vector is dependent upon
the method that will be used to transform host plants. The skilled
artisan is well aware of the genetic elements that must be present
on the plasmid vector in order to successfully transform, select
and propagate host cells containing the chimeric gene. The skilled
artisan will also recognize that different independent
transformation events will result in different levels and patterns
of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida
et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that
multiple events must be screened in order to obtain lines
displaying the desired expression level and pattern. Such screening
may be accomplished by Southern analysis of DNA, Northern analysis
of mRNA expression, Western analysis of protein expression, or
phenotypic analysis.
[0061] For some applications it may be useful to direct the instant
enzyme involved in lipid biosynthesis to different cellular
compartments, or to facilitate its secretion from the cell. It is
thus envisioned that the chimeric gene described above may be
further supplemented by altering the coding sequence to encode
delta-5 acyl-CoA desaturase or fatty acyl-CoA elongase with
appropriate intracellular targeting sequences such as transit
sequences (Keegstra, K. (1989) Cell 56:247-253), signal sequences
or sequences encoding endoplasmic reticulum localization
(Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.
42:21-53), or nuclear localization signals (Raikhel, N. (1992)
Plant Phys. 100:1627-1632) added and/or with targeting sequences
that are already present removed. While the references cited give
examples of each of these, the list is not exhaustive and more
targeting signals of utility may be discovered in the future.
[0062] The instant delta-5 acyl-CoA desaturase or fatty acyl-CoA
elongase (or portions thereof) may be produced in heterologous host
cells, particularly in the cells of microbial hosts, and can be
used to prepare antibodies to the these proteins by methods well
known to those skilled in the art. The antibodies are useful for
detecting delta-5 acyl-CoA desaturase or fatty acyl-CoA elongase in
situ in cells or in vitro in cell extracts. Preferred heterologous
host cells for production of the instant delta-5 acyl-CoA
desaturase or fatty acyl-CoA elongase are microbial hosts.
Microbial expression systems and expression vectors containing
regulatory sequences that direct high level expression of foreign
proteins are well known to those skilled in the art. Any of these
could be used to construct a chimeric gene for production of the
instant delta-5 acyl-CoA desaturase or fatty acyl-CoA elongase.
This chimeric gene could then be introduced into appropriate
microorganisms via transformation to provide high level expression
of the encoded enzyme involved in lipid biosynthesis. An example of
a vector for high level expression of the instant delta-5 acyl-CoA
desaturase or fatty acyl-CoA elongase in a bacterial host is
provided (Example 7).
[0063] All or a substantial portion of the nucleic acid fragments
of the instant invention may also be used as probes for genetically
and physically mapping the genes that they are a part of, and as
markers for traits linked to those genes. Such information may be
useful in plant breeding in order to develop lines with desired
phenotypes. For example, the instant nucleic acid fragments may be
used as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Maniatis) of restriction-digested plant genomic DNA
may be probed with the nucleic acid fragments of the instant
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et at., (1987) Genomics 1:174-181) in order to construct a genetic
map. In addition, the nucleic acid fragments of the instant
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the instant nucleic acid sequence in the
genetic map previously obtained using this population (Botstein, D.
et al., (1980) Am. J. Hum. Genet. 32:314-331).
[0064] The production and use of plant gene-derived probes for use
in genetic mapping is described in R. Bernatzky, R. and Tanksley,
S. D. (1986) Plant Mol. Biol. Reporter 4(1):37-41. Numerous
publications describe genetic mapping of specific cDNA clones using
the methodology outlined above or variations thereof. For example,
F2 intercross populations, backcross populations, randomly mated
populations, near isogenic lines, and other sets of individuals may
be used for mapping. Such methodologies are well known to those
skilled in the art.
[0065] Nucleic acid probes derived from the instant nucleic acid
sequences may also be used for physical mapping (i.e., placement of
sequences on physical maps; see Hoheisel, J. D., et al., In:
Nonmammalian Genomic Analysis: A Practical Guide, Academic press
1996, pp. 319-346, and references cited therein).
[0066] In another embodiment, nucleic acid probes derived from the
instant nucleic acid sequences may be used in direct fluorescence
in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends
Genet. 7:149-154). Although current methods of FISH mapping favor
use of large clones (several to several hundred KB; see Laan, M. et
al. (1995) Genome Research 5:13-20), improvements in sensitivity
may allow performance of FISH mapping using shorter probes.
[0067] A variety of nucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the instant
nucleic acid sequences. Examples include allele-specific
amplification (Kazazian, H. H. (1989) J. Lab. Clin. Med.
114(2):95-96), polymorphism of PCR-amplified fragments (CAPS;
Sheffield, V. C. et al. (1993) Genomics 16:325-332),
allele-specific ligation (Landegren, U. et al. (1988) Science
241:1077-1080), nucleotide extension reactions (Sokolov, B. P.
(1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping
(Walter, M. A. et al. (1997) Nature Genetics 7:22-28) and Happy
Mapping (Dear, P. H. and Cook, P. R. (1989) Nucleic Acid Res.
17:6795-6807). For these methods, the sequence of a nucleic acid
fragment is used to design and produce primer pairs for use in the
amplification reaction or in primer extension reactions. The design
of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
EXAMPLES
[0068] The present invention is further defined in the following
Examples, in which all parts and percentages are by weight and
degrees are Celsius, unless otherwise stated. It should be
understood that these Examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. From the above discussion and these Examples, one skilled in
the art can ascertain the essential characteristics of this
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions.
Example 1
Composition of cDNA Libraries
Isolation and Sequencing of cDNA Clones
[0069] cDNA libraries representing mRNAs from Limnanthes douglasii
embryo tissues were prepared in pcDNAII vectors according to the
manufacturer's protocol (Invitrogen Corporation, Carlsbad, Calif.).
cDNA inserts from randomly picked bacterial colonies containing
recombinant pcDNAII plasmids were amplified via polymerase chain
reaction using primers specific for vector sequences flanking the
inserted cDNA sequences or plasmid DNA was prepared from cultured
bacterial cells. Amplified insert DNAs or plasmid DNAs were
sequenced in dye-primer sequencing reactions to generate partial
cDNA sequences (expressed sequence tags or "ESTs"; see Adams, M. D.
et al., (1991) Science 252:1651). The resulting ESTs were analyzed
using a Perkin Elmer Model 377 fluorescent sequencer.
Example 2
Identification of cDNA Clones
[0070] ESTs encoding enzymes involved in lipid biosynthesis were
identified by conducting BLAST (Basic Local Alignment Search Tool;
Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also
www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences
contained in the BLAST "nr" database (comprising all non-redundant
GenBank CDS translations, sequences derived from the 3-dimensional
structure Brookhaven Protein Data Bank, the last major release of
the SWISS-PROT protein sequence database, EMBL, and DDBJ
databases). The cDNA sequences obtained in Example 1 were analyzed
for similarity to all publicly available DNA sequences contained in
the "nr" database using the BLASTN algorithm provided by the
National Center for Biotechnology Information (NCBI). The DNA
sequences were translated in all reading frames and compared for
similarity to all publicly available protein sequences contained in
the "nr" database using the BLASTX algorithm (Gish, W. and States,
D. J. (1993) Nature Genetics 3:266-272) provided by the NCBI. For
convenience, the P-value (probability) of observing a match of a
cDNA sequence to a sequence contained in the searched databases
merely by chance as calculated by BLAST are reported herein as
"pLog" values, which represent the negative of the logarithm of the
reported P-value. Accordingly, the greater the pLog value, the
greater the likelihood that the cDNA sequence and the BLAST "hit"
represent homologous proteins.
Example 3
Characterization of cDNA Clones Encoding Delta-5 Acyl-CoA
Desaturase Homologs
[0071] The BLASTX search using the EST sequences from clones
lde.pk0004.c10, lde.pk0012.e5 and lde.pk0012.g11, and the entire
cDNA insert from clone lde.pk0010.a8 revealed similarity of the
proteins encoded by the cDNAs to delta-9 acyl-lipid
desaturase/delta-9 acyl-CoA desaturase from Rosa hybrida (GenBank
Accession No. S80863; NCBI General Identifier No. 1911477). The
BLASTX search using the EST sequence from clone lde.pk0008.b9
revealed similarity of the protein encoded by the cDNA to a
fatty-acid desaturase from Rosa hybrida (GenBank Accession No.
D49383; NCBI General Identifier No. 2580425) The BLAST results for
each of these sequences are shown in Table 2:
TABLE-US-00002 TABLE 2 BLAST Results for Clones Encoding
Polypeptides Homologous to Desaturases GenBank Clone Accession No.
BLAST pLog Score lde.pk0004.c10 S80863 48.23 lde.pk0012.e5 S80863
23.64 lde.pk0012.g11 S80863 14.42 lde.pk0010.a8 S80863 9.89
lde.pk0008.b9 D49383 1.44
[0072] The sequence of the entire cDNA insert in clone
lde.pk0008.b9 was determined and is shown in SEQ ID NO:1; the
deduced amino acid sequence of this cDNA is shown in SEQ ID NO:2.
The EST sequences for clones lde.pk0004.c10, lde.pk0012.e5,
lde.pk0012.g11 and lde.pk0010.a8 are encompassed by the sequence
set forth in SEQ ID NO:1. The amino acid sequence set forth in SEQ
ID NO:2 was evaluated by BLASTP, yielding a pLog value of >250
versus the Arabidopsis thaliana delta-9 desaturase sequence (NCBI
General Identifier No. 2970034). FIG. 1 presents an alignment of
the amino acid sequences set forth in SEQ ID NO:2 and the
Arabidopsis thaliana delta-9 desaturase sequence (SEQ ID NO:3). The
amino acid sequence set forth in SEQ ID NO:2 is 47.9% similar to
the Arabidopsis thaliana sequence (SEQ ID NO:3). Sequence
alignments and percent identity calculations were performed using
the Megalign program of the LASARGENE bioinformatics computing
suite (DNASTAR Inc., Madison, Wis.). Pairwise alignment of the
amino acid sequences and percent similarity calculations were
performed using the Clustal method of alignment (Higgins, D. G. and
Sharp, P. M. (1989) CABIOS. 5: 151-153) with the default parameters
(GAP PENALTY=5, KTUPLE=1, WINDOW=5 and DIAGONALS SAVED=5).
[0073] Sequence alignments, BLAST scores and probabilities
suggested that the instant nucleic acid fragment encodes an entire
Limnanthes delta-9 acyl-CoA desaturase. However, the oil derived
from Limnanthes is composed mainly of very long-chain fatty acids
with a delta-5 cis double bond, suggesting that the instant nucleic
acid fragment may in fact encode a delta-5 acyl-CoA desaturase
rather than a delta-9 desaturase. As shown in Example 6, expression
of the Limnanthes desaturase in soybean embryos results in the
formation of oils containing 16:1 and 18:1 delta-5 fatty acids.
Accordingly, the instant nucleic acid fragments comprise the first
Limnanthes douglasii sequences encoding a delta-5 acyl-CoA
desaturase.
Example 4
Characterization of cDNAs Clones Encoding Fatty Acyl-CoA Elongase
Homologs
[0074] The BLASTX search using the EST sequences from clones
lde.pk0008.d5 and lde.pk0010.e4 revealed similarity of the proteins
encoded by the cDNAs to beta-ketoacyl-CoA synthase from Arabidopsis
thaliana (GenBank Accession No. AC003105; NCBI General Identifier
No. 2760830). The BLAST results for these ESTs are shown in Table
3:
TABLE-US-00003 TABLE 3 BLAST Results for a Clone Encoding a
Polypeptide Homologous to Beta-ketoacyl-CoA Synthase BLAST pLog
Score Clone AC003105 lde.pk0008.d5 78.05 lde.pk0010.e4 72.66
[0075] Further searching of the proprietary database indicated that
clone lde.pk0015.d10 also revealed similarity to beta-ketoacyl-CoA
synthase. The sequence of the entire cDNA insert in clone
lde.pk0008.d5 was determined and a contig was assembled with this
sequence and the sequence from a portion of the cDNA insert from
clone lde.pk0015.d10. The nucleotide sequence of this contig is
shown in SEQ ID NO:4; the deduced amino acid sequence of this
contig is shown in SEQ ID NO:5. A BLASTX search using the
nucleotide sequence set forth in SEQ ID NO:4 resulted in a pLog
value of >254 versus the Arabidopsis thaliana beta-ketoacyl-CoA
synthase sequence. The sequence of almost the entire cDNA insert
from clone lde.pk0010.e4 is shown in SEQ ID NO:6; the deduced amino
acid sequence of this cDNA is shown in SEQ ID NO:7. A BLASTX search
using the nucleotide sequences set forth in SEQ ID NO:6 resulted in
a pLog value of 132 versus the Arabidopsis thaliana sequence. The
amino acid sequence set forth in SEQ ID NO:5 is 74.5% similar to
the Arabidopsis thaliana sequence and the amino acid sequence set
forth in SEQ ID NO:7 is 80.3% similar to the Arabidopsis thaliana
sequence. The two Limnanthes sequences are 88.0% similar to each
other, suggesting that both Limnanthes sequences encode proteins of
similar function.
[0076] BLAST scores and probabilities indicated that the instant
nucleic acid fragments encoded a portion of a Limnanthes douglasii
beta-ketoacyl-CoA synthase homolog and an entire Limnanthes
douglasii beta-ketoacyl-CoA synthase homolog. However, the oil in
Limnanthes is composed mainly of very long-chain fatty acids with a
delta-5 cis double bond suggesting that the instant nucleic acid
fragments may in fact encode fatty acyl-CoA elongases rather than
beta-ketoacyl-CoA synthases. This is confirmed in Example 6 wherein
expression of the Limnanthes elongase in soybean embryos results in
an enrichment of 20:0 fatty acids. These sequences therefore
represent the first Lumnanthes douglasii sequences encoding fatty
acyl-CoA elongases.
Example 5
Expression of Chimeric Genes in Monocot Cells
[0077] A chimeric gene comprising a cDNA encoding an enzyme
involved in lipid biosynthesis in sense orientation with respect to
the maize 27 kD zein promoter that is located 5' to the cDNA
fragment, and the 10 kD zein 3' end that is located 3' to the cDNA
fragment, can be constructed. The cDNA fragment of this gene may be
generated by polymerase chain reaction (PCR) of the cDNA clone
using appropriate oligonucleotide primers. Cloning sites (NcoI or
SmaI) can be incorporated into the oligonucleotides to provide
proper orientation of the DNA fragment when inserted into the
digested vector pML103 as described below. Amplification is then
performed in a standard PCR. The amplified DNA is then digested
with restriction enzymes NcoI and SmaI and fractionated on an
agarose gel. The appropriate band can be isolated from the gel and
combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103.
Plasmid pML103 has been deposited under the terms of the Budapest
Treaty at ATCC (American Type Culture Collection, 10801 University
Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC
97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI
promoter fragment of the maize 27 kD zein gene and a 0.96 kb
SmaI-SalI fragment from the 3' end of the maize 10 kD zein gene in
the vector pGem9Zf(+) (Promega). Vector and insert DNA can be
ligated at 15.degree. C. overnight, essentially as described
(Maniatis). The ligated DNA may then be used to transform E. coli
XL1-Blue (Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial
transformants can be screened by restriction enzyme digestion of
plasmid DNA and limited nucleotide sequence analysis using the
dideoxy chain termination method (Sequenase.TM. DNA Sequencing Kit;
U.S. Biochemical). The resulting plasmid construct would comprise a
chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD
zein promoter, a cDNA fragment encoding an enzyme involved in lipid
biosynthesis, and the 10 kD zein 3' region.
[0078] The chimeric gene described above can then be introduced
into corn cells by the following procedure. Immature corn embryos
can be dissected from developing caryopses derived from crosses of
the inbred corn lines H99 and LH132. The embryos are isolated 10 to
11 days after pollination when they are 1.0 to 1.5 mm long. The
embryos are then placed with the axis-side facing down and in
contact with agarose-solidified N6 medium (Chu et al., (1975) Sci.
Sin. Peking 18:659-668). The embryos are kept in the dark at
27.degree. C. Friable embryogenic callus consisting of
undifferentiated masses of cells with somatic proembryoids and
embryoids borne on suspensor structures proliferates from the
scutellum of these immature embryos. The embryogenic callus
isolated from the primary explant can be cultured on N6 medium and
sub-cultured on this medium every 2 to 3 weeks.
[0079] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst
Ag, Frankfurt, Germany) may be used in transformation experiments
in order to provide for a selectable marker. This plasmid contains
the Pat gene (see European Patent Publication 0 242 236) which
encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT
confers resistance to herbicidal glutamine synthetase inhibitors
such as phosphinothricin. The pat gene in p35 S/Ac is under the
control of the 35S promoter from Cauliflower Mosaic Virus (Odell et
al. (1985) Nature 313:810-812) and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens.
[0080] The particle bombardment method (Klein T M et al. (1987)
Nature (London) 327:70-73) may be used to transfer genes to the
callus culture cells. According to this method, gold particles (1
.mu.m in diameter) are coated with DNA using the following
technique. Ten .mu.g of plasmid DNAs are added to 50 .mu.L of a
suspension of gold particles (60 mg per mL). Calcium chloride (50
.mu.L of a 2.5 M solution) and spermidine free base (20 .mu.L of a
1.0 M solution) are added to the particles. The suspension is
vortexed during the addition of these solutions. After 10 minutes,
the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the
supernatant removed. The particles are resuspended in 200 .mu.L of
absolute ethanol, centrifuged again and the supernatant removed.
The ethanol rinse is performed again and the particles resuspended
in a final volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of
the DNA-coated gold particles can be placed in the center of a
Kapton.TM. flying disc (Bio-Rad Labs). The particles are then
accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He
(Bio-Rad Instruments, Hercules Calif.), using a helium pressure of
1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0
cm.
[0081] For bombardment, the embryogenic tissue is placed on filter
paper over agarose-solidified N6 medium. The tissue is arranged as
a thin lawn and covered a circular area of about 5 cm in diameter.
The petri dish containing the tissue can be placed in the chamber
of the PDS-1000/He approximately 8 cm from the stopping screen. The
air in the chamber is then evacuated to a vacuum of 28 inches of
Hg. The macrocarrier is accelerated with a helium shock wave using
a rupture membrane that bursts when the He pressure in the shock
tube reaches 1000 psi.
[0082] Seven days after bombardment the tissue can be transferred
to N6 medium that contains gluphosinate (2 mg per liter) and lacks
casein or proline. The tissue continues to grow slowly on this
medium. After an additional 2 weeks the tissue can be transferred
to fresh N6 medium containing gluphosinate. After 6 weeks, areas of
about 1 cm in diameter of actively growing callus can be identified
on some of the plates containing the glufosinate-supplemented
medium. These calli may continue to grow when sub-cultured on the
selective medium.
[0083] Plants can be regenerated from the transgenic callus by
first transferring clusters of tissue to N6 medium supplemented
with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be
transferred to regeneration medium (Fromm et al., (1990)
Bio/Technology 8:833-839).
Example 6
Expression of Chimeric Genes in Dicot Cells
[0084] A seed-specific expression cassette composed of the promoter
and transcription terminator from the gene encoding the .beta.
subunit of the seed storage protein phaseolin from the bean
Phaseolus vulgaris (Doyle, J. J. et al. (1986) J. Biol. Chem.
261:9228-9238) can be used for expression of the instant enzymes
involved in lipid biosynthesis in transformed dicots. The phaseolin
cassette includes about 500 nucleotides upstream (5') from the
translation initiation codon and about 1650 nucleotides downstream
(3') from the translation stop codon of phaseolin. Between the 5'
and 3' regions are the unique restriction endonuclease sites Nco I
(which includes the ATG translation initiation codon), Sma I, Kpn I
and Xba I. The entire cassette is flanked by Hind III sites.
[0085] The cDNA fragment of this gene may be generated by
polymerase chain reaction (PCR) of the cDNA clone using appropriate
oligonucleotide primers. Cloning sites can be incorporated into the
oligonucleotides to provide proper orientation of the DNA fragment
when inserted into the expression vector. Amplification is then
performed as described above, and the isolated fragment is inserted
into a pUC18 vector carrying the seed expression cassette.
[0086] Dicot embroys may then be transformed with the expression
vector comprising sequences encoding enzymes involved in lipid
biosynthesis. To induce somatic embryos, cotyledons, 3-5 mm in
length dissected from surface sterilized, immature seeds of the
chosen dicot, can be cultured in the light or dark at 26.degree. C.
on an appropriate agar medium for 6-10 weeks. Somatic embryos which
produce secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos which multiplied as early, globular staged embryos,
the suspensions are maintained as described below.
[0087] Dicot embryogenic suspension cultures can maintained in 35
mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
fluorescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of
tissue into 35 mL of liquid medium.
[0088] Dicot embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein T. M.
et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050).
A DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can
be used for these transformations.
[0089] A selectable marker gene which can be used to facilitate
plant transformation is a chimeric gene composed of the 35S
promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz L et al. (1983) Gene 25:179-188) and
the 3' region of the nopaline synthase gene from the T-DNA of the
Ti plasmid of Agrobacterium tumefaciens. The seed expression
cassette comprising the phaseolin 5' region, the fragment encoding
the enzyme involved in lipid biosynthesis and the phaseolin 3'
region can be isolated as a restriction fragment. This fragment can
then be inserted into a unique restriction site of the vector
carrying the marker gene.
[0090] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.l
spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
.mu.L of the DNA-coated gold particles are then loaded on each
macro carrier disk.
[0091] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0092] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post
bombardment replaced with fresh media containing 50 mg/mL
hygromycin. This selective media can be refreshed weekly. Seven to
eight weeks post bombardment, green, transformed tissue may be
observed growing from untransformed, necrotic embryogenic clusters.
Isolated green tissue is removed and inoculated into individual
flasks to generate new, clonally propagated, transformed
embryogenic suspension cultures. Each new line may be treated as an
independent transformation event. These suspensions can then be
subcultured and maintained as clusters of immature embryos or
regenerated into whole plants by maturation and germination of
individual somatic embryos.
Expression of Limnanthes Delta-5 Acyl-CoA Desaturase and Fatty
acyl-CoA elongase in Soybean Embryos
[0093] To confirm the identity and activity of the nucleic acid
fragments set forth in SEQ ID NO:1 (encoding a Limnanthes delta-5
acyl-CoA lipid desaturase) and SEQ ID NO:4 (encoding a Limnanthes
fatty acyl-CoA elongase), these nucleic acids were cloned
individually into an in vivo expression vector. The cDNA inserts in
the library cloning vector pcDNAII are flanked by Not I sites
allowing for the removal of the entire cDNA insert by Not I
digestion. The delta-5 acyl-CoA desaturase and the fatty acyl-CoA
elongase-encoding plasmids were digested with Not I, the cDNA
fragment isolated, purified and ligated into the pKS67 vector
(described below) following standard molecular biology
techniques.
[0094] A plasmid, pZBL100, containing chimeric genes to allow
expression of hygromycin B phosphotransferase in certain bacteria
and in plant cells was constructed from the following genetic
elements: a) T7 promoter+Shine-Delgarno/hygromycin B
phosphotransferase (HPT)/T7 terminator sequence, b) 35S promoter
from cauliflower mosaic virus (CaMV)/hygromycin B
phosphotransferase (HPT)/nopaline synthase (NOS3' from
Agrobacterium tumefaciens T-DNA, and c) pSP72 plasmid vector
(Promega) with the b-lactamase coding region (ampicillin resistance
gene) removed.
[0095] The HPT gene was amplified by PCR from E. coli strain W677,
which contained a Klebsiella-derived plasmid pJR225. Starting with
the pSP72 vector the elements were assembled into a single plasmid
using standard cloning methods (Maniatis).
[0096] Plasmid pZBL 100 thus contains the T7 promoter/HPT/T7
terminator cassette for expression of the HPT enzyme in certain
strains of E. coli, such as NovaBlue (DE3) (Novagen), that are
lysogenic for lambda DE3 (which carries the T7 RNA Polymerase gene
under lacUV5 control). Plasmid pZBL100 also contains the
35S/HPT/NOS cassette for constitutive expression of the HPT enzyme
in plants, such as soybean. These two expression systems allow
selection for growth in the presence of hygromycin to be used as a
means of identifying cells that contain the plasmid in both
bacterial and plant systems. pZBL100 also contains three unique
restriction endonuclease sites suitable for the cloning of other
chimeric genes into this vector.
[0097] Plasmid pCW109 was derived from the commercially available
plasmid pUC18 (Gibco-BRL) by inserting into the Hind III site of
the cloning vector pUC18 a 555 bp 5' non-coding region (containing
the promoter region) of the b-conglycinin gene followed by the
multiple cloning sequence containing the restriction endonuclease
sites for Nco I, Sma I, Kpn I and Xba I, then 1174 bp of the common
bean phaseolin 3' untranslated region into the Hind III site. The
b-conglycinin promoter region used is an allele of the published
b-conglycinin gene (Doyle et al. (1986) J. Biol. Chem.
261:9228-9238) due to differences at 27 nucleotide positions. A
unique Not I site was introduced into the cloning region between
the -conglycinin promoter and the phaseolin 3' end in pCW109 by
digestion with Nco I and Xba I followed by removal of the single
stranded DNA ends with mung bean exonuclease. Not I linkers (New
England Biochemical catalog number NEB 1125) were ligated into the
linearized plasmid to produce plasmid pAW35.
[0098] Plasmid pML18 consists of the non-tissue specific and
constitutive cauliflower mosaic virus (35S) promoter (Odell, J. T.
et al. (1985) Nature 313:810-812; Hull et al. (1987) Virology
86:482-493), driving expression of the neomycin phosphotransferase
gene described in (Beck, E. et al. (1982) Gene 19:327-336) followed
by the 3' end of the nopaline synthase gene including nucleotides
848 to 1550 described by (Depicker et al. (1982) J. Appl. Genet.
1:561-574). This transcriptional unit was inserted into the
commercial cloning vector pGEM9Z (Gibco-BRL) and is flanked at the
5' end of the 35S promoter by the restriction sites Sal I, Xba I,
Bam HI and Sma I in that order. An additional Sal I site is present
at the 3' end of the NOS 3' sequence and the Xba I, Bam HI and Sal
I sites are unique. The single Not I site in pML18 was destroyed by
digestion with Not I, filling in the single stranded ends with
dNTPs and Klenow fragment followed by re-ligation of the linearized
plasmid. The modified pML18 was then digested with Hind III and
treated with calf intestinal phosphatase. The b-conglycinin:Not
I:phaseolin expression cassette in pAW35 was removed by digestion
with Hind III and the 1.8 kB fragment was isolated by agarose gel
electrophoresis and ligated into the modified and linearized pML18
construction described above. A clone with the desired orientation
was identified by digestion with Not I and Xba I to release a 1.08
kB fragment indicating that the orientation of the -conglycinin
transcription unit was the same as the selectable marker
transcription unit. The resulting plasmid was given the name
pBS19.
[0099] The pKS67 vector was prepared by isolating the
b-conglycinin-containing fragment from pBS19 by digestion with Hind
III, isolation by gel electrophoresis and ligation into the Hind
III-digested pZBL100, which had been treated with calf alkaline
phosphatase.
[0100] Soybean embryogenic suspension cultures were transformed
with the expression vectors by the method of particle gun
bombardment (Klein, T. M. et al. (1987) Nature (London) 327:70-73,
U.S. Pat. No. 4,945,050). Maintenance of transgenic embryos,
preparation of oils, and measurement of the fatty acid content by
gas chromatography was performed as indicated in PCT publication
WO93/11245 (incorporated herein by reference).
Demonstration of Elongase Activity
[0101] The percent accumulation of 16:0 fatty acids decreases in
soybean embryos expressing the fatty acyl-CoA elongase while the
levels of 20:0 fatty acids dramatically increase. FIG. 3 presents a
chromatographic analysis of oils derived wild-type, non-transgenic
soybean embryos (FIG. 3(A)) and from transgenic soybean embryos
expressing the Limnanthes fatty acyl-CoA elongase (FIG. 3(B)). The
peaks corresponding to the different fatty acids present in the
oils are indicated. The quantity of 16:0 and 18:2 fatty acids are
decreased in the oils from the fatty acyl-CoA elongase-expressing
soybean embryos when compared to oil derived wild-type,
non-transgenic soybean embryos. In addition, the quantity of 20:0
fatty acids is greatly enriched in the oils of the transgenic
embryos. The quantified distribution of 16:0 and 20:0 fatty acids
in wild-type soybean embryos and in soybean embryos expressing the
Limnanthes fatty acyl-CoA elongase is shown in Table 4. The levels
of 20:0 fatty acids in wild type embryos range from 0.2 to 0.6%
whereas in fatty acyl-CoA elongase-expressing embryos, the percent
of 20:0 fatty acids ranges from 7.7% to 11.0%.
TABLE-US-00004 TABLE 4 Percent Fatty Acid Distribution in
Transgenic Soybean Embryos Expressing Limnanthes Fatty Acyl-CoA
Elongase Embryo 16:0 20:0 4155 (wild-type) 12.9 0.2 323 (wild-type)
13.0 0.4 312 (wild-type) 18.4 0.6 4111 (wild-type) 15.4 0.6 4102
(wild-type) 15.2 0.6 195 (wild-type) 16.2 0.6 155 (transgenic) 7.4
9.3 161 (transgenic) 7.6 8.2 163 (transgenic) 7.9 7.7 175
(transgenic) 8.4 11.0 2211 (transgenic) 8.5 7.9 341 (transgenic)
6.4 10.8
[0102] FIG. 4 depicts the linear relationship between the decrease
in 16:0 fatty acid content and the increase in 20:0 fatty acid
content in transgenic soybean embryos expression the Limnanthes
fatty acyl-CoA elongase.
Demonstration of Delta-5 Desaturase Activity
[0103] Transgenic soybean embryos expressing the Limnanthes delta-5
acyl-CoA desaturase produce 16:1 fatty acids not seen in wild type
embryos. The fatty acid distribution in soybean embryos expressing
the delta-5 desaturase is illustrated in FIG. 5 which shows the
chromatograms corresponding to oils derived from wild type soybean
embryos (FIG. 5(A)) and soybean embryos expressing the Limnanthes
delta-5 acyl-CoA desaturase (FIG. 5(B)). Table 5 shows the
quantified percent distribution of 16:0, 16:1 delta-5, 18:0 and
18:1 delta-5 in wild-type embryos and transgenic embryos expressing
the Limnanthes delta-5 acyl-CoA desaturase:
TABLE-US-00005 TABLE 5 Percent Fatty Acid Distribution in
Transgenic Soybean Embryos Expressing Limnanthes Delta-5 Acyl-CoA
Desaturase Embryo 16:0 16:1.DELTA.5 18:0 18:1.DELTA.5 216.0
(wild-type) 13.03052 0 2.47064 0 216.1 (wild-type) 10.60185 0
1.76857 0 216.2 (wild-type) 11.95366 0 1.67544 0 218.0 (wild-type)
12.93328 0 2.15752 0 218.5 (wild-type) 11.57688 0 2.24243 0 220.0
(transgenic) 10.17740 3.63283 1.25350 0.57574 220.1 (transgenic)
8.99496 4.27946 1.22194 0.71544 220.2 (transgenic) 9.78203 2.86631
1.57083 nd 220.3 (transgenic) 9.47315 3.35682 1.49796 0.60828 220.4
(transgenic) 12.16690 2.46238 1.84877 0.45737 220.5 (transgenic)
12.22757 2.75365 2.38873 0.53878 220.6 (transgenic) 11.72778
2.43411 2.37860 0.57778 220.7 (transgenic) 9.31376 3.39302 1.33830
0.59855 220.8 (transgenic) 9.48067 3.66554 1.45045 0.66508 220.9
(transgenic) 9.37735 3.47590 0.95774 0.75598 217.1 (transgenic)
9.86364 3.56592 1.51745 0.64637 217.2 (transgenic) 11.03674 2.79068
1.92739 0.55140 217.3 (transgenic) 13.57543 2.45928 2.26611 0.56306
217.4 (transgenic) 11.33959 2.88931 1.73524 0.53747 217.5
(transgenic) 9.61358 3.40842 1.94986 0.73811 217.6 (transgenic)
10.54626 3.11490 1.69327 nd 217.7 (transgenic) 11.60064 3.34681
2.77254 0.78978 217.8 (transgenic) 13.76804 1.41011 3.41123 nd
217.9 (transgenic) 11.21888 2.98101 1.87345 0.61650 nd = not enough
18:1 delta-5 produced to be integrated by the instrument.
[0104] To confirm the location of the double bond catalyzed by the
desaturase, double bond positions of monounsaturated fatty acids
were established by GC-MS analysis of disulfide derivatives of
fatty acid methyl esters as described by Yamamoto, K. et al. (1991)
Chem Phys Lipids 60:39-50 and illustrated in FIG. 6.
[0105] Fatty acid methyl esters prepared from soybean embryos
expressing the Limnanthes delta-5 acyl-CoA desaturase were reacted
with dimethyl disulfide as previously described (Yamamoto, K. et
al. (1991) Chem. Phys. Lipids 60:39-50). This reaction converts the
double bonds of unsaturated fatty acid methyl esters to dimethyl
disulfide (DMDS) adducts. When analyzed by GC-MS, these derivatives
yield ions that are diagnostic for the positions of double bonds in
fatty acids. The DMDS derivatives of fatty acid methyl esters from
the transgenic soybean embryos were analyzed by GC-MS. These
derivatives were resolved using a 0.25 mm (inner diameter).times.30
m HP-INNOWax column (Hewlett Packard) with the oven temperature of
an HP6890 gas chromatograph temperature programmed from 185.degree.
C. (5 min hold) to 237.degree. C. (25 min hold) at a rate of
7.5.degree. C./min. The mass spectrum of the resolved DMDS
derivatives was obtained using an HP5973 mass selective detector
that was interfaced with the gas chromatograph. DMDS derivatives of
methyl hexadecenoic acid (16:1) were identified using a selected
ion scan for 362 m/z, which corresponds to the molecular ion of the
DMDS derivatives of methyl 16:1. This resulted in the
identification of two peaks with retention times between 18.5 and
19.5 minutes (FIG. 6(A)). The mass spectrum of the largest of these
peaks contained abundant ions with m/z of 161 and 201 (FIG. 6(B)).
The masses of these ions are consistent with the presence of the
double bond at the delta-5 carbon atom. The 161 m/z ion (Fragment
Y) is the expected mass for the carboxyl portion of the methyl
16:1.DELTA.5 DMDS derivative and the 201 m/z ion (Fragment X) is
the expected mass for the methyl end of the 16:1.DELTA.5 DMDS
derivative. Also consistent with the identification of this peak as
16:1.DELTA.5 DMDS derivative is the 129 m/z ion which is generated
by rearrangement of Fragment Y with the loss of 32 m/z. In general,
the Y-32 ion is considered a diagnostic ion for DMDS derivatives of
methyl esters of monounsaturated fatty acids (Francis, G. W. (1981)
Chem. Phys. Lipids 29:369-374). Of note, the second and smaller
peak in FIG. 6(A) was identified as the DMDS derivative of methyl
16:1.DELTA.9 (results not shown). This fatty acid, in contrast to
16:1.DELTA.5, is detectable in small amounts in virtually all plant
tissues.
[0106] Methyl octadecenoic acid (18:1) DMDS derivatives were
initially identified using a selected ion scan for 390 m/z (FIG.
6(C)), that corresponds to the mass of the molecular ion of these
adducts. As shown in FIG. 6(D), the fragmentation of the DMDS
derivative of methyl 18:1.DELTA.5 would be expected to generate
ions of 161 m/z, 229 m/z, and 129 m/z that correspond to Fragments
Y, X, and Y-32, respectively. These ions were detected in a
shoulder on the front of the peak corresponding to the derivative
of methyl 18:1.DELTA.9, the major monounsaturated fatty acid of
soybean embryos.
[0107] The results presented herein establish the occurrence of
16:1.DELTA.5 and 18:1.DELTA.5 in fatty acid methyl esters derived
from transgenic soybean embryos expressing the Limnanthes delta-5
acyl-CoA desaturase. Neither fatty acid was detectable in
derivatives prepared from wild-type soybean embryos.
[0108] These experiments show that when expressed in soybean
embryos, the Limnanthes fatty acyl-CoA elongase (SEQ ID NO:5)
catalyzes the production of arachidonate (20:0) from palmitate
(16:0). These experiments also show that the Limnanthes acyl-CoA
desaturase (SEQ ID NO:2) encodes a delta-5 desaturase which
produces 16:1 delta-5 and 18:1 delta-5 fatty acids when expressed
in transgenic soybean embryos. These experiments are the first
demonstration of the activity of the Limnanthes douglasii delta-5
acyl-CoA desaturase and the fatty acyl-CoA elongase whose sequences
are set forth in SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:7.
[0109] Expression of the Limnanthes fatty acyl-CoA elongase in
other oil producing crops will increase the amounts of C20:0 from
about less than 1% to over about 15%. Expression of the Limnanthes
fatty acyl-CoA elongase and delta-5 acyl-CoA desaturase in other
oil seed crops will have the result of producing 20:1 delta-5 oils
which may then be used in the production of industrially-useful
compounds.
Example 7
Expression of Chimeric Genes in Microbial Cells
[0110] The cDNAs encoding the instant enzyme involved in lipid
biosynthesis can be inserted into the T7 E. coli expression vector
pBT430. This vector is a derivative of pET-3a (Rosenberg et al.
(1987) Gene 56:125-135) which employs the bacteriophage T7 RNA
polymerase/T7 promoter system. Plasmid pBT430 was constructed by
first destroying the EcoR I and Hind III sites in pET-3a at their
original positions. An oligonucleotide adaptor containing EcoR I
and Hind III sites was inserted at the BamH I site of pET-3a. This
created pET-3aM with additional unique cloning sites for insertion
of genes into the expression vector. Then, the Nde I site at the
position of translation initiation was converted to an Nco I site
using oligonucleotide-directed mutagenesis. The DNA sequence of
pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in
pBT430.
[0111] Plasmid DNA containing a cDNA may be appropriately digested
to release a nucleic acid fragment encoding the protein. This
fragment may then be purified on a 1% NuSieve GTG.TM. low melting
agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium
bromide for visualization of the DNA fragment. The fragment can
then be purified from the agarose gel by digestion with GELase.TM.
(Epicentre Technologies) according to the manufacturer's
instructions, ethanol precipitated, dried and resuspended in 20
.mu.L of water. Appropriate oligonucleotide adapters may be ligated
to the fragment using T4 DNA ligase (New England Biolabs, Beverly,
Mass.). The fragment containing the ligated adapters can be
purified from the excess adapters using low melting agarose as
described above. The vector pBT430 is digested, dephosphorylated
with alkaline phosphatase (NEB) and deproteinized with
phenol/chloroform as described above. The prepared vector pBT430
and fragment can then be ligated at 16.degree. C. for 15 hours
followed by transformation into DH5 electrocompetent cells (GIBCO
BRL). Transformants can be selected on agar plates containing LB
media and 100 .mu.g/mL ampicillin. Transformants containing the
gene encoding the enzyme involved in lipid biosynthesis are then
screened for the correct orientation with respect to the T7
promoter by restriction enzyme analysis.
[0112] For high level expression, a plasmid clone with the cDNA
insert in the correct orientation relative to the T7 promoter can
be transformed into E. coli strain BL21(DE3) (Studier et al. (1986)
J. Mol. Biol. 189:113-130). Cultures are grown in LB medium
containing ampicillin (100 mg/L) at 25.degree. C. At an optical
density at 600 nm of approximately 1, IPTG
(isopropylthio-.beta.-galactoside, the inducer) can be added to a
final concentration of 0.4 mM and incubation can be continued for 3
h at 25.degree.. Cells are then harvested by centrifugation and
re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1
mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of
1 mm glass beads can be added and the mixture sonicated 3 times for
about 5 seconds each time with a microprobe sonicator. The mixture
is centrifuged and the protein concentration of the supernatant
determined. One .mu.g of protein from the soluble fraction of the
culture can be separated by SDS-polyacrylamide gel electrophoresis.
Gels can be observed for protein bands migrating at the expected
molecular weight.
Sequence CWU 1
1
711355DNALimnanthes douglasii 1gcttgagact ctctctctac ttccccatct
ctatatctct ctctctctct ctagaagcca 60tggcttcttt catcgcaacc acaacaccag
caatgccagc tttcgcttca gttcttgatc 120caaaaatacc cacaaaacca
gaacccaaaa ccgaaacccc caaaccaaaa gacgatctcg 180aacgcttccg
gacatcagaa gtcgtgttgg agaggaaatc caaaggattc tggcgccgga
240aatggaaccc tcgtgatatt caaaacgccg tcactttact ggtcctgcat
gctcttgcag 300cgatggcgcc cttttatttc agctgggatg cgttttggat
ctcttttatc ttgcttggtt 360tcgcaagcgg tgttcttggt atcactttgt
gcttccatag gtgtcttact catggcggtt 420tcaagcttcc taagttggtt
gagtacttct ttgcctactg tggctctctc gctcttcagg 480gagatcccat
ggaatgggtg agcaaccata ggtaccatca ccagttcgtc gatacagaaa
540gagatgttca tagtccaact caaggatttt ggttctgtca cattggttgg
gttcttgaca 600aagatttatt cgtcgaaaaa cgtggtggcc gaagaaacaa
tgtgaatgat ttgaagaaac 660aagccttcta cagattcctc cagaaaactt
atatgtacca tcaattggct ctaatagctc 720tactttacta cgtcggaggg
tttccataca ttgtctgggg aatgggtttt agattggtgt 780ttatgttcca
ttccactttc gctatcaact cagtttgtca taaatggggc ggaaggccat
840ggaatactgg agatttatcg accaacaata tgtttgttgc attgtgtgcg
tttggagagg 900gctggcataa caaccaccac gcattcgaac aatcagctcg
acacgggcta gaatggtggc 960agatcgatgt tacttggtac gttatcagga
ctctacaagc tattggattg gctaccaatg 1020tgaagctacc aactgaagct
cagaagcaaa agctcaaagc aaagagtgcc taaggagttt 1080gaagcatgta
ataagtgttt gtattcgata cctacttata tatgtttcta gagtcgtacg
1140tgtaatgaat aaagttcgag gcagctatat agactgtgtt cggatatgaa
aatcgttgta 1200ttcttgtatc tgatcgaaaa tagctgcctt gataggtgtt
cgataaaaca ttgttatgtt 1260gcttggtgta gttgtgtggg tcttgctttg
tactgtattg tgttgtgtca cgttttgaga 1320ttatatatag ttttcttgtg
ttcaaaaaaa aaaaa 13552356PRTLimnanthes douglasii 2Leu Arg Leu Ser
Leu Tyr Phe Pro Ile Ser Ile Ser Leu Ser Leu Ser1 5 10 15Leu Glu Ala
Met Ala Ser Phe Ile Ala Thr Thr Thr Pro Ala Met Pro 20 25 30Ala Phe
Ala Ser Val Leu Asp Pro Lys Ile Pro Thr Lys Pro Glu Pro 35 40 45Lys
Thr Glu Thr Pro Lys Pro Lys Asp Asp Leu Glu Arg Phe Arg Thr 50 55
60Ser Glu Val Val Leu Glu Arg Lys Ser Lys Gly Phe Trp Arg Arg Lys65
70 75 80Trp Asn Pro Arg Asp Ile Gln Asn Ala Val Thr Leu Leu Val Leu
His 85 90 95Ala Leu Ala Ala Met Ala Pro Phe Tyr Phe Ser Trp Asp Ala
Phe Trp 100 105 110Ile Ser Phe Ile Leu Leu Gly Phe Ala Ser Gly Val
Leu Gly Ile Thr 115 120 125Leu Cys Phe His Arg Cys Leu Thr His Gly
Gly Phe Lys Leu Pro Lys 130 135 140Leu Val Glu Tyr Phe Phe Ala Tyr
Cys Gly Ser Leu Ala Leu Gln Gly145 150 155 160Asp Pro Met Glu Trp
Val Ser Asn His Arg Tyr His His Gln Phe Val 165 170 175Asp Thr Glu
Arg Asp Val His Ser Pro Thr Gln Gly Phe Trp Phe Cys 180 185 190His
Ile Gly Trp Val Leu Asp Lys Asp Leu Phe Val Glu Lys Arg Gly 195 200
205Gly Arg Arg Asn Asn Val Asn Asp Leu Lys Lys Gln Ala Phe Tyr Arg
210 215 220Phe Leu Gln Lys Thr Tyr Met Tyr His Gln Leu Ala Leu Ile
Ala Leu225 230 235 240Leu Tyr Tyr Val Gly Gly Phe Pro Tyr Ile Val
Trp Gly Met Gly Phe 245 250 255Arg Leu Val Phe Met Phe His Ser Thr
Phe Ala Ile Asn Ser Val Cys 260 265 270His Lys Trp Gly Gly Arg Pro
Trp Asn Thr Gly Asp Leu Ser Thr Asn 275 280 285Asn Met Phe Val Ala
Leu Cys Ala Phe Gly Glu Gly Trp His Asn Asn 290 295 300His His Ala
Phe Glu Gln Ser Ala Arg His Gly Leu Glu Trp Trp Gln305 310 315
320Ile Asp Val Thr Trp Tyr Val Ile Arg Thr Leu Gln Ala Ile Gly Leu
325 330 335Ala Thr Asn Val Lys Leu Pro Thr Glu Ala Gln Lys Gln Lys
Leu Lys 340 345 350Ala Lys Ser Ala 3553305PRTArabidopsis thaliana
3Met Ser Leu Ser Ala Ser Glu Lys Glu Glu Asn Asn Lys Lys Met Ala1 5
10 15Ala Asp Lys Ala Glu Met Gly Arg Lys Lys Arg Ala Met Trp Glu
Arg 20 25 30Lys Trp Lys Arg Leu Asp Ile Val Lys Ala Phe Ala Ser Leu
Phe Val 35 40 45His Phe Leu Cys Leu Leu Ala Pro Phe Asn Phe Thr Trp
Pro Ala Leu 50 55 60Arg Val Ala Leu Ile Val Tyr Thr Val Gly Gly Leu
Gly Ile Thr Val65 70 75 80Ser Tyr His Arg Asn Leu Ala His Arg Ser
Phe Lys Val Pro Lys Trp 85 90 95Leu Glu Tyr Phe Phe Ala Tyr Cys Gly
Leu Leu Ala Ile Gln Gly Asp 100 105 110Pro Ile Asp Trp Val Ser Thr
His Arg Tyr His His Gln Phe Thr Asp 115 120 125Ser Asp Arg Asp Pro
His Ser Pro Asn Glu Gly Phe Trp Phe Ser His 130 135 140Leu Leu Trp
Leu Phe Asp Thr Gly Tyr Leu Val Glu Lys Cys Gly Arg145 150 155
160Arg Thr Asn Val Glu Asp Leu Lys Arg Gln Trp Tyr Tyr Lys Phe Leu
165 170 175Gln Arg Thr Val Leu Tyr His Ile Leu Thr Phe Gly Phe Leu
Leu Tyr 180 185 190Tyr Phe Gly Gly Leu Ser Phe Leu Thr Trp Gly Met
Gly Ile Gly Val 195 200 205Ala Met Glu His His Val Thr Cys Leu Ile
Asn Ser Leu Cys His Val 210 215 220Trp Gly Ser Arg Thr Trp Lys Thr
Asn Asp Thr Ser Arg Asn Val Trp225 230 235 240Trp Leu Ser Val Phe
Ser Phe Gly Glu Ser Trp His Asn Asn His His 245 250 255Ala Phe Glu
Ser Ser Ala Arg Gln Gly Leu Glu Trp Trp Gln Ile Asp 260 265 270Ile
Ser Trp Tyr Ile Val Arg Phe Leu Glu Ile Ile Gly Leu Ala Thr 275 280
285Asp Val Lys Leu Pro Ser Glu Ser Gln Arg Arg Arg Met Ala Met Val
290 295 300Arg30541807DNALimnanthes
douglasiiunsure(302)..(303)unsure(312)unsure(315)unsure(421)unsure(1727)
4ctcactctca cacctccttc tctctctttg tcggcttctc cggcgagata ctcaacggat
60tcaatcgaag ggtagtacaa tatgtcggag acaaaacctg agaaaccttt gatcgcaacc
120gtgaaaaaca cactacctga tttaaaacta tcaataaact taaaacacgt
gaaactcggt 180taccattacc tgatcaccca tggaatgtac ctgtgtctcc
ctcctctcgc actagtcctc 240ttcgctcaaa tctcaacttt gtccctcaaa
gatttcaacg acatctggga acagcttcag 300tnnaatctca tntcngtcgt
tgtttcatca acacttcttg tctccttact tatcctttac 360ttcatgactc
gtccgaggcc ggtttatttg atggatttcg cgtgctataa acccgacgaa
420nctcgaaaat ctactagaga acattttatg aagtgtggtg agagtttggg
ctcttttacg 480gaggataata tcgattttca gaggaaatta gtcgcacgat
ctggacttgg tgatgctacg 540tatttacctg aagctatcgg tactatcccg
gctcatccgt cgatgaaagc tgcgagaaga 600gaagctgagt tggtgatgtt
tggtgcgatt gatcaacttt tggagaagac aaaggtgaat 660ccgaaggata
tagggatctt ggttgttaat tgcagcctgt ttagtccgac tccgtccctc
720tcgtcgatga ttgttaacca ctataaactc cgtgggaaca ttataagcta
caatctaggc 780ggaatgggtt gcagtgctgg tttaatttcg gtcgacttag
ctaaaagact tctcgagaca 840aatccaaaca cttacgcttt agttatgagc
actgaaaata tcacactaaa ctggtacatg 900ggcaatgacc ggtccaaact
cgtgtccaat tgtcttttcc ggatgggagg agctgcggtc 960ttgttatcaa
acaaaacctc tgataagaaa agatcgaagt atcagttggt tactaccgtc
1020cgaagccaca aaggtgctga cgataattgc tacggttgca tattccaaga
agaagactcc 1080aacggcaaaa tcggtgtaag cctctccaaa aatctaatgg
cggtcgcagg ggacgcgctt 1140aagactaaca tcacgacgct tggtccgttg
gttttaccaa tgtcggaaca acttttgttt 1200ttcgccacgc tggttgctcg
aaaagttttc aagaagaaaa ttaagcccta cattccggac 1260tttaaactag
cttttgatca tttctgtatt catgcgggtg gtcgagctgt tttggacgag
1320cttgagaaga atttgcagtt gtcaagctgg catctagagc cgtcgagaat
gacgtttatc 1380cggtttggta atacgtcgag tagtactttg tggtacgagc
tggcgtattc ggaagccaaa 1440gggaggatta gaaaaggaga aagagtttgg
cagatagggt ttggttctgg gtttaaatgt 1500aatagtgctg tctggaaagc
cttaaagagc gttgatccaa agaaagagaa caatccatgg 1560atggatgaga
tccaccagtt tccggttgct gttgtctaag gttgtgtttt gatgtttaat
1620gtttggtgtg ttgatgcttg ctaattggtt agtgtaagaa gtacttggtt
gctgctgttt 1680caattactaa ctaaagagag tgttgaataa gcatagaaca
aagtaantaa ctggaaagtg 1740ctttgttgtt tgttcagtaa ctctattact
gctgaatttc tctcaagaga agaattatgt 1800ttaaaaa 18075505PRTLimnanthes
douglasiiUNSURE(74)UNSURE(77)UNSURE(114) 5Met Ser Glu Thr Lys Pro
Glu Lys Pro Leu Ile Ala Thr Val Lys Asn1 5 10 15Thr Leu Pro Asp Leu
Lys Leu Ser Ile Asn Leu Lys His Val Lys Leu 20 25 30Gly Tyr His Tyr
Leu Ile Thr His Gly Met Tyr Leu Cys Leu Pro Pro 35 40 45Leu Ala Leu
Val Leu Phe Ala Gln Ile Ser Thr Leu Ser Leu Lys Asp 50 55 60Phe Asn
Asp Ile Trp Glu Gln Leu Gln Xaa Asn Leu Xaa Ser Val Val65 70 75
80Val Ser Ser Thr Leu Leu Val Ser Leu Leu Ile Leu Tyr Phe Met Thr
85 90 95Arg Pro Arg Pro Val Tyr Leu Met Asp Phe Ala Cys Tyr Lys Pro
Asp 100 105 110Glu Xaa Arg Lys Ser Thr Arg Glu His Phe Met Lys Cys
Gly Glu Ser 115 120 125Leu Gly Ser Phe Thr Glu Asp Asn Ile Asp Phe
Gln Arg Lys Leu Val 130 135 140Ala Arg Ser Gly Leu Gly Asp Ala Thr
Tyr Leu Pro Glu Ala Ile Gly145 150 155 160Thr Ile Pro Ala His Pro
Ser Met Lys Ala Ala Arg Arg Glu Ala Glu 165 170 175Leu Val Met Phe
Gly Ala Ile Asp Gln Leu Leu Glu Lys Thr Lys Val 180 185 190Asn Pro
Lys Asp Ile Gly Ile Leu Val Val Asn Cys Ser Leu Phe Ser 195 200
205Pro Thr Pro Ser Leu Ser Ser Met Ile Val Asn His Tyr Lys Leu Arg
210 215 220Gly Asn Ile Ile Ser Tyr Asn Leu Gly Gly Met Gly Cys Ser
Ala Gly225 230 235 240Leu Ile Ser Val Asp Leu Ala Lys Arg Leu Leu
Glu Thr Asn Pro Asn 245 250 255Thr Tyr Ala Leu Val Met Ser Thr Glu
Asn Ile Thr Leu Asn Trp Tyr 260 265 270Met Gly Asn Asp Arg Ser Lys
Leu Val Ser Asn Cys Leu Phe Arg Met 275 280 285Gly Gly Ala Ala Val
Leu Leu Ser Asn Lys Thr Ser Asp Lys Lys Arg 290 295 300Ser Lys Tyr
Gln Leu Val Thr Thr Val Arg Ser His Lys Gly Ala Asp305 310 315
320Asp Asn Cys Tyr Gly Cys Ile Phe Gln Glu Glu Asp Ser Asn Gly Lys
325 330 335Ile Gly Val Ser Leu Ser Lys Asn Leu Met Ala Val Ala Gly
Asp Ala 340 345 350Leu Lys Thr Asn Ile Thr Thr Leu Gly Pro Leu Val
Leu Pro Met Ser 355 360 365Glu Gln Leu Leu Phe Phe Ala Thr Leu Val
Ala Arg Lys Val Phe Lys 370 375 380Lys Lys Ile Lys Pro Tyr Ile Pro
Asp Phe Lys Leu Ala Phe Asp His385 390 395 400Phe Cys Ile His Ala
Gly Gly Arg Ala Val Leu Asp Glu Leu Glu Lys 405 410 415Asn Leu Gln
Leu Ser Ser Trp His Leu Glu Pro Ser Arg Met Thr Phe 420 425 430Ile
Arg Phe Gly Asn Thr Ser Ser Ser Thr Leu Trp Tyr Glu Leu Ala 435 440
445Tyr Ser Glu Ala Lys Gly Arg Ile Arg Lys Gly Glu Arg Val Trp Gln
450 455 460Ile Gly Phe Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp
Lys Ala465 470 475 480Leu Lys Ser Val Asp Pro Lys Lys Glu Asn Asn
Pro Trp Met Asp Glu 485 490 495Ile His Gln Phe Pro Val Ala Val Val
500 5056844DNALimnanthes douglasii 6acacgggcaa tgaccgatcg
aaactcgtgt ctaattgtct tttccgtatg ggaggagctg 60cggttttatt atcaaacaaa
cattcggaca aaaaacgatc gaaataccag ttggttacta 120ccgtccgaag
ccacaaaggt gctgacgata attgctatgg ctgcatcttt caagaagagg
180actcgactgg aataagtggt gtaagtctct cgaaaaatct aatggcagtc
gcaggcgatg 240cactcaagac aaacatcacg acgatcggtc cgttagtttt
accaatgact gaacaacttt 300tgtattttgc ctccttggtc ggccgaaata
ttttcaaaat gaaaataaaa acctacgttc 360ccgattttaa actcgccttc
gagcatttct gtattcacgc aggtggtcga ggagtgttgg 420acgcgctgga
gaagaatttg cagttgtcgg agtggcatct tgagccatcg aggatgacgt
480tgtaccgatt tggtaatacg tcgagtagta gtttatggta tgagctggcg
tattcggaag 540ccaaagggag aattaagaag ggagagaggg tttggcagat
agggtttggt tcagggttta 600agtgtaatag tgtggtttgg aaagcgctac
ggacagtaga tccgaaggaa gagaataatc 660cttggacgga tgagatccac
cagtttccag ttgctgttgt ctgagtttat gttggatgtt 720tgaagtaaac
ttaatgtttt ggtctggtgt ccatgctgag attagtgcag caactctttt
780gcgaaataat aaatgcttag aaactgtttt gttgtttaaa aaaaaaaaaa
aaaaaaaaaa 840aaaa 8447233PRTLimnanthes douglasii 7Thr Gly Asn Asp
Arg Ser Lys Leu Val Ser Asn Cys Leu Phe Arg Met1 5 10 15Gly Gly Ala
Ala Val Leu Leu Ser Asn Lys His Ser Asp Lys Lys Arg 20 25 30Ser Lys
Tyr Gln Leu Val Thr Thr Val Arg Ser His Lys Gly Ala Asp 35 40 45Asp
Asn Cys Tyr Gly Cys Ile Phe Gln Glu Glu Asp Ser Thr Gly Ile 50 55
60Ser Gly Val Ser Leu Ser Lys Asn Leu Met Ala Val Ala Gly Asp Ala65
70 75 80Leu Lys Thr Asn Ile Thr Thr Ile Gly Pro Leu Val Leu Pro Met
Thr 85 90 95Glu Gln Leu Leu Tyr Phe Ala Ser Leu Val Gly Arg Asn Ile
Phe Lys 100 105 110Met Lys Ile Lys Thr Tyr Val Pro Asp Phe Lys Leu
Ala Phe Glu His 115 120 125Phe Cys Ile His Ala Gly Gly Arg Gly Val
Leu Asp Ala Leu Glu Lys 130 135 140Asn Leu Gln Leu Ser Glu Trp His
Leu Glu Pro Ser Arg Met Thr Leu145 150 155 160Tyr Arg Phe Gly Asn
Thr Ser Ser Ser Ser Leu Trp Tyr Glu Leu Ala 165 170 175Tyr Ser Glu
Ala Lys Gly Arg Ile Lys Lys Gly Glu Arg Val Trp Gln 180 185 190Ile
Gly Phe Gly Ser Gly Phe Lys Cys Asn Ser Val Val Trp Lys Ala 195 200
205Leu Arg Thr Val Asp Pro Lys Glu Glu Asn Asn Pro Trp Thr Asp Glu
210 215 220Ile His Gln Phe Pro Val Ala Val Val225 230
* * * * *
References