U.S. patent application number 14/715102 was filed with the patent office on 2015-11-12 for isolation and use of fad2 and fae1 from camelina.
The applicant listed for this patent is Global Clean Energy Holdings, Inc.. Invention is credited to Renata F. DITT, Carolyn HUTCHEON, Christine K. SHEWMAKER.
Application Number | 20150320002 14/715102 |
Document ID | / |
Family ID | 44657884 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150320002 |
Kind Code |
A1 |
HUTCHEON; Carolyn ; et
al. |
November 12, 2015 |
Isolation and Use of FAD2 and FAE1 From Camelina
Abstract
The present invention provides isolated FAD2 and FAE1 genes and
FAD2 and FAE1 protein sequences of Camelina species, e.g., Camelina
sativa, mutations in Camelina FAD2 and FAE1 genes, and methods of
using the same. In addition, methods of altering Camelina seed
composition and/or improving Camelina seed oil quality are
disclosed. Furthermore, methods of breeding Camelina cultivars
and/or other closely related species to produce plants having
altered or improved seed oil and/or meal quality are provided.
Inventors: |
HUTCHEON; Carolyn;
(Kirkland, WA) ; DITT; Renata F.; (Seattle,
WA) ; SHEWMAKER; Christine K.; (Woodland,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Global Clean Energy Holdings, Inc. |
Torrance |
CA |
US |
|
|
Family ID: |
44657884 |
Appl. No.: |
14/715102 |
Filed: |
May 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13072122 |
Mar 25, 2011 |
9035131 |
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14715102 |
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61318273 |
Mar 26, 2010 |
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61346410 |
May 19, 2010 |
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Current U.S.
Class: |
800/281 ;
435/189; 435/419; 435/468; 536/23.2; 800/306 |
Current CPC
Class: |
C12N 9/1029 20130101;
A01H 1/00 20130101; C12N 9/0004 20130101; C12Y 602/01003 20130101;
C12N 9/0083 20130101; C12N 15/8247 20130101; C12N 9/93 20130101;
A01H 1/06 20130101; A01H 5/10 20130101; C12N 9/0071 20130101 |
International
Class: |
A01H 5/10 20060101
A01H005/10; C12N 9/00 20060101 C12N009/00; C12N 9/02 20060101
C12N009/02 |
Claims
1. An isolated polynucleotide encoding a plant fatty acid synthesis
polypeptide, wherein the isolated polynucleotide encodes a plant
fatty acid desaturase (FAD) polypeptide, comprising a sequence
sharing at least 93% identity to SEQ ID NO: 1, 2, 3, 45, 46, 48,
51, 54, 55, 56, 60, or 61; or the isolated polynucleotide encodes a
plant fatty acid elongase (FAE) polypeptide comprising a sequence
sharing at least 91% identity to SEQ ID NO: 4, 5, 6, 47, 49, 50,
52, 53, 57, 58, 59, 62, or 63.
2. The isolated FAD polynucleotide of claim 1, wherein the
polynucleotide comprises a sequence selected from the group
consisting of SEQ ID NOs 1, 2, 3, 45, 46, 48, 51, 54, 55, 56, 60,
and 61; or the isolated FAE polynucleotide of claim 1, wherein the
polynucleotide comprises a sequence selected from the group
consisting of SEQ ID NOs 4, 5, 6, 47, 49, 50, 52, 53, 57, 58, 59,
62, and 63.
3. The isolated polynucleotide of claim 1, wherein the FAD
polynucleotide comprises one or more mutations listed in Tables 7
to 9; or wherein the FAE polynucleotide comprises one or more
mutations listed in Tables 10 to 12.
4. An isolated FAD polypeptide comprising an amino acid sequence
sharing at least 93% identity to a FAD polypeptide encoded by the
FAD polynucleotide of claim 1; or an isolated FAE polypeptide
comprising an amino acid sequence sharing at least 88% identity to
a FAE polypeptide encoded by the FAE polynucleotide of claim 1.
5. A plant cell, plant part, plant tissue culture or whole plant
comprising at least one FAD2 gene comprising at least one mutation
that disrupts and/or alters the function of the at least one FAD2
gene and/or at least one FAE1 gene comprising at least one mutation
that disrupts and/or alters the function of the at least one FAE1
gene.
6. The plant cell, plant part, plant tissue culture or whole plant
of claim 5 wherein the FAD2 gene is FAD2 A, FAD2 B and/or FAD2 C;
and wherein the FAE1 gene is FAE1 A. FAE1 Band/or FAE1 C.
7. The plant cell, plant part, plant tissue culture or whole plant
of claim 5, wherein the plant is a Camelina plant.
8. The Camelina plant cell, plant part, plant tissue culture or
whole plant of claim 5 further comprising one or more additional
genetic changes selected from the group consisting of: (a) one or
more non-FAD2, non-FAE1 fatty acid synthesis genes with at least
one mutation that disrupts and/or alters the function of that gene;
(b) a chimeric gene comprising a double stranded RNA region that is
both homologous and complementary to a region of one or more
non-FAD2, non-FAE1 fatty acid synthesis genes; and (c) a chimeric
gene comprising one or more non-FAD2, non-FAE1 fatty acid synthesis
genes, wherein the non-FAD2, non-FAE1 fatty acid synthesis genes
are operably linked to an overexpression promoter.
9. The plant cell, plant part, tissue culture or whole plant of
claim 8, wherein the additional fatty acid gene is selected from
the group consisting of FAD3, a hydroxylase and a thioesterase.
10. The plant cell, plant part, plant tissue culture or whole plant
of claim 5; wherein the mutation is selected from any one of
mutations listed in Tables 7 to 12 for a particular FAD2 or FAE1
gene.
11. A method of increasing the activity of a FAD2 and/or FAE1
protein in a Camelina plant cell, plant part, tissue culture or
whole plant comprising transforming the plant cell, plant part,
tissue culture or whole plant with a chimeric gene comprising one
FAD2 and/or FAE1 gene encoding the polypeptide of claim 4, or
functional variants thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 13/072,122, filed Mar. 25, 2011, now U.S. Pat.
No. 9,035,131; which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/318,273, filed Mar. 26, 2010, and U.S.
Provisional Patent Application Ser. No. 61/346,410, filed May 19,
2010. The contents of the above applications are hereby
incorporated by reference in their entireties for all purposes.
TECHNICAL FIELD
[0002] The invention relates to the identification, isolation and
use of nucleic acid sequences, including genes, and nucleic acid
fragments encoding fatty acid desaturase enzymes and/or fatty acid
elongases, mutants thereof, and methods of altering lipid
composition in Camelina species, e.g., Camelina sativa.
BACKGROUND
[0003] The current concern about our global dependence on fossil
fuels and the consequent impact on climate change have brought
biofuels to the forefront. This interest in biofuels has prompted
researchers to critically evaluate alternative feedstocks for
biofuel production. One important, emerging biofuel crop is
Camelina sativa L. Cranz (Brassicaceae), commonly referred to as
"false flax" or "gold-of-pleasure". Renewed interest in C. sativa
as a biofuel feedstock is due in part to its drought tolerance and
minimal requirements for supplemental nitrogen and other
agricultural inputs (Putnam, Budin et al. 1993; Zubr 1997;
Gehringer, Friedt et al. 2006; Gugel and Falk 2006). Similar to
other non-traditional, renewable oilseed feedstocks such as
Jatropha curcas L. ("jatropha"), C. sativa grows on marginal land.
Unlike jatropha, which is a tropical and subtropical shrub, C.
sativa is native to Europe and is naturalized in North America,
where it grows well in the northern United States and southern
Canada.
[0004] In addition to its drought tolerance and broad distribution,
several other aspects of C. sativa biology make it well suited for
development as an oilseed crop. First, C. sativa is a member of the
family Brassicaceae, and thus is a relative of both the genetic
model organism Arabidopsis thaliana and the oilseed crop Brassica
napus. The close relationship between C. sativa and A. thaliana
(Al-Shehbaz, Beilstein et al. 2006; Beilstein, Al-Shehbaz et al.
2006; Beilstein, Al-Shehbaz et al. 2008) makes the A. thaliana
genome an ideal reference point for the development of genetic and
genomic tools in C. sativa. Second, the oil content of C. sativa
seeds is comparable to that of B. napus, ranging from 30 to 40%
(w/w) (Budin, Breene et al. 1995; Gugel and Falk 2006), suggesting
that agronomic lessons from the cultivation of B. napus are
applicable to C. sativa cultivation. Finally, the properties of C.
sativa biodiesel are already well described (Rice, Frohlich et al.
1997; Frohlich and Rice 2005; Worgetter, Prankl et al. 2006), and
both seed oil and biodiesel from C. sativa were used as fuel in
engine trials with promising results (Bernardo, Howard-Hildige et
al. 2003; Frohlich and Rice 2005).
[0005] The quality of a biodiesel, regardless of its source, is
dependent upon the fatty acid methyl ester (FAME) composition,
which affects cold flow and oxidative stability (Knothe 2005;
Durrett, Benning et al. 2008). For example, saturated FAMEs have
poor cold flow properties since they can form crystals at lower
temperatures, while the FAMEs from polyunsaturated fatty acids
remain in solution at colder temperatures, and thus have good cold
flow properties (Stournas 1995; Serdari, Lois et al. 1999). In
contrast, the relationship between saturation and oxidative
stability is exactly opposite that of cold flow. Fatty acid
saturation is positively correlated with oxidative stability;
saturated fatty acids have the best oxidative stability and fatty
acids with 2 or greater double bonds have increasing oxidative
instability (Knothe and Dunn 2003; Knothe 2005; Durrett, Benning et
al. 2008). Additionally, polyunsaturated FAMEs can result in
increased NOx emissions, e.g., NO, NO.sub.2 et al (McCormick,
Graboski et al. 2001), and thus affect the production of a
monitored pollutant. Very long chain fatty acids (VLCFA; as used
herein, refers to those fatty acids containing greater than 18
carbons) result in a biodiesel with a high distillation temperature
that does not meet existing standards (American Society for Testing
and Materials, ASTM), reducing marketability. Given these
trade-offs, an ideal biodiesel blend is high in oleic acid (18:1;
carbons:double bonds), low in polyunsaturated FAMEs, and with few
long chain FAMEs. This blend is oxidatively stable, has a low cloud
point, and meets biodiesel standards (ASTM; Knothe 2005; Durrett,
Benning et al. 2008).
[0006] The naturally occurring oil composition of C. sativa
negatively affects its biodiesel properties. Polyunsaturated fatty
acids such as linoleic (18:2) and alpha-linolenic (18:3) acids
account for 52.1-54.7% of C. sativa seed oil (Ni Eidhin, Burke et
al. 2003; Abramovic and Abram 2005). This likely accounts for the
low oxidative stability of C. sativa FAMEs (Bernardo,
Howard-Hildige et al. 2003). C. sativa seeds also contain
21.4-22.4% VLCFA, of which 11-eicosenoic acid (20:1) at 14.9-16.2%
are especially abundant (Zubr 2002; Ni Eidhin, Burke et al. 2003;
Abramovic and Abram 2005), likely resulting in the high
distillation temperature of the FAMEs. Most desirable for biodiesel
is oleic acid (18:1), which accounts for 14.0-17.4% of C. sativa
seed oil (Budin, Breene et al. 1995; Zubr 2002; Ni Eidhin, Burke et
al. 2003; Abramovic and Abram 2005). There is therefore the
potential to optimize Camelina oil for biodiesel production by
decreasing both the amount of polyunsaturated fatty acids being
produced from oleic acid and decreasing the production of fatty
acids with chain length of 18 carbons or greater.
[0007] Genes affecting oil composition are well characterized in
Arabidopsis thaliana, a close relative of Camelina sativa, as well
as in some other plants. For example, oleic acid (18:1) is
converted to linoleic acid (18:2) in the endoplasmic reticulum by
the membrane bound delta-12-desaturase FATTY ACID DESATURASE 2
(FAD2). In Arabidopsis fad2 mutants, levels of 18:1 oleic acid in
the seeds increase by a factor of 2-3.4 while levels of 18:2
linoleic acids are decreased by a factor of 4-10 (Okuley, Lightner
et al. 1994). Thus, mutations affecting FAD2 have been shown to
lead to higher levels of oleic acid in A. thaliana and other
studies have shown FAD2 has a similar role in crops such as canola
(Hu, Sullivan-Gilbert et al. 2006), sunflower (Hongtrakul, Slabaugh
et al. 1998) and peanut (Patel, Jung et al. 2004).
[0008] Very long chain fatty acids are formed in the cytosol of A.
thaliana by sequential addition of 2 carbon units to 18 carbon
fatty acid CoA conjugates. The rate limiting step is thought to be
initial condensation, catalyzed in the seed by FATTY ACID ELONGASE
1 (FAE1) (James Jr, Lim et al. 1995) (Kunst, Taylor et al. 1992).
In wild type Arabidopsis, approximately 25% of fatty acids in seeds
are long chain fatty acids, while fae1 mutants contain less than 1%
long chain fatty acids. Interestingly, 18:1 content in seeds
increases by a factor of 2 in A. thaliana fae1 (Kunst, Taylor et
al. 1992). In Brassica napus, reductions in long chain fatty acids,
particularly erucic acid (22:1), are linked to changes in FAE1
activity (Han, Luhs et al. 2001; Katavic, Mietkiewska et al. 2002;
Wang, Wang et al. 2008; Wu, et al. 2008).
[0009] The close relationship between A. thaliana and C. sativa
suggests that FAD2 and FAE1 may play similar roles in both species,
making these genes good targets for manipulation of oil composition
in C. sativa. To our knowledge, FAD2 and FAE1 gene sequences have
not been previously reported for C. sativa. Indeed, published
studies detailing the biology of C. sativa and its closest
relatives in the genus Camelina are few. However, several important
findings can be drawn from the literature. Taxonomic treatments
describe 11 species in the genus with a center of diversity in
Eurasia (Akeroyd J: Camelina in Flora Europaea. 2nd edn. Cambridge,
UK: Cambridge University Press; 1993.) although C. sativa, C.
rumelica, C. microcarpa, and C. alyssum are naturalized weeds with
broad distributions. Camelina species can be annual or biennial,
with some species requiring vernalization to induce flowering
(Mirek Z: Genus Camelina in Poland--Taxonomy, Distribution and
Habitats. Fragmenta Floristica et Geobotanica 1981, 27:445-503.).
Chromosome counts range from n=6 in C. rumelica (Brooks R E:
Chromosome number reports LXXXVII Taxon 1985, 34:346-351; Baksay L:
The chromosome numbers and cytotaxonomical relations of some
European plant species. Ann Hist-Nat Mus Natl Hung 1957:169-174.)
or n=7 in C. hispida (Maassoumi A: Cruciferes de la flore d'Iran:
etude caryosystematique. Thesis. Strasbourg, France, 1980.),
upwards to n=20 in C. sativa, C. microcarpa, and C. alyssum
(Gehringer A, Friedt W, Luhs W, Snowdon R J: Genetic mapping of
agronomic traits in false flax (Camelina sativa subsp. sativa).
Genome 2006, 49:1555-1563; Francis A, Warwick S: The Biology of
Canadian Weeds. 142. Camelina alyssum (Mill.) Thell.; C. microcarpa
Andrz. ex D C.; C. sativa (L.) Crantz. Canadian Journal of Plant
Science 2009, 89:791-810.) Some Camelina species are interfertile;
crosses of C. saliva with C. alyssum, and C. sativa with C.
microcarpa, produce viable seed (Tedin O: Vererbung, Variation and
Systematik in der Gattung Camelina. Hereditas 1925, 6:19-386.) More
recently, plastid simple sequence repeat (SSR) markers (Flannery M
L, Mitchell F J, Coyne S, Kavanagh T A, Burke J I, Salamin N,
Dowding P, Hodkinson T R: Plastid genome characterisation in
Brassica and Brassicaceae using a new set of nine SSRs. Theor Appl
Genet 2006, 113:1221-1231.) and randomly amplified polymorphic DNA
(RAPD) markers have been reported and a mapping study using
amplified fragment length polymorphisms (AFLP) has been published
(Gehringer A, Friedt W, Luhs W, Snowdon R J: Genetic mapping of
agronomic traits in false flax (Camelina sativa subsp. sativa).
Genome 2006, 49:1555-1563). Additionally, the sequences of a few C.
sativa transcription factors are available from the literature
(Martynov V V, Tsvetkov I L, Khavkin E E: Orthologs of arabidopsis
CLAVATA 1 gene in cultivated Brassicaceae plants. Ontogenez 2004,
35:41-46.) and in GenBank.
[0010] As an oilseed crop in the Brassicaceae family, Camelina
sativa has inspired renewed interest due to its potential for
biofuels applications. Little is understood of the nature of the C.
sativa genome, however. A study was undertaken by the present
inventors to characterize two genes in the fatty acid biosynthesis
pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase
(FAE) 1.
SUMMARY OF THE INVENTION
[0011] Camelina sativa is a re-emerging oilseed with tremendous
potential as an alternative biofuel crop and for which genomic
information is becoming increasingly available. The inventors have
characterized two genes encoding fatty acid biosynthesis enzymes
and, in the process, have discovered unexpected complexity in the
C. sativa genome.
[0012] The present inventors disclose herewith the sequences of
three copies of both FAE1 and FAD2 recovered from C. sativa.
Southern blots were used to determine whether the recovered copies
are allelic or if they represent multiple loci. Moreover, the
inventors performed phylogenetic analyses to infer the evolutionary
history of the copies, and quantitative PCR (qPCR) to explore
whether there is evidence of functional divergence among them. To
better understand the C. sativa genome and to determine whether the
multiple copies recovered are the result of polyploidization, the
inventors also analyzed the genome sizes of C. sativa and its
closest relatives in the genus Camelina by flow cytometry.
Collectively the inventors' results indicate that C. sativa is an
allohexaploid whose oil composition is likely influenced by more
than one functional copy of FAE1 and FAD2. This should allow highly
specialized blends of oil to be produced from C. sativa with
mutations in FAE1 and FAD2, greatly increasing the utility of this
emerging biofuel crop.
[0013] The present inventors unexpectedly discovered by Southern
analysis that in C. sativa, there are three copies of both FAD2 and
FAE1 as well as LFY, a known single copy gene in other species. All
three copies of both FAD2 and FAE1 are expressed in developing
seeds, and sequence alignments show that previously described
conserved sites are present, suggesting that all three copies of
both genes could be functional. The regions downstream of FAD2 and
upstream of FAE1 demonstrate co-linearity with the Arabidopsis
genome. In addition, results from flow cytometry indicate that the
DNA content of C. sativa is approximately three-fold that of
diploid Camelina relatives. Phylogenetic analyses further support a
history of duplication and indicate that C. saliva and C.
microcarpa might share a parental genome. FAD2 and FAE1 sequences
from species in the tribe of Camelineae have been deposited in
Genbank at the NCBI [Genbank: GU929417-GU929441, SEQ ID NOs: 1 to
6, and SEQ ID NOs 45-63, as listed below, which are incorporated by
reference in their entireties.
TABLE-US-00001 GenBank SEQ access # Sequence Name ID NO GU929417
Camelina sativa FAD2 A (upstream, coding and downstream genomic 1
sequence) GU929418 Camelina sativa FAD2 B (upstream, coding and
downstream genomic 2 sequence) GU929419 Camelina sativa FAD2 C
(upstream, coding and downstream genomic 3 sequence) GU929420
Camelina sativa FAE1 A [upstream gene (KCS17), intergenic region
and 4 coding) GU929421 Camelina sativa FAEI B (upstream gene
(KCS17), intergenic region and 5 coding) GU929422 Camelina sativa
FAE1 C [upstream gene (KCS17), intergenic region and 6 coding)
GU929423 Capsella rubella FAD2 45 GU929424 Arabidopsis lyrata FAD2
46 GU929425 Arabidopsis lyrata FAE1 47 GU929426 Camelina hispida
FAD2 48 GU929427 Camelina hispida FAE1-1 49 GU929428 Camelina
hispida FAE1-2 50 GU929429 Camelina laxa FAD2 51 GU929430 Camelina
laxa FAE1-1 52 GU929431 Camelina laxa FAE1-2 53 GU929432 Camelina
microcarpa FAD2 A 54 GU929433 Camelina microcarpa FAD2 B 55
GU929434 Camelina microcarpa FAD2 C 56 GU929435 Camelina microcarpa
FAE1 A 57 GU929436 Camelina microcarpa FAE1 B 58 GU929437 Camelina
microcarpa FAE1 C 59 GU929438 Camelina rumelica FAD2-1 60 GU929439
Camelina rumelica FAD2-2 61 GU929440 Camelina rumelica FAE1-1 62
GU929441 Camelina rumelica FAE1-2 63
[0014] The C. sativa genome appears to be organized in three
copies, and can be considered to be an allohexaploid. The discovery
of triplication and divergence of genes that in known diploids are
present in single copy, the cytometrically determined genome size
of Camelina species, the pattern of relationship and inferred
duplication history in the gene trees, together with the previously
known chromosome counts for this taxon, indicate a likely
allohexaploid genomic constitution. The characterization of genes
encoding key functions of fatty acid biosynthesis lays the
foundation for future manipulations of this pathway in Camelina
sativa, which allows for the future manipulation of oil composition
of this emerging biofuel crop.
[0015] The present invention provides an isolated nucleic acid
sequence comprising a sequence selected from the group consisting
of SEQ ID NOs: 1 to 6 and 45 to 63, and fragments and variations
derived from thereof, which encode a plant fatty acid synthesis
gene.
[0016] In one embodiment, the present invention provides an
isolated polynucleotide encoding plant fatty acid desaturase,
comprising a nucleic acid sequence that shares at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, at least
99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least
99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least
99.9% identity to SEQ ID NO: 1, 2, 3, 45, 46, 48, 51, 54, 55, 56,
60, and/or 61.
[0017] In another embodiment, the present invention provides an
isolated polynucleotide encoding fatty acid elongase, comprising a
nucleic acid sequence that shares at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, at least 99.1%, at least
99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least
99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity
to SEQ ID NO: 4, 5, 6, 47, 49, 50, 52, 53, 57, 58, 59, 62, and/or
63.
[0018] The present invention further provides an isolated amino
acid sequence (e.g., a peptide, polypeptide and the like)
comprising a sequence selected from the group consisting of SEQ ID
NOs: 7 to 12, and fragments and variations derived from thereof,
which form a plant fatty acid synthesis protein.
[0019] In some embodiments, the present invention provides an
isolated amino acid sequence which forms a protein that shares an
amino acid sequence having at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, at least 99.1%, at least
99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least
99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity
to SEQ ID NO: 7, 8, 9, 64, 65, 67, 70, 73, 74, 75, 79, and/or
80.
[0020] In one embodiment, the present invention provides an
isolated amino acid sequence which forms a protein that shares an
amino acid having at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at
least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at
least 99.7%, at least 99.8%, or at least 99.9% identity to SEQ ID
NO: 10, 11, 12, 66, 68, 69, 71, 72, 76, 77, 78, 81, and/or 82.
[0021] The present invention also provides a chimeric gene
comprising the isolated nucleic acid sequence of any one of the
polynucleotides described above operably linked to suitable
regulatory sequences.
[0022] The present invention also provides a recombinant construct
comprising the chimeric gene as described above.
[0023] The present invention further comprises interfering RNA
(RNAi) based on the expression of the nucleic acid sequences of the
present invention, wherein such RNAi includes but is not limited to
microRNA (miRNA) and small interfering RNA (siRNA) which can be
used in gene silencing constructs.
[0024] The present invention also provides a transformed host cell
comprising the chimeric gene as described above. In one embodiment,
said host cell is selected from the group consisting of bacteria,
yeasts, filamentous fungi, algae, animals, and plants.
[0025] The present invention in another aspect, provides a plant
comprising in its genome one or more genes as described herein, one
or more genes with mutations as described herein, or the chimeric
genes as described herein.
[0026] The present invention in another aspect, provides a plant
seed obtained from the plants described herein, wherein the plants
comprise in their genomes one or more genes as described herein,
one or more genes with mutations as described herein, or the
chimeric genes as described herein.
[0027] The present invention in another aspect, provides Camelina
oil obtained from the seeds of a Camelina plant comprising the one
or more genes described herein, one or more genes with mutations as
described herein, or one or more chimeric genes as described
herein.
[0028] The present invention in another aspect, provides meals made
from Camelina plants comprising the one or more genes described
herein, one or more genes with mutations as described herein, or
one or more chimeric genes as described herein. In some
embodiments, the meal is a byproduct of the extraction of the oil
from said Camelina seeds. In some embodiments, said Camelina plant
has reduced level of erucic acid (22:1) compared to a wild type
Camelina plant. In some embodiments, said Camelina plant has less
than 4%, less than 3%, less than 2%, less than 1%, or less than
0.1% erucic acid (22:1) compared to the wild type. In further
embodiments, the Camelina meal is included in the diets of an
animal for about 0.1%, about 0.5%, about 1%, about 2%, about 3%,
about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or
about 10% of their feed on a weight or volume basis.
[0029] Thus, the present invention provides methods of altering
and/or improving Camelina fatty acids composition by disrupting
and/or altering one, two, or all three copies of one or more fatty
acid synthesis genes in Camelina. Methods of disrupting and/or
altering gene function include but are not limited to mutagenesis
(e.g., chemical mutagenesis, radiation mutagenesis, transposon
mutagenesis, insertional mutagenesis, signature tagged mutagenesis,
site-directed mutagenesis, and natural mutagenesis), antisense,
knock-outs, and/or RNA interference.
[0030] In some embodiments, the methods comprise introducing
mutations in one or more FAD2 genes and/or one or more FAE1 genes
of Camelina. In some embodiments, the methods disclosed herein
comprise utilizing Camelina mutants with mutations in all three
FAD2 genes (e.g., FAD2 A, FAD2 B, and FAD2 C), and/or Camelina
mutants with mutations in all three FAE1 genes (e.g., FAE1 A, FAE1
B, and FAE1 C).
[0031] The present invention provides mutants in FAD2 A, FAD2 B,
FAD2 C, FAE1 A, FAE1 B, and FAE1 C, including but not limited to
those as listed in Tables 7-12. In some embodiments, the methods of
altering and/or improving Camelina fatty acids composition comprise
utilizing one or more Camelina mutants for any one or more of the
mutations listed in Tables 7 to 12 and as described in Example 11.
In some embodiments, mutations in one or more copies of FAD2 genes
and/or mutations in one or more copies of FAE1 genes as described
in the Tables 7 to 12 are integrated together to create mutant
plants with double, triple, quadruple et al. mutations in one, two,
or all three copies of FAD2 and/or FAE1 genes. In some embodiments,
the mutations described in the Tables 7-12 can be integrated into
Camelina sativa cultivars other than Cs32 (commercial name as 5030)
or other Camelina species by classic breeding methods, with or
without the help of marker-facilitated inter-cultivar gene transfer
methods. In some embodiments, mutations described in the Tables
7-12 can be integrated into species closely related to Camelina
sativa. In still other embodiments, amino acids in conserved
domains or sites compared to FAD2 or FAE1 orthologs in other
species can be substituted or deleted to make mutants with reduced
or abolished activity, mutants that lead to loss-of-function (e.g.,
protein instability), and/or mutants that lead to gain-of-function
(e.g., more stable or more active protein).
[0032] In some embodiments, one, two, or all three copies of
Camelina FAD2 and/or FAE1 genes, and one, two, or all three copies
of other non-FAD, non-FAE fatty acid synthesis genes are disrupted.
In still some embodiments, one, two, or all three copies of
Camelina FAD2 and/or FAE1 genes are disrupted, while one or more
non-FAD, non-FAE fatty acid synthesis genes are overexpressed. In
still more embodiments, one, two, or all three copies of Camelina
FAD2 and/or FAE1 genes are disrupted, while one or more
non-fatty-acid-synthesis genes are disrupted and/or
overexpressed.
[0033] In another aspect, the present invention provides methods of
producing Camelina seed oil containing altered and/or increased
levels of oleic acid (18:1), and/or altered or reduced levels of
polyunsaturated fatty acids, and/or decreased very long chain fatty
acids. Such methods comprising utilizing the Camelina plants
comprising the chimeric genes as described herein, or Camelina
plants with disrupted FAD2 and/or FAE1 genes as described herein.
As used herein, the phrase "very long chain fatty acid" refers to a
fatty acid with more than 18 carbons.
[0034] The present invention also provides methods of increasing
the activity of a FAD2 and/or FAE1 protein in a Camelina plant
cell, plant part, tissue culture or whole plant comprising
transforming the plant cell, plant part, tissue culture or whole
plant with a chimeric gene comprising one FAD2 and/or FAE1 gene
encoding the polypeptide of the present invention, or functional
variants thereof. In one embodiment, the chimeric gene is
overexpressed. As used herein, a functional variant of a protein
refers to a polypeptide comprising one or more amino acid
modifications (e.g., substitution, deletion, modification, et al)
compared to the original protein, but still maintains the activity
of the original protein. In the present invention, "overexpression
promoter" means a promoter capable of causing strong expression
(large amount expression) of a gene that has been ligated thereto
in host plant cells. The overexpression promoter of the present
invention may be either an inducible promoter or a constitutive
promoter. A promoter is a DNA comprising an expression control
region generally located on the 5' upstream of a structural gene or
a modified sequence thereof. In the present invention, any
promoters appropriate for foreign gene expression in plant cells
can be used as overexpression promoters. Non-limiting examples of
such overexpression promoters to be used in the present invention
include, but are not limited to, a cauliflower mosaic virus (CaMV)
35S promoter, a rice actin promoter, a modified 35S promoter, or an
embryo-specific promoter. As used herein an "embryo-specific
promoter" refers to a promoter of an embryo-specific gene. An
embryo-specific gene is preferentially expressed during embryo
development in a plant. For purposes of the present disclosure,
embryo development begins with the first cell divisions in the
zygote and continues through the late phase of embryo development
(characterized by maturation, desiccation, dormancy), and ends with
the production of a mature and desiccated seed. Embryo-specific
genes can be further classified as "early phase-specific" and "late
phase-specific". Early phase-specific genes are those expressed in
embryos up to the end of embryo morphogenesis. Late phase-specific
genes are those expressed from maturation through to production of
a mature and desiccated seed. An early phase-specific promoter is a
promoter that initiates expression of a protein prior to day 7
after pollination in Arabidopsis or an equivalent stage in another
plant species. Non-limiting examples of promoter sequences that can
be used in the present invention include a promoter for the amino
acid permease gene (AAP1) (e.g., the AAP1 promoter from Arabidopsis
thaliana, Hirner et al, Plant J. 14:535-544, 1998), a promoter for
the oleate 12-hydroxylase:desaturase gene (e.g., the promoter
designated LFAH 12 from Lesquerellafendleri, Broun et al, Plant J.
13:201-210, 1998), a promoter for the 2S2 albumin gene (e.g., the
2S2 promoter from Arabidopsis thaliana, Guerche et al, Plant cell
2:469-478, 1990), a fatty acid elongase gene promoter (FAE1) (e.g.,
the FAE1 promoter from Arabidopsis thaliana, Rossak et al, Plant
MoI Biol. 46:717-715, 2001), and the leafy cotyledon gene promoter
(LEC2) (e.g., the LEC2 promoter from Arabidopsis thaliana, Kroj et
al Development 130:6065-6073, 2003). Other early embryo-specific
promoters of interest include, but are not limited to, seedstick
(Pinyopich et al, Nature 424:85-88, 2003), Fbp7 and Fbpl 1 (Petunia
Seedstick) (Colombo et al, Plant Cell. 9:703-715, 1997), Banyuls
(Devic et al, Plant J. 19:387-398, 1999), agl-15 and agl-18
(Lehti-Shiu et al, Plant MoI Biol. 58:89-107, 2005), Phel (Kohler
et al, Genes Develop. 17:1540-1553, 2003), Perl (Haslekas et al,
Plant MoI Biol. 36:833-845, 1998; Haslekas et al, Plant MoI Biol.
53:313-326, 2003), embl75 (Cushing et al, Planta 221:424-436,
2005), LIl (Kwong et al, Plant Cell 15:5-18, 2003), Lecl (Lotan et
al, Cell 93:1195-1205, 1998), Fusca3 (Kroj et al, Development
130:6065-6073, 2003), ttl2 (Debeaujon et al, Plant Cell 13:853-871,
2001), ttl6 (Nesi et al, Plant Cell 14:2463-2479, 2002), A-RZf (Zou
and Taylor, Gene 196:291-295, 1997), TtGl (Walker et al, Plant Cell
11:1337-1350, 1999; Tsuchiya et al, Plant J. 37:73-81, 2004), TtI
(Sagasser et al, Genes Dev. 16:138-149, 2002), TT8 (Nesi et al,
Plant Cell 12:1863-1878, 2000), Gea-8 (carrot) (Lin and Zimmerman,
J. Exp. Botany 50:1139-1147, 1999), Knox (rice) (Postma-Haarsma et
al, Plant MoI. Biol. 39:257-271, 1999), Oleosin (Plant et al, Plant
MoI Biol. 25:193-205, 1994; Keddie et al, Plant MoI Biol.
24:121-14$, 1994), ABI3 (Ng et al, Plant MoI Biol. 54:25-38, 2004;
Parcy et al, Plant Cell 6:1567-1582, 1994), and the like.
[0035] The present invention also provides methods of decreasing
the activity of a FAD2 and/or FAE1 protein in a Camelina plant
cell, plant part, tissue culture or whole plant comprising
contacting the plant cell, plant part, tissue culture or whole
plant with an inhibitory nucleic acid having complementarity to a
gene encoding the FAD2 and/or FAE1 protein.
[0036] In one aspect, the present invention provides methods of
breeding Camelina species producing altered levels of fatty acids
in the seed oil and/or meal. In one embodiment, such methods
comprise making a cross between a Camelina mutant with one or more
mutations listed in Tables 7-12 with a second Camelina cultivar to
produce an F1 plant; backcrossing the F1 plant to the second
Camelina cultivar; and repeating the backcrossing step to generate
an near isogenic line, wherein the one or more mutations are
integrated into the genome of the second Camelina cultivar; wherein
the near isogenic line derived from the second Camelina cultivar
with the integrated mutations has altered seed oil composition.
Optionally, such methods can be facilitated by molecular
markers.
[0037] In another aspect, the present invention provides methods of
breeding species close to Camelina sativa, wherein said species
produces altered levels of fatty acids in the seed oil and/or meal.
For example, intertribal somatic hybridizations are possible
between C. sativa and B. oleracea (see, e.g., Lise N. Hansen, 1998,
Euphytica, Volume 104, No. 3, pages 173-179). In one embodiment,
such methods comprise making a cross between the Camelina mutants
with one or more mutations listed in Tables 7-12 with a species
that is closely related to the Camelina species containing the
mutations to make an F1 plant; backcrossing the F1 plants to the
species that is closely related to the Camelina species containing
the mutations; and, repeating backcrossing step to generate an near
isogenic line, wherein the one or more mutations are integrated
into the genome of the species that is closely related to the
Camelina species containing the mutations; wherein the near
isogenic line derived from the species that is closely related to
the Camelina species containing the mutations has integrated these
mutations and has altered seed oil composition. Optionally, such
method can be facilitated by molecular markers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-1C depict Southern blot analysis of Camelina sativa
and Arabidopsis thaliana. A blot containing genomic DNA from C.
sativa and A. thaliana digested with EcoRI or a combination of
EcoRI and BamHI was hybridized with an .alpha.-32 P dCTP--labeled
(1A) FAD2 probe, (1B) FAE1 probe or (1C) LFY probe obtained from
PCR amplification of C. sativa DNA.
[0039] FIGS. 2A-2B depict FAD2 and FAE1 protein alignment. FIG. 2A
shows amino acid sequence comparison of the three Camelina sativa
FAD2 sequences, Arabidopsis thaliana FAD2 sequence [Genbank:
NM.sub.--112047], Brassica rapa FAD2 sequence [Genbank: AJ459107],
Glycine max FAD2-3 sequence [Genbank: DQ532371], Zea mays FAD2
sequence [Genbank: AB257309]. Blue underlines below the sequences
indicate amino acids conserved in all 50 FAD2 sequences (Belo,
Zheng et al. 2008) while the green underline indicates the ER
localization signal (McCartney, Dyer et al. 2004). The three His
boxes described by Tocher D R (1998) are indicated with red boxes.
FIG. 2B shows amino acid sequence comparison of the three Camelina
sativa FAE1 sequences, Arabidopsis thaliana FAE1 [Genbank:
NM.sub.--119617], Crambe abyssinica [Genbank: AAX22298], Brassica
rapa HEAC FAE1 [Genbank: Y14975], Brassica rapa LEAC FAE1 [Genbank:
Y14974], Limnanthes alba (meadow foam) [Genbank: AF247134],
Tropaeolum majus (nasturtium) [Genbank: ABD77097]. Blue underlines
below the sequence indicate the asparagine at position 424 and the
highly conserved histidine and cysteine residues described by
Ghanevati and Jaworski (Ghanevati and Jaworski 2001; Ghanevati and
Jaworski 2002). The red box indicates the region highly conserved
among condensing enzymes in very long chain fatty acid biosynthesis
(Moon, Smith et al. 2001) Abbreviations: Heac=High erucic acid,
Leac=Low erucic acid.
[0040] FIGS. 3A-3D depict FAD2 and FAE1 Expression in Developing
Seeds. Relative combined expression of all three copies of (3A)
FAD2 and (3B) FAE1 measured by real time quantitative PCR at 15,
20, 25, and 30 days post anthesis (DPA) and in 2 week old
seedlings. The 20 DPA sample, which expressed FAD2 and FAE1 at the
highest amount, was used as the calibrator. Error bars represent
the standard deviation of 3 replicate experiments. Sequenom SNP
analysis demonstrating the expression of each version of (3C) FAD2
or (3D) FAE1 relative to the other versions. Error bars represent
the standard deviation of three (for FAD2) or four (for FAE1) SNP
analyses. Because FAE1 is not expressed in C. sativa seedlings
(3B), the relative expression of the 3 copies of FAE1 in seedling
tissue is not shown (3D).
[0041] FIG. 4 depicts structure and conservation of the KCS17-FAE1
intergenic region in Camelina sativa. The three putative homologous
regions in allohexaploid C. sativa are aligned to the orthologous
region of Arabidopsis to display blocks of homology identified on a
dot matrix by perfect conservation of a sliding window of 9 bases.
The KCS17 and FAE1 gene, respectively blue and red, flank a
variable region in which conserved blocks common to two or more
genomes are marked by different shades of brown. Lined regions
display reduced or no conservation. The large variation in the
intergenic region of the triplicated KCS17-FAE1 DNA of C. sativa is
consistent with independent evolution before reunion of diverged
genomes by allohexaploidization.
[0042] FIG. 5 depicts genome content of Camelina species. 1C nuclei
were stained with propidium iodide and analyzed by flow cytometry.
Error bars represent the standard deviation of 2-4 replicate
samples.
[0043] FIGS. 6A-6B depict phylogenetic analyses of Camelineae FAD2
and FAE1. Maximum-likelihood trees showing branch length and
bootstrap support (100 bootstrap replicates) for (6A) 15 FAD2
sequences from species from the tribe Camelineae calculated using
the TVM+I+.GAMMA. model in PAUP* and rooted with Brassica rapa FAD2
(-LnL 3665.277); and for (6B) 15 FAE1 sequences from species from
the tribe Camelineae calculated using the HKY+I+.GAMMA. model in
PAUP* and rooted with Crambe abyssinica FAE1 (-LnL 5051.552).
Sequences obtained from Genbank are Capsella bursa-pastoris FAD2
[Genbank: DQ518293], Arabidopsis thaliana FAD2 [Genbank:
NM.sub.--112047], Brassica rapa FAD2 [Genbank: AJ459107],
Arabidopsis thaliana FAE1 [Genbank: NM.sub.--119617], and Crambe
abyssinica FAE1 [Genbank: AY793549].
[0044] FIG. 7 depicts a simplified version of fatty acid synthesis
pathways in plant.
[0045] FIGS. 8A-8B depict an exemplary field growth of EMS
mutagenized Camelina M2 population (upper-panel (8A)), and
exemplary mutant M2 plants with morphological changes (lower-panel
(8B)).
[0046] FIG. 9 depicts an exemplary LI-COR.RTM. gel identifying
mutants in Camelina FAD2 genes.
[0047] FIG. 10 depicts proximate locations of mutations in FAD2 A
and B, which were used in the preliminary GC analysis. "H"
identifies a His box.
[0048] FIG. 11 depicts a representative composition of Camelina
sativa seed oil.
[0049] FIGS. 12A-12B depict fatty acid compositions in FAD2 mutants
(12A) and in FAE1 mutants (12B) measured by gas chromatography.
Note: FIG. 12B is summarized FAE1 data from GC test 4 and replaces
FIG. 14B from U.S. Provisional Application No. 61/318,273, which
summarized FAE1 data from GC test 3.
[0050] FIG. 13 depicts lipid synthesis in the plastid and cytoplasm
of oilseeds. Key enzymes are in red text and boxed. ACCase=acetyl
co-A carboxylase, KAS=.beta.-ketoacyl-acyl carrier protein (ACP)
synthase, GPAT=glycerol phosphate acyltransferase,
LPAAT=lysophosphatidic acid acyltransferase, PAP=phosphatidate
phosphatase, DAGAT=diacylglycerol acyltransferase, R=fatty acyl
group, P=phosphate group, CPT=chloroplast
DETAILED DESCRIPTION
Definition
[0051] As used herein, the verb "comprise" as is used in this
description and in the claims and its conjugations are used in its
non-limiting sense to mean that items following the word are
included, but items not specifically mentioned are not
excluded.
[0052] As used herein, the term "plant" refers to any living
organism belonging to the kingdom Plantae (i.e., any genus/species
in the Plant Kingdom). This includes familiar organisms such as but
not limited to trees, herbs, bushes, grasses, vines, ferns, mosses
and green algae. The term refers to both monocotyledonous plants,
also called monocots, and dicotyledonous plants, also called
dicots. Examples of particular plants include but are not limited
to corn, potatoes, roses, apple trees, sunflowers, wheat, rice,
bananas, tomatoes, opo, pumpkins, squash, lettuce, cabbage, oak
trees, guzmania, geraniums, hibiscus, clematis, poinsettias,
sugarcane, taro, duck weed, pine trees, Kentucky blue grass,
zoysia, coconut trees, brassica leafy vegetables (e.g. broccoli,
broccoli raab, Brussels sprouts, cabbage, Chinese cabbage (Bok Choy
and Napa), cauliflower, cavalo, collards, kale, kohlrabi, mustard
greens, rape greens, and other brassica leafy vegetable crops),
bulb vegetables (e.g. garlic, leek, onion (dry bulb, green, and
Welch), shallot, and other bulb vegetable crops), citrus fruits
(e.g. grapefruit, lemon, lime, orange, tangerine, citrus hybrids,
pummelo, and other citrus fruit crops), cucurbit vegetables (e.g.
cucumber, citron melon, edible gourds, gherkin, muskmelons
(including hybrids and/or cultivars of cucumis melons),
water-melon, cantaloupe, and other cucurbit vegetable crops),
fruiting vegetables (including eggplant, ground cherry, pepino,
pepper, tomato, tomatillo, and other fruiting vegetable crops),
grape, leafy vegetables (e.g. romaine), root/tuber and corm
vegetables (e.g. potato), and tree nuts (almond, pecan, pistachio,
and walnut), berries (e.g., tomatoes, barberries, currants,
elderberries, gooseberries, honeysuckles, mayapples, nannyberries,
Oregon-grapes, see-buckthorns, hackberries, bearberries,
lingonberries, strawberries, sea grapes, lackberries, cloudberries,
loganberries, raspberries, salmonberries, thimbleberries, and
wineberries), cereal crops (e.g., corn, rice, wheat, barley,
sorghum, millets, oats, ryes, triticales, buckwheats, fonio, and
quinoa), pome fruit (e.g., apples, pears), stone fruits (e.g.,
coffees, jujubes, mangos, olives, coconuts, oil palms, pistachios,
almonds, apricots, cherries, damsons, nectarines, peaches and
plums), vine (e.g., table grapes, wine grapes), fibber crops (e.g.
hemp, cotton), ornamentals, and the like. For example, the plant is
a species in the tribe of Camelineae, such as C. alyssum, C.
anomala, C. grandiflora, C. hispida, C. laxa, C. microcarpa, C.
microphylla, C. persistens, C. rumelica, C. sativa, C.
Stiefelhagenii, or any hybrid thereof.
[0053] As used herein, the term "plant part" refers to any part of
a plant including but not limited to the shoot, root, stem, seeds,
stipules, leaves, petals, flowers, ovules, bracts, branches,
petioles, internodes, bark, pubescence, tillers, rhizomes, fronds,
blades, pollen, stamen, and the like. The two main parts of plants
grown in some sort of media, such as soil, are often referred to as
the "above-ground" part, also often referred to as the "shoots",
and the "below-ground" part, also often referred to as the
"roots".
[0054] The term "a" or "an" refers to one or more of that entity;
for example, "a gene" refers to one or more genes or at least one
gene. As such, the terms "a" (or "an"), "one or more" and "at least
one" are used interchangeably herein. In addition, reference to "an
element" by the indefinite article "a" or "an" does not exclude the
possibility that more than one of the elements are present, unless
the context clearly requires that there is one and only one of the
elements.
[0055] As used herein, the term "chimeric protein" refers to a
construct that links at least two heterologous proteins into a
single macromolecule (fusion protein).
[0056] As used herein, the term "nucleic acid" refers to a
polymeric form of nucleotides of any length, either ribonucleotides
or deoxyribonucleotides, or analogs thereof. This term refers to
the primary structure of the molecule, and thus includes double-
and single-stranded DNA, as well as double- and single-stranded
RNA. It also includes modified nucleic acids such as methylated
and/or capped nucleic acids, nucleic acids containing modified
bases, backbone modifications, and the like. The terms "nucleic
acid" and "nucleotide sequence" are used interchangeably.
[0057] As used herein, the terms "polypeptide," "peptide," and
"protein" are used interchangeably herein to refer to polymers of
amino acids of any length. These terms also include proteins that
are post-translationally modified through reactions that include
glycosylation, acetylation and phosphorylation.
[0058] As used herein, the term "homologous" or "homolog" or
"ortholog" is known in the art and refers to related sequences that
share a common ancestor or family member and are determined based
on the degree of sequence identity. The terms "homology",
"homologous", "substantially similar" and "corresponding
substantially" are used interchangeably herein. They refer to
nucleic acid fragments wherein changes in one or more nucleotide
bases do not affect the ability of the nucleic acid fragment to
mediate gene expression or produce a certain phenotype. These terms
also refer 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 alter the functional
properties of the resulting nucleic acid fragment relative to the
initial, unmodified fragment. It is therefore understood, as those
skilled in the art will appreciate, that the invention encompasses
more than the specific exemplary sequences. These terms describe
the relationship between a gene found in one species, subspecies,
variety, cultivar or strain and the corresponding or equivalent
gene in another species, subspecies, variety, cultivar or strain.
For purposes of this invention homologous sequences are compared.
"Homologous sequences" or "homologs" or "orthologs" are thought,
believed, or known to be functionally related. A functional
relationship may be indicated in any one of a number of ways,
including, but not limited to: (a) degree of sequence identity
and/or (b) the same or similar biological function. Preferably,
both (a) and (b) are indicated. The degree of sequence identity may
vary, but in one embodiment, is at least 50% (when using standard
sequence alignment programs known in the art), at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
at least 90%, at least about 91%, at least about 92%, at least
about 93%, at least about 94%, at least about 95%, at least about
96%, at least about 97%, at least about 98%, or at least 98.5%, or
at least about 99%, or at least 99.5%, or at least 99.8%, or at
least 99.9%. Homology can be determined using software programs
readily available in the art, such as those discussed in Current
Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987)
Supplement 30, section 7.718, Table 7.71. Some alignment programs
are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus
(Scientific and Educational Software, Pennsylvania). Other
non-limiting alignment programs include Sequencher (Gene Codes, Ann
Arbor, Mich.), AlignX, and Vector NTI (Invitrogen, Carlsbad,
Calif.).
[0059] As used herein, the term "nucleotide change" or "nucleotide
modification" refers to, e.g., nucleotide substitution, deletion,
and/or insertion, as is well understood in the art. For example,
mutations containing alterations that produce silent substitutions,
additions, or deletions, but do not alter the properties or
activities of the encoded protein or how the proteins are made.
[0060] As used herein, the term "protein modification" refers to,
e.g., amino acid substitution, amino acid modification, deletion,
and/or insertion, as is well understood in the art.
[0061] As used herein, the term "derived from" refers to the origin
or source, and may include naturally occurring, recombinant,
unpurified, or purified molecules. A nucleic acid or an amino acid
derived from an origin or source may have all kinds of nucleotide
changes or protein modification as defined elsewhere herein.
[0062] As used herein, the term "at least a portion" of a nucleic
acid or polypeptide means a portion having the minimal size
characteristics of such sequences, or any larger fragment of the
full length molecule, up to and including the full length molecule.
For example, a portion of a nucleic acid may be 12 nucleotides, 13
nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17
nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 22
nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30
nucleotides, 32 nucleotides, 34 nucleotides, 36 nucleotides, 38
nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55
nucleotides, and so on, going up to the full length nucleic acid.
Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino
acids, 6 amino acids, 7 amino acids, and so on, going up to the
full length polypeptide. The length of the portion to be used will
depend on the particular application. A portion of a nucleic acid
useful as hybridization probe may be as short as 12 nucleotides; in
one embodiment, it is 20 nucleotides. A portion of a polypeptide
useful as an epitope may be as short as 4 amino acids. A portion of
a polypeptide that performs the function of the full-length
polypeptide would generally be longer than 4 amino acids.
[0063] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences which differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well-known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988).
[0064] As used herein, the term "suppression" or "disruption" of
regulation refers to reduced activity of regulatory proteins, and
such reduced activity can be achieved by a variety of mechanisms
including antisense, mutation knockout or RNAi. Antisense RNA will
reduce the level of expressed protein resulting in reduced protein
activity as compared to wild type activity levels. A mutation in
the gene encoding a protein may reduce the level of expressed
protein and/or interfere with the function of expressed protein to
cause reduced protein activity.
[0065] As used herein, the terms "polynucleotide", "polynucleotide
sequence", "nucleic acid sequence", "nucleic acid fragment", and
"isolated nucleic acid fragment" are used interchangeably herein.
These terms encompass nucleotide sequences and the like. A
polynucleotide may be a polymer of RNA or DNA that is single- or
double-stranded, that optionally contains synthetic, non-natural or
altered nucleotide bases. A polynucleotide in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found
in their 5'-monophosphate form) are referred to by a single letter
designation as follows: "A" for adenylate or deoxyadenylate (for
RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate,
"G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for
deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C
or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and
"N" for any nucleotide.
[0066] The term "primer" as used herein refers to an
oligonucleotide which is capable of annealing to the amplification
target allowing a DNA polymerase to attach, thereby serving as a
point of initiation of DNA synthesis when placed under conditions
in which synthesis of primer extension product is induced, i.e., in
the presence of nucleotides and an agent for polymerization such as
DNA polymerase and at a suitable temperature and pH. The
(amplification) primer is preferably single stranded for maximum
efficiency in amplification. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
agent for polymerization. The exact lengths of the primers will
depend on many factors, including temperature and composition (A/T
and G/C content) of primer. A pair of bi-directional primers
consists of one forward and one reverse primer as commonly used in
the art of DNA amplification such as in PCR amplification.
[0067] As used herein, "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.
[0068] As used herein, "regulatory sequences" may include, but are
not limited to, promoters, translation leader sequences, introns,
and polyadenylation recognition sequences.
[0069] As used herein, "promoter" refers to a DNA sequence capable
of controlling the expression of a coding sequence or functional
RNA. 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 that 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.
It is further recognized that since in most cases the exact
boundaries of regulatory sequences have not been completely
defined, DNA fragments of some variation may have identical
promoter activity. Promoters that cause a gene to be expressed in
most cell types at most times are commonly referred to as
"constitutive promoters".
[0070] As used herein, the "3' non-coding sequences" or "3' UTR
(untranslated region) sequence" 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, I. L., et al. (1989) Plant Cell
1:671-680.
[0071] As used herein, 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 regulated by the other. For
example, a promoter is operably linked with a coding sequence when
it is capable of regulating 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 a sense or antisense orientation. In
another example, the complementary RNA regions of the invention can
be operably linked, either directly or indirectly, 5' to the target
mRNA, or 3' to the target mRNA, or within the target mRNA, or a
first complementary region is 5' and its complement is 3' to the
target mRNA.
[0072] As used herein, the term "cross", "crossing", "cross
pollination" or "cross-breeding" refer to the process by which the
pollen of one flower on one plant is applied (artificially or
naturally) to the ovule (stigma) of a flower on another plant.
[0073] As used herein, the term "gene" refers to any segment of DNA
associated with a biological function. Thus, genes include, but are
not limited to, coding sequences and/or the regulatory sequences
required for their expression. Genes can also include nonexpressed
DNA segments that, for example, form recognition sequences for
other proteins. Genes can be obtained from a variety of sources,
including cloning from a source of interest or synthesizing from
known or predicted sequence information, and may include sequences
designed to have desired parameters.
[0074] As used herein, the term "vector", "plasmid", or "construct"
refers broadly to any plasmid or virus encoding an exogenous
nucleic acid. The term should also be construed to include
non-plasmid and non-viral compounds which facilitate transfer of
nucleic acid into virions or cells, such as, for example,
polylysine compounds and the like. The vector may be a viral vector
that is suitable as a delivery vehicle for delivery of the nucleic
acid, or mutant thereof, to a cell, or the vector may be a
non-viral vector which is suitable for the same purpose. Examples
of viral and non-viral vectors for delivery of DNA to cells and
tissues are well known in the art and are described, for example,
in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94:12744-12746).
Examples of viral vectors include, but are not limited to,
recombinant plant viruses. Non-limiting examples of plant viruses
include, TMV-mediated (transient) transfection into tobacco (Tuipe,
T-H et al (1993), J. Virology Meth, 42: 227-239), ssDNA genomes
viruses (e.g., family Geminiviridae), reverse transcribing viruses
(e.g., families Caulimoviridae, Pseudoviridae, and Metaviridae),
dsNRA viruses (e.g., families Reoviridae and Partitiviridae), (-)
ssRNA viruses (e.g., families Rhabdoviridae and Bunyaviridae), (+)
ssRNA viruses (e.g., families Bromoviridae, Closteroviridae,
Comoviridae, Luteoviridae, Potyviridae, Sequiviridae and
Tombusviridae) and viroids (e.g., families Pospiviroldae and
Avsunviroidae). Detailed classification information of plant
viruses can be found in Fauquet et al (2008, "Geminivirus strain
demarcation and nomenclature". Archives of Virology 153:783-821,
incorporated herein by reference in its entirety), and Khan et al.
(Plant viruses as molecular pathogens; Publisher Routledge, 2002,
ISBN 1560228954, 9781560228950). Examples of non-viral vectors
include, but are not limited to, liposomes, polyamine derivatives
of DNA, and the like.
Camelina Sativa
[0075] Camelina is a genus of flowering plants belonging to the
Brassicaceae family. Camelina sativa is a particular species of the
genus Camelina that is important historically and is a source of
oil that can be used in, for example, biofuels and lubricants. C.
sativa is being investigated for both biofuel and human utility. It
is a crop that has not benefited much from molecular investigation
in the past and as such, there is relatively little sequence
information available. The utility of a plant oil either for
biodiesel or food depends on its fatty acid composition. Camelina
has a fatty acid composition with high levels of both
polyunsaturated fatty acids such as 18:2 and 18:3 (52-54%) as well
as long chain fatty acids such as 20:1 (11-15%) and 22:1 (2-5%).
For biodiesel, the optimum fatty acid is 18:1 (oleic). Oleic has
the best balance of characteristics for cloud point vs. oxidative
stability. Polyunsaturated fatty acids such as 18:2 and 18:3 have
poor oxidative stability. The long chain fatty acids such as 20:1
and 22:1 contribute to out of range distillation temperatures in
biodiesel. For biodiesel utility it is therefore desirable to lower
the level of polyunsaturated fatty acids and to lower the level of
long chain fatty acids. The ultimate goal is to increase the
percentage of 18:1 fatty acid. 18:1 is also considered a good fatty
acid for food utility.
[0076] Camelina has not been intensively bred and the germplasm is
somewhat limited genetically. An in-house field study of a
significant number of cultivars showed little variation in the
fatty acid composition. This agrees with published literature
(e.g., Putnam et al., 1993. Camelina: A promising low-input
oilseed. p. 314-322. In: J. Janick and J. E. Simon (eds.), New
crops. Wiley, New York.)
Fatty Acids Synthesis in Plants
[0077] Fatty acid biosynthesis in plants takes place within the
endoplasmic reticulum and plastids, the latter of which is an
organelle widely thought to have originated from a photosynthetic
bacterial symbiont. Fatty acid metabolism in plants closely
resembles that of bacteria.
[0078] During fatty acid biosynthesis, a repeated series of
reactions incorporates acetyl moieties of acetyl-CoA into an acyl
group 16 or 18 carbons long. The enzymes involved in this synthesis
are acetyl-CoA carboxylase (ACCase), malonyl-CoA:ACP transacylase,
3-ketoacyl-ACP synthase I and III (KAS I and KAS III),
3-ketoacyl-ACP reductase, 2,3-trans-Enoyl-ACP reductase,
3-hydroxyacyl-ACP dehydratase (all referred as fatty acid synthase
(FAS), except for ACCase). The name fatty acid synthase refers to a
complex of several individual enzymes that catalyze the conversion
of acetyl-CoA and malonyl-CoA to 16:0 and 18:0 fatty acids.
Acyl-carrier protein (ACP), an essential protein cofactor, is
generally considered a component of FAS.
[0079] The last three steps of the fatty acids synthesis cycle
reduce a 3-ketoacyl substrate to form a fully saturated acyl chain.
Each cycle of fatty acid synthesis adds two carbons to the acyl
chain. Typically, fatty acid synthesis ends at 16:0 or 18:0, when
one of several reactions stops the process. The most common
reactions are hydrolysis of acyl moiety from ACP by a thioesterase,
transfer of the acyl moiety from ACP directly onto a glycerolipid
by an acyl transferase, or double-bond formation on the acyl moiety
by an acyl-ACP desaturase. The thioesterase reaction yields a
sulfhydryl ACP.
[0080] Two principal types of acyl-ACP thioesterases occur in
plants. For making storage lipids (triglycerides) in the ER, the
FAT enzymes convert the fatty acid-ACP to a fatty acid-Co-A. The
substrate for FAE1 is an R-CoA and it is an R-CoA that is added to
various positions in the glycerol backbone during the Kennedy
pathway portion of the synthesis of Triglycerides in the ER (FIG.
7). The major class, designated FatA, is most active with 18:1
delta9-ACP. A second class designated FatB, typified by 16:0-ACP
thioesterase, is most active with shorter-chain, saturated
acyl-ACPs. Thioesterases play important role in plants that have
unusually short fatty acids, such as coconut, many species of
Cuphea, and California bay. These plants have thioesterases that
are especially active with C10 to C12 acyl-ACPs, by prematurely
terminating fatty acid biosynthesis.
[0081] Unsaturated fatty acids are produced by desaturation of
saturated lipids with the help of desaturases (FAD enzymes). Most
fatty acid desaturases (FADs) in plants are integral membrane
proteins, with the exception that plant contains a soluble,
plastid-localized stearoyl-ACP desaturase. The number and
properties of different FADs in plants are known from the isolation
of a comprehensive collection of Arabidopsis mutants with defects
in each of eight desaturase genes. The enzymes encoded by these
genes differ in substrate specificity, subcellular location, mode
of regulation, or some combination of these. A summary of the
Arabidopsis FADs is shown below:
TABLE-US-00002 Site of subcellular Fatty acid double-bond Name
location substrates insertion Notes FAD2 ER 18:1.DELTA.9 .DELTA.12
preferred substrate is phosphatidylcholine, oleate desaturase FAD3
ER 18:2.DELTA.9,12 .omega.3 preferred substrate is
phosphatidylcholine, linoleate desaturase FAD4 Chloroplast 16:0
.DELTA.3 produces 16:1-trans at sn-2 of phosphatidylglycerol FADS
Chloroplast 16:0 .DELTA.7 desaturates 16:0 at sn-2 of
monogalactosyldiacylglycerol FAD6 Chloroplast 16:1.DELTA.7 and
.omega.6 acts on all chloroplast glycerolipids, oleate 18:1.DELTA.9
desaturase FAD7 Chloroplast 16:247,11 and .omega.3 acts on all
chloroplast glycerolipids, linoleate 18:2.DELTA.9,12 desaturase
FAD9 Chloroplast 16:247,11 and .omega.3 isoenzyme of FAD7 induced
by low temperature, 18:2.DELTA.9,12 linoleate desaturase FAB2
Chloroplast 18:0 .DELTA.9 stromal stearoyl-ACP desaturase
[0082] The biochemical defect of each class of mutants is shown by
breaks in the pathway on page 480 of Buchanan et al., Biochemistry
and Molecular Biology of Plants, American Society of Plant
Physiologists, 2000, ISBN 0943088372, 9780943088372, which is
incorporated by reference in its entirety.
[0083] Extensive surveys of the fatty acid composition of seed oils
from different plant species have resulted in the identification of
more than 200 naturally occurring fatty acids, which can broadly be
classified into 18 structural classes, such as laballenic acid,
stearolic acid, sterculynic acid, chaulmoogric acid, ricinoleic
acid, vernolic acid, furan-containing fatty acid, et al. Less is
known about the mechanisms responsible for the synthesis and
accumulation of unusual fatty acids, or of their significance to
the fitness of the plants that accumulate them. However, recent
studies indicate that enzymes involved in the synthesis of unusual
fatty acids are structurally similar to the desaturases and
hydroxylases. Unusual fatty acids occur almost exclusively in seed
oils and may serve a defense function.
[0084] Synthesis of structural lipids (e.g. cutin, suberin,
epicuticular wax) has also been studied in Arabidopsis. Proposed
pathways related to this is shown on page 512 of Buchanan et al.,
Biochemistry and Molecular Biology of Plants, American Society of
Plant Physiologists, 2000, ISBN 0943088372, 9780943088372, which is
incorporated by reference in its entirety.
[0085] Thus, as used herein, the phrase "fatty acid synthesis
genes" or "FAS gene" refers to any genes that are involved in
synthesis of fatty acids, cuticle, and wax as described above. For
example, such genes include, but are not limited to,
malonyl-CoA:ACP transacylase, 3-ketoacyl-ACP synthase I and III
(KAS I and KAS III), 3-ketoacyl-ACP reductase, 2,3-trans-Enoyl-ACP
reductase, 3-hydroxyacyl-ACP dehydratase, acyl-ACP thioesterases,
fatty acid desaturases (e.g., FAD2, FADS), fatty acid elongases
(e.g., FAE1), hydroxylases, and enzymes displayed in FIGS. 7 and
13.
[0086] Seed oil of Camelina sativa contains high levels (up to 45%)
of omega-3 fatty acids, which is uncommon in vegetable sources.
Over 50% of the fatty acids in cold pressed Camelina oil are
polyunsaturated. The major components are alpha-linolenic
acid--C18:3 (omega-3-fatty acid, approx 35-45%) and linoleic
acid--C18:2 (omega-6 fatty acid, approx 15-20%). FIG. 11 shows a
representative composition of Camelina seed oil. The oil is also
very rich in natural antioxidants, such as tocopherols, making this
highly stable oil very resistant to oxidation and rancidity. It has
1-3% erucic acid. The vitamin E content of Camelina oil is
approximately 110 mg/100 g. The present invention relates to
increasing oleic acid (18:1) level, decreasing the level of long
chain fatty acids, and/or improving the seed oil quality of
Camelina. As used herein, the term "level" refers to the relative
percentage of a component in a mixture.
[0087] In the endoplasmic reticulum, oleic acid (18:1) is converted
to linoleic acid (18:2) by a delta-12-desaturase, fatty acid
desaturase 2 (FAD2). Mutations in Arabidopsis thaliana FAD2 have
been shown to increase the levels of 18:1 in the seeds 2-3.4 fold
while decreasing the levels of 18:2 fatty acids 4-10 fold. (Levels
of 20:1 also increased approximately 1.5 fold--Okuley 1994.)
[0088] Very long chain fatty acids are synthesized in the cytosol
by extension of an 18 carbon fatty acid. The rate limiting step is
thought to be the initial condensation step, catalyzed in the seed
by fatty acid elongase 1 (FAE1, Kunst 1992). In Arabidopsis, where
approximately 25% of seed fatty acids can be long chain fatty
acids, mutants in FAE1 have less than 1%. Interestingly,
Arabidopsis fae1 mutants show a greater than 2-fold increase in
18:1 content in the seeds. (Katavic et al. (2002). "Restoring
enzyme activity in nonfunctional low erucic acid Brassica napus
fatty acid elongase 1 by a single amino acid substitution." Eur J
Biochem 269(22): 5625-31.)
FAD2 and FAE1 Genes of Camelina Sativa
[0089] The invention discloses the full genomic sequence of three
FAD2 genes and three FAE1 genes from Camelina sativa with both
upstream and downstream regions for FAD2 and upstream regions for
FAE1 (deposited in Genbank at the NCBI, Genbank IDs:
GU929417-GU929422, SEQ ID NOs. 1-6). These sequences include both
the coding region as well as several hundred base pairs upstream
and downstream of the genes. The coding sequences for the Camelina
sativa FAD2 were obtained using primers from Arabidopsis thaliana
FAD2 while the coding regions for the Camelina sativa FAE1 were
obtained using primers from Crambe abyssinica FAE1. Also obtained
are coding sequences for FAD2 and FAE1 genes from Capsell rubella,
A. Lyrata, Camelina hispida, Camelina laxa, Camelina microcarpa,
and Camelina rumelica (GU929423-GU929441, SEQ ID NOs 45-63), which
were amplified using C. Sativa primers. The upstream regions for
all the genes were obtained using a combination of RACE PCR and PCR
with primers from upstream Arabidopsis sequences in conjunction
with primers to Camelina sequences. The downstream regions of FAD2
were obtained using PCR with primers designed from a combination of
downstream Arabidopsis sequence in conjunction with primers to
Camelina sequences. The Camelina sativa FAD2 and FAE1 genes are
highly homologous to both Arabidopsis and Brassica napus (e.g.,
canola, oilseed rape) FAD2 and FAE1. However, the disclosed
sequences are specific to Camelina sativa.
[0090] The present invention provides an isolated nucleic acid
sequence comprising a sequence selected from the group consisting
of SEQ ID NOs: 1 to 6 and SEQ ID NOs: 45-63, and fragments and
variations derived from thereof. In one embodiment, the present
invention provides an isolated polynucleotide encoding plant fatty
acid desaturase, comprising a nucleic acid sequence that shares at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, at least 99.1%, at least 99.2%, at least 99.3%, at least
99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least
99.8%, or at least 99.9% identity to SEQ ID NO: 1, 2, 3, 45, 46,
48, 51, 54, 55, 56, 60, and/or 61. In another embodiment, the
present invention provides an isolated polynucleotide encoding
fatty acid elongase, comprising a nucleic acid sequence that shares
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least
99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least
99.8%, or at least 99.9% identity to SEQ ID NO: 4, 5, 6, 47, 49,
50, 52, 53, 57, 58, 59, 62, and/or 63.
[0091] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman (Adv. Appl. Math., 2:482, 1981);
Needleman and Wunsch (J. MoI. Biol., 48:443, 1970); Pearson and
Lipman (Proc. Natl. Acad. Sci., 85:2444, 1988); Higgins and Sharp
(Gene, 73:237-44, 1988); Higgins and Sharp (CABIOS, 5:151-53,
1989); Corpet et al. (Nuc. Acids Res., 16:10881-90, 1988); Huang et
al. (Comp. Appls Biosci., 8:155-65, 1992); and Pearson et al.
(Meth. Mol. Biol., 24:307-31, 1994). Altschul et al. (Nature
Genet., 6:119-29, 1994) presents a detailed consideration of
sequence alignment methods and homology calculations.
[0092] The present invention also provides a chimeric gene
comprising the isolated nucleic acid sequence of any one of the
polynucleotides described above operably linked to suitable
regulatory sequences.
[0093] The present invention also provides a recombinant construct
comprising the chimeric gene as described above. In one embodiment,
said recombinant construct is a gene silencing construct, such as
used in RNAi gene silencing.
[0094] The expression vectors of the present invention will
preferably include at least one selectable marker. Such markers
include dihydrofolate reductase, G418 or neomycin resistance for
eukaryotic cell culture and tetracycline, kanamycin or ampicillin
resistance genes for culturing in E. coli and other bacteria.
Vectors that can be used with the invention comprise vectors for
use in bacteria, which comprise pQE70, pQE60 and pQE-9, pBluescript
vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among preferred
eukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44, pXT1 and
pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors will be
readily apparent to the skilled artisan.
[0095] The present invention also provides a transformed host cell
comprising the chimeric gene as described above. In one embodiment,
said host cell is selected from the group consisting of bacteria,
yeasts, filamentous fungi, algae, animals, and plants.
[0096] These sequences allow the design of gene-specific primers
and probes for both FAD2 and FAE1. Additional data demonstrates
that all three copies of each gene are expressed in the seed, i.e.
no one copy is silent in the seed.
[0097] Primers are short nucleic acid molecules, for instance DNA
oligonucleotides, usually 7 nucleotides or more in length, for
example that hybridize to contiguous complementary nucleotides or a
sequence to be amplified. Longer DNA oligonucleotides may be about
15, 20, 25, 30 or 50 nucleotides or more in length. Primers can be
annealed to a complementary target DNA strand by nucleic acid
hybridization to form a hybrid between the primer and the target
DNA strand, and then the primer extended along the target DNA
strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification of a nucleic acid sequence, for example, by the PCR
or other nucleic-acid amplification methods known in the art, as
described above.
[0098] A probe comprises an identifiable, isolated nucleic acid
that recognizes a target nucleic acid sequence. A probe includes a
nucleic acid that is attached to an addressable location, a
detectable label or other reporter molecule and that hybridizes to
a target sequence. Typical labels include radioactive isotopes,
enzyme substrates, co-factors, ligands, chemiluminescent or
fluorescent agents, haptens, and enzymes. Methods for labelling and
guidance in the choice of labels appropriate for various purposes
are discussed, for example, in Sambrook et al. (ed.), Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 and Ausubel
et al. Short Protocols in Molecular Biology, 4.sup.th ed., John
Wiley & Sons, Inc., 1999.
[0099] Methods for preparing and using nucleic acid probes and
primers are described, for example, in Sambrook et al. (ed.),
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989; Ausubel et al. Short Protocols in Molecular Biology, 4.sup.th
ed., John Wiley & Sons, Inc., 1999; and Innis et al. PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc., San Diego, Calif., 1990. Amplification primer pairs can be
derived from a known sequence, for example, by using computer
programs intended for that purpose such as PRIMER (Version 0.5,
1991, Whitehead Institute for Biomedical Research, Cambridge,
Mass.). One of ordinary skill in the art will appreciate that the
specificity of a particular probe or primer increases with its
length. Thus, in order to obtain greater specificity, probes and
primers can be selected that comprise at least 20, 25, 30, 35, 40,
45, 50 or more consecutive nucleotides of a target nucleotide
sequences.
[0100] The inventor also obtained Real Time qPCR expression data
that shows that FAD2 and FAE1 genes are expressed in the seed. In
addition, SNP expression data demonstrated that all three copies of
FAD2 and of FAE1 are expressed. Data that supports these SNP
results was also obtained from sequencing a cDNA library from
developing Camelina seed.
[0101] The invention also provides an EMS mutant library that has
been created in Camelina sativa variety CS32 (commercial name as
SO30). Initial TILLING.RTM. using primers designed to the three
FAD2 genes yielded mutants in all three FAD2 genes (Hutcheon et
al., TILLING.RTM. for Altered Fatty Acid Profiles in Camelina
sativa, July 2009, American Society of Plant Biologists Annual
Meeting, which is herein incorporated by reference in its entirety
for all purposes). Preliminary analysis on lipid composition of
these mutants using Gas Chromatography-Flame Ionization Detector
(GC-FID) has also been conducted. In addition, Tilling mutants have
been identified in FAE1 and preliminary analysis of lipid
composition using GC-FID has been conducted on these mutants
(Tables 19-20).
[0102] The close relationship between C. species and the model
plant Arabidopsis thaliana (Al-Shehbaz, Beilstein et al. 2006;
Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al.
2008) facilitates the manipulation of known pathways, such as the
one regulating fatty acid biosynthesis. C. sativa seed oil is high
in both polyunsaturated and long chain fatty acids (Budin, Breene
et al. 1995; Zubr 1997; Gugel and Falk 2006), suggesting that both
FAD2 and FAE1 are present and active. Three copies each of the FAD2
and FAE1 genes were isolated from an agronomic accession of
Camelina sativa using primers designed from Arabidopsis thaliana or
Crambe abyssinica sequence, Previously identified conserved sites
in FAD2 (Tocher D R 1998; McCartney, Dyer et al. 2004; Belo, Zheng
et al. 2008) and FAE1 (Ghanevati and Jaworski 2001; Moon, Smith et
al. 2001; Ghanevati and Jaworski 2002) are present in all three
copies of each gene and a 5' intron shown to be important in
regulating FAD2 expression in sesame (Kim, Kim et al. 2006) was
identified in all three CsFAD2 copies. Real Time qPCR data and
Sequenom MassARRAY SNP analysis of the FAD2 and FAE1 cDNA showed
that all three copies of each gene are expressed in developing
seeds. Thus, it seems likely that all three copies of FAD2 and FAE1
in C. sativa are functional.
[0103] The cloning of three copies of FAD2 and FAE1 from the C.
sativa genome, as well as the observation of three LFY
hybridization signals by Southern analysis could be explained by at
least two possible scenarios: segmental duplications of selected
regions within a diploid genome either through tandem duplications
or through transpositions, or whole genome duplications resulting
from polyploidization. The possibility that ancient segmental
duplications or transpositions affected all three examined loci
seems less probable than polyploidy. Furthermore, no evidence of
recent segmental duplication involving multiple genes has been
observed in sequenced plant genomes (Arabidopsis genome (TAIR 2009,
2010); rice genome (TIGR Rice Database); maize genome (Maize Genome
Browser 2010); and Soybean Genome (Phytozome, 2010)).
FAD2 and FAE1 Proteins of Camelina Sativa
[0104] The present invention also provides polypeptides and amino
acid sequences comprising at least a portion of the isolated
protein selected from the group consisting of SEQ ID NOs: 7-12, and
all variants thereof.
[0105] The present invention also provides an isolated amino acid
sequence comprising a sequence selected from the group consisting
of SEQ ID NOs: 7 to 12, and fragments and variations derived from
thereof. In some embodiments, the present invention provides an
isolated polypeptide comprising an amino acid sequence that shares
at least about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%,
about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%,
about 99.7%, about 99.8%, or about 99.9% identity to SEQ ID NO: 7,
8, 9, 64, 65, 67, 70, 73, 74, 75, 79, and/or 80. In one embodiment,
the present invention provides an isolated polypeptide comprising
an amino acid sequence which encodes an amino acid sequence that
shares at least about 85%, about 86%, about 87%, about 88%, about
89%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about
99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about
99.7%, about 99.8%, or about 99.9% identity to SEQ ID NO: 10, 11,
12, 66, 68, 69, 71, 72, 76, 77, 78, 81, and/or 82.
[0106] The invention also encompasses variants and fragments of
proteins of FAD2 and FAE1 isolated in the present invention. The
variants may contain alterations in the amino acid sequences of the
constituent proteins. The term "variant" with respect to a
polypeptide refers to an amino acid sequence that is altered by one
or more amino acids with respect to a reference sequence. The
variant can have "conservative" changes, or "nonconservative"
changes, e.g., analogous minor variations can also include amino
acid deletions or insertions, or both.
[0107] Functional fragments and variants of a polypeptide include
those fragments and variants that maintain one or more functions of
the parent polypeptide. It is recognized that the gene or cDNA
encoding a polypeptide can be considerably mutated without
materially altering one or more of the polypeptide's functions.
First, the genetic code is well-known to be degenerate, and thus
different codons encode the same amino acids. Second, even where an
amino acid substitution is introduced, the mutation can be
conservative and have no material impact on the essential
function(s) of a protein. See, e.g., Stryer Biochemistry 3rd Ed.,
1988. Third, part of a polypeptide chain can be deleted without
impairing or eliminating all of its functions. Fourth, insertions
or additions can be made in the polypeptide chain for example,
adding epitope tags, without impairing or eliminating its functions
(Ausubel et al. J. Immunol. 159(5): 2502-12, 1997). Other
modifications that can be made without materially impairing one or
more functions of a polypeptide can include, for example, in vivo
or in vitro chemical and biochemical modifications or the
incorporation of unusual amino acids. Such modifications include,
but are not limited to, for example, acetylation, carboxylation,
phosphorylation, glycosylation, ubiquination, labelling, e.g., with
radionucleotides, and various enzymatic modifications, as will be
readily appreciated by those well skilled in the art. A variety of
methods for labelling polypeptides, and labels useful for such
purposes, are well known in the art, and include radioactive
isotopes such as .sup.32P, ligands which bind to or are bound by
labelled specific binding partners (e.g., antibodies),
fluorophores, chemiluminescent agents, enzymes, and anti-ligands.
Functional fragments and variants can be of varying length. For
example, some fragments have at least 10, 25, 50, 75, 100, 200, or
even more amino acid residues. These mutations can be natural or
purposely changed. In some embodiments, mutations containing
alterations that produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
proteins or how the proteins are made are an embodiment of the
invention.
[0108] Conservative amino acid substitutions are those
substitutions that, when made, least interfere with the properties
of the original protein, that is, the structure and especially the
function of the protein is conserved and not significantly changed
by such substitutions. Conservative substitutions generally
maintain (a) the structure of the polypeptide backbone in the area
of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Further
information about conservative substitutions can be found, for
instance, in Ben Bassat et al. (J. Bacterial., 169:751-757, 1987),
O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein
Sci., 3:240-247, 1994), Hochuli et al. (Bio/Technology,
6:1321-1325, 1988) and in widely used textbooks of genetics and
molecular biology. The Blosum matrices are commonly used for
determining the relatedness of polypeptide sequences. The Blosum
matrices were created using a large database of trusted alignments
(the BLOCKS database), in which pairwise sequence alignments
related by less than some threshold percentage identity were
counted (Henikoff et al., Proc. Natl. Acad. Sci. USA,
89:10915-10919, 1992). A threshold of 90% identity was used for the
highly conserved target frequencies of the BLOSUM90 matrix. A
threshold of 65% identity was used for the BLOSUM65 matrix. Scores
of zero and above in the Blosum matrices are considered
"conservative substitutions" at the percentage identity selected.
The following table shows exemplary conservative amino acid
substitutions.
TABLE-US-00003 Very Highly- Highly Conserved Original Conserved
Substitutions (from the Conserved Substitutions Residue
Substitutions Blosum90 Matrix) (from the Blosum65 Matrix) Ala Ser
Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg Lys Gln, His, Lys Asn,
Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys, Ser, Thr Arg,
Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu,
Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg, Asn,
Asp, Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp,
Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln,
Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met,
Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val Lys
Arg; Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met Leu;
Ile Gln, Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met; Leu; Tyr
Leu, Trp, Tyr Ile, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr Ala,
Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala, Asn,
Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; Phe His, Phe, Trp His,
Phe, Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu, Met, Thr
[0109] In some examples, variants can have no more than 3, 5, 10,
15, 20, 25, 30, 40, 50, or 100 conservative amino acid changes
(such as very highly conserved or highly conserved amino acid
substitutions). In other examples, one or several hydrophobic
residues (such as Leu, Ile, Val, Met, Phe, or Trp) in a variant
sequence can be replaced with a different hydrophobic residue (such
as Leu, Ile, Val, Met, Phe, or Trp) to create a variant
functionally similar to the disclosed FAD2 and FAE1 proteins.
[0110] In one embodiment, variants may differ from the disclosed
sequences by alteration of the coding region to fit the codon usage
bias of the particular organism into which the molecule is to be
introduced. In other embodiments, the coding region may be altered
by taking advantage of the degeneracy of the genetic code to alter
the coding sequence such that, while the nucleotide sequence is
substantially altered, it nevertheless encodes a protein having an
amino acid sequence substantially similar to the disclosed FAD2 and
FAE1 proteins.
Camelina Sativa as an Allohexaploid Plant
[0111] The present inventors for the first time in the art
demonstrates that Camelina sativa is an allohexaploid plant.
[0112] While not wishing to be bound to any particular theory,
triplication of the C. sativa genome likely occurred through whole
genome duplication, either through autopolyploidization or through
allopolyploidization. An autopolyploidy event might have
triplicated a single diploid genome resulting in an autohexaploid
with a haploid genome of 18, 21, or 24 chromosomes. Given that C.
sativa has a chromosome count of n=20, chromosome splitting or
fusion could then have increased the chromosomes from 18 to 20, or
decreased the chromosomes from 21 or 24 to 20.
[0113] Alternatively, triplication of the C. sativa genome might
have resulted from two allopolyploidy events, resulting in first a
tetraploid then a hexaploid, similar to the origin of cultivated
wheat. According to this hypothesis, the three copies of each gene
diverged in different diploid genomes before converging through
polyploidy events. Taking into consideration the reported
chromosome counts of various Camelina species, the basal chromosome
number of the diploid parental species contributing to the C.
sativa haploid genome of 20 chromosomes could be 7+7+6 or 8+6+6.
The allopolyploid hypothesis is supported by the observation that
C. sativa demonstrates diploid inheritance (Gehringer, Friedt et
al. 2006; Lu. 2008), as would be expected for an allopolyploid
(Sybenga 1996). A hexaploid C. sativa could also be derived from
the combination of an autotetraploid and a diploid species if, in
an autopolyploidized genome, homologous chromosomes differentiated
so that the subsequent chromosome-specific pairing mimicked an
allopolyploid genome in its diploid inheritance patterns.
Regardless of its evolutionary path, the C. sativa genome appears
organized in three redundant and differentiated copies and can be
formally considered to be an allohexaploid.
[0114] Results from the inventors' phylogenetic analyses support a
history of duplication for both FAD2 and FAE1 in Camelina. For
FAD2, duplications were only recovered for C. sativa, C.
microcarpa, and C. rumelica. These data are consistent with genome
size data, which indicate that all three genomes are larger than C.
laxa and C. hispida, from which only a single FAD2 copy was
recovered. Taken together, the results suggest that C. sativa, C.
microcarpa, and C. rumelica are likely polyploids. Given the
slightly smaller genome size of C. rumelica, and the fact that only
two FAD2 copies were recovered from it, the C. rumelica sampled may
be tetraploid while C. saliva and C. microcarpa are hexaploid.
Interestingly, in both the FAD2 and FAE1 trees, one copy each of C.
rumelica and C. microcarpa are strongly supported as sister. Thus,
trees from these genes indicate that C. rumelica and C. microcarpa
are closely related. The various placement of C. microcarpa FAD2
and FAE1 copies can be explained if C. microcarpa is the result of
a hybridization event between C. rumelica and a currently
unsampled, and thus unidentified species of Camelina. Two of the
three copies of both FAD2 and FAE1 are identical, or nearly
identical, in C. sativa and C. microcarpa, suggesting that C.
sativa and C. microcarpa share a parental genome. Thus, the
inventors suggest that an unsampled Camelina species contributed
its genome to the hybrid formation of both C. saliva and C.
microcarpa. In the case of C. microcarpa, the hybridization event
likely involved C. rumelica. Given the chromosome count of n=6 for
C. rumelica, the other putative parent would be expected to have an
x=7 genome, and furthermore to be tetraploid at n=14. Such a cross
would result in the observed C. microcarpa genome, with chromosome
count n=20. Interestingly, C. hispida is the only species we
sampled with a chromosome count of n=7; however no strong
relationship between C. hispida and C. microcarpa is inferred in
either gene tree. However, a weak relationship between C. sativa
and C. hispida is inferred from the FAE1 tree, and thus the
possibility that C. hispida is involved in the polyploid formation
of C. sativa should be explored further.
[0115] The likely allohexaploid nature of the Camelina sativa
genome has multiple implications. Its vigor and adaptability to
marginal growth conditions may result at least in part from
polyploidy. Polyploids are thought to be more adaptable to new or
harsh environments, with the ability to expand into broader niches
than either progenitor (Brochmann, Brysting et al. 2004; Salmon
2005). Indeed, C. hispida and C. laxa, both of which are likely
diploids, are found only in Turkey, Iran, Armenia, and Azerbaijan,
while C. microcarpa and C. sativa are distributed throughout Asia,
Europe, and North Africa and are naturalized in North America
(GRIN; Akeroyd 1993). The mechanisms behind this increased
adaptability are not completely understood, but have been
attributed to heterosis, genetic and regulatory network
redundancies, and epigenetic factors (Comai 2005; Hegarty and
Hiscock 2008).
[0116] Allohexaploidy might also affect any potential manipulations
of the C. sativa genome, such as introgression of geimplasm or
induced mutations. Introgression of an exotic germplasm could be
facilitated by the type of polyploidy-dependent manipulations that
are possible in wheat, a potentially comparable allohexaploid (Gill
and Friebe 1998; Dubcovsky and Dvorak 2007). In addition,
polyploids have displayed excellent response to reverse genomics
approaches such as Targeting Induced Local Lesions in Genomes
(TILLING.RTM.) (Slade, Fuerstenberg et al. 2005; Cooper, Till et
al. 2008). As in wheat, any recessive induced mutations could be
masked by redundant homologous loci that have maintained function
(Stadler 1929; Swaminathan and Rao 1960). This implies that
multiple knockout alleles at different homologous sites can be
combined to achieve partial or complete suppression of a targeted
function (Muramatsu 1963; Li, Huang et al. 2008). We also expect
that single locus traits, whether transgenic or not, will display
diploid inheritance due to preferential intragenomic pairing.
Methods of Altering and/or Improving Camelina Seed Oil
Composition
[0117] In light of the discovery that Camelina is an allohexaploid
plant, the present invention provides methods of altering and/or
improving Camelina seed oil composition. As used herein, the term
"altering" refers to any change of fatty acid composition in the
seed oil, including but not limited to compound structure,
distribution, relative ratio, and yield, et al. The term
"improving" refers to any change in seed oil composition that makes
the seed oil composition better in one or more qualities for
industrial or nutritional applications. Such improvement includes,
but is not limited to, improved quality as meal, improved quality
as raw material to produce biofuel, biodiesel, lubricant, more
monounsaturated fatty acids and less polyunsaturated fatty acids,
increased stability, lower cloud point, less NOx emissions, reduced
trans-fatty acids, et al.
[0118] The quality of a biodiesel is greatly dependent upon its
composition (Conley S P, Tao B: Biodiesel quality: Is All biodiesel
Created Equal? Purdue University Extension; 2006). Polyunsaturated
fatty acid methyl esters (FAME) have been shown to
disproportionately increase oxidation of biodiesel. The
temperatures at which biodiesel forms crystals (the cloud point)
and at which it can no longer be poured (the pour point) are also
affected by composition: saturated FAMEs and long chain FAMEs
greatly increase cloud point and pour point. Biodiesel higher in
unsaturated FAMEs are therefore better in colder environments, but
have a lower oxidative stability than biodiesel higher in saturated
FAMEs. Polyunsaturated FAMEs have also been shown to result in
increased NOx emissions while long chain fatty acids result in a
biodiesel with too high of a distillation temperature by ASTM
standards. A biodiesel high in 18:1 and low in polyunsaturated
FAMEs and long chain FAMEs is thought to be the best compromise,
resulting in higher oxidative stability with a low enough cloud
point and a high enough cetane number to meet biodiesel standards
(ASTM D6751).
[0119] Meal is a significant byproduct of the extraction of the oil
from oilseeds for biofuel. To be able to take advantage of this
byproduct as a protein supplement for livestock is essential
economically for biofuel producers. In order for meal from a
particular oilseed to be included in livestock feed in the US, it
must be approved by the Association of American Feed Control
Officials (AAFCO). The approval takes into account feeding studies
in livestock and published studies on the quality of the meal and
formulates a definition for the meal that is included in the
annually updated AAFCO manual. Currently soybean meal is the best
source for animal feed because of its favorable amino acid content
and high digestibility. Another widely used meal comes from Canola,
an oilseed rape that has been bred to contain <2% erucic acid
(22:1) and <30 .mu.mol/g of glucosinolates. High amounts of
erucic acid have been linked to fatty deposits in the heart muscles
of animals and glucosinolates lend an unpalatable taste and confer
adverse effects on growth in animals. Camelina oil has about 1-4%
erucic acid, so the development of lines with consistently <2%
erucic acid is still desirable. Thus the identification of FAE1
mutants with reduced very long chain fatty acids (VLCFA) such as
22:1 is valuable for the potential to create Camelina varieties
having oil, and thus meal, with <2% erucic acid. Camelina meal
has been tested at least in poultry, goat, cattle (Pilgeram et al.,
Camelina sativa, A montana omega-3 and fuel crop, Issues in new
crops and new uses. 2007. J. Janick and A. Whipkey (eds.) and
turkeys (Frame et al., Use of Camelina sativa in the Diets of Young
Turkeys; J. Appl. Poult. Res. 16:381-386). ASHS Press, Alexandria,
Va.). Camelina meal can currently be included in the diets of
broiler chickens and feedlot beef cattle at no more than 10% (FDA,
November 2009). Future feeding studies may enable the expansion of
Camelina meal to swine, laying hens and dairy cattle.
[0120] In one embodiment, the methods relate to increasing
monounsaturated fatty acids (e.g., oleic acids (18:1)) level and/or
reducing polyunsaturated fatty acids level in the seed oil, wherein
the method comprises disrupting and/or altering one or more copies
of one or more Camelina fatty acids synthesis genes. In some
embodiments, one, two, or all three copies of Camelina FAD2 and/or
FAE1 genes are disrupted. For example, the methods comprise
utilizing one or more Camelina mutants in any one of the mutations
listed in Tables 7 to 12 described in Example 11.
[0121] In some embodiments, the methods related to increasing
monounsaturated fatty acids (e.g., oleic acids (18:1)) level and/or
decreasing very long chain fatty acids (>18 carbons), wherein
the methods comprise disrupting and/or altering one or more copies
of two or more Camelina fatty acids synthesis genes. In some
embodiments, one, two, or all three copies of Camelina FAD2 and
one, two, or all three FAE1 genes are disrupted.
[0122] In some embodiments, mutations in one or more copies of FAD2
genes and/or one or more copies of FAE1 genes described in the
Tables 7 to 12 are integrated together to create mutant plants with
double, triple, quadruple et al. mutations. Such mutants can be
created by classic breeding methods.
[0123] In some embodiments, mutations described in the Tables 7-12
can be integrated into Camelina cultivars other than Cs32 by
classic breeding methods, with or without the help of
marker-facilitated gene transfer methods.
[0124] In some embodiments, mutations described in the Tables 7-12
can be integrated into species closely related to Camelina sativa,
such as other species in the Brassicaceae family, such as Brassica
oleracea (cabbage, cauliflower, etc.), Brassica rapa (turnip,
Chinese cabbage, etc.), Brassica napus (rapeseed, etc.), Raphanus
sativus (common radish), Armoracia rusticana (horseradish),
Matthiola (stock), and many others, with or without the help of
marker-facilitated inter-cultivar gene transfer methods.
[0125] In one embodiment, mutants in Tables 7 to 12, wherein the
mutants are in evolutionarily conserved regions or sites can be
used to produce Camelina plants with improved or altered seed oil.
In one embodiment, mutants in Table 7 to 12, wherein the mutant is
due to nonsense mutation (premature stop codon), can be used to
produce Camelina plants with improved or altered seed oil.
[0126] In one embodiment, mutants in Tables 7 to 12, wherein the
mutants are not in evolutionarily conserved regions or sites, can
also be used to produce Camelina plants with improved or altered
seed oil. Non-limiting examples of improved seed oil are those
having increased oleic acid, increased fatty acids of C18 or less
(C.ltoreq.18), decreased very long chain fatty acid (C>18),
and/or decreased polyunsaturated fatty acids, in ratio and/or in
absolute weight. As used herein, the term "C.ltoreq.18" refers to a
chemical compound having not more than 18 carbons; as used herein,
the term C>18 refers to a chemical compound that has more than
18 carbons.
[0127] In other embodiments, amino acids in conserved domains or
sites of Camelina FAD2 or FAE1 proteins can be compared to FAD2 or
FAE1 orthologs in other species, e.g., closely related Brassicaceae
species, or plant species with known FAD/FAE sequences, which do
not contain mutations listed in Tables 7 to 12. Then, the FAD/FAE
genes in these related species can be substituted or deleted to
make mutants with reduced or abolished activity.
[0128] In one embodiment, the oleic acid level in the seed oil
produced from the Camelina plants of the present invention is
increased as compared to the same plants known in the prior art
(e.g., comparable wild type plant). For example, the level of oleic
acid in the seed oil is increased by about 1%, about 2%, about 3%,
about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about
10%, about 12%, about 14%, about 16%, about 18%, about 19%, about
20%, about 22%, about 24%, about 26%, about 28%, about 30%, about
32%, about 34%, about 36%, about 38%, about 40%, about 42%, about
44%, about 46%, about 48%, about 50%, about 52%, about 54%, about
56%, about 58%, about 59%, about 60%, about 62%, about 64%, about
66%, about 68%, about 70%, about 75%, about 80%, about 85%, about
90%, about 95%, about 100%, about 150%, about 200%, about 250%,
about 300%, about 350%, about 400%, about 450%, or about 500%.
[0129] In another embodiment, the oleic acid yield in the seed oil
produced per Camelina plant of the present invention is increased
as compared to the same plants known in the prior art (e.g.,
comparable wild type plant). As used herein, the term "yield"
refers to amount of one or more types of fatty acids produced per
plant, or per acre. For example, the yield of oleic acid in the
seed oil is increased by about 1%, about 2%, about 3%, about 4%,
about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about
12%, about 14%, about 16%, about 18%, about 19%, about 20%, about
22%, about 24%, about 26%, about 28%, about 30%, about 32%, about
34%, about 36%, about 38%, about 40%, about 42%, about 44%, about
46%, about 48%, about 50%, about 52%, about 54%, about 56%, about
58%, about 59%, about 60%, about 62%, about 64%, about 66%, about
68%, about 70%, about 75%, about 80%, about 85%, about 90%, about
95%, about 100%, about 150%, about 200%, about 250%, about 300%,
about 350%, about 400%, about 450%, or about 500%.
[0130] In another embodiment, the polyunsaturated fatty acid level
and/or yield in the seed oil produced from the Camelina plants of
the present invention is decreased as compared to the same plants
known in the prior art (e.g., comparable wild type plant). For
example, the level and/or yield of polyunsaturated fatty acid in
the seed oil is decreased by about 1%, about 2%, about 3%, about
4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,
about 12%, about 14%, about 16%, about 18%, about 19%, about 20%,
about 22%, about 24%, about 26%, about 28%, about 30%, about 32%,
about 34%, about 36%, about 38%, about 40%, about 42%, about 44%,
about 46%, about 48%, about 50%, about 52%, about 54%, about 56%,
about 58%, about 59%, about 60%, about 62%, about 64%, about 66%,
about 68%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, about 100%, about 150%, about 200%, about 250%, about
300%, about 350%, about 400%, about 450%, or about 500%.
[0131] In another embodiment, the very long chain fatty acid
(C>18) level and/or yield in the seed oil produced from the
Camelina plants of the present invention is decreased as compared
to the same plants known in the prior art (e.g., comparable wild
type plant). For example, the level and/or yield of very long chain
fatty acid in the seed oil is decreased by about 1%, about 2%,
about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about
9%, about 10%, about 12%, about 14%, about 16%, about 18%, about
19%, about 20%, about 22%, about 24%, about 26%, about 28%, about
30%, about 32%, about 34%, about 36%, about 38%, about 40%, about
42%, about 44%, about 46%, about 48%, about 50%, about 52%, about
54%, about 56%, about 58%, about 59%, about 60%, about 62%, about
64%, about 66%, about 68%, about 70%, about 75%, about 80%, about
85%, about 90%, about 95%, about 100%, about 150%, about 200%,
about 250%, about 300%, about 350%, about 400%, about 450%, or
about 500%.
[0132] In another embodiment, the fatty acids of C18 or less level
and/or yield in the seed oil produced from the Camelina plants of
the present invention is increased as compared to the same plants
known in the prior art (e.g., comparable wild type plant). For
example, the level and/or yield of fatty acids of C18 or less in
the seed oil is increased by about 1%, about 2%, about 3%, about
4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,
about 12%, about 14%, about 16%, about 18%, about 19%, about 20%,
about 22%, about 24%, about 26%, about 28%, about 30%, about 32%,
about 34%, about 36%, about 38%, about 40%, about 42%, about 44%,
about 46%, about 48%, about 50%, about 52%, about 54%, about 56%,
about 58%, about 59%, about 60%, about 62%, about 64%, about 66%,
about 68%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, about 100%, about 150%, about 200%, about 250%, about
300%, about 350%, or about 400%.
[0133] Molecular markers are used for the visualization of
differences in nucleic acid sequences. This visualization is
possible due to DNA-DNA hybridization techniques (RFLP) and/or due
to techniques using the polymerase chain reaction (e.g. STS,
microsatellites, AFLP, SNP, IMP et al.). All differences between
two parental genotypes will segregate in a mapping population based
on the cross of these parental genotypes. The segregation of the
different markers may be compared and recombination frequencies can
be calculated. The recombination frequencies of molecular markers
on different chromosomes is generally 50%. Between molecular
markers located on the same chromosome the recombination frequency
depends on the distance between the markers. A low recombination
frequency corresponds to a low distance between markers on a
chromosome. Comparing all recombination frequencies will result in
the most logical order of the molecular markers on the chromosomes.
This most logical order can be depicted in a linkage map.
[0134] Molecular markers for the present invention, for example,
can be generated by analyzing progeny of a cross between e.g., Cs32
cultivar to another Camelina species, e.g., Camelina microcarpa.
The present inventors have generated such progeny and more Inter
MITE Polymorphisms (IMP) markers can be generated following the
procedures outlined in the present application. IMP markers are
developed by and exclusive to DNA LandMarks Inc. IMP markers are
based on Miniature Inverted-repeat Transposable Elements (MITEs),
which are short interspersed DNA transposons with terminal inverted
repeats (TIRs). They are small in size (<500 bp), conserved
TIRs, high A+T content, and consist of several distinct families
such as Tourist-like, Stowaway-like. They present in plants, fungi,
vertebrates, fishes, insects. In plants, they are highly associated
with genes (flanking regions, introns). They are also abundant in
plants (several thousand copies per genome). IMP markers have many
unique advantages:
[0135] Naturally multiplexed--Greatly lowers cost/data point
[0136] Reliable--PCR based, reproducible results
[0137] Portable--Markers are cross-applicable in all crops
[0138] Practical--Useful in a variety of marker-assisted breeding
functions
Similarly, Cs32 can be crossed to other species in the Brassicaceae
family to generate molecular markers for further applications.
[0139] In some other embodiments, one, two, or all three copies of
Camelina FAD2 and/or FAE1 genes, and one, two, or all three copies
of other non-FAD2, non-FAE1 fatty acid synthesis genes are
disrupted. As used herein, the phrase "non-FAD, non-FAE fatty acid
synthesis genes" refers to polynucleotides encoding polypeptides
that are involved in plant fatty acid synthesis, but share less
than 95% identity to FAD2 or FAE1 polypeptide disclosed in the
present invention. In still some embodiments, one, two, or all
three copies of Camelina FAD2 and/or FAE1 genes are disrupted,
while one or more non-FAD, non-FAE fatty acid synthesis genes are
overexpressed. In still more embodiments, one, two, or all three
copies of Camelina FAD2 and/or FAE1 genes are disrupted, while one
or more non-fatty-acid-synthesis genes are overexpressed and/or
disrupted. As used herein, the phrase "non-fatty-acid-synthesis
genes" refers to polynucleotides encoding polypeptides that are not
directly involved in the synthesis of fatty acids.
[0140] According to the present invention, one skilled in the art
will be able to pick preferred target genes and decide when
disruption or overexpression is needed to achieve certain goals,
e.g., an induction or reduction of certain fatty acids composition,
based on the plant fatty acid metabolic pathways and metabolic
analysis tools known in the art (e.g., MetaCyc and AraCyc database,
see Zhang et al., Plant Physiology, 2005, 138:27-37). For example,
one skilled in the art would be able to combine FAD2 and/or FAE1
loss-of-function mutants (e.g., mutants with reduced, or abolished
FAD2 and/or FAE1 protein activity), FAD2 and/or FAE1
gain-of-function mutants (e.g., mutants with altered or increased
FAD2 and/or FAE1 protein activity), or FAD2 and/or FAE1
overexpression with overexpression or disruption of non-FAD,
non-FAE fatty acid genes to modulate the fatty acid synthesis in a
plant. While not wishing to be bound by any particular theory,
knock-down of FAD2 can potentially lower 18:2 fatty acid;
knock-down of FAD3 can potentially lower 18:3 fatty acid;
overexpressing plastidial enzyme .DELTA.9 will give higher 18:1;
knock-down of both FAD2 and FAD3 will contribute to a higher cloud
point of the oil; knock-down of thioesterases (e.g., FAT A and/or
FAT B) will lower the amount of 16:0 fatty acids; knock-down of
fatty acid elongase (FAE) will lower the amount of long-chain fatty
acids; a dominant negative KRP protein or a REV protein can
increase cell size and thus increase oil production (see US
2008/263727 and US 2007/056058, incorporated by reference in their
entireties).
[0141] In addition, using the compositions and methods of the
present invention, one skilled in the art will be able to combine
disruption of FAD2 and/or FAE1 genes with other mutants and/or
transgenes which can generally improve plant health, plant biomass,
plant resistance to biotic and abiotic factors, plant yields,
wherein the final preferred fatty acid production is increased.
Such mutants and/or transgenes include, but are not limited to,
cell cycle controlling genes, cell size controlling genes, cell
division controlling genes, pathogen resistance genes, and genes
controlling plant traits related to seed yield, which are well
known to one skilled in the art (e.g., REV genes, KRP genes).
[0142] Methods of disrupting and/or altering a target gene have
been known to one skilled in the art. These methods include, but
are not limited to, mutagenesis (e.g., chemical mutagenesis,
radiation mutagenesis, transposon mutagenesis, insertional
mutagenesis, signature tagged mutagenesis, site-directed
mutagenesis, and natural mutagenesis), knock-outs/knock-ins,
antisense and RNA interference. Various types of mutagenesis can be
used to produce and/or isolate variant nucleic acids that encode
for protein molecules and/or to further modify/mutate the proteins
of the present invention. They include but are not limited to
site-directed, random point mutagenesis, homologous recombination
(DNA shuffling), mutagenesis using uracil containing templates,
oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA
mutagenesis, mutagenesis using gapped duplex DNA or the like.
Additional suitable methods include point mismatch repair,
mutagenesis using repair-deficient host strains,
restriction-selection and restriction-purification, deletion
mutagenesis, mutagenesis by total gene synthesis, double-strand
break repair, and the like. Mutagenesis, e.g., involving chimeric
constructs, is also included in the present invention. In one
embodiment, mutagenesis can be guided by known information of the
naturally occurring molecule or altered or mutated naturally
occurring molecule, e.g., sequence, sequence comparisons, physical
properties, crystal structure or the like. For more information of
mutagenesis in plants, such as agents, protocols, see Acquaah et
al. (Principles of plant genetics and breeding, Wiley-Blackwell,
2007, ISBN 1405136464, 9781405136464, which is herein incorporated
by reference in its entity). Methods of disrupting plant genes
using RNA interference is described later in the specification.
[0143] The present invention provides methods of producing Camelina
seed oil containing altered and/or increased levels of oleic acid
(18:1), and/or altered or reduced levels of polyunsaturated fatty
acids, and/or decreased very long chain fatty acids (C>18). Such
methods comprise utilizing the Camelina plants comprising the
chimeric genes as described above, or Camelina plants with
disrupted FAD2 and/or FAE1 genes as described above.
[0144] The present invention also provides methods of breeding
Camelina species producing altered levels of fatty acids in the
seed oil and/or meal. In one embodiment, such methods comprise
i) making a cross between the Camelina mutants with mutations as
described above to a second Camelina species to make F1 plants; ii)
backcrossing said F1 plants to said second Camelina species; iii)
repeating backcrossing step until said mutations are integrated
into the genome of said second Camelina species. Optionally, such
method can be facilitated by molecular markers.
[0145] The present invention provides methods of breeding species
close to Camelina sativa, wherein said species produces altered
levels of fatty acids in the seed oil and/or meal. In one
embodiment, such methods comprise
i) making a cross between the Camelina mutants with mutations as
described above to a species close to Camelina sativa to make F1
plants; ii) backcrossing said F1 plants to said species that is
close to Camelina sativa; iii) repeating backcrossing step until
said mutations are integrated into the genome of said species that
is close to Camelina sativa. Special techniques (e.g., somatic
hybridization) may be necessary in order to successfully transfer a
gene from Camelina sativa to another species and/or genus, such as
to B. oleracea. Optionally, such method can be facilitated by
molecular markers.
Plant Transformation
[0146] The present polynucleotides of the present invention can be
transformed into a Camelina plant, or other plants.
[0147] The most common method for the introduction of new genetic
material into a plant genome involves the use of living cells of
the bacterial pathogen Agrobacterium tumefaciens to literally
inject a piece of DNA, called transfer or T-DNA, into individual
plant cells (usually following wounding of the tissue) where it is
targeted to the plant nucleus for chromosomal integration. There
are numerous patents governing Agrobacterium mediated
transformation and particular DNA delivery plasmids designed
specifically for use with Agrobacterium--for example, U.S. Pat. No.
4,536,475, EP0265556, EP0270822, WO8504899, WO8603516, U.S. Pat.
No. 5,591,616, EP0604662, EP0672752, WO8603776, WO9209696,
WO9419930, WO9967357, U.S. Pat. No. 4,399,216, WO8303259, U.S. Pat.
No. 5,731,179, EP068730, WO9516031, U.S. Pat. No. 5,693,512, U.S.
Pat. No. 6,051,757 and EP904362A1. Agrobacterium-mediated plant
transformation involves as a first step the placement of DNA
fragments cloned on plasmids into living Agrobacterium cells, which
are then subsequently used for transformation into individual plant
cells. Agrobacterium-mediated plant transformation is thus an
indirect plant transformation method. Methods of
Agrobacterium-mediated plant transformation that involve using
vectors with no T-DNA are also well known to those skilled in the
art and can have applicability in the present invention. See, for
example, U.S. Pat. No. 7,250,554, which utilizes P-DNA instead of
T-DNA in the transformation vector.
[0148] Direct plant transformation methods using DNA have also been
reported. The first of these to be reported historically is
electroporation, which utilizes an electrical current applied to a
solution containing plant cells (M. E. Fromm et al., Nature, 319,
791 (1986); H. Jones et al., Plant Mol. Biol., 13, 501 (1989) and
H. Yang et al., Plant Cell Reports, 7, 421 (1988). Another direct
method, called "biolistic bombardment", uses ultrafine particles,
usually tungsten or gold, that are coated with DNA and then sprayed
onto the surface of a plant tissue with sufficient force to cause
the particles to penetrate plant cells, including the thick cell
wall, membrane and nuclear envelope, but without killing at least
some of them (U.S. Pat. No. 5,204,253, U.S. Pat. No. 5,015,580). A
third direct method uses fibrous forms of metal or ceramic
consisting of sharp, porous or hollow needle-like projections that
literally impale the cells, and also the nuclear envelope of cells.
Both silicon carbide and aluminium borate whiskers have been used
for plant transformation (Mizuno et al., 2004; Petolino et al.,
2000; U.S. Pat. No. 5,302,523 US Application 20040197909) and also
for bacterial and animal transformation (Kaepler et al., 1992;
Raloff, 1990; Wang, 1995). There are other methods reported, and
undoubtedly, additional methods will be developed. However, the
efficiencies of each of these indirect or direct methods in
introducing foreign DNA into plant cells are invariably extremely
low, making it necessary to use some method for selection of only
those cells that have been transformed, and further, allowing
growth and regeneration into plants of only those cells that have
been transformed.
[0149] For efficient plant transformation, a selection method must
be employed such that whole plants are regenerated from a single
transformed cell and every cell of the transformed plant carries
the DNA of interest. These methods can employ positive selection,
whereby a foreign gene is supplied to a plant cell that allows it
to utilize a substrate present in the medium that it otherwise
could not use, such as mannose or xylose (for example, refer U.S.
Pat. No. 5,767,378; U.S. Pat. No. 5,994,629). More typically,
however, negative selection is used because it is more efficient,
utilizing selective agents such as herbicides or antibiotics that
either kill or inhibit the growth of nontransformed plant cells and
reducing the possibility of chimeras. Resistance genes that are
effective against negative selective agents are provided on the
introduced foreign DNA used for the plant transformation. For
example, one of the most popular selective agents used is the
antibiotic kanamycin, together with the resistance gene neomycin
phosphotransferase (nptII), which confers resistance to kanamycin
and related antibiotics (see, for example, Messing & Vierra,
Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)).
However, many different antibiotics and antibiotic resistance genes
can be used for transformation purposes (refer U.S. Pat. No.
5,034,322, U.S. Pat. No. 6,174,724 and U.S. Pat. No. 6,255,560). In
addition, several herbicides and herbicide resistance genes have
been used for transformation purposes, including the bar gene,
which confers resistance to the herbicide phosphinothricin (White
et al., Nuel Acids Res 18: 1062 (1990), Spencer et al., Theor Appl
Genet 79: 625-631(1990), U.S. Pat. No. 4,795,855, U.S. Pat. No.
5,378,824 and U.S. Pat. No. 6,107,549). In addition, the dhfr gene,
which confers resistance to the anticancer agent methotrexate, has
been used for selection (Bourouis et al., EMBO J. 2(7): 1099-1104
(1983).
[0150] The expression control elements used to regulate the
expression of a given protein can either be the expression control
element that is normally found associated with the coding sequence
(homologous expression element) or can be a heterologous expression
control element. A variety of homologous and heterologous
expression control elements are known in the art and can readily be
used to make expression units for use in the present invention.
Transcription initiation regions, for example, can include any of
the various opine initiation regions, such as octopine, mannopine,
nopaline and the like that are found in the Ti plasmids of
Agrobacterium tumefaciens. Alternatively, plant viral promoters can
also be used, such as the cauliflower mosaic virus 19S and 35S
promoters (CaMV 19S and CaMV 35S promoters, respectively) to
control gene expression in a plant (U.S. Pat. Nos. 5,352,605;
5,530,196 and 5,858,742 for example). Enhancer sequences derived
from the CaMV can also be utilized (U.S. Pat. Nos. 5,164,316;
5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and
5,858,742 for example). Lastly, plant promoters such as prolifera
promoter, fruit specific promoters, Ap3 promoter, heat shock
promoters, seed specific promoters, etc. can also be used.
[0151] Either a gamete-specific promoter, a constitutive promoter
(such as the CaMV or Nos promoter), an organ-specific promoter
(such as the E8 promoter from tomato), or an inducible promoter is
typically ligated to the protein or antisense encoding region using
standard techniques known in the art. The expression unit may be
further optimized by employing supplemental elements such as
transcription terminators and/or enhancer elements. For example,
the 5' introns of FAD2 gene in sesame have been demonstrated to
increase and/or regulate expression of certain genes (Kim et al.
2006. Mol Genet Genomics 276(4): 351-68). Thus, the 5' intron
sequences of the FAD2 genes of the present invention can be used to
increase expression of either a FAD2 or a non-FAD2 gene. The
expression cassette can comprise, for example, a seed-specific
promoter (e.g. the phaseolin promoter (U.S. Pat. No. 5,504,200).
The term "seed-specific promoter", means that a gene expressed
under the control of the promoter is predominantly expressed in
plant seeds with no or no substantial expression, typically less
than 10% of the overall expression level, in other plant tissues.
Seed specific promoters have been well known in the art, for
example, U.S. Pat. Nos. 5,623,067, 5,717,129, 6,403,371, 6,566,584,
6,642,437, 6,777,591, 7,081,565, 7,157,629, 7,192,774, 7,405,345,
7,554,006, 7,589,252, 7,595,384, 7,619,135, 7,642,346, and US
Application Publication Nos. 20030005485, 20030172403, 20040088754,
20040255350, 20050125861, 20050229273, 20060191044, 20070022502,
20070118933, 20070199098, 20080313771, and 20090100551.
[0152] Thus, for expression in plants, the expression units will
typically contain, in addition to the protein sequence, a plant
promoter region, a transcription initiation site and a
transcription termination sequence. Unique restriction enzyme sites
at the 5' and 3' ends of the expression unit are typically included
to allow for easy insertion into a pre-existing vector.
[0153] In the construction of heterologous promoter/structural gene
or antisense combinations, the promoter is preferably positioned
about the same distance from the heterologous transcription start
site as it is from the transcription start site in its natural
setting. As is known in the art, however, some variation in this
distance can be accommodated without loss of promoter function.
[0154] In addition to a promoter sequence, the expression cassette
can also contain a transcription termination region downstream of
the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes. If the
mRNA encoded by the structural gene is to be efficiently processed,
DNA sequences which direct polyadenylation of the RNA are also
commonly added to the vector construct. Polyadenylation sequences
include, but are not limited to the Agrobacterium octopine synthase
signal (Gielen et al., EMBO J 3:835-846 (1984)) or the nopaline
synthase signal (Depicker et al., Mol. and Appl. Genet. 1:561-573
(1982)). The resulting expression unit is ligated into or otherwise
constructed to be included in a vector that is appropriate for
higher plant transformation. One or more expression units may be
included in the same vector. The vector will typically contain a
selectable marker gene expression unit by which transformed plant
cells can be identified in culture. Usually, the marker gene will
encode resistance to an antibiotic, such as G418, hygromycin,
bleomycin, kanamycin, or gentamicin or to an herbicide, such as
glyphosate (Round-Up) or glufosinate (BASTA) or atrazine.
Replication sequences, of bacterial or viral origin, are generally
also included to allow the vector to be cloned in a bacterial or
phage host; preferably a broad host range for prokaryotic origin of
replication is included. A selectable marker for bacteria may also
be included to allow selection of bacterial cells bearing the
desired construct. Suitable prokaryotic selectable markers include
resistance to antibiotics such as ampicillin, kanamycin or
tetracycline. Other DNA sequences encoding additional functions may
also be present in the vector, as is known in the art. For
instance, in the case of Agrobacterium transformations, T-DNA
sequences will also be included for subsequent transfer to plant
chromosomes.
[0155] To introduce a desired gene or set of genes by conventional
methods requires a sexual cross between two lines, and then
repeated back-crossing between hybrid offspring and one of the
parents until a plant with the desired characteristics is obtained.
This process, however, is restricted to plants that can sexually
hybridize, and genes in addition to the desired gene will be
transferred.
[0156] Recombinant DNA techniques allow plant researchers to
circumvent these limitations by enabling plant geneticists to
identify and clone specific genes for desirable traits, such as
improved fatty acid composition, and to introduce these genes into
already useful varieties of plants. Once the foreign genes have
been introduced into a plant, that plant can then be used in
conventional plant breeding schemes (e.g., pedigree breeding,
single-seed-descent breeding schemes, reciprocal recurrent
selection) to produce progeny which also contain the gene of
interest.
[0157] Genes can be introduced in a site directed fashion using
homologous recombination. Homologous recombination permits
site-specific modifications in endogenous genes and thus inherited
or acquired mutations may be corrected, and/or novel alterations
may be engineered into the genome. Homologous recombination and
site-directed integration in plants are discussed in, for example,
U.S. Pat. Nos. 5,451,513; 5,501,967 and 5,527,695.
[0158] Methods of producing transgenic plants are well known to
those of ordinary skill in the art. Transgenic plants can now be
produced by a variety of different transformation methods
including, but not limited to, electroporation; microinjection;
microprojectile bombardment, also known as particle acceleration or
biolistic bombardment; viral-mediated transformation; and
Agrobacterium-mediated transformation. See, for example, U.S. Pat.
Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318;
5,641,664; 5,736,369 and 5,736369; International Patent Application
Publication Nos. WO2002/038779 and WO/2009/117555; Lu et al.,
(Plant Cell Reports, 2008, 27:273-278); Watson et al., Recombinant
DNA, Scientific American Books (1992); Hinchee et al., Bio/Tech.
6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988);
Toriyama et al., Bio/Tech. 6:1072-1074 (1988); Fromm et al.,
Bio/Tech. 8:833-839 (1990); Mullins et al., Bio/Tech. 8:833-839
(1990); Hiei et al., Plant Molecular Biology 35:205-218 (1997);
Ishida et al., Nature Biotechnology 14:745-750 (1996); Zhang et
al., Molecular Biotechnology 8:223-231 (1997); Ku et al., Nature
Biotechnology 17:76-80 (1999); and, Raineri et al., Bio/Tech.
8:33-38 (1990)), each of which is expressly incorporated herein by
reference in their entirety.
[0159] Agrobacterium tumefaciens is a naturally occurring bacterium
that is capable of inserting its DNA (genetic information) into
plants, resulting in a type of injury to the plant known as crown
gall. Most species of plants can now be transformed using this
method, including cucurbitaceous species.
[0160] Microprojectile bombardment is also known as particle
acceleration, biolistic bombardment, and the gene gun
(Biolistic.RTM. Gene Gun). The gene gun is used to shoot pellets
that are coated with genes (e.g., for desired traits) into plant
seeds or plant tissues in order to get the plant cells to then
express the new genes. The gene gun uses an actual explosive (.22
caliber blank) to propel the material. Compressed air or steam may
also be used as the propellant. The Biolistic.RTM. Gene Gun was
invented in 1983-1984 at Cornell University by John Sanford, Edward
Wolf, and Nelson Allen. It and its registered trademark are now
owned by E. I. du Pont de Nemours and Company. Most species of
plants have been transformed using this method.
[0161] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome,
although multiple copies are possible. Such transgenic plants can
be referred to as being hemizygous for the added gene. A more
accurate name for such a plant is an independent segregant, because
each transformed plant represents a unique T-DNA integration event
(U.S. Pat. No. 6,156,953). A transgene locus is generally
characterized by the presence and/or absence of the transgene. A
heterozygous genotype in which one allele corresponds to the
absence of the transgene is also designated hemizygous (U.S. Pat.
No. 6,008,437).
Breeding Methods
[0162] Classic breeding methods can be included in the present
invention to introduce one or more mutations of the present
invention into other Camelina varieties, or other close-related
species of the Brassicaceae family that are compatible to be
crossed with Camelina. In one embodiment, the mutations are on the
FAD2 A, FAD2 B, and/or FAD2 C genes. In one embodiment, the
mutations are on the FAE1 A, FAE1 B, and/or FAE1 C genes. In one
embodiment, the mutations are on any FAD2 gene and/or any FAE1
gene.
[0163] Open-Pollinated Populations.
[0164] The improvement of open-pollinated populations of such crops
as rye, many maizes and sugar beets, herbage grasses, legumes such
as alfalfa and clover, and tropical tree crops such as cacao,
coconuts, oil palm and some rubber, depends essentially upon
changing gene-frequencies towards fixation of favorable alleles
while maintaining a high (but far from maximal) degree of
heterozygosity. Uniformity in such populations is impossible and
trueness-to-type in an open-pollinated variety is a statistical
feature of the population as a whole, not a characteristic of
individual plants. Thus, the heterogeneity of open-pollinated
populations contrasts with the homogeneity (or virtually so) of
inbred lines, clones and hybrids.
[0165] Population improvement methods fall naturally into two
groups, those based on purely phenotypic selection, normally called
mass selection, and those based on selection with progeny testing.
Interpopulation improvement utilizes the concept of open breeding
populations; allowing genes to flow from one population to another.
Plants in one population (cultivar, strain, ecotype, or any
germplasm source) are crossed either naturally (e.g., by wind) or
by hand or by bees (commonly Apis mellifera L. or Megachile
rotundata F.) with plants from other populations. Selection is
applied to improve one (or sometimes both) population(s) by
isolating plants with desirable traits from both sources.
[0166] There are several primary methods of open-pollinated
population improvement. First, there is the situation in which a
population is changed en masse by a chosen selection procedure. The
outcome is an improved population that is indefinitely propagable
by random-mating within itself in isolation. Second, the synthetic
variety attains the same end result as population improvement but
is not itself propagable as such; it has to be reconstructed from
parental lines or clones. Third, a method used in plant species
that are largely self pollinated in nature, such as soybeans,
wheat, rice, safflower, camelina and others is pedigree selection.
In this situation, crosses are made and individual plants and lines
from individual plants are selected for desired traits. These lines
are thn advanced as genetically homogeneous varieties. Since the
individuals are largely self pollinated these lines are analogous
to an inbred line with favorable agronomic characteristics. These
plant breeding procedures for improving open-pollinated populations
are well known to those skilled in the art and comprehensive
reviews of breeding procedures routinely used for improving
cross-pollinated plants are provided in numerous texts and
articles, including: Allard, Principles of Plant Breeding, John
Wiley & Sons, Inc. (1960); Simmonds, Principles of Crop
Improvement, Longman Group Limited (1979); Hallauer and Miranda,
Quantitative Genetics in Maize Breeding, Iowa State University
Press (1981); and, Jensen, Plant Breeding Methodology, John Wiley
& Sons, Inc. (1988).
[0167] Mass Selection.
[0168] In mass selection, desirable individual plants are chosen,
harvested, and the seed composited without progeny testing to
produce the following generation. Since selection is based on the
maternal parent only, and there is no control over pollination,
mass selection amounts to a form of random mating with selection.
As stated above, the purpose of mass selection is to increase the
proportion of superior genotypes in the population.
[0169] Synthetics.
[0170] A synthetic variety is produced by crossing inter se a
number of genotypes selected for good combining ability in all
possible hybrid combinations, with subsequent maintenance of the
variety by open pollination. Whether parents are (more or less
inbred) seed-propagated lines, as in some sugar beet and beans
(Vicia) or clones, as in herbage grasses, clovers and alfalfa,
makes no difference in principle. Parents are selected on general
combining ability, sometimes by test crosses or toperosses, more
generally by polycrosses. Parental seed lines may be deliberately
inbred (e.g. by selfing or sib crossing). However, even if the
parents are not deliberately inbred, selection within lines during
line maintenance will ensure that some inbreeding occurs. Clonal
parents will, of course, remain unchanged and highly
heterozygous.
[0171] Whether a synthetic can go straight from the parental seed
production plot to the farmer or must first undergo one or two
cycles of multiplication depends on seed production and the scale
of demand for seed. In practice, grasses and clovers are generally
multiplied once or twice and are thus considerably removed from the
original synthetic.
[0172] While mass selection is sometimes used, progeny testing is
generally preferred for polycrosses, because of their operational
simplicity and obvious relevance to the objective, namely
exploitation of general combining ability in a synthetic.
[0173] The number of parental lines or clones that enter a
synthetic vary widely. In practice, numbers of parental lines range
from 10 to several hundred, with 100-200 being the average. Broad
based synthetics formed from 100 or more clones would be expected
to be more stable during seed multiplication than narrow based
synthetics.
[0174] Pedigreed Varieties.
[0175] A pedigreed variety is a superior genotype developed from
selection of individual plants out of a segregating population
followed by propagation and seed increase of self pollinated
offspring and careful testing of the genotype over several
generations. This is an open pollinated method that works well with
naturally self pollinating species. This method can be used in
combination with mass selection in variety development. Variations
in pedigree and mass selection in combination are the most common
methods for generating varieties in self pollinated crops.
[0176] Hybrids.
[0177] A hybrid is an individual plant resulting from a cross
between parents of differing genotypes. Commercial hybrids are now
used extensively in many crops, including corn (maize), sorghum,
sugarbeet, sunflower and broccoli. Hybrids can be formed in a
number of different ways, including by crossing two parents
directly (single cross hybrids), by crossing a single cross hybrid
with another parent (three-way or triple cross hybrids), or by
crossing two different hybrids (four-way or double cross
hybrids).
[0178] Strictly speaking, most individuals in an out breeding
(i.e., open-pollinated) population are hybrids, but the term is
usually reserved for cases in which the parents are individuals
whose genomes are sufficiently distinct for them to be recognized
as different species or subspecies. Hybrids may be fertile or
sterile depending on qualitative and/or quantitative differences in
the genomes of the two parents. Heterosis, or hybrid vigor, is
usually associated with increased heterozygosity that results in
increased vigor of growth, survival, and fertility of hybrids as
compared with the parental lines that were used to form the hybrid.
Maximum heterosis is usually achieved by crossing two genetically
different, highly inbred lines.
[0179] The production of hybrids is a well-developed industry,
involving the isolated production of both the parental lines and
the hybrids which result from crossing those lines. For a detailed
discussion of the hybrid production process, see, e.g., Wright,
Commercial Hybrid Seed Production 8:161-176, In Hybridization of
Crop Plants.
RNA Interference (RNAi)
[0180] RNA interference (RNAi) is the process of sequence-specific,
post-transcriptional gene silencing or transcriptional gene
silencing in animals and plants, initiated by double-stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene. The
preferred RNA effector molecules useful in this invention must be
sufficiently distinct in sequence from any host polynucleotide
sequences for which function is intended to be undisturbed after
any of the methods of this invention are performed. Computer
algorithms may be used to define the essential lack of homology
between the RNA molecule polynucleotide sequence and host,
essential, normal sequences.
[0181] The term "dsRNA" or "dsRNA molecule" or "double-strand RNA
effector molecule" refers to an at least partially double-strand
ribonucleic acid molecule containing a region of at least about 19
or more nucleotides that are in a double-strand conformation. The
double-stranded RNA effector molecule may be a duplex
double-stranded RNA foamed from two separate RNA strands or it may
be a single RNA strand with regions of self-complementarity capable
of assuming an at least partially double-stranded hairpin
conformation (i.e., a hairpin dsRNA or stem-loop dsRNA). In various
embodiments, the dsRNA consists entirely of ribonucleotides or
consists of a mixture of ribonucleotides and deoxynucleotides, such
as RNA/DNA hybrids. The dsRNA may be a single molecule with regions
of self-complementarity such that nucleotides in one segment of the
molecule base pair with nucleotides in another segment of the
molecule. In one aspect, the regions of self-complementarity are
linked by a region of at least about 3-4 nucleotides, or about 5,
6, 7, 9 to 15 nucleotides or more, which lacks complementarity to
another part of the molecule and thus remains single-stranded
(i.e., the "loop region"). Such a molecule will assume a partially
double-stranded stem-loop structure, optionally, with short single
stranded 5' and/or 3' ends. In one aspect the regions of
self-complementarity of the hairpin dsRNA or the double-stranded
region of a duplex dsRNA will comprise an Effector Sequence and an
Effector Complement (e.g., linked by a single-stranded loop region
in a hairpin dsRNA). The Effector Sequence or Effector Strand is
that strand of the double-stranded region or duplex which is
incorporated in or associates with RISC. In one aspect the
double-stranded RNA effector molecule will comprise an at least 19
contiguous nucleotide effector sequence, preferably 19 to 29, 19 to
27, or 19 to 21 nucleotides, which is a reverse complement to the
RNA of Camelina genes (e.g., FAD2 and FAE1 genes), or an opposite
strand replication intermediate. In one embodiment, said
double-stranded RNA effector molecules are provided by providing to
a Camelina plant, plant tissue, or plant cell an expression
construct comprising one or more double-stranded RNA effector
molecules. In one embodiment, the expression construct comprises a
double-strand RNA derived from any one of SEQ ID NOs 1-6 and SEQ ID
NOs 45-63. In other embodiments, the expression construct comprises
a double-strand RNA derived from more than one sequences of SEQ ID
NOs 1-6 and SEQ ID NOs 45-63. In further embodiments, the
expression construct comprises a double-strand RNA derived from
more than one sequences of SEQ ID NOs 1-6 and SEQ ID NOs 45-63, and
one or more other genes involved in plant fatty acid synthesis. One
skilled in the art will be able to design suitable double-strand
RNA effector molecule based on the nucleotide sequences of Camelina
FAD2 and FAE1 in the present invention and other Camelina fatty
acid synthesis genes known in the art.
[0182] In some embodiments, the dsRNA effector molecule of the
invention is a "hairpin dsRNA", a "dsRNA hairpin", "short-hairpin
RNA" or "shRNA", i.e., an RNA molecule of less than approximately
400 to 500 nucleotides (nt), or less than 100 to 200 nt, in which
at least one stretch of at least 15 to 100 nucleotides (e.g., 17 to
50 nt, 19 to 29 nt) is based paired with a complementary sequence
located on the same RNA molecule (single RNA strand), and where
said sequence and complementary sequence are separated by an
unpaired region of at least about 4 to 7 nucleotides (or about 9 to
about 15 nt, about 15 to about 100 nt, about 100 to about 1000 nt)
which forms a single-stranded loop above the stem structure created
by the two regions of base complementarity. The shRNA molecules
comprise at least one stem-loop structure comprising a
double-stranded stem region of about 17 to about 500 bp; about 17
to about 50 bp; about 40 to about 100 bp; about 18 to about 40 bp;
or from about 19 to about 29 bp; homologous and complementary to a
target sequence to be inhibited; and an unpaired loop region of at
least about 4 to 7 nucleotides, or about 9 to about 15 nucleotides,
about 15 to about 100 nt, about 250-500 bp, about 100 to about 1000
nt, which forms a single-stranded loop above the stem structure
created by the two regions of base complementarity. It will be
recognized, however, that it is not strictly necessary to include a
"loop region" or "loop sequence" because an RNA molecule comprising
a sequence followed immediately by its reverse complement will tend
to assume a stem-loop conformation even when not separated by an
irrelevant "stuffer" sequence.
[0183] The expression construct of the present invention comprising
DNA sequence which can be transcribed into one or more
double-stranded RNA effector molecules can be transformed into a
Camelina plant, wherein the transformed plant produces different
fatty acid compositions than the untransformed plant. The target
sequence to be inhibited by the dsRNA effector molecule include,
but are not limited to, coding region, 5' UTR region, 3' UTR region
of fatty acids synthesis genes. In one embodiment, the target
sequence is from one or more Camelina FAD2 and/or FAE1 genes.
[0184] The effects of RNAi can be both systemic and heritable in
plants. In plants, RNAi is thought to propagate by the transfer of
siRNAs between cells through plasmodesmata. The heritability comes
from methylation of promoters targeted by RNAi; the new methylation
pattern is copied in each new generation of the cell. A broad
general distinction between plants and animals lies in the
targeting of endogenously produced miRNAs; in plants, miRNAs are
usually perfectly or nearly perfectly complementary to their target
genes and induce direct mRNA cleavage by RISC, while animals'
miRNAs tend to be more divergent in sequence and induce
translational repression. Detailed methods for RNAi in plants are
described in David Allis et al (Epigenetics, CSHL Press, 2007, ISBN
0879697245, 9780879697242), Sohail et al (Gene silencing by RNA
interference: technology and application, CRC Press, 2005, ISBN
0849321417, 9780849321412), Engelke et al. (RAN Interference,
Academic Press, 2005, ISBN 0121827976, 9780121827977), and Doran et
al. (RNA Interference: Methods for Plants and Animals, CABI, 2009,
ISBN 1845934105, 9781845934101), which are all herein incorporated
by reference in their entireties for all purposes.
[0185] The present invention is further illustrated by the
following examples that should not be construed as limiting. The
contents of all references, patents, and published patent
applications cited throughout this application, as well as the
Figures, are incorporated herein by reference in their entirety for
all purposes.
EXAMPLE
Example 1
Methods and Materials
Southern Blot
[0186] Camelina sativa Cs32 and Cs11, and Arabidopsis thaliana
ecotype Col-0 (Table 2) seeds were germinated on Arabidopsis Growth
Media (1.times. Murashige and Skoog (MS) mineral salts, 0.5 g/L
MES, 0.8% PhytaBlend.TM. all from Caisson Labs, North Logan, Utah;
pH5.7) and allowed to grow for .about.2 weeks under 16/8 hours
day/night, 22/18.degree. C., and .about.130 .mu.E m.sup.-2 s.sup.-1
light intensity. Genomic DNA was isolated according to the CTAB
method (Saghai-Maroof, Soliman et al. 1984) and 10 .mu.g was
digested overnight (.about.16 h) with EcoRI or a combination of
EcoRI plus BamHI. DNA electrophoresis and blotting were carried out
using standard molecular biology techniques (Tom Maniatis 1982).
The probe was labelled with .alpha.-32P dCTP according to
instructions of the DECAprime II kit (Ambion, Austin, Tex.).
Hybridization was carried out overnight at 42.degree. C. The blot
was washed (30 minutes each) at 42.degree. C. in 2.times.SSC, 0.1%
SDS, followed by 55.degree. C. in 2.times.SSC, 0.1% SDS, and then
55.degree. C. in 0.1.times.SSC, 1% SDS, and exposed to a
phosphorimager screen. The blot was hybridized with different
probes after stripping the membrane in boiling 0.1% SDS for 20
minutes each time.
Cloning of C. sativa FAD2 and FAE1 Genes and Upstream Regions.
[0187] FAD2 and FAE1 genes were amplified from C. sativa Cs32 DNA
isolated as described above, using Phusion polymerase (New England
Biolabs, Ipswich, Mass.) and the primers listed in Table 3,
according to the manufacturer's directions. The amplified fragments
were cloned using the Zero Blunt PCR Cloning kit (Invitrogen,
Carlsbad, Calif.)
FAD2 and FAE1 Sequence Alignments
[0188] Translated amino acid FAD2 and FAE1 sequences were aligned
with AlignX (Invitrogen), with a gap opening penalty of 15, a gap
extension penalty of 6.66, and a gap separation penalty range of 8.
Alignments were imported into Boxshade (EMBnet) to highlight the
conserved residues.
RNA Isolation and cDNA Preparation
[0189] C. sativa Cs32 plants were grown under 24/18.degree. C.
day/night conditions with a 16/8 hour photoperiod. Flowers were
tagged and embryos harvested at the time points indicated. RNA was
then isolated using the urea LiCl method described by Tai et al
(Tai, Pelletier et al. 2004). cDNA were prepared from 0.5 .mu.g of
DNAsed RNA that was reverse transcribed with the High Capacity cDNA
RT kit (Applied Biosystems, Foster City, Calif.) using random
primers according to the manufacturer's instructions.
Quantitative Real-Time PCR
[0190] Relative expression of FAD2 and FAE1 cDNA was measured by
real-time quantitative PCR and calculated according to the
comparative C.sub.T method (2.sup.-.DELTA..DELTA.CT). In brief,
separate reactions were prepared in duplicate or triplicate for
each of the genes to be measured. Each reaction contained 8 .mu.l
of the appropriate primers (200 nM each) and probe (900 nM) for Cs
ACTIN (reference gene) or Cs FAD2 or FAE1 (target gene); 10 .mu.l
of Applied Biosystems 2.times. fast Taqman PCR mix; 2 .mu.l of
cDNA. The reactions were run on an Applied Biosystems 7900HT
according to the manufacturer's fast PCR method. Real-time primers
and probes are listed in Table 4.
Relative Expression Analysis
[0191] Three single nucleotide polymorphisms (SNPs) for each of
FAD2 A, B, and C and FAE1 A, B, and C were identified. Each
identified SNP distinguishes one copy from the other two. An
additional SNP, which distinguishes FAE1 A, B, and C copies from
each other, was also identified (Table 5). SNP frequencies were
determined in cDNA isolated as described above by the Sequenom
MassARRAY.TM. allele-specific expression analysis method with no
competitor, as described in Park et al (Park, Correll et al.
2004).
Genome Size Estimation
[0192] Camelina lines (Table 2) were grown in the greenhouse at
temperatures fluctuating between 16 and 26 C with 16 hour day
length supplemented by halogen lights. The nuclei were extracted
from leaves according to Henry et al [74]. Nuclei were also
extracted from approximately 50 seeds of all species, except C.
laxa and C. hispida, which are late flowering. The seeds were
crushed with a pestle in 1.4 mL of the same extraction buffer used
for the leaves. The fluid was then drawn through four layers of
cheesecloth and strained and processed as for the leaf nuclei.
Nuclei of diploid and tetraploids of Arabidopsis thaliana accession
Col-0 (1 C genome size 157 Mb, and 314 Mb, respectively [75]), and
tetraploid Arabidopsis arenosa accession Care-1 (1C genome size 480
Mb [Dilkes, unpublished results]) were used as standards for DNA
content. Data was collected on two different days and normalized
separately to account for daily fluctuations in flow cytometer
performance. The 2C, 4C, and 8C nuclear peaks were used in a
regression analysis of measured fluorescence intensity versus
nuclear DNA content, producing equations of genome size versus
fluorescence that were used to estimate the 2C content of Camelina
nuclei.
Phylogenetic Inference
[0193] FAD2 and FAE1 were PCR amplified from several Camelina
species and other species from the tribe Camelineae (Table 2) using
primers designed from C. sativa FAD2 and FAE1 sequences (Table 3).
Amplified fragments for FAD2 and FAE1 were cloned as described for
C. sativa above, then aligned by translated amino acids sequences
using MacClade 4.05 (Maddison 2004.) ModelTest 3.7 (Posada and
Crandall 1998) in PAUP*4.0 b (Swofford 2001) was used to determine
the model of sequence evolution favored by the data for each gene.
Subsequent maximum likelihood (ML) analyses were performed in
PAUP*4.0 b using a heuristic search with tree bisection
reconnection (TBR) branch swapping. ML clade support using 100
bootstrap data sets were assessed and this support is presented on
the most likely tree recovered from the ML heuristic search.
Camelina Alkaline Transesterification for FAMES Composition and Gas
Chromatography (GC/FID) Analysis of Camelina seeds
[0194] Approximately 50 mg of seeds were ground up in liquid
nitrogen with mortar and pestle. 5 mL of 0.2M KOH in methanol was
added to each vial containing the ground seeds. Samples were
capped, heated at 37 C. for 1 hr and vortexed every 10 minutes.
Reaction was stopped with addition of 1 mL 1M acetic acid and 2 mL
heptanes. Samples were vortexed, and then centrifuged for 10 min at
room temp at 2990 rpm and the upper organic phase was collected.
Before GC analysis, samples were diluted 1/10 in heptanes.
[0195] The supernatant was transferred to a GC vial, in which 1
.mu.L was used for GC analysis. Analysis was carried out on GC/FID
7890A series with a SP.sub.--2330 column. Injector and detector
temperature were 250.degree. C. and 300.degree. C. respectively;
oven temperature was held at 50.degree. C. for 2 min, then
programmed to 180.degree. C. at a heating rate of 10.degree.
C./min, then programmed to hold for 5 min followed by an increase
of 5.degree. C./min to 240.degree. C. Total run time was 32.5 min.
Flow rates for hydrogen and air to the FID were 30 and 450 mL/min
respectively. Helium as the carrier gas flowed at a rate of 1.69
mL/min and nitrogen as the make-up gas at 30 mL/min.
Example 2
Southern Blot Hybridizations Show Multiple Copies of Genes in
Camelina Sativa
[0196] As a first step to characterize genes involved in fatty acid
biosynthesis, the inventors determined the copy number of FAD2 and
FAE1 by Southern blot analysis. Since C. sativa is closely related
to Arabidopsis thaliana (Al-Shehbaz, Beilstein et al. 2006;
Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al.
2008), the inventors designed primers based on Arabidopsis that
amplified conserved regions of FAD2 and FAE1. Using these primers,
the inventors PCR amplified products of 225 base pairs (bp) (FAD2)
and 403 by (FAE1) from Arabidopsis and from C. sativa. The C.
sativa products were cloned, sequenced, and compared with
Arabidopsis FAD2 and FAE1 sequences (TAIR 2009) to confirm their
identities. The inventors used the C. sativa fragments as probes in
Southern blot experiments (FIG. 1). Results of the Southern blots
revealed three bands in C. sativa for both FAD2 (FIG. 1A) and FAE1
(FIG. 1B), whereas hybridization revealed only a single band in
Arabidopsis for both genes (FIGS. 1A & B). These results
suggest that FAD2 and FAE1 occur in at least three copies in C.
sativa, while they are single copy in Arabidopsis (TAIR 2009).
Fatty acid genes can be multi-copy in many species, including
soybean (Schlueter, Lin et al. 2007), Brassica napes (Scheffler,
Sharpe et al. 1997), olive (Olea europaea) (Hernandez, Mancha et
al. 2005), maize (Mikkilineni and Rocheford 2003), and sunflower
(Martinez-Rivas, Sperling et al. 2001). Therefore, the inventors
designed a probe for Southern blot hybridization of the gene LEAFY
(LFY), which is known to be single copy in a wide variety of
species from several plant families (Frohlich and Estabrook 2000).
Three bands were observed following hybridization using the LFY
probe, suggesting LFY also exists as three copies in C. sativa
(FIG. 1C).
Example 3
Copies of C. Sativa FAD2 and FAE1 are Highly Similar to Each Other
and to their Putative Orthologs from Arabidopsis
[0197] The inventors cloned and sequenced the full length genomic
and cDNA sequences of C. sativa FAD2 and FAE1 (SEQ ID NOs: 1 to 6).
Using primers designed from Arabidopsis FAD2 and Crambe abyssinica
FAE1, the inventors PCR amplified a band of approximately 1.2 kb
for FAD2 and 1.5 kb for FAE1 from C. sativa. For each gene, the
inventors sequenced more than 60 clones. Three different versions
of both FAD2 and FAE1 were recovered and designated A, B, and C. It
should be noted that the A, B, and C copies were named
independently for FAD2 and FAE1, and thus are not associated with a
particular genome.
[0198] The three copies of C. sativa FAD2 are 1155 by long, lack
introns in the coding regions, are 97% identical at the nucleotide
level, and encode proteins that are 99% identical in sequence
(Table 1). One of the FAD2 copies contains a BamHI site, and thus
this copy likely produced the .about.1.3 kb fragment in the
Southern blot hybridization of FAD2 (FIG. 1A; BamHI+ EcoRI digest).
The C. sativa nucleotide sequences of FAD2 are greater than 93%
identical to Arabidopsis FAD2, and the putative encoded proteins
from the two species share greater than 96% identity (Table 1).
[0199] An approximately 1.4 kb intron found within the 5'
untranslated region was also recovered from all three copies of C.
sativa FAD2. A similarly sized intron is present in Arabidopsis
(TAIR 2009) and in Sesamum indicum (sesame) where it has been shown
to be involved in regulating FAD2 expression (Kim et al. 2006).
[0200] All three copies of FAE1 in C. sativa are 1518 by long and
lack introns. When the nucleotide sequences and the putative
encoded proteins of the three copies are compared they are more
than 96% identical (Table 1). In comparison to Arabidopsis, the
nucleotide sequences are more than 90% identical, while the encoded
proteins are more than 91% identical (Table 1). Thus, the three
copies of C. sativa FAD2 and the three copies of FAE1 are highly
similar to each other and to their putative orthologs from
Arabidopsis.
Example 4
Alignments of FAD2 and FAE1 Protein Sequences from Several Species
Reveals Conserved and Non-Conserved Domains
[0201] The inventors aligned translated amino acid sequences from
the three copies of C. sativa FAD2 with the FAD2 protein sequences
from Arabidopsis; Brassica rapa, an agronomically important member
of the Brassicaceae family; Glycine max, an agronomically important
dicot; and Zea mays, an agronomically important monocot (FIG. 2A).
All three copies of C. sativa FAD2 have the three conserved HIS
boxes found in all membrane-bound desaturases (Tocher D R 1998) as
well as the ER localization signal described by McCartney et al
(Belo, Zheng et al. 2008)(McCartney, Dyer et al. 2004).
Furthermore, the conserved amino acids identified in an alignment
of the FAD2 sequences from 34 different species [49] are also
present in C. sativa with the exception of a positively-charged
histidine at position number 44, which is substituted by a polar,
uncharged glutamine in C. sativa. When the inventors amplified the
FAD2 gene from several species in the tribe Camelineae (Table 2)
and aligned the translated amino acid sequences, the inventors
found that the FAD2 proteins from Capsella rubella, Camelina
microcarpa, Camelina laxa, and one copy from Camelina rumelica
contain a glutamine at amino acid position 44, while the FAD2
proteins from Arabidopsis lyrata, Camelina hispida, and a second
copy from Camelina rumelica contained a histidine (data not
shown).
TABLE-US-00004 TABLE 1 Nucleotide and Amino Acid identity of
Camelina sativa and Arabidopsis thaliana FAD2 and FAE1 genes. Gene
% Nucleotide Identity* % Amino Acid Identity AtFAD2 CsFAD2A CsFAD2B
CsFAD2C AtFAD2 CsFAD2A CsFAD2B CsFAD2C FAD2 AtFAD2 100 93.6 93.8
93.4 100 96.9 96.6 96.4 CsFAD2A 100 97.3 98.3 100 99.0 99.5 CsFAD2B
100 97.7 100 99.5 CsFAD2C 100 100 AtFAE1 CsFAE1A CsFAE1B CsFAE1C
AtFAE1 CsFAE1A CsFAE1B CsFAE1C FAE1 AtFAE1 100 90.7 91.2 91.0 100
91.9 91.7 91.7 CsFAE1A 100 97.8 96.8 100 97.6 96.4 CsFAE1B 100 97.2
100 96.8 CsFAE1C 100 100 *Nucleotide identity is in coding region
only.
TABLE-US-00005 TABLE 2 Plant species and sources Species Source
Catalogue number Camelina sativa Cs32 USDA PI 311732 Camelina
sativa Cs11 Ames 26668 Arabidopsis thaliana, ABRC CS28166 ecotype
Col-0 Arabidopsis lyrata ABRC CS22696 Camelina laxa USDA PI 650132
Camelina microcarpa wild collection; number "01-22" Harvard
Herbarium Camelina microcarpa USDA PI 633188 Capsella bursa
pastoris Wild collection; number "08-188" Harvard Herbarium
collection Capsella rubella ABRC CS22561 Camelina hispida var Ames
21324 grandiflora Camelina alyssum Ames 26658 Camelina rumelica
Ames 21327
TABLE-US-00006 TABLE 3 Primers used for amplification of genomic
regions of C. sativa Primer Name Primer sequence (5'-3') Southern
FAD2_631F TCAACAACCCTCTTGGACGCATCA analysis of (SEQ ID NO: 13) FAD2
FAD2_832R CTTGTGCAGCAGCGTAACGGTAAA (SEQ ID NO: 14) Southern AtFAE1
probe F AGACGGTCCAAGTACAAGCTAGTTC analysis of (SEQ ID NO: 15) FAE1
AtFAE1 probe R CCAAATCTATGTAACGTTGATCT (SEQ ID NO: 16) Southern
AtLFY probe F GATGCGGCGGGGAATAACGGCGGAG analysis of (SEQ ID NO: 17)
LFY AtLFY probe R CCTGAAGAAGGAACTCACGGCATT (SEQ ID NO: 18) Cloning
of AtFAD2_start AACATGGGTGCAGGTGGAAGAATG FAD2 coding (SEQ ID NO:
19) region AtFAD2_stop2 TCATAACTTATTGTTGTACCAGTAC (SEQ ID NO: 20)
Cloning of CaFAE1 start ATGACGTCCATTAACGTAAAGCTC FAE1 coding (SEQ
ID NO: 21) region CaFAE1 stop TTAGGACCGACCGTTTTGGGC (SEQ ID NO: 22)
KCS17-FAE1 AtKCS F GGGTGGCTCTTCGCAATGTCGAGCCC intergenic (SEQ ID
NO: 23) region "A" and CsFAE1 5' RACE GAGGCTTTTCCGGCAAGTAACGCCG "C"
(initial (SEQ ID NO: 24) clones) KCS17-FAE1 AtKCS cons F
GGTATGAATTGGCTTACACGGAAG intergenic (SEQ ID NO: 25) region "A"
CsKCSA_F TATGAATTGGCTTACACGGAAGCC (SEQ ID NO: 26) CsFAE1A_R2
TATATTGCCAATATAAGTATTAAAGGTCC (SEQ ID NO: 27) KCS17-FAE AtKCS cons
F GGTATGAATTGGCTTACACGGAAG intergenic (SEQ ID NO: 28) region "B"
CsFAE1B_R TATATTGCCAATATAAGTATTAAAGGTCC (SEQ ID NO: 29) KCS17-FAE
AtKCS cons F GGTATGAATTGGCTTACACGGAAG intergenic (SEQ ID NO: 30)
region "C" CsFAE1C_R GGTAGAGATCGTTTGTGGTAAGCG (SEQ ID NO: 31)
Camelinae CsFAD2 start ATGGGTGCAGGTGGAAGAATGC FAD2 (SEQ ID NO: 32)
CsFAD2 stop TCATAACTTATTGTTGTACCAGTACACACC (SEQ ID NO: 33)
Camelinae CsFAE1 start ATGACGTCCGTTAACGCAAAGCTC FAE1 (SEQ ID NO:
34) CsFAE1 stop TTAGGACCGACCGTTTTTGACATG (SEQ ID NO: 35)
TABLE-US-00007 TABLE 4 Primers used for qPCR analyses Primer or
Probe Name Sequence (5'-3') qPCR of CsACT For ACA ATT TCC CGC TCT
GCT GTT GTG CsACTIN (SEQ ID NO: 36) CsACT Rev AGG GTT TCT CTC TTC
CAC ATG CCA (SEQ ID NO: 37) CsACT probe FAM - TGT TTC AAA CGC TCT
ATC CCT CGC TC - IABLFQ (SEQ ID NO: 38) qPCR of CsFAD2 A For1 CTG
CGA GAA ACC ACC GTT CAC CC CsFAD2 (SEQ ID NO: 39) CsFAD2 all Rev
CAC GAG TAG TCA ACG AGG TAA ACC GG (SEQ ID NO: 40) CsFAD2 all FAM -
CCA CTT CTA TTC CCA TCT CCA probe ACA CAA CC - IABLFQ (SEQ ID NO:
41) qPCR of CsFAE1 all For AAC CTT TGC TTG TTT CCG TTA ACG CsFAE1
GC (SEQ ID NO: 42) CsFAE1 all Rev CAC GAG TAG TCA ACG AGG TAA ACC
GG (SEQ ID NO: 43) CsFAE1 all FAM - CCA CTT CTA TTC CCA TCT CCA
probe ACA CAA CC - IABLFQ (SEQ ID NO: 44)
TABLE-US-00008 TABLE 5 SNPs distinguishing each copy of FAD2 and
FAE1 Nucleotide position from beginning of SNP_ID coding region
FAD2_A4 51 FAD2_A2 453 FAD2_A6 549 FAD2_B4 288 FAD2_B5 687 FAD2_B8
1109 FAD2_C1 78 FAD2_C5 615 FAD2_C3 966 FAE1_A4 624 FAE1_A3 1368
FAE1_A7 1475 FAE1_B4 414 FAE1_B5 783 FAE1_B8 1438 FAE1_C1 336
FAE1_C2 721 FAE1_C7 1419 FAE1_ABC1 104
[0202] The inventors aligned the translated amino acid sequences
from the three copies of C. sativa FAE1 with the seed-specific FAE1
proteins from A. thaliana, Crambe abyssinica, a high and low erucic
acid Brassica rapa, Limnanthes alba, and Tropaeolum majus (FIG.
2B). L. alba and T. majus are both in the order Brassicales and
their seeds accumulate high levels of very long chain fatty acids
(Cahoon, Marillia et al. 2000; Mietkiewska, Giblin et al. 2004).
Four conserved histidine residues and six conserved cysteine
residues, including the active site at cysteine 223, as well as an
asparagine residue at 424 required for FAE1 activity were
previously identified by Ghanevati and Jaworski (Ghanevati and
Jaworski 2001; Ghanevati and Jaworski 2002). All conserved residues
were found to be present in all three copies of C. saliva FAE1.
More differences were apparent between the three C. saliva FAE1
sequences and the other FAE1 sequences than observed in the FAD2
comparison (FIGS. 2A and B), an observation consistent with the
level of amino acid identity seen between Arabidopsis and C. saliva
FAD2 versus FAE1 (Table 1).
Example 5
All Three Copies of FAD2 and FAE1 are Expressed in Developing Seeds
of C. Sativa
[0203] The conservation of amino acids as well as the presence of
the 5' regulatory intron in FAD2 suggests that all three copies of
FAD2 and of FAE1 could be functional. To determine whether these
genes are also expressed, the inventors first evaluated total FAD2
and FAE1 gene expression in developing seeds and in seedling tissue
using quantitative real time PCR (qPCR) with primer/probe
combinations designed to detect all three copies of each gene. FAD2
expression in seedling tissue is present but minimal (0.4% of that
seen in seeds at 20 days post-anthesis (DPA)), while FAE1
expression could not be detected in seedlings (FIGS. 3A and B). In
developing seeds, both FAD2 and FAE1 expression peaks at 20 DPA and
is reduced by 30 DPA (FIGS. 3A and B). In Arabidopsis, FAD2 peaks
earlier and decreases sooner than FAE1 (Ruuska, Girke et al.
2002).
[0204] The inventors wondered whether the expression of each of the
FAD2 and FAE1 copies present in C. sativa are equally or
differentially expressed in the seed. Duplicated genes are
frequently silenced either throughout the plant or in a
tissue-specific manner (Comai, Tyagi et al. 2000; Kashkush, Feldman
et al. 2002; He, Friebe et al. 2003; Adams, Percifield et al.
2004); hence the inventors hypothesized that one or more of the
copies of each gene could be significantly down-regulated. The
inventors used the Sequenom MassARRAY.TM. method for determining
allele-specific expression of a gene (Park, Correll et al. 2004) to
evaluate the relative expression of each of the copies of FAD2 and
FAE1. The inventors identified at least three single nucleotide
polymorphisms (SNPs) specific to each of the FAD2 A, B, and C and
the FAE1 A, B, and C copies and then calculated the frequency of
each SNP in seed cDNA. Controls consisting of the cloned FAE1 A, B,
and C copies combined to known frequencies showed that the method
is greater than 80% accurate (Table 6). No evidence of silencing of
any particular copy of either FAE1 or FAD2 was discovered. The
inventors did observe differential expression, especially of FAE1
A, which accounts for approx 40-50% of FAE1 expression in seeds at
20-30 DPA (FIGS. 3C and D).
[0205] Six cloned DNA positive controls were also included in the
analysis and the relative amount of "B" version in each measured
with the FAE1_B5 SNP and all 3 versions with the FAE1_ABC SNP:
TABLE-US-00009 TABLE 6 Expression level of FAE1 genes relative to
FAE1 B FAE1_B5 FAE1 ABC relative relative relative relative B A B C
C1 100% Version A 0.00 1 0 0 C2 100% Version B 1.00 0 1 0 C3 100%
Version C 0.00 0 0 1 C4 60% A, 20% B, 20% C 0.20 0.59 0.20 0.22 C5
20% A, 60% B, 20% C 0.54 0.29 0.48 0.23 C6 20% A, 20% B, 60% C 0.20
0.24 0.28 0.48
[0206] As the results indicate, all three FAE1 genes are expressed
in the seed. A dosage effect may still be expected, however, since
FAE1 B appears to account for only approximately 25-30% of FAE1
expression in the seeds. A mutation in FAE1 A would be expected to
have a greater effect on fatty acids composition in the seeds since
it accounts for .about.41-48% of FAE1 expression.
Example 6
Characterization of Sequences Upstream of C. Sativa FAE1 and
Downstream of C. Sativa FAD2 Suggests Colinearity with A.
Thaliana
[0207] To investigate whether the different copies of C. sativa
FAD2 and FAE1 are the result of allelic variation or are in fact
independent loci, the inventors obtained sequence from the region
upstream of FAE1 and downstream of FAD2. Assuming colinearity
between C. sativa and Arabidopsis for the region around FAE1, the
inventors PCR amplified the region 5' to FAE1 using a forward
primer for the upstream gene KCS17 with reverse primers for C.
sativa FAE1. The resulting sequences obtained for the putative C.
saliva KCS17 were highly similar to the last 189 bp of Arabidopsis
KCS17, suggesting that the inventors had in fact amplified the
orthologous C. sativa region upstream of FAE1, confirming
colinearity between the two species. The inventors then used a dot
plot (see details for Nucleic Acid Dot Plots in Maizel et al.,
1981; Pustell et al. 1982; Quigley et al., 1984) to compare the
three C. sativa upstream sequences to each other and to Arabidopsis
with parameters set for perfect match on a sliding window of 9
bases. The coordinates from the dot plot were used to define blocks
of homology between Arabidopsis and the three C. sativa copies
(FIG. 4). The results show a variable intergenic region containing
conserved blocks common to two or more genomes.
[0208] Co-linearity with Arabidopsis was also found for a region
downstream of FAD2 containing the ACTIN11 (ACT11) gene for two out
of the three C. sativa copies (data not shown). For the third copy,
the region downstream of FAD2 A could have been missed if the
length of the amplified product was too large. Alternatively, the
region downstream of FAD2 A might not exhibit colinearity with
Arabidopsis.
Example 7
The Genomes of C. Sativa, C. Alyssum, and C. Microcarpa are Larger
than the Genomes of Other Camelina Species
[0209] The inventors calculated DNA content in several accessions
of C. sativa and related species from flow cytometry analyses using
propidium iodide-stained nuclei. The inventors used Arabidopsis
accession Col-0 (2.times.) and its tetraploid (4.times.) derivative
as genome size standards. C. sativa, C. alyssum, and C. microcarpa
diploid (2C) genomes had a haploid content between 650 and 800 Mb
(FIG. 5). C. sativa accessions uniformly displayed a genome size
close to 750 Mb. North American isolates of C. sativa, C. alyssum,
and C. microcarpa have reported chromosome counts of n=20 (Francis
and Warwick 2009). The genomes of C. rumelica (600 Mb), C. hispida
(300 Mb) and C. laxa (210 Mb) are smaller than those of C. sativa,
C. alyssum, and C. microcarpa. Chromosome counts of both n=6
(Baksay 1957; Brooks 1985) and n=12 (Maassoumi 1980) have been
recorded for C. rumelica, while only a single count of n=7 exists
for C. hispida (Maassoumi 1980). To our knowledge, no published
counts exist for C. laxa.
Example 8
Phylogenetic Analysis of FAD2 and FAE1 Indicate that C. Sativa and
C. Microcarpa are Closely Related
[0210] To understand the duplication history of the multiple FAD2
and FAE1 copies recovered from C. sativa, the inventors amplified
the FAD2 and FAE1 genes from several species in the tribe
Camelineae (Table 2), and inferred phylogeny for each gene. The
sampling of taxa chosen allowed the inventors to test whether FAD2
and FAE1 duplication events occurred after Camelina diverged from
its closest relatives or within the genus. Results from the
evaluation of 55 different models of sequence evolution using
Modeltest 3.7 (Posada and Crandall 1998) indicated that the FAD2
sequence data are best described by the TVM+I+.GAMMA. model, while
the FAE1 data are best described by the HKY+I+.GAMMA. model.
Likelihood phylogenetic analyses in PAUP*4.0 (Swofford 2001)
produced a single FAD2 tree (-LnL 3665.277; FIG. 6A), and a single
FAE1 tree (-LnL 5051.552; FIG. 6B).
[0211] Phylogenies inferred from FAD2 and FAE1 data indicate a
history of duplication for both markers. Both C. microcarpa and C.
sativa have three distinct copies of FAD2 and FAE1. Moreover, for
FAD2, the A and C copies from these two species are monophyletic
with strong (100%) bootstrap support (bs); for FAE1 the A and B
copies from these species are strongly monophyletic (100% bs). In
contrast, neither the FAD2 B copies of C. sativa and C. microcarpa,
nor the FAE1 C copies of these species form a monophyletic group
with each other. Instead, results indicate that C. rumelica has two
distinct copies of FAD2 and that one of these copies (FAD2-2) is
strongly monophyletic with C. microcarpa FAD2 B. The inventors
recovered only a single FAD2 copy for C. laxa and C. hispida. In
contrast, at least two distinct copies of FAE1 were recovered from
all sampled Camelina species. The FAE1-1 copy of C. laxa, C.
hispida, and C. rumelica form a monophyletic group (91% bs), with
the former two species sister to one another with strong support
(100% bs). Similar to the results from FAD2, C. rumelica FAE1-2 is
sister to one of the C. microcarpa copies (FAE1 C; 99% bs). Neither
the C. sativa FAD2 B copy, nor the C. sativa FAE1 C copy, shows a
well supported sister relationship to other FAD2 or FAE1 sequences.
However, in the FAE1 tree, C. sativa FAE1 C is very weakly
supported as sister to C. hispida FAE1-2 (53%). Finally, all
recovered FAD2 and FAE1 copies from species of the genus Camelina
are monophyletic and sister to other sampled members of the tribe
Camelineae, consistent with phylogenies based on other markers
(Beilstein, Al-Shehbaz et al. 2006; Beilstein, Al-Shehbaz et al.
2008).
Example 9
Camelina Breeding Program
[0212] Since Camelina has not been intensively bred and the
germplasm is somewhat limited genetically, the inventors
established three strategies for long term development of Camelina
germplasm. These three, non-mutually exclusive strategies for
Camelina germplasm enhancement include: transgenic approach,
classical and molecular breeding, and mutation breeding. The long
term goals are to achieve increased yield, increased seed oil
content and improved fatty acid composition (e.g., higher
percentage oleic acid (18:1), which is an optimal fatty acid for
biodiesel and/or lower percentage of very long chain fatty acids
(VLCFA, such as 20:1, 20:2, 22:1, etc)).
[0213] In the transgenic approach, REV and KRP yield technology (US
2008/263727 and US 2007/056058, incorporated by reference in their
entireties) can be introduced into Camelina to obtain events with
increased seed yield or seed size, agronomic properties beneficial
to obtaining Camelina germplasm with increased oil yield per unit
land for biofuel purposes. Efficient transformation of Camelina has
been established before (WO 2009/117555, incorporated by reference
in its entirety).
[0214] In the classical and molecular breeding approach, broad
field evaluations of more than 100 accessions of Camelina in
Northern United States and Canada was initiated across multiple
field locations and over multiple years. Different accessions were
evaluated for seed yield, oil yield, fatty acid composition, and
agronomic performance under different environmental conditions.
Superior lines with higher yield identified in the evaluations are
used in the breeding program. In addition, molecular breeding
studies are also in progress. Preliminary results show that
existing Camelina cultivars are closely related, as indicated by
AFLP analysis in which 379 markers were scored. Jaccard analysis
suggested there is more than 90% genetic similarity across existing
cultivars. Therefore, there is much room for improvement of
Camelina germplasm, which will be realized by classical and
molecular breeding programs. In the mutation breeding approach, an
EMS mutagenized population was created in a selected Camelina
cultivar, and Targeting Induced Local Lesions In Genomes
(TILLING.RTM.) method was used to find mutations in known gene
sequences. Especially, mutations with altered fatty acid
compositions and improved yield as expressed in amount of oil
produced per acre are of the most interest. M2 plants/M3 seed were
harvested, and gene sequences for select targets were isolated and
characterized. Preferred fatty acids include 16:1 and 18:1
monounsaturated fatty acids, since they have the best combination
of proper cetane number, cloud point, oxidative stability, and less
NOx emissions, as compared to saturated fatty acids (e.g., 12:0,
14:0, 16:0, 18:0, 20:0, and 22:0), or poly unsaturated fatty acids
(e.g., 18:2, 18:3).
Example 10
TILLING.RTM. Method to Isolate Camelina Mutants in FAD2 and FAE1
Genes
[0215] As described above, the goal is to improve Camelina sativa
fatty acid composition for biodiesel. For example, since oleic acid
(18:1) is optimal for fatty acid biodiesel, one specific goal is to
increase 18:1 and decrease polyunsaturated fatty acids and long
chain fatty acids. One way is to lower the activity of FAD2 and of
FAE1, as indicated by the fatty acids synthesis pathway shown in
FIG. 7.
[0216] An EMS mutant library has been created in Camelina sativa
line CS32. This library has a population of about 8000 mutants and
was used to screen for mutants of FAD2 genes (FIG. 8). Initial
TILLING.RTM. using primers designed to the three FAD2 genes yielded
mutants in all three FAD2 genes. Later, TILLING.RTM. using primers
designed to the three FAE1 genes also yielded mutants in all three
FAE1 genes. Lu et al (Camelina sativa: A Potential Oilseed Crop for
Biofuels and Genetically Engineered Products, Information Systems
for Biotechnology New Report, January 2008) describes a preliminary
mutant screen where a random screen was carried out for fatty acid
composition Camelina mutant using gas chromatography (GC). The
TILLING.RTM. method of the present invention is superior to this
because it is not necessary to GC screen thousands of mutants;
rather, mutants in known fatty acid genes are identified (Hutcheon
et al., TILLING.RTM. for Altered Fatty Acid Profiles in Camelina
sativa, July 2009, American Society of Plant Biologists Annual
Meeting, which is herein incorporated by reference in its entirety
for all purposes). Also the identification of Camelina sequences
allows for the design of gene-specific TILLING.RTM. primers which
can make it much easier to get mutations in all three versions of
any given gene, FAD2 or FAE1.
[0217] A non-limiting exemplary protocol of TILLING.RTM. is
described below: [0218] 1. Seeds are mutagenized to induce point
mutations throughout the genome. [0219] 2. A founder population is
grown from mutagenized seeds. [0220] 3. Founder population is
self-fertilized to produce a crossed population. [0221] 4. DNA
samples from the crossed population are collected in 96-well plates
and seeds from the crossed population are stored. [0222] 5. Up to
eight 96-well plates are pooled into one and the samples (768)
subjected to PCR with two gene-specific primers labelled with
different IRDye.RTM. infrared dyes. [0223] 6. Resulting amplicons
are heated and cooled, resulting in heteroduplexes between wild
type and mutant samples. [0224] 7. CEL I nuclease is used to cleave
at base mismatches. [0225] 8. Samples are denatured and
electrophoresed on a LI-COR.RTM. DNA Analysis System. [0226] 9. In
lanes that have a mutation in the pool, a band will be visible
below the wild type band on the IRDye.RTM. 700 infrared dye image.
A counterpart band will be visible in the same lane on the
IRDye.RTM. 800 infrared dye image. This band is the cleavage
product labeled with IRDye.RTM. 800 infrared dye from the
complementary DNA strand. The sum of the length of the two
counterpart bands is equal to the size of the amplicon, which makes
it easy to distinguish mutations from amplification artefacts. An
exemplary LI-COR gel identifying mutants in Camelina FAD2 genes is
shown in FIG. 9. [0227] 10. After detection of a mutation in a pool
(lane), the individual DNA samples in the pool are screened again
to find out which of the eight pooled samples from the crossed
population has the mutation.
[0228] More information on TILLING.RTM. is described by Colbert et
al. (2001. High Throughput Screening for Induced Point Mutations.
Plant Physiology 126: 480-484.); McCallum et al. (2000. Target
Induced Local Lesions In Genomes (TILLING) for Plant Functional
Genomics. Plant Physiology 123:439-442); Henikoff et al.
(Single-Nucleotide Mutations for Plant Functional Genomics. Annual
Review of Plant Biology. 54:15.1-15.27.); and Till et al. (2003.
Large-Scale Discovery of Induced Point Mutations With
High-Throughput TILLING. Genome Research 13:524-530).
[0229] A pilot study determined that the mutation density of the
inventors' mutant Camelina population was 1/25 kb. TILLING.RTM. of
an initial 768 M2 individuals for FAD2 has identified 60 mutants,
60% of which are non-silent mutations. Of the non-silent mutations,
about 30% are predicted to be severe missense or truncation
mutations. Mutations were identified in all 3 copies of Camelina
FAD2. The inventors' previous finding that Camelina sativa may be
polyploid is further supported by the high density of lesions this
plant is willing to tolerate in its genome. The mutant M3 plants
were grown and a preliminary analysis of their fatty acid profiles
by GC was performed.
Example 11
Mutations of Camelina FAD2 and FAE1 Genes Identified in
TILLING.RTM.
[0230] Initial screening of the TILLING.RTM. population for FAD2
mutants resulted in plants with silent, STOP (nonsense) and/or
severe missense mutations in FAD2 A, B, and C; and FAE1 A, B and C
genes.
[0231] Positions and effects of mutations in FAD2 A, B, and C genes
and FAE1 A, B and C genes are displayed in Tables 7 to 12 below
(*indicates the mutation results in a stop codon,=indicates silent
mutation).
TABLE-US-00010 TABLE 7 Summary of Camelina FAD2 A mutants
Nucleotide Change Effect Primer set Plant ID Mutation Score G1516A
G35R FAD2A 2480 severe missense C1645T L78F FAD2A 2487 severe
missense C1746T H111= FAD2A 2782 silent C1813T P134S FAD2A 2085
severe missense G1844A R144H FAD2A 2764 severe missense C1977T
V188= FAD2A 2484 silent G2015A G201D FAD2A 2993 severe missense
C2099T S229F FAD2A 2579 severe missense G2155A G248R FAD2A 2200
severe missense G1495A, E28K, E287K FAD2A 2983 missense, severe
G2272A missense G2138A R242H FAD2A 2986 missense
TABLE-US-00011 TABLE 8 Summary of Camelina FAD2 B mutants
Nucleotide Change Effect Primer set Plant ID Mutation Score C207T
S53F FAD2B 2474 or 2199 severe missense C213T S55F FAD2B 3142
Severe Missense G785A A246T FAD2B 3363 Missense C476T R143C FAD2B
3314 Severe Missense C176T P43S FAD2B 3325 Severe Missense G462A
W138* FAD2B 3489 Nonsense G498A G150E FAD2B 3702 Severe Missense
G779A A244T FAD2B 3732 Missense G737A D230N FAD2B 3814 Missense
C812T L255F FAD2B 4245 Missense C882T P278L FAD2B 4408 Missense
G410A D121N FAD2B 4875 Missense G675A C209Y FAD2B 4916 Missense
C459T S137F FAD2B 5155 Severe Missense C528T P160L FAD2B 5746
Severe Missense C987T T313M FAD2B 6023 Severe Missense C284T P79S
FAD2B 6107 Severe Missense G416A V123I FAD2B 6122 Severe Missense
G650A G201S FAD2B 6105 Severe Missense C656T P203S FAD2B 6277
Missense C203T R52C FAD2B 6493 Severe Missense G582A G178E FAD2B
6486 Severe Missense G372A C108Y FAD2B 6479 Severe Missense G322A
W91* FAD2B 6490 Nonsense G374A G109S FAD2B 6752 Severe Missense
G926A G293R FAD2B 6778 Severe Missense C490T S147= FAD2B 3207
silent C940T T297= FAD2B 3423 silent G148A T33= FAD2B 3521
silent
TABLE-US-00012 TABLE 9 Summary of Camelina FAD2 C mutants
Nucleotide Change Effect Primer set plant ID Mutation Score G1429A
E28K FAD2C 6431 Missense C1501T R52C FAD2C 3168 Severe Missense
C1542T S65= FAD2C 5756 silent C1576T L77F FAD2C 5550 Missense
C1582T P79S FAD2C 5655 Severe Missense G1607A W87* FAD2C 4506
Nonsense C1609T P88S FAD2C 3210 Severe Missense G1619A W91* FAD2C
3284 Nonsense G1672A G109S FAD2C 3690 Severe Missense G1717A G124S
FAD2C 5644 Severe Missense C1720T L125F FAD2C 4933 Missense C1741T
L132F FAD2C 4995 Missense G1795A G150R FAD2C 3147 Severe Missense
G1796A G150E FAD2C 4608 Severe Missense C1799T S151F FAD2C 3275
Severe Missense G1808A R154K FAD2C 3490 Missense G1810A D155N FAD2C
2578, 2586 Severe Missense C1857T G170= FAD2C 4716 silent C1873T
P176S FAD2C 3267 Severe Missense G1880A G178E FAD2C 5903 Severe
Missense G1883A R179H FAD2C 4846 Severe Missense G1890A M181I FAD2C
4400 Missense G1915A G190R FAD2C 5524 Severe Missense G1948A G201S
FAD2C 6120 Severe Missense G1963A G206R FAD2C 4556 Missense C2029T
L228F FAD2C 4802 Missense G2072A R242H FAD2C 5122 Missense G2080A
A245T FAD2C 3152 Missense C2081T A245V FAD2C 5318 Missense C2084T
A246V FAD2C 4884 Missense C2096T A250V FAD2C 3318 Missense C2110T
L255F FAD2C 5734 Missense C2112T L255= FAD2C 4677 silent G2117A
G257E FAD2C 5491 Severe Missense G2117A G257E FAD2C 6470 Severe
Missense G2140A A265T FAD2C 3924 Missense G2149A V268I FAD2C 6068
Severe Missense C2188T P281S FAD2C 4864 Severe Missense C2204T
S286F FAD2C 5183 Severe Missense G2255A G303E FAD2C 4467 Severe
Missense G2268A K307= FAD2C 6509 silent C2285T T313M FAD2C 5426
Severe Missense C2293T H316Y FAD2C 2785, 2487, Severe Missense
2488, or 2786 C2315T S323L FAD2C 6060 Severe Missense G2422A E359K
FAD2C 4997 Severe Missense G2443A V366I FAD2C 6579 Missense C1595T
S83F FAD2C 4138 Severe Missense C2383T Q346* FAD2C 6077
Nonsense
TABLE-US-00013 TABLE 10 Summary of Camelina FAE1 A mutants
Nucleotide Change Effect Primer set Plant ID Mutation Score G621A
V55I FAE1-A 4696 Missense C695T L79= FAE1-A 3920 silent C714T L86F
FAE1-A 4489 Severe Missense G798A V114M FAE1-A 5495 Missense G801A
A115T FAE1-A 3436 Missense G805A C116Y FAE1-A 3533 Missense G810A
D118N FAE1-A 3424 Missense G810A D118N FAE1-A 5977 Missense C817T
S120F FAE1-A 3821 Severe Missense C820T S121L FAE1-A 4703 Missense
G821A S121= FAE1-A 6126 silent G867A E137K FAE1-A 6361 Missense
G877A S140N FAE1-A 3284 Severe Missense G997A R180K FAE1-A 3390
Missense G997A R180K FAE1-A 5346 Missense G1005A G183S FAE1-A 6655
Severe Missense C1042T T195I FAE1-A 5557 Severe Missense G1061A
M201I FAE1-A 4088 Severe Missense G1065A V203I FAE1-A 4469 Missense
C1072T T205I FAE1-A 4500 Severe Missense C1083T R209* FAE1-A 3395
Nonsense C1091T N211= FAE1-A 5486 silent G1120A G221D FAE1-A 6386
Severe Missense C1141T A228V FAE1-A 4467 Severe Missense C1167T
H237Y FAE1-A 4164 Severe Missense C1167T H237Y FAE1-A 4318 Severe
Missense G1254A V266I FAE1-A 3365 Missense G1258A S267N FAE1-A 3783
Severe Missense C1272T R272C FAE1-A 5401 Severe Missense G1311A
G285R FAE1-A 3799 Missense G1354A R299Q FAE1-A 5095 Severe Missense
G1366A G303E FAE1-A 3820 Severe Missense G1387A R310Q FAE1-A 6528
Missense G1390A C311Y FAE1-A 3631 Severe Missense G1401A G315R
FAE1-A 4257 Missense G1402A G315E FAE1-A 6186 Missense G1402A G315E
FAE1-A 6446 Missense G1407A D317N FAE1-A 3897 Severe Missense
G1416A G320S FAE1-A 5197 Severe Missense G1426A G323E FAE1-A 5680
Severe Missense G1426A G323E FAE1-A 6284 Severe Missense C1450T
T331I FAE1-A 4412 Missense G1463A G335= FAE1-A 5117 silent G1518A
E354K FAE1-A 3597 Severe Missense
TABLE-US-00014 TABLE 11 Summary of Camelina FAE1 B mutants
Nucleotide Change Effect Primer set PLANT ID Mutation Score C710T
P76L FAE1B 5778 Severe Missense C718T L79F FAE1B 5840 Severe
Missense G724A D81N FAE1B 6324 Severe Missense C731T S83L FAE1B
4318 Severe Missense C817T R112W FAE1B 4140 Severe Missense G823A
V114M FAE1B 3768 Missense G823A V114M FAE1B 5966 Missense C845T
S121L FAE1B 3758 Missense G858A L125= FAE1B 3709 silent G887A G135D
FAE1B 4015 Severe Missense C907T Q142* FAE1B 5951 Nonsense C928T
P149S FAE1B 5107 Severe Missense C952T R157C FAE1B 4840 Severe
Missense G953A R157H FAE1B 4239 Severe Missense G958A E159K FAE1B
6322 Severe Missense G969A Q162= FAE1B 3529 silent G988A E169K
FAE1B 3734 Severe Missense C1019T P179L FAE1B 3873 Severe Missense
G1031A G183D FAE1B 4135 Missense G1042A V187M FAE1B 6517 Severe
Missense C1063T P194S FAE1B 6478 Severe Missense C1082T A200V FAE1B
3986 Severe Missense G1086A M201I FAE1B 3895 Missense G1109A R209Q
FAE1B 4139 Severe Missense C1154T A224V FAE1B 3352 Severe Missense
C1229T T249I FAE1B 4169 Severe Missense G1231A E250K FAE1B 6678
Severe Missense C1271T S263F FAE1B 3829 Severe Missense C1271T
S263F FAE1B 6700 Severe Missense G1275A M264I FAE1B 6308 Severe
Missense G1306A G275R FAE1B 5333 Severe Missense C1310T A276V FAE1B
3241 Severe Missense G1314A A277= FAE1B 4884 silent C1310T A276V
FAE1B 3284 Severe Missense C1325T S281F FAE1B 5343 Severe Missense
G1337A G285E FAE1B 3358 Missense G1337A G285E FAE1B 3821 Missense
G1343A R287Q FAE1B 5930 Silent C1352T S290F FAE1B 4882 Severe
Missense C1384T H301Y FAE1B 4687 Severe Missense C1389T T302= FAE1B
5840 silent G1412A R310Q FAE1B 3936 Missense G1417A V312M FAE1B
3173 Severe Missense G1427A G315E FAE1B 3926 Missense G1435A E318K
FAE1B 6479 Missense G1441A G320S FAE1B 3842 Severe Missense C1493T
A337V FAE1B 4630 Severe Missense C1522T P347S FAE1B 3912 Severe
Missense
TABLE-US-00015 TABLE 12 Summary of Camelina FAE1 C mutants
Nucleotide Change Effect Primer set Plant ID Mutation Score A506T
T15S FAE1-C 3688 Missense A506T T15S FAE1-C 4325 Missense A506T
T15S FAE1-C 4907 Missense A506T TI5S FAE1-C 6025 Missense A506T
TI5S FAE1-C 6695 Missense C564T S34F FAE1-C 4965 Missense C605T
L48F FAE1-C 6835 Missense G704A D81N FAE1-C 4510 Severe Missense
C719T L86F FAE1-C 5015 Severe Missense G798A R112Q FAE1-C 4184
Missense C802T N113= FAE1-C 6130 silent C822T SI2OF FAE1-C 3886
Severe Missense C825T 5121L FAE1-C 4255 Missense G840A R126K FAE1-C
5936 Missense G855A R131H FAE1-C 3725 Severe Missense G855A R131H
FAE1-C 4813 Severe Missense C858T 5132L FAE1-C 5951 Severe Missense
C887T P142S FAE1-C 3918 Missense C887T P142S FAE1-C 4198 Missense
C906T P148L FAE1-C 4068 Severe Missense C911T Q150* FAE1-C 5566
Nonsense C911T Q150* FAE1-C 6139 Nonsense G926A A155T FAE1-C 3923
Missense G933A R157H FAE1-C 5576 Severe Missense G982A E173= FAE1-C
3367 silent C987T T175I FAE1-C 3247 Severe Missense G1010A G1835
FAE1-C 3365 Severe Missense C1047T T195I FAE1-C 5891 Severe
Missense G1067A V202I FAE1-C 5975 Missense C1088T R209* FAE1-C 6476
Nonsense G1115A G218R FAE1-C 3970 Severe Missense G1137A G225D
FAE1-C 3911 Severe Missense C1154T L231F FAE1-C 6643 Severe
Missense G1175A V238I FAE1-C 3380 Missense C1251T 5263F FAE1-C 5793
Severe Missense C1252T S263= FAE1-C 3885 silent G1255A M264I FAE1-C
5422 Severe Missense G1283A G274S FAE1-C 4945 Severe Missense
G1287A G275E FAE1-C 3749 Severe Missense C1305T 5281F FAE1-C 3401
Severe Missense C1305T 5281F FAE1-C 4608 Severe Missense G1316A
G285R FAE1-C 4123 Missense C1353T T297M FAE1-C 3427 Severe Missense
G1359A R299Q FAE1-C 3166 Severe Missense C1400T Q313* FAE1-C 5114
Nonsense C1403T Q314* FAE1-C 4162 Nonsense G1406A G315R FAE1-C 3776
Missense G1472A A337T FAE1-C 4852 Missense C1486T N341= FAE1-C 4399
silent C1494T T344M FAE1-C 5013 Severe Missense C1502T P347S FAE1-C
6553 Severe Missense
[0232] As tables 7-12 indicate, multiple mutants were isolated in
each FAD2 or FAE1 gene copy. The types of mutants include missense,
severe missense, nonsense and silent mutations.
Example 12
Fatty Acids Composition in FAD2 and FAE1 Mutants
[0233] Fatty acid methyl ester (FAME) composition in Camelina FAD2
mutants was analyzed in a preliminary test by gas chromatography
(GC) following the protocol described in Example 1. The results
were shown in Table 13.
TABLE-US-00016 TABLE 13 % FAME content in Camelina FAD2 mutants Cs
32 Combined FAD2A FAD2A missense FAD2A missense wild Null Q44*
G150E S229F FAD2B W91* Mutation type Population HOMO HOMO NULL HOMO
NULL HOMO NULL sample size 10 14 8 7 4 5 4 6 6 C18:1 14.4 .+-. 0.4
14.3 .+-. 2.0 22.6 .+-. 1.2 24.0 .+-. 1.2 17.1 .+-. 0.9 19.2 .+-.
1.3 13.9 .+-. 0.9 18.4 .+-. 0.6 12.8 .+-. 0.5 C18:2 21.4 .+-. 0.8
28.8 .+-. 2.8 20.3 .+-. 1.1 19.0 .+-. 0.5 26.9 .+-. 1.2 22.2 .+-.
1.1 26.8 .+-. 1.4 28.7 .+-. 0.4 31.5 .+-. 2.0 C18:3 33.7 .+-. 0.6
25.4 .+-. 1.9 26.2 .+-. 1.8 26.1 .+-. 1.6 26.0 .+-. 1.1 25.7 .+-.
1.2 26.5 .+-. 1.3 23.8 .+-. 1.5 24.2 .+-. 2.2 C20:1 15.5 .+-. 1.0
10.7 .+-. 1.4 12.2 .+-. 1.0 13.1 .+-. 1.4 10.5 .+-. 1.3 13.4 .+-.
1.0 10.8 .+-. 1.3 10.2 .+-. 1.6 10.7 .+-. 1.2 % increase in 56.9%
66.7% 33.3% 27.8% 18:1 relative to wild type seeds Note: HOMO means
the plants are all homozygous mutants at the specified locus. NULL
means there is no mutation at the specified locus. % means % of
FAME composition
[0234] As the results indicate, an obvious increase of oleic acid
(18:1) was observed in certain FAD2 mutants tested compared to NULL
control plants. Thus, the data supports the inventors' prediction
very well that disruption in one, two or more FAD2 gene in Camelina
is sufficient to alter its fatty acid composition, and more
specifically, to increase the oleic acid (18:1) concentration.
[0235] More mutants in FAD2 genes and FAE1 genes were subjected to
GC analysis. To select mutants with potentially the most profound
phenotype, FAD2 A, B, and C, or FAE1 A, B, and C protein sequences
were analyzed against orthologs in Arabidopsis, Crambe, B. rapa
HEAC, B. rapa LEAC, meadow foam, and nasturtium. It is preferred
that a mutation happens at the position which is conserved through
reference species, and/or a position described before as conserved
in orthologs or close-related genes in other species (e.g., see
reference 52, Ghanevati and Jaworski, 2002, and Jet et al.,
Dissection of malonyl-coenzyme A decarboxylation from polyketide
formation in the reaction mechanism of a plant polyketide synthase,
Biochemistry, 39:890-902). For example, the G150E, Q44* (nonsense),
S229F and W91* (nonsense) mutations in FAD2 genes are potentially
very promising as are the following mutants in FAE1: Q142*
(nonsense), R209* (nonsense), G221D and H301Y.
[0236] Two independent GC analyses of fatty acid compositions in
FAD2 A and FAD2 B mutants were conducted, and the results are shown
in Tables 14 and 15. Mutants with clear increases in oleic acid
were selected, and their results from both GC runs were averaged
together to produce Table 16. Some of these tested mutations have
obvious increased oleic acid (18:1), such as FAD2 A mutants G150E,
Q44*, S229F, and FAD2 B mutants W91* compared to NULL population or
wild-type Cs32 control plants, while no significant difference was
found between NULL population and wild type Cs32 plants. Table 17
shows the fatty acid compositions of selected FAD2 mutants for one
of the independent GC analyses. The result of Table 16 is further
summarized in FIG. 12A. As the results indicate, these mutants have
evident increased oleic acid (18:1) and reduced polyunsaturated
fatty acids (e.g., 18:2 and 18:3) in seed oil, just as the
inventors predicted.
[0237] A third independent GC analysis was conducted in which FAD2
C mutants were included. This was a preliminary analysis where
seeds from heterozygous plants were used, resulting in a mixed
population containing null, heterozygous and homozygous seeds. The
results (see Table 18 in U.S. Provisional Application No.
61/318,273, incorporated by reference in its entirety) showed that
all tested FAD2 C mutants do not have significant induction of 18:1
fatty acid, as compared to Cs32 control plants. While not wishing
to be bound by any particular theory, the results suggest that any
potential increase in 18:1 in a FAD2 C mutant plant is not
detectable in progeny from heterozygous plants, where the mixture
of wild type, heterozygous and homozygous seeds may dilute the
effects of the homozygous seed.
[0238] The same preliminary third GC run analyzed mutants at the
FAE1 loci. Though results (see Table 19 and Table 20 in U.S.
Provisional Application No. 61/318,273) showed that some of these
tested mutants, for example FAE1 A mutant R272C, FAE1 B mutants
S281F and R209Q, and FAE1C mutants Q313* and Q150* had obvious
decreased 20:1 and/or 22:1 in seed oil relative to wild type Cs32
plants, the inclusion of a significant number of heterozygous lines
may have confounded the results as was the case with the FAD2 C
results above.
[0239] A fourth independent GC analysis (results shown in Tables
18a and 19a of the present specification) was conducted on M4 or M5
generation FAD2 A, FAD2 B, FAD2 C, FAE1 A, FAE1 B and FAE1 C
mutants. This analysis included multiple homozygous lines for a
given FAD2 or FAE1 mutation, which conferred more confidence in the
results due to multiple samples for a given mutation. In addition,
the inventors limited the number of heterozygous lines analyzed
where the seeds were a mixture of homozygous (designated `hom`),
heterozygous (designated `het`) and null because of ambiguous
results in the third GC run. In test 4, Arabidopsis FAD2 and FAE1
mutants, wild type Camelina sativa CS32, and null sibling lines not
carrying a FAD2 or FAE1 mutation were included as controls. From
this analysis, some FAD2 A Q44* and G150E, FAD2 B W91* and G150E,
and FAD2C W87* homozygous or heterozygous lines clearly had greater
18:1 fatty acid levels compared to their null sibling lines or the
CS32 control.
[0240] For FAE1, some FAE1 A G221D, FAE1 B Q142* and H301Y, and
FAE1 C R209* homozygous or heterozygous lines clearly had lower
20:1 fatty acid levels and/or lower 22:1 fatty acid levels compared
to their null sibling lines or the CS32 control. The FAE1 data from
the fourth GC run is summarized in FIG. 12B (this Figure replaces
FIG. 14B from U.S. Provisional Application No. 61/318,273, which
summarized FAE1 data from the third GC run). This data supports the
inventors' prediction that disruption in one, two or more FAE1
genes in Camelina is sufficient to alter its fatty acid
composition, and more specifically, to decrease the very long chain
(for example 20:1 and 22:1) fatty acid content.
[0241] The fourth GC analysis did not include some FAD2 C, FAE1 A,
FAE1 B and FAE1 C mutants included in the third GC analysis due to
pursuance of a select number of mutant lines in the breeding
program for FAD2 (A, B and C) and FAE1 (A, B and C) mutants. In
particular, FAD2 C mutants Q346*, G150R, R242H, G190R were included
in the third but not the fourth GC analysis. Similarly, FAE1 A
mutants G183S, R272C, C311Y, FAE1 B mutants P76L, L79F, R157H,
R209Q, E250K, W91*, and FAE1 C mutants R157H, G225D, L231F, G274S,
Q313*, Q314* were included in the third but not the fourth GC
analysis. The FAE1 C Q150* mutant, which was analyzed in test 3 but
not test 4, will be tested for fatty acid composition in future GC
runs. In test 3, homozygous FAE1 C Q150* mutant plants were used
for analysis. According to the GC data in test 3, the fatty acids
composition in homozygous FAE1 C Q150* mutant is as following:
C16:0, 6.19%; C18:0, 2.74%; C18:1, 13.81%; C18:2, 24.05%; C20:0,
0.97%; C18:3, 33.28%; C20:1, 14.25%; C20:2, 1.79%; C20:3, 0.70%;
and C22:1, 2.23%.
TABLE-US-00017 TABLE 14 Fatty Acids Composition in FAD2 mutants,
sorted by mutation, Test No. 1 muta- geno- Line Gene SNP tion Plant
# type C16:0 C18:0 C18:1 C18:2 C20:0 C18:3 C20:1 C20:2 C20:3 C22:1
2362 FAD2A 5 G150E 4 HOMO 7.9% 4.3% 22.9% 19.1% 1.9% 28.1% 12.3%
0.8% 0.7% 2.1% 2362 FAD2A 5 G150E 5 HOMO 7.8% 3.8% 23.0% 19.1% 1.8%
28.1% 13.1% 0.7% 0.6% 2.1% 2362 FAD2 A 5 G150E 1 HET 7.3% 3.8%
22.3% 20.7% 2.1% 25.7% 14.3% 0.9% 0.6% 2.3% 2362 FAD2 A 5 G150E 2
HET 11.0% 5.4% 13.6% 15.0% 2.8% 34.6% 12.9% 1.4% 0.8% 2.4% 2362
FAD2A 5 G150E 11 Null 8.3% 5.2% 17.9% 25.3% 2.7% 27.4% 10.0% 1.0%
0.5% 1.7% 2362 FAD2A 5 G150E 18 Null 9.0% 5.1% 17.8% 28.2% 3.2%
24.9% 8.9% 1.2% 0.6% 1.1% 2510 FAD2A 4 S147F 3 HOMO 7.2% 5.1% 15.9%
24.4% 3.8% 28.4% 10.3% 1.3% 0.8% 2.7% 2510 FAD2A 4 S147F 10 HOMO
9.0% 6.4% 15.9% 29.2% 3.5% 25.0% 7.1% 1.2% 0.6% 2.1% 2510 FAD2A 4
S147F 1 HET 8.2% 5.5% 15.1% 27.2% 3.3% 27.9% 8.0% 1.3% 0.8% 2.8%
2510 FAD2A 4 S147F 4 HET 8.4% 5.7% 15.8% 30.1% 3.7% 23.7% 8.5% 1.3%
0.3% 2.4% 2510 FAD2A 4 S147F 2 Null 8.5% 4.7% 12.6% 28.2% 3.5%
27.7% 9.2% 1.7% 0.9% 2.9% 2510 FAD2A 4 S147F 5 Null 7.7% 3.5% 14.0%
26.9% 2.5% 29.3% 10.7% 1.5% 0.9% 3.0% 2579 FAD2A 7 S229F 1 HOMO
7.3% 3.4% 19.3% 23.6% 1.7% 26.5% 13.0% 1.2% 0.7% 3.2% 2579 FAD2A 7
S229F 5 HOMO 8.0% 4.4% 20.9% 22.3% 2.2% 27.4% 10.6% 1.1% 0.4% 2.8%
2579 FAD2A 7 S229F 2 HET 7.5% 3.6% 17.0% 25.8% 0.1% 31.7% 10.1%
1.6% 0.4% 2.3% 2579 FAD2A 7 S229F 3 HET 7.7% 4.6% 16.5% 25.0% 0.1%
30.5% 11.2% 1.3% 0.8% 2.4% 2579 FAD2A 7 S229F 10 Null 8.4% 3.6%
12.0% 26.8% 2.6% 28.9% 11.9% 1.9% 0.9% 3.1% 2579 FAD2A 7 S229F 12
Null 8.0% 5.2% 14.4% 26.1% 3.5% 28.9% 8.9% 1.5% 0.8% 2.8% 2764
FAD2A 3 R144H 5 HOMO 7.5% 3.8% 22.1% 26.3% 0.1% 26.0% 10.5% 1.1%
0.5% 2.1% 2764 FAD2A 3 R144H 8 HOMO 7.5% 3.3% 17.0% 21.6% 0.1%
34.4% 11.3% 1.2% 0.9% 2.9% 2764 FAD2A 3 R144H 10 HOMO 6.9% 4.3%
17.6% 22.5% 2.9% 28.4% 12.3% 1.2% 0.8% 3.2% 2764 FAD2A 3 R144H 16
HOMO 6.5% 3.0% 18.7% 24.5% 1.7% 26.9% 13.3% 1.3% 0.7% 3.4% 2764
FAD2A 3 R144H 1 HET 6.9% 3.4% 18.4% 24.5% 1.5% 27.7% 12.7% 1.3%
0.5% 3.0% 2764 FAD2A 3 R144H 2 HET 7.7% 3.9% 15.7% 25.5% 0.1% 32.5%
10.1% 1.4% 0.8% 2.3% 2764 FAD2A 3 R144H 3 HET 7.3% 3.5% 17.6% 26.5%
0.1% 29.1% 11.4% 1.4% 0.7% 2.4% 2764 FAD2A 3 R144H 6 HET 7.7% 3.3%
16.8% 22.3% 0.1% 34.5% 10.8% 1.1% 0.9% 2.6% 2764 FAD2A 3 R144H 4
Null 6.8% 3.5% 17.1% 27.8% 1.7% 24.9% 12.8% 1.5% 0.7% 3.1% 2764
FAD2A 3 R144H 7 Null 7.1% 3.2% 17.1% 25.2% 1.5% 28.2% 12.5% 1.5%
0.5% 3.3% 2785 FAD2C 11 H316Y 3 HOMO 8.1% 3.7% 16.6% 20.5% 2.6%
32.1% 11.2% 1.3% 0.8% 3.2% 2785 FAD2C 11 H316Y 10 HOMO 8.3% 3.7%
15.2% 16.7% 2.3% 34.2% 14.3% 1.2% 1.1% 3.1% 2785 FAD2C 11 H316Y 12
HOMO 8.5% 3.5% 14.3% 18.0% 2.7% 32.8% 14.1% 1.2% 1.2% 3.7% 2785
FAD2C 11 H316Y 18 HOMO 8.1% 3.9% 14.8% 19.1% 2.1% 35.0% 11.0% 1.3%
1.3% 3.4% 2785 FAD2C 11 H316Y 1 HET 8.8% 4.0% 14.3% 22.0% 2.0%
33.3% 9.9% 1.6% 0.8% 3.4% 2785 FAD2C 11 H316Y 6 HET 8.9% 3.9% 14.4%
20.5% 0.1% 36.0% 11.1% 1.3% 1.1% 2.8% 2785 FAD2C 11 H316Y 7 HET
8.2% 3.9% 14.4% 20.5% 0.1% 36.1% 11.1% 1.3% 1.1% 3.1% 2785 FAD2C 11
H316Y 8 HET 9.0% 3.9% 12.6% 21.0% 0.1% 38.2% 10.2% 1.5% 1.0% 2.5%
2785 FAD2C 11 H316Y 2 Null 8.6% 3.8% 11.3% 20.6% 2.8% 35.1% 11.1%
1.6% 1.4% 3.7% 2785 FAD2C 11 H316Y 5 Null 8.8% 4.7% 12.8% 20.7%
3.0% 34.2% 10.3% 1.4% 1.1% 3.0% 2812 FAD2B 9 H145Y 1 HOMO 8.3% 4.1%
15.1% 27.4% 2.9% 24.6% 12.4% 1.5% 0.7% 3.1% 2812 FAD2B 9 H145Y 2
HOMO 9.9% 3.6% 13.3% 28.7% 2.4% 27.0% 9.3% 1.5% 0.8% 3.5% 2812
FAD2B 9 H145Y 12 HET 7.8% 3.9% 16.6% 29.1% 2.6% 24.6% 10.7% 1.5%
0.7% 2.6% 2812 FAD2B 9 H145Y 25 HET 7.8% 4.1% 15.9% 30.5% 2.2%
25.8% 9.0% 1.6% 0.7% 2.4% 2826 FAD2A 2 Q44* 1 HOMO 7.7% 4.9% 22.2%
20.1% 2.1% 28.4% 11.0% 0.8% 0.6% 2.1% 2826 FAD2A 2 Q44* 2 HOMO 7.8%
4.9% 19.9% 19.2% 2.6% 29.5% 11.7% 1.0% 0.8% 2.6% 2826 FAD2A 2 Q44*
3 HOMO 7.5% 4.6% 22.9% 19.8% 2.1% 27.7% 11.7% 0.8% 0.6% 2.3% 2826
FAD2A 2 Q44* 4 HOMO 7.7% 5.4% 24.7% 20.3% 2.5% 25.7% 10.6% 0.7%
0.5% 1.9% 2826 FAD2A 2 Q44* 5 HOMO 7.6% 5.2% 22.9% 19.1% 2.2% 28.1%
11.5% 0.8% 0.6% 2.0% 2826 FAD2A 2 Q44* 37 HET 7.3% 4.8% 22.9% 19.1%
2.0% 28.5% 12.2% 0.7% 0.6% 1.9% 3006 FAD2B 8 W91* 1 HOMO 7.9% 4.6%
18.9% 28.5% 1.7% 26.8% 8.9% 1.0% 0.5% 1.1% 3006 FAD2B 8 W91* 2 HOMO
8.2% 5.3% 18.8% 29.2% 1.7% 26.3% 7.8% 0.9% 0.4% 1.3% 3006 FAD2B 8
W91* 3 HOMO 8.0% 5.5% 18.4% 28.9% 2.7% 24.4% 8.6% 1.1% 0.5% 1.8%
3006 FAD2B 8 W91* 4 HOMO 7.5% 5.1% 18.0% 28.9% 2.9% 22.9% 10.6%
1.3% 0.5% 2.3% 3006 FAD2B 8 W91* 5 Null 7.8% 4.2% 12.3% 33.5% 2.9%
23.8% 9.3% 2.1% 0.7% 3.5% 3006 FAD2B 8 W91* 7 Null 7.9% 4.4% 17.6%
30.0% 2.1% 24.9% 9.7% 1.2% 0.5% 1.6% 3006 FAD2B 8 W91* 8 HET 8.0%
3.9% 13.4% 30.6% 2.2% 26.9% 9.3% 2.0% 0.5% 3.1% Note: *stands for
nonsense mutation; HOMO means the plants are all homozygous mutants
at the specified locus. HET means the plants are heterozygous
mutants at the specified locus. NULL means there is no mutation at
the specified locus. % means % of FAME composition
TABLE-US-00018 TABLE 15 Fatty Acids Composition in FAD2 mutants,
sorted by mutation, Test No. 2 muta- Plant geno- # of Gene SNP tion
# type samples C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2
C20:3 C22:1 none none 1 CS32 10 5.8% 2.3% 14.9% 20.3% 34.0% 1.2%
16.2% 1.8% 0.9% 2.7% controls none none 2 CS32 controls 6.1% 2.3%
14.6% 21.3% 33.3% 1.2% 15.6% 1.8% 1.2% 2.6% none none 3 CS32
controls 6.2% 2.3% 14.1% 21.7% 34.2% 1.1% 15.5% 1.8% 1.0% 2.1% none
none 4 CS32 controls 6.0% 2.4% 14.2% 20.8% 34.5% 1.2% 15.8% 1.6%
1.0% 2.4% none none 5 CS32 controls 6.7% 2.6% 14.6% 23.1% 33.5%
0.7% 12.9% 1.8% 1.3% 2.7% none none 6 CS32 controls 6.2% 2.5% 14.6%
21.6% 33.4% 1.0% 15.4% 1.8% 1.1% 2.5% none none 7 CS32 controls
5.9% 2.4% 14.6% 22.2% 32.7% 1.3% 15.7% 1.8% 1.0% 2.5% none none 8
CS32 controls 6.0% 2.3% 13.6% 21.0% 34.3% 1.4% 16.0% 1.9% 1.0% 2.6%
none none 9 CS32 controls 5.9% 2.4% 14.3% 20.6% 33.6% 1.3% 16.2%
1.9% 1.1% 2.7% none none 10 CS32 controls 6.2% 2.4% 13.9% 21.4%
33.2% 1.3% 15.9% 1.9% 1.2% 2.6% FAD2B/ 51 D60N Y1 605 1 8.7% 2.5%
10.4% 29.0% 28.4% 1.7% 13.3% 2.1% 0.9% 3.0% C FAD2A 5 G150E 4 HOMO
1 8.0% 4.4% 22.5% 18.8% 27.5% 2.2% 12.7% 0.5% 1.7% 1.7% FAD2A 5
G150E 5 HOMO 1 7.8% 4.2% 23.2% 18.4% 26.6% 2.2% 14.2% 0.7% 0.6%
2.2% FAD2A G150E 20 Null 2 new 8.7% 4.2% 16.2% 26.7% 25.7% 2.2%
12.0% 1.3% 0.4% 2.4% FAD2A G150E 24 Null 8.7% 5.1% 16.4% 27.4%
26.0% 2.2% 10.9% 1.1% 0.6% 1.6% FAD2A G150E 6 HOMO 5 new 7.7% 4.2%
26.1% 18.8% 25.2% 1.9% 13.3% 0.6% 0.5% 1.7% FAD2A G150E 8 HOMO 8.1%
4.6% 24.2% 18.7% 28.1% 2.2% 10.2% 0.7% 0.5% 2.7% FAD2A G150E 14
HOMO 8.2% 4.5% 24.6% 19.4% 24.7% 2.0% 13.7% 0.6% 0.3% 1.9% FAD2A
G150E 23 HOMO 8.6% 4.7% 23.0% 19.0% 26.7% 2.1% 13.1% 0.6% 0.5% 1.8%
FAD2A G150E 25 HOMO 8.0% 4.0% 24.2% 20.0% 23.7% 1.8% 14.6% 0.7%
0.5% 2.5% FAD2A G150E 3 Het 3 new 7.9% 4.4% 21.1% 22.2% 27.2% 2.0%
12.4% 0.8% 0.3% 1.7% at least FAD2A G150E 7 Het 8.5% 4.5% 19.1%
24.3% 24.8% 2.1% 12.8% 1.0% 0.6% 2.2% FAD2A G150E 9 Het 8.4% 4.7%
17.9% 23.3% 27.5% 2.5% 12.2% 1.0% 0.6% 1.8% FAD2A 2 Q44* 2 HOMO 1
8.3% 5.1% 22.8% 20.8% 26.7% 2.0% 11.1% 0.8% 0.4% 2.0% FAD2A 2 Q44*
1 HOMO 1 7.8% 4.9% 19.7% 18.9% 28.3% 2.6% 13.9% 0.9% 0.7% 2.4%
FAD2A 2 Q44* 4 HOMO 1 7.6% 5.1% 23.7% 20.2% 25.7% 2.4% 12.8% 0.6%
0.4% 1.5% FAD2A Q44* 40 Null 1 8.2% 6.1% 25.6% 20.4% 24.3% 2.4%
10.8% 0.6% 0.3% 1.4% FAD2A Q44* 36 HOMO 3 7.8% 5.5% 23.2% 21.4%
23.8% 2.5% 13.0% 0.7% 0.2% 1.8% FAD2A Q44* 38 HOMO 8.0% 6.2% 22.3%
20.4% 25.4% 2.9% 12.4% 0.7% 0.3% 1.5% FAD2A Q44* 39 HOMO 9.2% 5.6%
22.9% 22.0% 23.7% 2.3% 11.0% 0.7% 0.4% 2.0% FAD2A 3 R144H 16 HOMO 1
6.9% 2.9% 18.5% 26.2% 26.5% 1.6% 13.1% 1.2% 0.3% 2.9% FAD2A 3 R144H
5 HOMO 1 7.1% 4.0% 21.7% 26.0% 23.2% 2.4% 12.1% 0.9% 0.3% 2.3%
FAD2A R144H 19 Null 2 (34) 7.1% 3.0% 17.8% 27.1% 26.0% 1.7% 12.8%
1.3% 0.4% 2.7% FAD2A R144H 25 Null 7.4% 4.3% 15.2% 25.6% 29.6% 2.4%
11.5% 1.2% 0.4% 2.4% FAD2A 7 S229F 5 HOMO 1 7.6% 3.8% 19.6% 21.4%
27.4% 2.0% 13.9% 1.0% 0.6% 2.6% FAD2A 7 S229F 3 HET 1 7.9% 5.1%
18.2% 26.0% 25.7% 2.0% 10.4% 1.2% 0.7% 2.6% FAD2A 7 S229F 12 Null 1
8.6% 5.6% 15.0% 27.8% 26.6% 3.4% 8.2% 1.4% 0.7% 2.8% FAD2A 7 S229F
10 Null 1 9.3% 5.7% 12.8% 28.1% 25.4% 3.7% 10.1% 1.8% 0.4% 2.7%
FAD2A S229F 7 HOMO 3 8.4% 5.8% 18.4% 22.7% 24.5% 3.5% 11.8% 1.1%
0.6% 3.2% FAD2A S229F 9 HOMO 7.3% 5.1% 17.8% 22.4% 25.2% 3.6% 14.0%
1.1% 0.6% 2.9% FAD2A S229F 19 HOMO 6.9% 5.0% 21.0% 20.9% 24.8% 2.7%
14.3% 0.9% 0.6% 2.9% FAD2A S229F 13 Null 2 7.8% 5.6% 14.1% 26.4%
25.6% 3.6% 12.2% 1.5% 0.7% 2.5% FAD2A S229F 14 Null 7.6% 4.8% 13.8%
25.0% 28.3% 3.2% 12.6% 1.2% 0.8% 2.6% FAD2A S229F 4 Het 3 (46) 7.6%
3.5% 15.6% 23.8% 27.3% 2.3% 14.2% 1.6% 0.8% 3.2% FAD2A S229F 6 Het
7.7% 2.8% 14.8% 24.5% 29.4% 1.9% 13.5% 1.5% 0.8% 3.1% FAD2A S229F 8
Het 7.3% 5.0% 14.2% 22.6% 28.2% 3.7% 13.5% 1.4% 1.0% 3.2% FAD2A
S229F Y1 610 7 5.3% 2.3% 16.5% 21.8% 29.8% 1.5% 16.2% 2.0% 1.2%
3.4% FAD2A S229F Y2 610 5.5% 2.3% 16.1% 22.0% 30.6% 1.2% 16.2% 1.9%
1.2% 2.9% FAD2A S229F Y3 610 6.5% 2.2% 17.0% 23.3% 29.5% 1.1% 15.3%
1.7% 0.9% 2.4% FAD2A S229F Y4 610 5.9% 2.0% 14.8% 21.8% 34.2% 0.9%
14.7% 2.0% 1.1% 2.5% FAD2A S229F Y5 610 5.9% 2.0% 14.4% 22.3% 34.7%
1.0% 14.7% 1.8% 1.0% 2.3% FAD2A S229F Y6 610 5.5% 2.2% 16.7% 21.6%
31.9% 1.2% 15.2% 1.8% 1.1% 2.8% FAD2A S229F Y7 610 6.3% 2.5% 17.8%
23.7% 28.4% 1.4% 14.8% 1.6% 0.9% 2.6% FAD2B W91* 1 HOMO 1 7.7% 5.1%
19.4% 28.6% 23.9% 1.9% 10.9% 0.9% 0.4% 1.2% FAD2B W91* 8 HET 1 7.8%
5.5% 18.5% 27.5% 24.4% 2.5% 10.9% 0.9% 0.5% 1.5% FAD2B W91* 7 Null
1 7.6% 3.9% 12.4% 32.4% 23.9% 2.6% 11.5% 1.9% 0.7% 3.1% FAD2B W91*
5 Null 1 7.9% 4.2% 12.8% 29.4% 26.1% 2.6% 11.8% 1.7% 0.7% 2.9%
FAD2B W91* 10 Null 4 8.4% 5.0% 12.1% 28.8% 26.9% 2.8% 11.4% 1.7%
0.8% 2.0% FAD2B W91* 13 Null 7.6% 4.8% 13.0% 33.7% 21.9% 2.8% 10.8%
1.8% 0.6% 2.9% FAD2B W91* 23 Null 8.3% 5.8% 13.4% 31.9% 21.4% 4.0%
10.6% 1.7% 0.4% 2.6% FAD2B W91* 24 Null 9.1% 5.1% 13.1% 32.8% 24.8%
2.2% 8.4% 1.8% 0.3% 2.5% FAD2B W91* 21 HOMO 2 7.9% 5.0% 17.9% 28.5%
23.5% 2.2% 11.5% 1.1% 0.5% 1.8% FAD2B W91* 22 HOMO 7.7% 6.0% 18.1%
28.1% 21.8% 3.3% 11.5% 1.1% 0.4% 2.0% FAD2B W91* Y1 1105 9 5.9%
2.5% 19.7% 22.9% 33.6% 0.6% 12.3% 1.0% 0.7% 0.8% FAD2B W91* Y2 1105
6.4% 2.5% 19.0% 24.5% 30.4% 1.0% 13.0% 1.2% 0.7% 1.3% FAD2B W91* Y3
1105 6.8% 2.7% 18.8% 25.4% 31.6% 0.6% 11.5% 1.2% 0.5% 1.0% FAD2B
W91* Y4 1105 7.1% 3.0% 19.8% 26.8% 28.0% 1.0% 11.9% 1.0% 0.5% 0.9%
FAD2B W91* Y5 1105 6.4% 2.4% 19.8% 25.1% 29.7% 0.8% 12.7% 1.1% 0.6%
1.5% FAD2B W91* Y6 1105 6.8% 2.6% 18.1% 26.1% 29.6% 1.1% 12.7% 1.3%
0.5% 1.3% FAD2B W91* Y7 1105 6.0% 2.7% 14.6% 24.5% 30.3% 1.0% 14.9%
2.0% 1.1% 2.9% FAD2B W91* Y8 1105 5.9% 2.5% 19.0% 23.0% 32.0% 0.9%
13.7% 1.2% 0.5% 1.2% FAD2B W91* Y9 1105 6.5% 2.5% 17.2% 24.2% 31.7%
1.1% 13.4% 1.1% 0.7% 1.5% Note: *stands for nonsense mutation; HOMO
means the plants are all homozygous mutants at the specified locus.
HET means the plants are heterozygous mutants at the specified
locus. NULL means there is no mutation at the specified locus. %
means % of FAME composition
TABLE-US-00019 TABLE 16 Fatty Acids Composition in selected FAD2
mutants, sorted by mutation, Average of Test No. 1 and Test No. 2
muta- Plant # of Gene SNP tion # genotype samples C16:0 C18:0 C18:1
C18:2 C18:3 C20:0 C20:1 C20:2 C20:3 C22:1 none none CS32 1 CS32 10
5.79% 2.28% 14.91% 20.26% 34.01% 1.20% 16.16% 1.75% 0.92% 2.71%
controls controls none none CS32 2 CS32 6.07% 2.30% 14.63% 21.35%
33.27% 1.19% 15.64% 1.80% 1.17% 2.56% controls controls none none
CS32 3 CS32 6.17% 2.30% 14.13% 21.74% 34.22% 1.08% 15.46% 1.79%
1.02% 2.09% controls controls none none CS32 4 CS32 6.03% 2.40%
14.23% 20.78% 34.53% 1.23% 15.81% 1.56% 1.01% 2.41% controls
controls none none CS32 5 CS32 6.73% 2.57% 14.63% 23.08% 33.52%
0.75% 12.87% 1.83% 1.34% 2.67% controls controls none none CS32 6
CS32 6.22% 2.46% 14.57% 21.59% 33.42% 0.98% 15.40% 1.84% 1.05%
2.47% controls controls none none CS32 7 CS32 5.88% 2.42% 14.58%
22.23% 32.70% 1.26% 15.70% 1.79% 0.97% 2.46% controls controls none
none CS32 8 CS32 6.03% 2.26% 13.61% 20.96% 34.27% 1.35% 15.97%
1.89% 1.04% 2.62% controls controls none none CS32 9 CS32 5.93%
2.38% 14.34% 20.60% 33.61% 1.26% 16.23% 1.85% 1.11% 2.70% controls
controls none none CS32 10 CS32 6.19% 2.42% 13.93% 21.40% 33.20%
1.35% 15.85% 1.88% 1.20% 2.57% controls controls FAD2A Cs32 14.36%
21.40% 33.67% 15.51% AVE FAD2A Cs32 SD 0.39% 0.83% 0.57% 0.96%
FAD2A 5 G150E 4 HOMO 1 7.98% 4.39% 22.51% 18.83% 27.52% 2.20%
12.69% 0.47% 1.71% 1.71% FAD2A 5 G150E 5 HOMO 1 7.80% 4.19% 23.22%
18.43% 26.56% 2.16% 14.17% 0.68% 0.56% 2.24% FAD2A G150E 6 HOMO 5
new 7.68% 4.22% 26.13% 18.78% 25.20% 1.94% 13.30% 0.55% 0.46% 1.74%
FAD2A G150E 8 HOMO 8.09% 4.58% 24.20% 18.71% 28.10% 2.24% 10.23%
0.73% 0.46% 2.66% FAD2A G150E 14 HOMO 8.24% 4.46% 24.63% 19.43%
24.68% 2.02% 13.72% 0.64% 0.29% 1.89% FAD2A G150E 23 HOMO 8.60%
4.73% 22.96% 19.00% 26.72% 2.07% 13.07% 0.58% 0.52% 1.76% FAD2A
G150E 25 HOMO 8.05% 4.01% 24.16% 19.98% 23.65% 1.84% 14.60% 0.72%
0.52% 2.46% FAD2A G150E Homo 23.97% 19.02% 26.06% 13.11% AVE FAD2A
G150E Homo SD 1.22% 0.52% 1.60% 1.43% FAD2A G150E 20 Null 2 new
8.73% 4.20% 16.24% 26.75% 25.73% 2.17% 12.03% 1.30% 0.44% 2.41%
FAD2A 5 G150E 11 Null 8.29% 5.23% 17.87% 25.33% 27.41% 2.72% 9.99%
0.98% 0.49% 1.69% FAD2A 5 G150E 18 Null 8.98% 5.13% 17.82% 28.18%
24.86% 3.19% 8.92% 1.20% 0.57% 1.14% FAD2A G150E 24 Null 8.74%
5.08% 16.38% 27.40% 26.04% 2.17% 10.88% 1.09% 0.63% 1.59% FAD2A
G150E Null AVE 17.08% 26.91% 26.01% 10.46% FAD2A G150E null SD
0.89% 1.21% 1.06% 1.32% FAD2A 2 Q44* 2 HOMO 1 8.29% 5.10% 22.83%
20.76% 26.73% 2.03% 11.13% 0.78% 0.39% 1.96% FAD2A 2 Q44* 1 HOMO 1
7.80% 4.89% 19.67% 18.86% 28.30% 2.61% 13.90% 0.87% 0.73% 2.36%
FAD2A 2 Q44* 4 HOMO 1 7.63% 5.05% 23.75% 20.19% 25.73% 2.39% 12.83%
0.60% 0.38% 1.45% FAD2A 2 Q44* 5 HOMO 7.61% 5.25% 22.91% 19.10%
28.07% 2.20% 11.49% 0.78% 0.62% 1.95% FAD2A 2 Q44* 3 HOMO 7.50%
4.63% 22.86% 19.84% 27.72% 2.09% 11.66% 0.80% 0.63% 2.26% FAD2A
Q44* 36 HOMO 3 7.79% 5.55% 23.24% 21.36% 23.80% 2.46% 13.03% 0.71%
0.22% 1.84% FAD2A Q44* 38 HOMO 7.98% 6.21% 22.35% 20.37% 25.41%
2.87% 12.40% 0.66% 0.28% 1.47% FAD2A Q44* 39 HOMO 9.19% 5.64%
22.93% 22.02% 23.75% 2.34% 10.96% 0.74% 0.42% 2.00% FAD2A Q44* Homo
22.57% 20.31% 26.19% 12.18% AVE FAD2A Q44* homo SD 1.24% 1.07%
1.82% 1.04% FAD2A 7 S229F 5 HOMO 1 7.60% 3.79% 19.62% 21.37% 27.42%
2.01% 13.94% 0.97% 0.63% 2.64% FAD2A 7 S229F 1 HOMO 7.31% 3.36%
19.34% 23.61% 26.47% 1.74% 13.03% 1.25% 0.73% 3.17% FAD2A S229F 7
HOMO 3 8.44% 5.78% 18.39% 22.68% 24.53% 3.52% 11.78% 1.09% 0.56%
3.22% FAD2A S229F 9 HOMO 7.32% 5.15% 17.75% 22.35% 25.15% 3.56%
14.04% 1.11% 0.65% 2.91% FAD2A S229F 19 HOMO 6.92% 4.96% 21.02%
20.89% 24.77% 2.73% 14.28% 0.92% 0.63% 2.87% FAD2A S229F 20 HOMO
19.23% 22.18% 25.67% 13.42% AVE FAD2A S229F 21 HOMO 1.25% 1.08%
1.23% 1.03% SD FAD2A 7 S229F 12 Null 1 8.62% 5.59% 14.98% 27.76%
26.56% 3.41% 8.20% 1.36% 0.69% 2.82% FAD2A 7 S229F 10 Null 1 9.35%
5.67% 12.84% 28.10% 25.37% 3.68% 10.10% 1.77% 0.41% 2.71% FAD2A
S229F 13 Null 2 7.77% 5.64% 14.08% 26.36% 25.64% 3.59% 12.18% 1.49%
0.75% 2.51% FAD2A S229F 14 Null 7.57% 4.84% 13.78% 24.96% 28.33%
3.24% 12.64% 1.21% 0.85% 2.59% FAD2A S229F 15 Null AVE 13.92%
26.80% 26.47% 10.78% FAD2A S229F 16 Null SD 0.88% 1.44% 1.34% 2.05%
FAD2B W91* 1 HOMO 1 7.72% 5.07% 19.39% 28.57% 23.91% 1.93% 10.91%
0.92% 0.44% 1.15% FAD2B W91* 21 HOMO 2 7.85% 5.02% 17.95% 28.51%
23.54% 2.22% 11.50% 1.10% 0.53% 1.78% FAD2B W91* 22 HOMO 7.73%
5.99% 18.08% 28.11% 21.75% 3.34% 11.49% 1.09% 0.44% 1.97% FAD2B 8
W91* 2 HOMO 8.23% 5.35% 18.80% 29.20% 26.29% 1.68% 7.84% 0.86%
0.41% 1.34% FAD2B 8 W91* 3 HOMO 8.05% 5.50% 18.37% 28.87% 24.40%
2.69% 8.63% 1.15% 0.53% 1.82% FAD2B 8 W91* 4 HOMO 7.54% 5.09%
17.96% 28.89% 22.95% 2.92% 10.62% 1.25% 0.51% 2.27% FAD2B W91* 5
HOMO 18.43% 28.69% 23.80% 10.17% AVE FAD2B W91* 6 HOMO 0.57% 0.38%
1.52% 1.55% SD FAD2B W91* 7 Null 1 7.61% 3.94% 12.42% 32.37% 23.94%
2.61% 11.48% 1.87% 0.68% 3.09% FAD2B W91* 5 Null 1 7.93% 4.16%
12.77% 29.39% 26.08% 2.58% 11.78% 1.68% 0.74% 2.89% FAD2B W91* 10
Null 4 8.35% 5.04% 12.13% 28.77% 26.89% 2.84% 11.44% 1.69% 0.82%
2.04% FAD2B W91* 13 Null 7.61% 4.83% 12.99% 33.71% 21.94% 2.81%
10.77% 1.84% 0.62% 2.88% FAD2B W91* 23 Null 8.31% 5.84% 13.38%
31.92% 21.35% 4.00% 10.55% 1.66% 0.36% 2.62% FAD2B W91* 24 Null
9.08% 5.10% 13.10% 32.82% 24.75% 2.16% 8.39% 1.77% 0.34% 2.49%
FAD2B W91* 25 Null AVE 12.80% 31.50% 24.16% 10.73% FAD2B W91* 26
Null SD 0.46% 1.97% 2.21% 1.24% Note: *stands for nonsense
mutation; HOMO means the plants are all homozygous mutants at the
specified locus. HET means the plants are heterozygous mutants at
the specified locus. NULL means there is no mutation at the
specified locus. % means % of FAME composition
TABLE-US-00020 TABLE 17 Fatty Acids Composition in selected FAD2
mutants, sorted by mutation, Test 2 muta- Plant # of Gene SNP tion
# genotype samples C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2
C20:3 C22:1 none none CS32 1 CS32 10 5.8% 2.3% 14.9% 20.3% 34.0%
1.2% 16.2% 1.8% 0.9% 2.7% controls controls none none CS32 2 CS32
controls 6.1% 2.3% 14.6% 21.3% 33.3% 1.2% 15.6% 1.8% 1.2% 2.6%
controls none none CS32 3 CS32 controls 6.2% 2.3% 14.1% 21.7% 34.2%
1.1% 15.5% 1.8% 1.0% 2.1% controls none none CS32 4 CS32 controls
6.0% 2.4% 14.2% 20.8% 34.5% 1.2% 15.8% 1.6% 1.0% 2.4% controls none
none CS32 5 CS32 controls 6.7% 2.6% 14.6% 23.1% 33.5% 0.7% 12.9%
1.8% 1.3% 2.7% controls none none CS32 6 CS32 controls 6.2% 2.5%
14.6% 21.6% 33.4% 1.0% 15.4% 1.8% 1.1% 2.5% controls none none CS32
7 CS32 controls 5.9% 2.4% 14.6% 22.2% 32.7% 1.3% 15.7% 1.8% 1.0%
2.5% controls none none CS32 8 CS32 controls 6.0% 2.3% 13.6% 21.0%
34.3% 1.4% 16.0% 1.9% 1.0% 2.6% controls none none CS32 9 CS32
controls 5.9% 2.4% 14.3% 20.6% 33.6% 1.3% 16.2% 1.9% 1.1% 2.7%
controls none none CS32 10 CS32 controls 6.2% 2.4% 13.9% 21.4%
33.2% 1.3% 15.9% 1.9% 1.2% 2.6% controls FAD2A 5 G150E 4 HOMO 1
8.0% 4.4% 22.5% 18.8% 27.5% 2.2% 12.7% 0.5% 1.7% 1.7% FAD2A 5 G150E
5 HOMO 1 7.8% 4.2% 23.2% 18.4% 26.6% 2.2% 14.2% 0.7% 0.6% 2.2%
FAD2A G150E 6 HOMO 5 new 7.7% 4.2% 26.1% 18.8% 25.2% 1.9% 13.3%
0.6% 0.5% 1.7% FAD2A G150E 8 HOMO 8.1% 4.6% 24.2% 18.7% 28.1% 2.2%
10.2% 0.7% 0.5% 2.7% FAD2A G150E 14 HOMO 8.2% 4.5% 24.6% 19.4%
24.7% 2.0% 13.7% 0.6% 0.3% 1.9% FAD2A G150E 23 HOMO 8.6% 4.7% 23.0%
19.0% 26.7% 2.1% 13.1% 0.6% 0.5% 1.8% FAD2A G150E 25 HOMO 8.0% 4.0%
24.2% 20.0% 23.7% 1.8% 14.6% 0.7% 0.5% 2.5% FAD2A 2 Q44* 2 HOMO 1
8.3% 5.1% 22.8% 20.8% 26.7% 2.0% 11.1% 0.8% 0.4% 2.0% FAD2A 2 Q44*
1 HOMO 1 7.8% 4.9% 19.7% 18.9% 28.3% 2.6% 13.9% 0.9% 0.7% 2.4%
FAD2A 2 Q44* 4 HOMO 1 7.6% 5.1% 23.7% 20.2% 25.7% 2.4% 12.8% 0.6%
0.4% 1.5% FAD2A 2 Q44* 5 HOMO FAD2A 2 Q44* 3 HOMO FAD2A Q44* 36
HOMO 3 7.8% 5.5% 23.2% 21.4% 23.8% 2.5% 13.0% 0.7% 0.2% 1.8% FAD2A
Q44* 38 HOMO 8.0% 6.2% 22.3% 20.4% 25.4% 2.9% 12.4% 0.7% 0.3% 1.5%
FAD2A Q44* 39 HOMO 9.2% 5.6% 22.9% 22.0% 23.7% 2.3% 11.0% 0.7% 0.4%
2.0% FAD2A 7 S229F 5 HOMO 1 7.6% 3.8% 19.6% 21.4% 27.4% 2.0% 13.9%
1.0% 0.6% 2.6% FAD2A 7 S229F 1 HOMO FAD2A S229F 7 HOMO 3 8.4% 5.8%
18.4% 22.7% 24.5% 3.5% 11.8% 1.1% 0.6% 3.2% FAD2A S229F 9 HOMO 7.3%
5.1% 17.8% 22.4% 25.2% 3.6% 14.0% 1.1% 0.6% 2.9% FAD2A S229F 19
HOMO 6.9% 5.0% 21.0% 20.9% 24.8% 2.7% 14.3% 0.9% 0.6% 2.9% FAD2B
W91* 1 HOMO 1 7.7% 5.1% 19.4% 28.6% 23.9% 1.9% 10.9% 0.9% 0.4% 1.2%
FAD2B W91* 21 HOMO 2 7.9% 5.0% 17.9% 28.5% 23.5% 2.2% 11.5% 1.1%
0.5% 1.8% FAD2B W91* 22 HOMO 7.7% 6.0% 18.1% 28.1% 21.8% 3.3% 11.5%
1.1% 0.4% 2.0% FAD2B 8 W91* 2 HOMO FAD2B 8 W91* 3 HOMO FAD2B 8 W91*
4 HOMO FAD2A G150E 20 Null 2 8.7% 4.2% 16.2% 26.7% 25.7% 2.2% 12.0%
1.3% 0.4% 2.4% FAD2A 5 G150E 11 Null 8.3% 5.2% 17.9% 25.3% 27.4%
2.7% 10.0% 1.0% 0.5% 1.7% FAD2A 5 G150E 18 Null 9.0% 5.1% 17.8%
28.2% 24.9% 3.2% 8.9% 1.2% 0.6% 1.1% FAD2A G150E 24 Null 8.7% 5.1%
16.4% 27.4% 26.0% 2.2% 10.9% 1.1% 0.6% 1.6% FAD2A 7 S229F 12 Null 1
8.6% 5.6% 15.0% 27.8% 26.6% 3.4% 8.2% 1.4% 0.7% 2.8% FAD2A 7 S229F
10 Null 1 9.3% 5.7% 12.8% 28.1% 25.4% 3.7% 10.1% 1.8% 0.4% 2.7%
FAD2A S229F 13 Null 2 7.8% 5.6% 14.1% 26.4% 25.6% 3.6% 12.2% 1.5%
0.7% 2.5% FAD2A S229F 14 Null 7.6% 4.8% 13.8% 25.0% 28.3% 3.2%
12.6% 1.2% 0.8% 2.6% FAD2B W91* 7 Null 1 7.6% 3.9% 12.4% 32.4%
23.9% 2.6% 11.5% 1.9% 0.7% 3.1% FAD2B W91* 5 Null 1 7.9% 4.2% 12.8%
29.4% 26.1% 2.6% 11.8% 1.7% 0.7% 2.9% FAD2B W91* 10 Null 4 8.4%
5.0% 12.1% 28.8% 26.9% 2.8% 11.4% 1.7% 0.8% 2.0% FAD2B W91* 13 Null
7.6% 4.8% 13.0% 33.7% 21.9% 2.8% 10.8% 1.8% 0.6% 2.9% FAD2B W91* 23
Null 8.3% 5.8% 13.4% 31.9% 21.4% 4.0% 10.6% 1.7% 0.4% 2.6% FAD2B
W91* 24 Null 9.1% 5.1% 13.1% 32.8% 24.8% 2.2% 8.4% 1.8% 0.3% 2.5%
Note: *stands for nonsense mutation; HOMO means the plants are all
homozygous mutants at the specified locus. HET means the plants are
heterozygous mutants at the specified locus. NULL means there is no
mutation at the specified locus. % means % of FAME composition
TABLE-US-00021 TABLE 18a Fatty Acids Composition in selected FAD2
mutants, sorted by gene, Test 4 Seed Geno- gene- Sample type ration
gene mutation C16:0 C18:0 C18:1 C18:2 C20:0 C18:3 C20:1 C20:2 C20:3
C22:1 2362-Q10 HOM M5 FAD2 A G150E 8.0% 4.3% 21.6% 16.2% 2.3% 30.3%
13.4% 0.8% 0.7% 2.2% 2362-Q11 HOM M5 FAD2 A G150E 8.0% 3.6% 20.4%
17.1% 1.8% 32.3% 13.1% 0.8% 0.7% 2.2% 2362-Q12 HOM M5 FAD2 A G150E
8.3% 3.6% 19.4% 17.9% 1.5% 32.5% 13.0% 0.9% 0.7% 2.2% 2362-Q13 HOM
M5 FAD2 A G150E 8.2% 4.2% 20.4% 17.3% 2.3% 31.0% 13.1% 0.9% 0.6%
2.1% 2826-P1 Het M5 FAD2 A Q44* 7.1% 3.4% 16.3% 16.3% 2.2% 35.1%
14.6% 1.2% 1.1% 2.8% 2826-P2 Het M5 FAD2 A Q44* 7.7% 4.0% 17.2%
16.4% 2.5% 33.7% 14.1% 1.0% 0.9% 2.5% 2826-P3 Het M5 FAD2 A Q44*
8.4% 4.0% 17.8% 19.8% 2.7% 28.9% 12.4% 1.4% 1.2% 3.3% 2826-P4 Het
M5 FAD2 A Q44* 7.7% 3.9% 15.5% 16.6% 2.5% 34.4% 14.5% 1.2% 1.0%
2.6% 3006-R1 HOM M5 FAD2 B W91* 7.7% 3.5% 11.9% 21.5% 2.3% 35.6%
12.3% 1.6% 1.1% 2.6% 3006-R2 HOM M5 FAD2 B W91* 7.8% 3.6% 12.1%
22.6% 2.3% 33.4% 12.7% 1.6% 1.0% 2.7% 3006-R3 HOM M5 FAD2 B W91*
8.1% 3.5% 12.3% 22.5% 2.1% 34.5% 12.0% 1.6% 1.1% 2.4% 3006-R4 HOM
M5 FAD2 B W91* 7.8% 3.6% 12.7% 22.2% 2.1% 34.4% 12.2% 1.6% 1.0%
2.4% 3489-N2 HOM M4 FAD2 B W138* 8.0% 3.4% 11.9% 23.2% 2.5% 31.7%
12.8% 1.9% 1.1% 3.6% 3489-N5 HOM M4 FAD2 B W138* 8.2% 3.5% 11.5%
23.7% 2.6% 31.0% 12.8% 2.0% 1.0% 3.6% 3489-N9 HOM M4 FAD2 B W138*
7.9% 3.3% 12.3% 22.2% 2.6% 31.0% 13.7% 1.9% 1.2% 3.8% 3489-N12 HOM
M4 FAD2 B W138* 7.8% 3.4% 11.9% 22.9% 2.6% 30.7% 14.0% 2.0% 1.1%
3.8% 3489-N16 HOM M4 FAD2 B W138* 7.8% 3.5% 11.9% 22.7% 2.6% 31.2%
13.6% 2.0% 1.1% 3.8% 3702-O2 HOM M4 FAD2 B G150E 7.7% 4.2% 12.8%
23.5% 3.0% 31.1% 12.0% 1.8% 1.0% 2.9% 3702-O3 HOM M4 FAD2 B G150E
9.7% 5.6% 17.1% 34.2% 3.7% 18.0% 8.2% 1.0% 0.3% 2.0% 3702-O4 Het M4
FAD2 B G150E 7.5% 4.4% 11.6% 24.7% 4.5% 31.1% 10.4% 1.8% 0.9% 3.2%
3702-O6 HOM M4 FAD2 B G150E 7.8% 4.4% 12.0% 24.4% 3.0% 31.4% 11.5%
1.8% 0.9% 2.7% 3702-O7 HOM M4 FAD2 B G150E 8.0% 5.8% 13.6% 25.2%
4.2% 29.7% 9.1% 1.4% 0.7% 2.2% 3702-O9 Het M4 FAD2 B G150E 7.1%
4.0% 12.1% 23.7% 3.2% 31.2% 12.4% 1.9% 1.0% 3.3% 6490-M1 HOM M4
FAD2 B W91* 6.3% 3.2% 13.2% 21.8% 2.3% 32.1% 14.4% 1.8% 1.2% 3.7%
6490-M2 HOM M4 FAD2 B W91* 6.1% 2.9% 12.0% 20.2% 2.2% 34.2% 14.8%
2.1% 1.3% 4.1% 6490-M3 HOM M4 FAD2 B W91* 6.2% 3.0% 12.5% 20.8%
2.3% 33.5% 14.5% 2.0% 1.3% 4.0% 6490-M4 HOM M4 FAD2 B W91* 8.3%
3.2% 12.3% 21.3% 2.3% 32.0% 14.0% 1.8% 1.2% 3.6% 6490-M5 HOM M4
FAD2 B W91* 8.0% 2.5% 12.0% 20.5% 1.8% 33.8% 14.7% 1.9% 1.3% 3.6%
6490-M10 null M4 FAD2 B W91* 9.0% 2.6% 10.9% 18.5% 2.1% 35.5% 14.4%
2.1% 1.4% 3.3% 3284-B11 null M4 FAD2 C W91* 8.9% 4.7% 10.1% 22.3%
2.8% 35.8% 10.3% 2.0% 1.2% 2.0% 3284-B12 Het M4 FAD2 C W91* 8.6%
5.2% 11.9% 23.2% 3.2% 33.4% 10.2% 1.6% 0.9% 1.7% 3284-B13 Het M4
FAD2 C W91* 8.1% 4.4% 11.6% 21.1% 2.7% 36.3% 11.1% 1.7% 1.2% 1.9%
3284-B15 null M4 FAD2 C W91* 9.0% 4.2% 9.3% 22.1% 2.9% 36.9% 10.5%
1.9% 1.3% 1.9% 3284-B21 Het M4 FAD2 C W91* 8.4% 4.5% 10.6% 20.3%
2.9% 36.5% 11.7% 1.7% 1.2% 2.2% 4506-A2 null M4 FAD2 C W87* 7.5%
3.5% 11.9% 23.6% 3.0% 30.8% 12.9% 2.1% 1.1% 3.5% 4506-A10 Hom M4
FAD2 C W87* 6.9% 3.6% 14.7% 20.7% 2.6% 31.5% 14.1% 1.6% 1.0% 3.3%
4506-A12 Hom M4 FAD2 C W87* 7.3% 4.0% 15.9% 20.4% 2.9% 30.0% 14.1%
1.4% 0.9% 3.2% 4506-A15 Hom M4 FAD2 C W87* 8.2% 3.3% 7.1% 24.1%
2.5% 33.8% 14.0% 1.9% 1.1% 4.1% 4506-A16 null M4 FAD2 C W87* 8.0%
3.5% 12.8% 23.6% 3.0% 30.9% 12.5% 1.7% 1.0% 3.1% 4608-C4 Hom M4
FAD2 C G150E 8.8% 3.5% 9.2% 23.3% 2.9% 35.0% 11.2% 1.9% 1.2% 2.9%
4608-C12 Hom M4 FAD2 C G150E 9.3% 4.0% 10.0% 25.9% 3.0% 31.6% 10.8%
1.9% 1.0% 2.7% 4608-C13 Het M4 FAD2 C G150E 9.0% 3.9% 9.2% 25.4%
2.8% 32.8% 11.0% 2.1% 1.1% 2.8% 4608-C15 null M4 FAD2 C G150E 8.9%
3.9% 8.9% 26.0% 2.9% 32.7% 10.7% 2.2% 1.1% 2.7% 4608-C17 Het M4
FAD2 C G150E 9.0% 3.8% 8.7% 23.6% 3.1% 34.1% 11.1% 2.2% 1.3% 3.1%
Cs32-1 7.8% 4.7% 12.4% 27.1% 4.3% 25.5% 12.1% 2.0% 0.8% 3.3% Cs32-2
8.0% 4.5% 12.0% 26.7% 3.9% 27.1% 11.8% 2.0% 0.8% 3.2% Cs32-3 8.0%
4.1% 12.1% 26.6% 3.7% 27.7% 11.7% 2.1% 0.9% 3.2% Cs32-4 7.9% 3.9%
12.2% 26.2% 3.4% 28.7% 11.7% 2.0% 0.9% 3.0% At FAD2_1 5.4% 3.0%
49.9% 4.2% 1.5% 10.3% 24.2% 0.0% 0.0% 1.5% At FAD2_2 5.6% 3.6%
50.5% 4.5% 0.0% 10.3% 24.1% 0.1% 0.0% 1.4% At FAE1_1 10.3% 4.7%
28.8% 34.2% 1.0% 20.9% 0.1% 0.0% 0.0% 0.0% At FAE1_2 10.2% 5.2%
28.7% 33.9% 1.0% 20.9% 0.1% 0.0% 0.0% 0.0% Note: *stands for
nonsense mutation; Hom means the plants are all homozygous mutants
at the specified locus. Het means the plants are heterozygous
mutants at the specified locus. Null means there is no mutation at
the specified locus. % means % of FAME composition Gene indicates
in which gene the mutation is located
TABLE-US-00022 TABLE 19a Fatty Acids Composition in selected FAE1
mutants, sorted by gene, Test 4 Seed Geno- gener- Sample type ation
gene mutation C16:0 C18:0 C18:1 C18:2 C20:0 C18:3 C20:1 C20:2 C20:3
C22:1 3395-D10 Hom M4 FAE1 A R209* 9.7% 4.2% 13.2% 20.8% 2.4% 33.6%
11.4% 1.6% 1.1% 2.0% 3395-D12 Hom M4 FAE1 A R209* 8.3% 4.7% 15.8%
21.0% 2.4% 32.7% 11.0% 1.3% 1.0% 1.6% 3395-D13 Hom M4 FAE1 A R209*
7.8% 4.2% 14.1% 20.7% 2.2% 35.7% 11.0% 1.5% 1.1% 1.7% 3395-D17 null
M4 FAE1 A R209* 9.8% 3.4% 11.7% 20.8% 2.1% 34.6% 11.7% 1.8% 1.3%
2.8% 3395-D18 null M4 FAE1 A R209* 7.4% 4.3% 14.0% 21.4% 2.5% 33.5%
11.6% 1.6% 1.2% 2.6% 3395-D19 null M4 FAE1 A R209* 7.7% 3.5% 14.2%
20.2% 2.4% 33.5% 12.7% 1.6% 1.1% 3.1% 3395-D20 Hom M4 FAE1 A R209*
7.8% 4.4% 14.8% 21.4% 2.4% 33.8% 11.3% 1.4% 1.0% 1.7% 6386-F1 Het
M4 FAE1 A G221D 11.1% 5.0% 10.6% 32.7% 3.6% 23.4% 9.2% 1.7% 0.5%
2.1% 6386-F2 HOM M4 FAE1 A G221D 9.2% 4.7% 13.3% 26.8% 2.2% 31.3%
9.1% 1.3% 0.7% 1.2% 6386-F7 Hom M4 FAE1 A G221D 8.8% 4.6% 12.7%
26.3% 2.4% 32.7% 9.0% 1.5% 0.8% 1.1% 6386-F9 Hom M4 FAE1 A G221D
9.0% 4.4% 11.5% 26.8% 2.6% 30.9% 10.2% 1.8% 0.9% 1.9% 6386-F13 null
M4 FAE1 A G221D 8.9% 4.6% 12.7% 25.4% 2.3% 33.6% 9.0% 1.4% 0.8%
1.2% 6386-F15 null M4 FAE1 A G221D 8.1% 4.2% 11.2% 25.3% 3.4% 30.1%
11.8% 1.9% 1.0% 2.9% 6386-F19 Het M4 FAE1 A G221D 8.2% 4.2% 11.4%
24.8% 2.7% 32.8% 10.9% 1.8% 1.0% 2.2% 4687-I4 HOM M4 FAE1 B H301Y
7.1% 2.6% 14.6% 20.7% 1.2% 41.0% 9.2% 1.3% 1.1% 1.3% 4687-I10 null
M4 FAE1 B H301Y 7.3% 3.2% 14.5% 21.0% 1.8% 37.8% 10.5% 1.4% 1.1%
1.5% 4687-I11 HOM M4 FAE1 B H301Y 7.7% 3.2% 15.9% 21.8% 1.4% 38.9%
8.2% 1.1% 0.9% 0.9% 4687-I14 null M4 FAE1 B H301Y 7.6% 3.6% 16.0%
21.6% 1.8% 34.1% 11.5% 1.4% 1.0% 1.5% 4687-I17 HOM M4 FAE1 B H301Y
7.2% 3.0% 14.7% 19.6% 1.3% 43.0% 8.2% 1.1% 1.0% 0.9% 5343-H6 null
M4 FAE1 B S281F 7.6% 3.5% 11.5% 19.6% 2.5% 36.5% 12.6% 1.8% 1.4%
3.0% 5343-H7 null M4 FAE1 B S281F 7.8% 3.6% 11.6% 20.1% 2.6% 35.7%
12.5% 1.8% 1.2% 3.1% 5343-H10 HOM M4 FAE1 B S281F 7.9% 4.0% 13.2%
22.9% 2.3% 34.5% 10.6% 1.5% 1.0% 2.1% 5343-H14 HOM M4 FAE1 B S281F
8.3% 3.2% 11.2% 20.6% 1.8% 39.3% 10.6% 1.7% 1.3% 2.1% 5343-H15 HOM
M4 FAE1 B S281F 8.1% 4.0% 12.0% 22.1% 2.2% 36.5% 10.4% 1.6% 1.1%
2.0% 5343-H16 HOM M4 FAE1 B S281F 7.9% 3.0% 11.0% 19.4% 1.7% 40.6%
10.9% 1.8% 1.5% 2.3% 5951-G1 HOM M4 FAE1 B Q142* 8.7% 3.6% 14.1%
22.4% 1.1% 43.0% 4.9% 0.8% 0.7% 0.7% 5951-G2 HOM M4 FAE1 B Q142*
8.1% 3.8% 14.4% 22.3% 1.4% 40.7% 6.6% 0.9% 0.8% 0.9% 5951-G3 HOM M4
FAE1 B Q142* 7.9% 3.1% 11.3% 20.0% 2.1% 39.0% 11.0% 1.7% 1.5% 2.4%
5951-G4 HOM M4 FAE1 B Q142* 9.4% 3.3% 14.3% 23.8% 0.9% 44.0% 3.0%
0.5% 0.5% 0.3% 5951-G5 HOM M4 FAE1 B Q142* 8.0% 2.8% 10.5% 20.4%
2.2% 39.0% 11.4% 1.7% 1.4% 2.7% 6476-K2 Hom M4 FAE1 C R209* 7.8%
3.2% 9.2% 23.2% 2.2% 38.7% 9.4% 1.9% 1.3% 2.9% 6476-K4 HOM M4 FAE1
C R209* 7.3% 3.8% 10.6% 23.1% 2.4% 38.3% 9.1% 1.7% 1.2% 2.3%
6476-K6 HOM M4 FAE1 C R209* 8.3% 4.0% 10.2% 22.9% 2.5% 36.3% 9.8%
1.9% 1.2% 2.9% 6476-K7 null M4 FAE1 C R209* 7.1% 3.6% 9.8% 23.6%
3.0% 32.1% 13.0% 2.5% 1.2% 4.0% 6476-K15 HOM M4 FAE1 C R209* 7.5%
4.0% 11.2% 21.6% 2.2% 35.6% 11.7% 1.9% 1.2% 3.0% Cs32-1 7.8% 4.7%
12.4% 27.1% 4.3% 25.5% 12.1% 2.0% 0.8% 3.3% Cs32-2 8.0% 4.5% 12.0%
26.7% 3.9% 27.1% 11.8% 2.0% 0.8% 3.2% Cs32-3 8.0% 4.1% 12.1% 26.6%
3.7% 27.7% 11.7% 2.1% 0.9% 3.2% Cs32-4 7.9% 3.9% 12.2% 26.2% 3.4%
28.7% 11.7% 2.0% 0.9% 3.0% At FAE1_1 10.3% 4.7% 28.8% 34.2% 1.0%
20.9% 0.1% 0.0% 0.0% 0.0% At FAE1_2 10.2% 5.2% 28.7% 33.9% 1.0%
20.9% 0.1% 0.0% 0.0% 0.0% Note: *stands for nonsense mutation; Hom
means the plants are all homozygous mutants at the specified locus.
Het means the plants are heterozygous mutants at the specified
locus. Null means there is no mutation at the specified locus. %
means % of FAME composition Gene indicates in which gene the
mutation is located
Example 13
Fatty Acids Composition in Plants with Multiple Mutations in FAD2
and/or FAE1 Genes
[0242] To further increase the oleic acid (18:1) level and/or yield
and improve Camelina seed oil quality, mutations in one or more
copies of FAD2 genes and/or one or more copies of FAE1 genes are
integrated together to create mutant plants with double, triple,
quadruple et al. mutations. Such mutants can be created by classic
breeding methods. Table 20 below shows a list of non-limiting
examples of such mutants.
TABLE-US-00023 TABLE 20 Plants with more than one mutation in Fatty
Acid Synthesis Genes Genotype Plant ID FAD2 A FAD2 B FAD2 C FAE1 A
FAE1 B FAE1 C A1 HOMO HOMO NULL NULL NULL NULL A2 HOMO NULL HOMO
NULL NULL NULL A3 NULL HOMO HOMO NULL NULL NULL A4 HOMO HOMO HOMO
NULL NULL NULL A5 NULL NULL NULL HOMO HOMO NULL A6 NULL NULL NULL
HOMO NULL HOMO A7 NULL NULL NULL NULL HOMO HOMO A8 NULL NULL NULL
HOMO HOMO HOMO A9 HOMO NULL NULL HOMO NULL NULL A10 HOMO NULL NULL
HOMO HOMO NULL A11 HOMO NULL NULL HOMO HOMO HOMO A12 HOMO HOMO NULL
HOMO NULL NULL A13 HOMO HOMO NULL HOMO HOMO NULL A14 HOMO HOMO NULL
HOMO HOMO HOMO A15 HOMO HOMO HOMO HOMO NULL NULL A16 HOMO HOMO HOMO
HOMO HOMO NULL A17 HOMO HOMO HOMO HOMO HOMO HOME Note: HOMO means
the plants are all homozygous mutants at the specified locus. NULL
means there is no mutation at the specified locus.
[0243] Fatty acid compositions in these mutants are then analyzed
by gas chromatography (GC). The results will show that one or more
of these mutants produce seed oil with higher oleic acid (18:1)
levels and/or lower VLCFA levels when compared to Cs32 control
plants or to one or more single mutants that have only one mutation
in a FAD2 gene and/or a FAE1 gene.
[0244] Thus, mutations in more than one FAD2 and/or FAE1 genes
further increase oleic acid (18:1) levels and/or lower VLCFA
levels, and improve Camelina seed oil quality.
Example 14
Fatty Acids Composition in RNAi Transgenic Camelina Plants
[0245] As described in the present invention, RNAi technology can
be used to disrupt one or more fatty acid synthesis genes (e.g.,
FAD2, FAE1, and other genes) in Camelina to obtain an increase in
oleic acid (18:1) and/or a decrease in VLCFA in the seed oil as
measured by relative percent or absolute yield. The advantage of
this method is that an RNAi expression vector can contain a double
strand RNA that simultaneously suppresses one or more homologous
genes. This is extremely helpful in Camelina as the inventors
proved it is an allohexaploid species.
[0246] Using RNAi technology to knock down expression of all FAD2
genes and/or all FAE1 genes may be more convenient than classic
breeding method. Whereas both sense- and antisense-mediated gene
silencing have proven fruitful for PTGS in plant cells, RNAi
induction can be more efficiently achieved by specialized
expression cassettes that produce self-complementary hairpin
(hp)-like RNA molecules. Such cassettes typically include plant
promoter and terminator sequences that control the expression of
two inversely repeated sequences of the target genes that are
separated by a specific spacer.
[0247] Upon delivery to plant cells, expression of an RNAi cassette
will result in a dsRNA molecule composed of two distinct regions: a
single-stranded loop, encoded by the spacer region, and a
double-stranded stem, encoded by the inverted repeats. It is the
stem region that is used as a substrate by the dicer, but, because
the spacer itself can potentially be recognized as a substrate as
well, intron sequences are often used in the construction of such
RNAi vectors (e.g. Meyer et al., 2004, Vectors for RNAi technology
in poplar. Plant Biol (Stuttg) 6: 100-103). These vectors include,
but are not limited to, pHANNIBAL, pHANNIBAL, pHELLSGATE, pSAT,
pCAMBIA, pGREEN, et al. RNAi (or hpRNA) plant expression can
potentially be delivered to plant cells by various means of
transformation but are typically used by incorporating into binary
plasmids to be delivered to plant cells by Agrobacterium-mediated
transformation.
[0248] Fatty acid synthesis genes that are potential targets
include, any one of FAD2 genes and/or any one of FAE1 genes as
provided in the present invention, or alternatively along with any
other genes involved in Camelina fatty acid synthesis as described
herein or elsewhere.
[0249] A non-limiting example of using RNAi technology to suppress
Camelina FAD2 genes is described below. A complete hpRNA expression
cassette is composed of four distinct regions: a promoter and
terminator sequence, the ChsA intron sequence, and a dual MCS. The
dual MCS results from cloning of the ChsA intron sequence into
pSAT6-MCS and dividing the original MCS into two new, distinct
regions, designated MCS-I and MCS-II, which contain the following
unique restriction endonuclease recognition sites: NcoI, BspEI,
BglII, XhoI, SacI, and EcoRI in MCS-I and PstI, SalI, KpnI, SacII,
ApaI, XmaI, SmaI, BamHI, and XbaI in MCS-II. The two MCS regions
allow the successive cloning of the target gene sequence in reverse
orientation and assembly of a hpRNA sequence. In pSAT6.35S.RNAi,
expression of hpRNA is controlled by tandem CaMV 35S promoter
(35SP) and CaMV 35S terminator (35ST), conferring a complete
expression cassette. In pSAT6.Napin.RNAi, expression of hpRNA is
controlled by Napin plant seed-specific promoter. hpRNA designed
according to conserved, specific 19 to 29, 19 to 27, or 19 to 21
polynucleotides of FAD2 A, FAD2 B, and FAD2 C genes, which does not
share homology to other genes, are introduced into either
pSAT6.35S.RNAi or pSAT6.Napin.RNA vector to make the final RNAi
construct. Such conserved, specific 15-21 polynucleotides sequences
can be designed by one of ordinary skill in the art based on FAD2
genes disclosed in the present invention and known Camelina
non-FAD2 gene sequences deposited in the GenBank.
[0250] Further, pSAT6.35S.RNAi or pSAT6.Napin.RNA vector containing
the hpRNA targeting FAD2 genes is transformed into Camelina plant
using the method described in WO2009/117555, and positive
transformants are selected. Northern blot or qPCR is used to verify
if one or more FAD2 genes are suppressed in the transformants. The
transgenic lines with the most efficient suppression in all FAD2
genes are subjected to fatty acid composition analysis by GC, and
the results indicate such transgenic Camelina plants have an
increased oleic acid (18:1) level and/or reduced polyunsaturated
fatty acids level in the seed oil compared to transgenic Camelina
plants with empty control vector.
[0251] In addition, another pSAT6.35S.RNAi or pSAT6.Napin.RNA
vector containing the hpRNA targeting FAE1 genes is transformed
into Camelina plant using the method described in WO2009/117555,
and positive transformants are selected. Northern blot or qPCR is
used to verify if one or more FAE1 genes are suppressed in the
transformants. The transgenic lines with the most efficient
suppression in all FAE1 genes are subjected to fatty acid
composition analysis by GC, and the results indicate such
transgenic Camelina plants have a decreased long chain fatty acid
level, and/or reduced long chain polyunsaturated fatty acids level
in the seed oil compared to transgenic Camelina plants with empty
control vector.
Example 15
Fatty Acid Composition in Camelina Plants Having Suppressed FAD2
and/or FAE1 Gene Functions in Combination with Overexpression or
Suppression of Other Non-FAD and Non-FAE Fatty Acid Synthesis
Genes
[0252] Other fatty acid synthesis enzymes may be manipulated in the
fatty acid synthesis pathways to increase the amount of oleic acid
(18:1) or decrease the amount of palmitic acid (16:0) to create
Camelina oil with fatty acid profiles optimal for biodiesel
production. Lower amounts of 16:0 saturated fatty acid and higher
amounts of 18:1 monounsaturated fatty acid is desirable for a good
balance of proper cetane number, cloud point, oxidative stability,
and reduced NOx emissions, as mentioned in the Background and
Example 9.
[0253] Three key enzymes regulate the amount of 16:0, 18:0 and 18:1
fatty acids (FIG. 13): acyl-acyl carrier protein thioesterase (also
known as FATB), .beta.-ketoacyl-acyl carrier protein (ACP) synthase
II (KAS II) and .DELTA.-9 desaturase. FATB hydrolyzes the fatty
acyl group from acyl carrier protein (ACP) and thus determines the
amount and type of fatty acid that is exported from the plastid.
Suppression of FATB leads to a reduction in 16:0 and 18:0 (stearic
acid) released to the cytoplasm. KAS II converts palmitoyl-ACP
(16:0-ACP) to stearoyl-ACP (18:0 ACP), and thus the overexpression
of KAS II leads to an increase in the amount of 16:0 being
converted to 18:0. .DELTA.-9 desaturase converts 18:0-ACP to
oleoyl-ACP (18:1-ACP), and thus the overexpression of .DELTA.-9
desaturase leads to an increase in the amount of 18:0 being
converted to 18:1. Since the product of KAS II activity (18:0-ACP)
is the substrate for .DELTA.-9 desaturase, the overexpression of
both KAS II and .DELTA.-9 desaturase will lead to a further
decrease in 16:0 and 18:0 and an increase in 18:1.
[0254] Camelina lines having suppressed FAD2 and/or FAE1 gene
functions, as described in the present invention, obtained either
by TILLING or transgenic means (e.g., antisense, RNAi), may be
combined with overexpression or suppression of the non-FAD and
non-FAE genes described in this example to create new Camelina
lines with even greater percentages of 18:1 fatty acid and/or
lesser percentages of 16:0 and/or 18:0 fatty acids compared to
lines with only FAD2/FAE1 modifications or only non-FAD/non-FAE
modifications.
[0255] For example, Camelina FAD2 and/or FAE1 mutant plants,
permutations of which are described in Example 13, may be combined
by breeding with a Camelina plant overexpressing KAS II in a
seed-specific manner to create a new Camelina line where the amount
of 18:1 is higher and the amount of 16:0 is lower compared to
either parent plant alone. The seed-specific overexpression of KAS
II may also indirectly decrease the amount of 18:2 and/or 18:3
polyunsaturated fatty acids.
[0256] Alternatively, Camelina FAD2 and/or FAE1 mutant plants may
be combined by breeding with a Camelina plant overexpressing
.DELTA.-9 desaturase in a seed-specific manner to create a new
Camelina line where the amount of 18:1 is higher and the amount of
16:0 is lower compared to either parent plant alone. This
combination may also decrease the amount of very long chain fatty
acids.
[0257] In addition, Camelina FAD2 and/or FAE1 mutant plants may be
combined by breeding with a Camelina plant overexpressing both KAS
II and .DELTA.-9 desaturase in a seed-specific manner to create a
new Camelina line where the amount of 18:1 is higher and the amount
of 16:0 is lower compared to any of the original parent
modifications (FAD2/FAE1 suppression, KAS II overexpression or
.DELTA.-9 desaturase overexpression) alone.
[0258] Optionally, Camelina FAD2 and/or FAE1 mutant plants may be
combined by breeding with a Camelina plant knocked out for FATB
function (either by TILLING or transgenic means with a
seed-specific promoter) to create a new Camelina line where the
amount of 18:1 is higher and the amount of 16:0 is lower compared
to either parent plant alone. Arabidopsis FATB knockout plants are
compromised in growth and produce less viable seeds (Bonaventure et
al, The Plant Cell, Vol. 15, 1020-1033, April 2003). This
detrimental phenotype may be alleviated in a polyploid like
Camelina, where the presence of multiple copies for a given gene
may allow greater flexibility in manipulating the levels of
camelina FATB. Alternatively, the detrimental FATB knockout
phenotype may be alleviated by only suppressing or knocking out
FATB function using a FATB antisense or RNAi construct driven by a
seed-specific promoter.
[0259] Other combinations of FAD2/FAE1 suppression, KAS II
seed-specific overexpression, .DELTA.-9 desaturase seed-specific
overexpression and/or FATB suppression may be envisioned to obtain
Camelina plants with increased 18:1 and decreased 16:0 and/or
18:0.
Example 16
Camelina Plants Having Mutations in FAD2 and/or FAE1 Genes in
Combination with Overexpression of REV/KRP Genes for Altered Fatty
Acid Composition and Increased Seed Yield
[0260] The purpose of suppressing Camelina FAD2 and/or FAE1
functions is to obtain an altered fatty acid profile of Camelina
oil more suitable for conversion to biodiesel. Another attribute
that would contribute to improvement of the oilseed crop for
biofuel purposes would be an increase in seed yield, either by an
increase in total seed number or seed size, in order to increase
the amount of oil recovered per unit of land. Two yield
technologies, REV and KRP dominant negative, have been described
(US 2008/263727 and US 2007/056058, incorporated by reference in
their entireties) that give increased seed yield when overexpressed
under early embryo-specific promoters.
[0261] Camelina FAD2 and/or FAE1 mutant plants, permutations of
which are described in Example 13, may be combined by breeding with
a Camelina plant overexpressing REV in an early embryo-specific
manner to create a new Camelina line with greater seed yield and
high 18:1 and/or low VLCFAs compared to either parent plant
alone.
[0262] Similarly, Camelina FAD2 and/or FAE1 mutant plants may be
combined by breeding with a Camelina plant overexpressing KRP
dominant negative in an early embryo-specific or constitutive
manner to create a new Camelina line with greater seed yield and
high 18:1 and/or low VLCFAs compared to either parent plant
alone.
[0263] Additionally, Camelina FAD2 and/or FAE1 mutant plants may be
combined by breeding with a Camelina plant overexpressing both REV
and KRP dominant negative in an early embryo-specific (or
constitutive for KRP) manner to create a new Camelina line with
greater seed yield and high 18:1 and/or low VLCFAs compared to any
of the original parent modifications (FAD2/FAE1 suppression, early
embryo-specific REV overexpression or embryo-specific or
constitutive KRP dominant negative overexpression) alone.
[0264] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only examples and
should not be taken as limiting the scope of the invention.
[0265] Unless defined otherwise, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Definitions of common terms in molecular biology may be found in
Benjamin Lewin, Genes IX, published by Oxford University Press,
2007 (ISBN-10 0131439812); Kendrew et al. (eds.), The Encyclopedia
of Molecular Biology, published by Blackwell Science Ltd., 1994
(ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology
and Biotechnology: A Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8); Oxford Dictionary of
Biochemistry and Molecular Biology, Revised Edition, 2000. Although
any methods and materials, similar or equivalent to those described
herein, can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein.
[0266] All publications, patents, and patent publications cited are
incorporated by reference herein in their entirety for all
purposes. Also incorporated by reference herein are nucleic acid
sequences and polypeptide sequences deposited into the GenBank,
which are cited in this specification.
[0267] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0268] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth and as follows in the scope of the appended
claims.
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Sequence CWU 1
1
8212791DNACamelina sativa 1gcggaggagc ttcttcctcg tagggggttt
cttcttcttc atcgttttta acgaaccatc 60gttaagtcaa atctcccccc ccccctacgt
cagctccagg tccgtccttc tcatttccga 120tttcgattca tttacgtctc
gtctggtctg ttctgtgttt ttttttttct ttttctttct 180cccgcactat
ctcatttccg atgttttttt aaataaaaac cgatttcatt atatagatct
240ggcttatatg tcttgcattc aaccttagat ctggtctcga tgctctgttt
ttttctttag 300ttgagaaatc tgatgttgtt acaatgagtt cttattcata
taatgattac tagtgccttg 360ggtcatccat gaaaacgata tgttaatgct
atgatttttt tatttgtttt ctttgtcaaa 420aatgaagtgc tgctttgacc
cattcttctt tagatatttt tattttattt ttgttgggtt 480ggtagaatag
tgagaatcac cataaaattc tcttatcagt ttcacgtccc tggttttttt
540tattttttat ttatttaaag atctgtaata tattcagttt tccctaattt
tgtttgtgta 600aaatttgctt tgaattgccg tgttgaatct cttatggatt
tgacctatgc ctaccgggtc 660ttaagattga tgataaaact ttttaaatag
acaaaaaaaa aagtttcact gattgattct 720cataaacttt acaatgaagt
tggaattagg gtaattcagg atcagatgcg tagtagattc 780agatgcaaaa
taatgagttg catgacttgt taatattata gatccataaa gacatattta
840aatatctgac atatgatgtc ggcaaaattc ggtggtatat agtatacatc
acttagaaac 900tgtttccttt ggacttgttt gccaacttgg ttgtattcag
gaggatttgt gattttgatt 960gatccattta ctttctctat tgttttttct
ttttcttttg gggtctactt ggtggtattc 1020ataagagaac ttttgttttt
gattgaattt aattacaaga aactgatgat gataaccaca 1080ataaagagat
tgtgacctgt cgtattgaaa tcttattagt agtagcagtc gtgttctcaa
1140cgtcaatggg tttcctttct ttggtttctt actttacgcc gcttctctgc
tctttttgtt 1200cgttttggtc cacgcacttt ccttttttgt ggcaatccct
ttcacaacct catctctgaa 1260taataataat tattactagt ttgttgattt
gatcattacc acctcgtttt ctagtgcatg 1320caaaatttgt caattagtgt
gataaacaaa caattccttt cttgagtttc agctttttga 1380tttttctttg
ctctatgttt cttttgcaga atcatgggtg caggtggaag aatgccagtt
1440ccttcttctt cttccaagaa atctgaaacc gatgccataa agcgtgtgcc
ctgcgagaaa 1500ccaccgttca cgctgggaga tctgaagaaa gcaatcccac
cgcagtgttt caaacgctct 1560atccctcgct ctttctccta ccttatcact
gacatcatta ttgcctcctg cttctactac 1620gtcgccacca attacttttc
tctcctccct cagcctctct cttacttggc ttggcccctc 1680tattgggctt
gtcaaggctg tgtcctaacc ggtgtctggg tcatagccca cgaatgcggt
1740caccacgcat tcagcgacta ccagtggctc gatgacacag tcggtcttat
cttccattcc 1800ttccttctcg tcccttactt ctcctggaag tacagtcatc
gccgtcacca ttccaacaca 1860ggatccctcg aaagagatga agtatttgtc
ccaaagcaga agtccgctat caagtggtat 1920ggcaaatacc tcaacaaccc
tgctggacgc atcatgatgt tgaccgtcca gtttgtcctc 1980gggtggccct
tgtacttggc ctttaacgtc tccggcagac catacgacgg gttcgcttgc
2040catttcttcc ccaacgctcc catctacaac gaccgtgaac gcctccagat
atatctctct 2100gatgccggta ttctagcagt ctgttttggg ctttaccgtt
acgccgctgc acaaggattg 2160gcctcgatga tctgcctcta cggagtacca
cttctgatag tgaacgcgtt cctcgtcttg 2220atcacttact tgcagcacac
tcatcctgcg ttgcctcact acgattcatc cgagtgggat 2280tggcttaggg
gagctttggc taccgtagac agagactatg gaatcttgaa taaggtgttc
2340cacaacatca cggacacaca tgtggctcat catctgttct cgacaatgcc
gcattataat 2400gcgatggaag ctacaaaggc gataaagcca atactcggtg
actattacca gttcgacgga 2460acaccatggt atgtggccat gtatagggag
gcaaaggagt gtatctatgt agaaccggac 2520agggaaggtg acaagaaagg
tgtgtactgg tacaacaata agttatgagg atgatggtga 2580aagaacactg
aagaaattgt cgatctttct ctagtctggt tctcttttgt ttaagaagtt
2640atgttttgtt tcaataattt cagtgtccat tttgttgtgt tatgacattt
tggcaaatta 2700tgtgatgtgg gaagttagtg ttcaaatgtt ttgtgtctgt
attgttcttc tcatcgctgt 2760tttgttggga tcgtagaaat gtgaccttcg g
279123875DNACamelina sativamisc_feature(3864)..(3864)n is a, c, g,
or t 2gtggaggagc ttcttcctcg tagggggttt cttcttcttc atcgttatta
acgaaccatc 60gttgagtcaa atctcccaac cccctacgtc agctccaggt ccgtcctttc
tcatttcata 120ttccgattca tttacgtctc gtctgatctg ttctttgttt
ttattttttt tttctttctc 180ccgcactatc tcattttcga ttcttttttt
ttttagaacc gatttgatga tatagatctg 240gcttatatgt cttgcattca
accttagatc tggtctcgat gctctgtttt ttctttagtt 300gagaaatctg
atgttgttgt tacaatgagt tcttattcat aataatgatt actagtgcct
360tgggtcatcc atgaaaacga tatgttgtta tactatgatt ttttatttgt
caaaaatgaa 420gtgctgcttt gacccattct ctttagattt attatttttg
ttgggttggt agaatagtga 480gaatcaccat aaaattctct tatcagtttc
acgtcctgtt tttttttcaa aaagatccgt 540aatatattca gttttttttt
atttgtgtgt aaaatttgct ttgtattgcc gtgttgaatc 600tcttatggat
ttgacctatg cctaccgggt cttatggatt gatgatataa ctttttaaac
660agacaaaata agtttcactg aatgattctc ataaactata caataaagtt
ggaattaggg 720taattcagga tcagatgcgt agattcagat gcaaataatg
agttgcatga cattttatta 780ttatagatcc gtaaccgtaa agacatatta
tgttctgttt taaatatctg atatatgatg 840tcggcaaatt tcggtggttt
atacatcact taaaaactgt ttcctttgga cttgtttgcc 900aacttggtgc
tattcaggag gatttctgat tttgattgat ccatttactt tctctattgt
960tttttttttt tttgggggtc tacttgttgg tattcataaa agaatttttg
atcttgattg 1020aatttaatta caagaaactg ctgatgataa ccacaataaa
gagattgtga cctgtcgtat 1080tgaaatctta ttagtagtag tagtcgtgtt
ctcaacgtca atgggttttt ctttctttgg 1140tttcttactt tacgccgctt
ctctgctctt tttattcctt ttggtccacg ctctctcctt 1200ttgtggcaat
ccctttcaca acctcatctc tgaataacaa taattattac tagtttgttg
1260atttcatcat taccactcgt tttctagtgc atgcaaaatt tgtcaattag
tgataaactg 1320aaaattcctt tcttgatttt tctttgctct tggtttgttg
cagaatcatg ggtgcaggtg 1380gaagaatgcc agttccttct tcttcttcca
agaaatcaga aaccgatgcc ataaagcgtg 1440tgccctgcga gaaaccaccg
ttcacgttgg gagaattgaa gaaagcaatc ccaccgcagt 1500gtttcaaacg
ctctatccct cgctctttct cctaccttat cactgacatc attgttgcct
1560cctgcttcta ctacgtcgcc accaattact tctctctcct ccctcagcct
ctctcttact 1620tggcttggcc tctctactgg gcttgtcaag gctgcgtcct
aaccggtgtc tgggtcatag 1680ctcacgaatg cggtcaccac gcattcagcg
actaccaatg gcttgatgac acagttggtc 1740ttatcttcca ttccttcctt
ctcgtccctt acttctcctg gaagtacagt catcgccgtc 1800accattccaa
cacaggatct ctcgaaagag atgaagtatt tgtcccaaag cagaaatcag
1860ctatcaagtg gtatggcaaa tacctcaaca accctcctgg acgcatcatg
atgttaaccg 1920tccagtttgt cctcgggtgg cccttgtact tggcctttaa
cgtctcgggc agaccgtacg 1980acgggttcgc ttgccatttc ttccccaacg
ctcccatcta caacgaccgt gaacgcctcc 2040agatatatct ctcggatgcc
ggtattctag cagtctgttt tgggctttac cgttacgctg 2100ctgcacaagg
aatggcctcg atgatctgcc tctacggagt accgcttctg atagtgaacg
2160cgttcctcgt cttgatcact tacttgcagc acactcatcc tgcgttgcct
cactacgatt 2220catccgagtg ggattggctt aggggagctt tggctaccgt
tgacagagac tatggaatct 2280tgaacaaggt gttccacaac atcacggaca
cacatgtggc tcatcatctg ttctcgacaa 2340tgccacatta taatgcgatg
gaagctacaa aggcgataaa gccaatactc ggtgactact 2400accagttcga
cggaacaccg tggtatgtgg cgatgtatag ggaggcaaag gagtgtatct
2460atgtagaacc ggacagagaa ggtgacaaga aaggtgtgta ctggtacaac
aataagttat 2520gaggatgatg atggtgaaag aacactgaag aaattgtcga
actttctcta gtctggttct 2580cttttgttta agaagttatg ttttgtttca
ataatttcaa tgtccatttt gttgtgttat 2640gacattttgg caaattatgt
gatgtgggaa gttagtgttt aaatgttttg tgtctgtatt 2700gttcttctca
tcgctgtttt gctgggatcg tagaaatgtg accttcggac agtaaaactc
2760tactaaaact atcttccttt cggtatcttc aaaagtgtta acttaactat
gatgcacgta 2820gtgaatcctg acttaaataa tcgacttctg tttaagacct
atcaactgta agagggttac 2880acgaatgttt ctttaacaaa ataaacataa
caattgctct ctctaaatta ggttcgatgt 2940ttttgtctgt ttgtttgatg
catggtagtc ggagtagctg tcatgttcaa gttcaatctt 3000cagtttagaa
cttgtttcca ttgttttatg actagcactg aattccattg tactctctgt
3060ctgtgatact gaagccaagc gtgacaaatg ttgaacatgc catgtcgatg
tattaaaggg 3120gattgagtta atagtgctgt tttggctggc aggtcacaat
cacaattctt tcacactcca 3180atcatgtggt taggcttacg attccttttt
tttagaatta gcttttggta aagaactgag 3240acctctggcc tttacatatg
aaatatgaaa cccctttaac taaaattata tagcacgcca 3300aatccattac
ctctggtttc atctttgaga gggaacatta agagtaaaag aaagagaaat
3360aaataaaaaa taattttttc cattacagaa tcaccaaaag agaaggacaa
caagaaagaa 3420atgaggtgaa gaaacataga aaacaaaaga atgttctgta
accaagtcga tcgatgaaca 3480aaaggcttta ccaatacgga aacaatcttt
catcccttcg atttaagcat aaacttagaa 3540gcatttcctg tggactatgg
atggccctga ctcatcatac tcaccctttg atatccacat 3600ctgtaaagca
acaacattgt gtatgattaa caaatttcaa atgggtaaca aagtaagtaa
3660aaaagcacaa aaactcatag agaataaaga atgaagatat tacctgttgg
aaggtactga 3720gggaagctaa aatggagcct ccaatccaga cactgtactt
cctctcaggt ggtgcaacca 3780ccttaatctt catgctgctt ggagccaaag
cagtgatctc tttgctcatt ctatcagcaa 3840ttccagggaa catggtggtt
ccancantga gcaag 387532725DNACamelina sativa 3gcggaggacc ttcttcctcg
tagggggttt cttcttcttc atcgttatta acgaaccatc 60gttaagtcaa atctcccccc
ccccccccta cgtcagctcc aggtccgtcc ttctcatttc 120cgatttcgat
tctttaacgt atcgtctggt ctgttctgtg tttttatttt tttctttctt
180tctcccgcac tatctcattt tcgatttttt tttaataaaa aaaaaaccga
tttcgtgata 240tagatctggc ttatatgtct tgcattcaac cttagatctg
gtctcgatgc tctgttttgt 300tttttttttt ttgttgagaa atctgatgtt
gttacaatga gttcttattc atataatgat 360tactagtgcc ttgggtcatc
catgataacg atatgttata ctatgatttt tattttattt 420ttcttttgtc
aaaaatgaag tgctgctttg acccattctc tttagatatt tttatttttt
480tatttttgtt gggttggtag aatagtgaga atcaccataa aattctctta
tcagtttcac 540gtcctggctt tttttcttgt ttttgttttt ttttaaagat
ctgtaatata ttcagttttc 600cctatttttg tttttgtaaa atttgctttg
aattgccgtg ttgaatctct cttatggatt 660tgacctatgc ctaccgaggt
cttatggatt gatgatatga ctttttaaat aaagttggaa 720ttagggtaat
tcaggatcag atgcgtagta gattcagatg caaataatga gttgcatgac
780ttgaaaatat tatagatccg taaagacata tttaaatatc tgacatatga
tgtcggcaaa 840tttcggtggt ttatacatca ctcaaaactt aaaactgttt
cttttggact tgtttggcaa 900cttggtggta ttcaagagga tttgtgattt
tgattgatcc atttactttc tctattgttt 960tttcttttgg ggtctacttg
ttggtattca taagagaact ttgtgatctt gattaaattt 1020aattacaaga
aactgatgat gatatccaca ataaagagat tgtgacctgt cgtattgaat
1080atcttattag tagtagtagt cgtgttctca acgtcaatgg gtttctttct
ttggtttctt 1140actttacgcc gcttctctgc tctttttatt ccttttggtc
cacgcacttt ccttttgtgg 1200caatcccttt cacaacctaa tcttcaattt
ggatcatttc tctgaataat aataacaact 1260agtttgttga tttgatcact
accactcgtt ttctagtcca tgcaaaattt gtcaattcct 1320ttattccttt
gatttttttg cagaaacatg ggtgcaggtg gaagaatgcc ggttccttct
1380tcttcttcca agaaatcaga aaccgatgcc ataaagcgtg tgccttgcga
gaaaccgccg 1440ttcacactgg gagaattgaa gaaagcgatc ccaccgcagt
gtttcaaacg ctctatccct 1500cgctctttct cctaccttat cactgacatc
attgttgcct cctgcttcta ctacgtcgcc 1560accaattact tctctctcct
ccctcagcct ctctcttact tggcttggcc cctctattgg 1620gcttgtcaag
gctgtgtcct aaccggtgtc tgggtcatag cccacgaatg cggtcaccac
1680gcattcagcg actaccaatg gcttgatgac acagttggtc ttatcttcca
ttccttcctt 1740ctcgtccctt acttctcctg gaagtacagt catcgccgtc
accattccaa cacaggatct 1800ctcgaaagag atgaagtatt tgtcccaaag
cagaagtccg ctatcaagtg gtatggcaaa 1860tacctcaaca accctgctgg
acgcatcatg atgttaaccg tccagtttgt cctcgggtgg 1920cccttgtact
tggcctttaa cgtctcgggc agaccatacg atgggttcgc ttgccatttc
1980ttccccaacg ctcccatcta caacgaccgt gaacgcctcc agatatatct
ctctgatgcc 2040ggtattctag cagtctgttt tgggctttac cgttacgccg
ctgcacaagg attggcctcg 2100atgatctgcc tctacggagt accacttctg
atagtaaacg cgttcctcgt cttgatcact 2160tacttgcagc acactcatcc
tgcgttgcct cactacgatt catccgagtg ggattggctt 2220aggggagctt
tggctaccgt agacagagac tatggaatct tgaacaaggt gttccacaac
2280atcacggaca cacatgtggc tcatcatctg ttttcgacaa tgccgcatta
taatgcgatg 2340gaagctacaa aggcgataaa gccaatactc ggtgactatt
accagttcga cggaacacca 2400tggtatgtgg ccatgtatag ggaggcaaag
gagtgtatct atgtagaacc ggacagggaa 2460ggtgacaaga aaggtgtgta
ctggtacaac aataagttat gaggatgatg gtgaaagaac 2520actgaagaaa
ttgtcgatct ttctctagtc tggttctctt ttgtttaaga agttatgttt
2580tatttcaata attccagtgt ccattttgtt gtgttatgac attttggcaa
attatgtgat 2640gtgggaagtt agtgttcaaa tgttttgtgt ctgtattgtt
cttctcatcg ctgttttgtt 2700gggatcgtag aaatgtgacc ttcgg
272545288DNACamelina sativa 4ggtatgaatt ggcttacacg gaagccaaag
gaagaatgag gaaagggaac agagtttggc 60agattgcttt tggaagcggg tttaagtgta
acagcgcggt ttgggtggct ctccgcgatg 120tcaagccctc ggttaacaat
ccttgggaac attgcatcca tagatatcca gttaagatcg 180atctttgaac
tcgtaagaac cggtccgaaa acatggttag tccccctcca tgtaccaaaa
240aaaaaaagtt taactcttat atttttagtt ctttaccaat gggtcaagaa
attctgttga 300aggtaacact taaatgtatg tatgtgttta ttatattatt
tattataatt aaaaaaatag 360ttttatttcc ggatatacca taagttgaat
ttttaaaaac aaatataaat tgttcaatct 420ataaaatatt caagctttag
taatattatt ctttaaaaat aatatctatt gaattaaaaa 480atttactaag
taacgggtca aaatttgaat atttaaattc aatttcatat tttttgttaa
540attttataat tttatataat ataataaact ttaaataatt tattttgaaa
tatttttaat 600atattgaaac ttgatataaa agttagaaac tataaacact
ctattgaaat aaaagttatc 660aataatattt cactataata tgaaagaatt
tcaagaaacc aagtatatta aaacaaaacg 720tataaaaata tattaaatta
ctcataatat aatataactc actttttaaa aaccaaattt 780acaaaattat
gttttataat gacattcaag tcatgatgta gaatatacat tgttgaaata
840atttcacata cataaactaa tataacatat taaaatttta ttttaaaata
taaaatatac 900ataattttgt ataaataaaa tttaaccagt gttatagcac
gggtaattat ctagaatcat 960taacaatata aaacttataa aataagtttt
ttttttcaag ctatcatatt tagcatatga 1020ttttattgca taatgtttta
tccaaaaaac ttttaaaaac aagtatataa attacatttt 1080atataaaaat
tacaatataa ataaaataaa tttcaacacg tactctaaca cggatcttaa
1140tctagtatgt tgtgtataaa ggtaacacta aaatgaccaa gaatggttac
gaagtcaaaa 1200gatgggacca aaagcgttga caaaatttta gttcttttct
aaaaataaaa ttgtttgtat 1260aataaaaatt gttaggtaga acttagaaca
ctcaactaat atatccttgc gtacgaaaac 1320atgtgttaag tgaagtgacc
ttaatgtagt ggccaaaagt aagtctttaa tgcacaagac 1380accatcacac
cagagatcga gtctcgttcc ctacgaatgt agggattagt gtaatggtcc
1440gacaaaaaaa acaaaacatg tattaaattt agaatacatg aaattactca
caagttgcga 1500atacatgtac ccctcggctc tgggatgcta aaccgggttc
gtcttatact caaatataac 1560atttaagttt gaaatttttt tttgtcaata
aaaataattt gtatactaaa aaatttagtt 1620aaaagtttaa aacaagtttt
acttaaaaaa aaatatttgt gaaaaaatcc gaagtgttaa 1680atttagaaca
cacaaaaata tgttggactt atgacacgaa gcttacagat ccgaaatgtt
1740tagtcagatt aatcttcaag cgtatttaag tttaacactt aagtttgtaa
atatacgaaa 1800atattggtta aaaaacctta aattttagta tcgacaaaaa
aaaaatctta aattttagtt 1860taaaatctga aaagaaaaag tgaaaacctt
tttgtttatt tgtaggaaaa atcaatcact 1920taaaactaaa aaaaactaga
aaaagaaata aagaaagcaa tatacctctg cttgtgatat 1980ggaagtggaa
gaaatacaag atttgatcga ttctcatcat cttgagatga ctgaatagtc
2040ggaaccatgg aaacaccaaa cacacaggat catgactggt tctctactct
cactcgtcct 2100cgttcctgca ttctcgcatc cttgagccgt cgttgatata
tccaccgtgt gaaaacgttt 2160cggtgatcgt agagaaaatt ttgacctaat
ttaaatcgat gacgatcgga tgaagatgat 2220gtagtcacga tttaacaaga
gagattaatc acgaaaagag atgctctccg ctccgatcaa 2280atatcaaaaa
gagatcatct ctttttgtga ttgtgtaaga agttagagag aagacttgac
2340gtcctgtaat cgtgatctcg atcgaatgaa gatatgtcgt cacgattaac
atgaggatgg 2400cgatcaatca atcacgaaca gagtcagaga tgcttcccct
aatcttgaca gagatcacca 2460acgcaaagct ttggatttgg tttctcaaag
aagaagaaga agagagattt ggaaaacttt 2520gacaaagaag aaaatgagag
aatgaatgaa tgatgtgatg ttggagattt tttaacctaa 2580tgactaaatg
agccgtcttt tatatacgca acagctatat taatattttt ttatttttta
2640ttttctaatt tcagcaaatt taaataacga aattgtgatt gtttcctttg
ttttcttttc 2700cccacgagtt ctcaagttgt ttgttttctt attttggttg
caaccaaaaa aaacaagaca 2760gaaaaaacaa tttatctcaa gagtaacaaa
aaggagattc gagatccttt ggagttactc 2820aattacattc atatgtcacg
agataaaaag gttaaacaat cactggatca gtgtgctcat 2880ggtgttccag
gtccaaaaca tgtgtcacga gataaaaaca aagagaaaca caactgaaaa
2940tgattaccaa gatcacaact atatataaag gacttataaa aaatgaattt
gaaagtggtt 3000aaactaagtg attataagtg ttattgcagt taccccctta
taggtttggt gaatcttatt 3060agagataact tattcttaag atagttgcaa
ttaaccaaaa aaaaaaattg tccggatagt 3120ttgatgcaat taaatgatta
atgagtgttc tatagggtct gattcttaat atttcgaaat 3180atttggcctt
aactaaactt ccaccatgat ttatttactg atctagttcg gggacagact
3240ttgcgaataa aactcattac cgagaaacat tcatcccata attgctattt
agtcagaggc 3300taatcgacta tggcctttca gccaatcaaa gctacgaaca
cgaatctccc taaaacatcc 3360tcaagtattt tatttaatac acatgtatcg
tattgagcac cactcataaa ctaatttcat 3420acatttatca tactctttat
ttgtaataat aaaagcatca acatattgta ggcaattaga 3480atcaaaacaa
aacatttttt ttttctttcc aaattttcaa aattggtaaa cgaaacttgg
3540acctttaata cttatattgg caatataata atattgcaga gtggactatt
tcccttattt 3600tggcaacttt cagtggacta gtaatttatt tcaatgtgga
tgcttgcatg agtgtgaata 3660tacacatgtc tatatgcatg cctgcaaatc
gtaacggacc acaaaaaagg atccatacaa 3720atacctctta acggctcctc
tctatcatac tctccgacac aaactgagca atgacgtccg 3780ttaacgcaaa
gctcctttac cattacgtcc taaccaactt tttcaacctt tgcttgtttc
3840cgttaacggc gttacttgcc ggaaaagcct ctaggcttac ctcaaacgat
ctctaccact 3900tctattccca tctccaacac aaccttataa ccgtaatttt
actctttgct ttcaccgctt 3960tcggtttggt tctctacatt gtaacccggc
ccaaaccggt ttacctcgtt gactactcgt 4020gctaccttcc accaccgcat
ctcaaagtta gtgtttccaa ggcgatggat attttctacc 4080aaataagaaa
agctgatacc tcacggaacg tggcatgcga tgatccatcc tcgcttgatt
4140tcctgaggaa gattcaagaa cgttcaggtc taggtgatga aacctacagt
ccccagggac 4200tcattaacgt gcccccacga aagacctttg cagcttcacg
tgaagagaca gagcaggtaa 4260tcatcggtgc gctagataag ctattcgaga
ataccaaagt taaccctaga gaaattggta 4320tacttgtggt caactcaagc
atgtttaatc caactccttc gctatctgcg atggtcgtta 4380atactttcaa
gcttcgaagc aacatcaaaa gctttagtct cggaggaatg ggttgtagtg
4440ctggtgtcat cgccattgat cttgcaaagg acttgttgca tgttcataaa
aacacttatg 4500cacttgtggt gagcactgag aacatcactc aaggcattta
tgctggcgaa aatagatcca 4560tgatggttag caattgcttg ttccgtgttg
gtggcgcagc gattttgctc tccaacaagc 4620caggagatcg gagacggtcc
aagtacaagt tatgtcatac tgttcgaacg cataccggag 4680ctgatgacat
gtcttttcga tgtgtgcaac aaggagacga tgagagcggt aaaatcggag
4740tttgtctgtc aaaggacata accgttgttg cggggatagc gcttaagaaa
aacatagcaa 4800cgttgggtcc gttgattctt cctttaagcg aaaaatttct
gtttttagta accttcatcg 4860ccaagaaact tttgaaggac aagatcaagc
actattacgt cccggatttc aagcttgcta 4920ttgaccattt ctgtattcat
gcgggaggca gagccgtgat cgatgtgctt gagaagagct 4980taggactatc
tccaatcgat gtggaggcat ctagatcaac gttacacaga tttgggaata
5040cttcgtctag ctcaatttgg tatgaattgg catacataga agcaaaagga
aggatgaaga 5100aagggaatag agcttggcag attgctttag ggtcaggatt
taagtgtaac agtgcggttt 5160gggtggctct atgcaatgtc aaggcttcgg
cgaatagtcc ttgggaacat tgcatcgata 5220gatatccggt tcaaattgat
tctggttcat caaaatcaga tactcatgtc aaaaacggtc 5280ggtcctaa
528855097DNACamelina sativamisc_feature(12)..(12)n is a, c, g, or t
5ggtatgaatt gncttacacg gaagccaaag gaagaatgag gaaagggaac
agagtttggc 60agattgcttt tggaagcggg tttaaatgta acagcgcggt ttgggtggct
ctccgcgatg 120tcgagccctc gtttaaaaat ccttgggaac attgcatcga
tagatatccg gttaagatcg 180atctttgaac tcgtaagaac ggtagattgg
tctggaaaca tggttagtcc tccatgtacc 240aaaaaaaaaa ggttaactct
tatatctttt gttctttacc aaggggtcaa gaaaatcagt 300ggaaggttaa
tgtatgtttg tatatgttgt gtataaaggt aacccttaat taatgaccaa
360gaatggttat gaagtcaaaa gataggacca aaagtgttga cctattaaaa
tttaaaattt 420tagttttttg gaaaaattaa atcatttgtg aaataaaaat
ggtgagttag aaaaatcaaa 480acaatttact tacttacaaa gaaccttaaa
attaaactta agtgttaaat ttagaataaa 540tgaaaaaatg ctaaacttat
gacaccaaaa cctacgaact cttataagga cggggatact 600aaatttgttt
gtttgtttgt tttttttttt ttttttgtaa atcaatccgg tttgttttca
660atgtattcaa attaaagtcg ggttatgaca ccaacaccaa gcttacggat
ctgaggaaga 720cgggatgcta agtcgggtta gtcatcaagc gtattcaaat
aactctgaag tcttagaact 780ttttttgtta ataaaatatt ttgtataata
aaaaaaattt agttaaaaaa atttaaaaca 840atttttacat aaaatattat
ttgtgaaaga actttttaag tgttaaattt agaacactcg 900aaaacatgtt
agacttgtga caccaagctt attccaattt aacgcttaag tttgaaaact
960ttttttgtca ataaaaaata tactatatga aaagattggt tataaaaacc
ttaaatttag 1020tttaaagtct gaaaataaaa ataaaaaata aaaaaaatta
ttagttggaa aggaaataaa 1080gaaagcaata tacctctggt tgtgatgtgg
aagaaacaca agatttgatc gattctcatc 1140agcttcagat gactgaatga
atagtcggaa ccatggaaac accaccaaac acacaggatc 1200atgactggtt
cactactctc actcgtcctc gttcccgcat tctcgcatcc ttgagccgtc
1260tttgacagat ggttaaaaca cgaacagagc tgaataagta gaagaaaaaa
gagagagttt 1320gagaagtttg atcgaatgaa taagaagaaa gacttttttt
gacctggaat cgatctcgat 1380tgaacagaga tgttttcctc tttaccgcat
tagatcagat caatccaccg tgtgaaaacg 1440tttcggtgat cgtagagcag
attttgacct aattaattaa gatcgatcac gtacgatcgg 1500atgacgataa
tgtaagagat caatcacgaa aagagatgct ccgatcaaat atcaaaggga
1560taataaagtt gatgcagaga tcatctctgt tcgtggttgt ttgagattag
ggagaagact 1620tgacctggaa tcaatctcga tcgaatgaag atgttgtcac
gattaacatg gggatggcga 1680tcagtcaatc acgaacagag tctgatatga
ttccccctcc gatcttgaca gagatcacca 1740acgcaaagct ttggatttgg
tttctcaaag aagaagaaga agagagattt ggagaaactt 1800tgatggtttt
acaaagaaga agagagaatg aatgaatgat ttggagattt tttaacctaa
1860tgactaataa taatgagccg agtgtatgaa tgtatttaat ataagcaacg
gctatttttt 1920cttcttttct tttaacttta agcaaattta gaaaaggaaa
ttgtgattgt ttcctttttt 1980ccccacgagc tctcaagttg aatccttttc
ttatttgagt tgcaaccaat aataaaaaaa 2040aaagatagaa aaacggaaat
ttatcacaag agtaacaaaa aggagattcg agatccttta 2100gagtttactc
aattacattc atatgtgcta gttgtgggag tgagaggagt tttctccttc
2160cgaagtgatt tatgtatgga ggagtttatc accgttaaga gttccgaatt
gaaagagact 2220atttgattgc taaaattgta tattactgtc tgagagaaaa
aatattcgat cccccacaaa 2280gtctcccccc ccttatatat ttatacagac
caattaatta cccaattaat gcgtaattaa 2340ttttacacga ttctcgcgtc
ctaattaatt cacttgaatg ctagaatctt tgaccgaggt 2400cgaactgcga
gctgactaat atgacgcctc tccgcgctct cttcttctct ctgggcctga
2460tgggccgacc agcgctacac ctttgacccg tttagcgtgc ccggcccagg
tcctctgcct 2520gttatccgag atgacgaatc gtgggtacaa cactagtgta
tgaccttttg cctttatagg 2580cactaaaaaa ctacccaaaa aaaatatgaa
gaagaaaaaa aggttaaaca atcactggat 2640cagtgtgctc atggtgttcc
aggtccaaag catgtgtgtc acgagataaa aatagagaga 2700aacacaactg
attatgatca ccaagatcac gaatatatat aaaggactta taaaaaatga
2760atttgaaagt ggttaaacta agtgattata tgtgtgattg ctcttagccc
cttaggtgtg 2820gtgaatctta ttatagagat gacttatttt aaagatagtt
gcaattaaaa aaaaaaatca 2880gtgtccggat agtttgatgc aattaattag
tgttctatag ggtctgattc ttaatattta 2940tgcaaatatt atagtatttc
aaagtatttg gccttaacta aacttccacc tgatttattt 3000actgatctag
ttcggggaca gactttgcga ataaaactcg ttcccgagaa acattcatcc
3060cataactgct atttagtcag aggctaatcg actatagcct ttcagccaat
caaatctacg 3120aacacgaatc cccctaaaac atcctcaagt atttatttaa
tacacatgta tcgtattgag 3180caccactcat aaactatttt tttttttgtt
tttaacaaaa aaaatttatc atactctttt 3240gtaataatag atgcatcaac
atattgtagg caacgttgaa gaaccagtac attctttttt 3300tttttgctcc
aaattttcaa aattggaaaa tgaaacttgg acgaaataaa tttaacactc
3360tgtatatatt ggcaatataa tattgcagag tggactattt accttatttt
ggcaactttc 3420agtggactag taatttattt caatgtgtat gcttgcatga
gtgtgaatat acacatgtct 3480atatgcatgc ttgcaaatcg taacggacca
caaaaaagga tccatacaaa tacctcttaa 3540cggctcctct ctatcatact
ctccgacaca aactgagcaa tgacgtccgt taacgcaaag 3600ctcctttacc
attacgttct aaccaacttt ttcaaccttt gcttgtttcc gttaacggcg
3660ttacttgccg gaaaagcctc taagcttaca gcaaacgatc tctaccactt
ctattcccat 3720ctccaacaca accttataac cgtaatttta ctctttgctt
tcaccgcttt cggtttggtt 3780ctctacattg taacccggcc caaaccggtt
tacctcgttg actactcgtg ctaccttcca 3840ccaccgcatc tcaaagttag
tgtttccaag gcgatggata ttttctacca aataagaaaa 3900gctgatacct
cacggaacgt ggcatgcgat gatccatcct cgcttgattt cctgaggaag
3960attcaagaac gttcaggtct aggtgatgaa acgtacagtc cccagggact
cattaacgtg 4020cccccacaaa agacctttgc agcttcacgt gaagagacag
agcaggtaat catcggtgcg 4080ctagaaaagc tattcgagaa caccaaagta
aaccctagag agattggtat acttgtggtg 4140aactcaagca tgtttaatcc
aactccttcg ctatctgcga tggtcgttaa cactttcaag 4200ctccgaagca
acatcaaaag ctttagtctc ggaggaatgg gttgtagtgc tggtgttatc
4260gccattgatc ttgcaaagga cttgttgcat gttcataaaa acacttatgc
acttgtggtg 4320agcactgaga acatcactca aggcatttat gctggcgaaa
acagatccat gatggttagc 4380aattgcttgt ttcgtgttgg tggggcagcg
attttgctct ccaacaaact gggagatcgg 4440agacggtcca agtacaagct
atgtcatact gttcgaacgc ataccggagc tgatgacaag 4500tcttttcgat
gtgtgcaaca aggagacgat gagggcggta aaatcggagt ttgtctgtca
4560aaggacataa ccgttgttgc ggggacagcg cttaagaaaa acatagcaac
gttgggtccg 4620ttgattcttc ctttaagcga aaagtttctg tttttagtta
ccttcatcgc caagaaactt 4680ttgaaggaca agatcaagca ctgttacgtc
ccggatttca agcttgctat cgaccatttc 4740tgtattcatg cgggaggcag
agccgtgatc gatgtgcttg agaagagctt aggactatcg 4800ccaatcgatg
tggaggcatc tagatcaacg ttacatagat ttgggaatac ttcgtctagc
4860tcaatttggt atgaattggc atacatagaa gcaaaaggaa ggatgaagaa
agggaataga 4920gcttggcaga ttgctttagg gtcagggttt aagtgtaaca
gtgcggtttg ggtggctcta 4980tgcaatgtca aggcttcggc gaatagtcct
tgggaagatt gcatcgatag atatccggtt 5040caaattgatt ctgattcatc
aaaatcagag actcatgtca aaaacggtcg gtcctaa 509763583DNACamelina
sativa 6gaggcgtcta gaatgacttt gcatagattt ggaaacactt cctcgagctc
gatatggtat 60gaattggctt acacggaagc taaaggaaga atgaggaaag ggaacagagt
ttggcagatt 120gcttttggaa gcgggtttaa gtgtaacagc gcggtttgga
tggctctccg cgatgtcgag 180ccctcgttta aaatccttgg gaacattgca
tcgatagata tccggttaag atcgatcttt 240gaactcgtaa gaacggtgga
ttggtccgga aacatggtta gtcctccatg taccaaaaaa 300gaaagtgaac
ttatatctct agttctttac taaggggtca agaaatcagt tgaaggttaa
360tgtatgttta tatatgttgt gtataaaggt aacccttaaa tgaccaagac
tgttgaccta 420ttaaaattta aaatttaagt tctctcgaaa aattaaatcg
tttgtaaaat aaaaattgtg 480agttagaaaa atcaaaacaa tttacttacc
tacaagaacc ttaaaattaa acttaagtat 540taaattttga atacatgaaa
aaatgttaaa cttatgacac caaaatttac gaactctgag 600gacggaaatg
cttaattcag tttgtttgtt tttttgtttt gtaaatcaat ctggtttgtt
660ttcaatgtat ctaaactaaa gtcgggttat gacaccaaca ccaagcttat
ggagacagga 720tgttaagtcg ggttagtcat caaggtattc aaataagtaa
taacttttaa gttttagaac 780ttttttgtca gtaaaatatt ttgtataata
aaaaagttta gttaaaacat taaaacagtt 840tttactgaaa atattatttg
tgaaaaaact tcttttaagt gttaaattta gaacacgtaa 900aaacatgtta
gacttatgac accaagctta tagacccgaa aaggaaactg tgattgtttc
960cttttcccca cgagttctaa gttgtttcct ttacttattt gggttgcaac
caatataaaa 1020agaaagaaaa atggaaattt atcacaaaaa ggagagtcga
gatctctaga gttacattca 1080tatgtgctag tgtctgacct tttgccttaa
tttataggca ctaataaaaa aactaccaaa 1140aaaaaaattg aagaagaaca
aaaggttaaa caatcactgg atcagtgtgc tcatggtgtt 1200ccaggtccaa
aacatgtgtc acgagattaa aaaagagaga aacacaactg aaactattca
1260ccaagatcac aactatatat ataaaggact tataaaaaat caatttgaaa
gtggttaaac 1320taaacgatta taagtgtgat tgcacttacc cccttatagg
tttggtgaat cttattagag 1380ataacttatt tttaagatag ttgcaattaa
aaaaaaaaaa aaattgtccg gatagtttga 1440tgcaattaat gagtgttcta
tatggtctga ttcttaatat ttatgcaaat attatagtat 1500ttcaaagtat
ttggccttaa ctgacagact ttgcaaataa aactcattcc cgagaaacat
1560tcatcccata attggtattt agtcagaggc taatcgacta tggcctttca
gccaatcaaa 1620gctacgaaca cgaatccccc taaaacatcc tcaagtattt
atttaataca tcgtattgag 1680caccactcat aaactaattc catacattta
tcatactgtt tatttgtaat aataaaagca 1740gcaacatatt gtagtttgta
ggcaataaga aacaaaacaa aacatttttt tttctctcca 1800aattttcaaa
attggaaaac gaaacttgga ccttcaatac ttatatatta tatttgcaat
1860ataaaattgc agagtggact atttccctta ttttggcaac tttcagtgga
ctagtaattt 1920atttcaatgt gtatgcttgc atgagtgtga atatacacat
gtctatatgc atgcctgcaa 1980atcgtaacgg accacaaaaa aggatccata
caaatatacc tctcaacggc tcctctctat 2040tatgctctcc gacacaaact
gagaaatgac gtccgttaac gcaaagctcc tttaccatta 2100cgtcctaacc
aactttttca acctttgctt gtttccgtta acggcgttac ttgccggaaa
2160agcctctacg cttaccacaa acgatctcta ccacttctat tcccatctcc
aacacaacct 2220tgtaaccgta attttactct ttgctttctc ctctttcggt
ttggttctct acgttgtaac 2280ccggcgcaga ccggtttacc tcgttgacta
ctcgtgctac cttccaccac cgcatctcaa 2340agttagtgtt tctaaggtca
tggatatttt ctaccaaata agaaaagctg atacctcacg 2400aaacgtggca
tgcgatgatc catcctcgct tgatttcctg aggaagattc aagaacgttc
2460aggtctaggt gatgaaacct acagtccccc gggactcatt cacgtgcccc
cacaaaagac 2520ttttgcagct tcacgtgaag agacagagca ggtaatcatc
ggtgcgttag aaaagttatt 2580cgagaacacc aaagttaacc ctagagagat
tggtatactt gtggtcaact caagcatgtt 2640taatccaact ccttcgctat
ctgcgatggt cgttaacact ttcaagctcc gaagcaacat 2700caaaagcttt
agtctcggag gaatgggttg tagtgctggt gtcatcgcca ttgatcttgc
2760aaaggacttg ttgcatgttc ataaacacac ttatgcactt gtggtgagca
ctgagaacat 2820cactcaaggc atttatgctg gcgaaaatag atccatgatg
gttagcaatt gcttgtttcg 2880tgttggtggg gcagcgattt tgctatccaa
caagccggga gatcggagac ggtccaagta 2940caagctatgt cacacggttc
ggactcatac cggagctgat gacaagtctt ttcgatgtgt 3000gcaacaagga
gacgatgaga gcggtaaaat cggagtttgt ctgtcaaagg acataacagt
3060tgttgcgggg acagcgctta agaaaaacat agcaacgtta ggtccgttga
ttcttccttt 3120aagcgaaaag tttctgttct tagttacctt catcgccaag
aaacttttga aggacaagat 3180caagcactat tacgtcccgg atttcaagct
tgctattgac catttctgta ttcatgccgg 3240aggcagagcc gtaatcgatg
tgcttgagaa gagcttagga ctatcgccaa tcgatgtgga 3300ggcatctaga
tcaacgttac atagatttgg gaatacttcg tctagctcaa tttggtatga
3360attggcatac atagaagcaa aaggaaggat gaagaaaggg aatagagctt
ggcagattgc 3420tttagggtca gggtttaagt gtaacagtgc ggtttgggtg
gctctatgca atgtcaaggc 3480ttccgcgaat agtccttggg aacattgcat
cgatagatat ccggttcaaa ttgattctga 3540ttcatcaaaa tcagagactc
atgtcaaaaa cggtcggtcc taa 35837384PRTCamelina sativa 7Met Gly Ala
Gly Gly Arg Met Pro Val Pro Ser Ser Ser Ser Lys Lys 1 5 10 15 Ser
Glu Thr Asp Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro Phe 20 25
30 Thr Leu Gly Asp Leu Lys Lys Ala Ile Pro Pro Gln Cys Phe Lys Arg
35 40 45 Ser Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile
Ile Ala 50 55 60 Ser Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser
Leu Leu Pro Gln 65 70 75 80 Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr
Trp Ala Cys Gln Gly Cys 85 90 95 Val Leu Thr Gly Val Trp Val Ile
Ala His Glu Cys Gly His His Ala 100 105 110 Phe Ser Asp Tyr Gln Trp
Leu Asp Asp Thr Val Gly Leu Ile Phe His 115 120 125 Ser Phe Leu Leu
Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg 130 135 140 His His
Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro 145 150 155
160 Lys Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro
165 170 175 Ala Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly
Trp Pro 180 185 190 Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr
Asp Gly Phe Ala 195 200 205 Cys His Phe Phe Pro Asn Ala Pro Ile Tyr
Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Leu Ser Asp Ala Gly
Ile Leu Ala Val Cys Phe Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala
Ala Gln Gly Leu Ala Ser Met Ile Cys Leu Tyr 245 250 255 Gly Val Pro
Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu
Gln His Thr His Pro Ala Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280
285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile
290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala
His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met
Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr
Gln Phe Asp Gly Thr Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu
Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp
Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380
8384PRTCamelina sativa 8 Met Gly Ala Gly Gly Arg Met Pro Val Pro
Ser Ser Ser Ser Lys Lys 1 5 10 15 Ser Glu Thr Asp Ala Ile Lys Arg
Val Pro Cys Glu Lys Pro Pro Phe 20 25 30 Thr Leu Gly Glu Leu Lys
Lys Ala Ile Pro Pro Gln Cys Phe Lys Arg 35 40 45 Ser Ile Pro Arg
Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile Val Ala 50 55 60 Ser Cys
Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln 65 70 75 80
Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys 85
90 95 Val Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His
Ala 100 105 110 Phe Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu
Ile Phe His 115 120 125 Ser Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys
Tyr Ser His Arg Arg 130 135 140 His His Ser Asn Thr Gly Ser Leu Glu
Arg Asp Glu Val Phe Val Pro 145 150 155 160 Lys Gln Lys Ser Ala Ile
Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro 165 170 175 Pro Gly Arg Ile
Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro 180 185 190 Leu Tyr
Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala 195 200 205
Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210
215 220 Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly
Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Ala Gln Gly Met Ala Ser Met
Ile Cys Leu Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Ala Phe
Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ala Leu
Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala
Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val
Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Leu
Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala 325 330
335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Trp
340 345 350 Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val
Glu Pro 355 360 365 Asp Arg Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr
Asn Asn Lys Leu 370 375 380 9384PRTCamelina sativa 9Met Gly Ala Gly
Gly Arg Met Pro Val Pro Ser Ser Ser Ser Lys Lys 1 5 10 15 Ser Glu
Thr Asp Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro Phe 20 25 30
Thr Leu Gly Glu Leu Lys Lys Ala Ile Pro Pro Gln Cys Phe Lys Arg 35
40 45 Ser Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile Val
Ala 50 55 60 Ser Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu
Leu Pro Gln 65 70 75 80 Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp
Ala Cys Gln Gly Cys 85 90 95 Val Leu Thr Gly Val Trp Val Ile Ala
His Glu Cys Gly His His Ala 100 105 110 Phe Ser Asp Tyr Gln Trp Leu
Asp Asp Thr Val Gly Leu Ile Phe His 115 120 125 Ser Phe Leu Leu Val
Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg 130 135 140 His His Ser
Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro 145 150 155 160
Lys Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro 165
170 175 Ala Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp
Pro 180 185 190 Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp
Gly Phe
Ala 195 200 205 Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg
Glu Arg Leu 210 215 220 Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala
Val Cys Phe Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Ala Gln Gly
Leu Ala Ser Met Ile Cys Leu Tyr 245 250 255 Gly Val Pro Leu Leu Ile
Val Asn Ala Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr
His Pro Ala Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp
Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300
Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305
310 315 320 Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr
Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp
Gly Thr Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu
Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp Lys Lys Gly
Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380 10505PRTCamelina sativa
10Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly
Lys 20 25 30 Ala Ser Arg Leu Thr Ser Asn Asp Leu Tyr His Phe Tyr
Ser His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe
Ala Phe Thr Ala Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg
Pro Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Ala Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Gln Gly Leu 130 135
140 Ile Asn Val Pro Pro Arg Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Asp Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255
Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg
Arg 275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Met Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Val Val Ala Gly Ile 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp
Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Ser Gly Ser Ser Lys Ser 485 490 495 Asp
Thr His Val Lys Asn Gly Arg Ser 500 505 11505PRTCamelina sativa
11Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly
Lys 20 25 30 Ala Ser Lys Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr
Ser His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe
Ala Phe Thr Ala Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg
Pro Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Ala Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Gln Gly Leu 130 135
140 Ile Asn Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255
Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Leu Gly Asp Arg
Arg 275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Gly Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Val Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp
Lys Ile Lys His Cys Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu Asp 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Ser Asp Ser Ser Lys Ser 485 490 495 Glu
Thr His Val Lys Asn Gly Arg Ser 500 505 12505PRTCamelina sativa
12Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly
Lys 20 25 30 Ala Ser Thr Leu Thr Thr Asn Asp Leu Tyr His Phe Tyr
Ser His Leu 35 40 45 Gln His Asn Leu Val Thr Val Ile Leu Leu Phe
Ala Phe Ser Ser Phe 50 55 60 Gly Leu Val Leu Tyr Val Val Thr Arg
Arg Arg Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Pro Gly Leu 130 135
140 Ile His Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 His Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255
Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg
Arg 275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Val Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp
Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Ser Asp Ser Ser Lys Ser 485 490 495 Glu
Thr His Val Lys Asn Gly Arg Ser 500 505 1324DNAArtificial
SequencePrimer FAD2 631F 13tcaacaaccc tcttggacgc atca
241424DNAArtificial SequencePrimer FAD2 832R 14cttgtgcagc
agcgtaacgg taaa 241525DNAArtificial SequencePrimer AtFAE1 probe F
15agacggtcca agtacaagct agttc 251623DNAArtificial SequencePrimer
AtFAE1 probe R 16ccaaatctat gtaacgttga tct 231725DNAArtificial
SequencePrimer AtLFY probe F 17gatgcggcgg ggaataacgg cggag
251824DNAArtificial SequencePrimer AtLFY probe R 18cctgaagaag
gaactcacgg catt 241924DNAArtificial SequencePrimer AtFAD2 start
19aacatgggtg caggtggaag aatg 242025DNAArtificial SequencePrimer
AtFAD2 stop2 20tcataactta ttgttgtacc agtac 252124DNAArtificial
SequencePrimer CaFAE1 start 21atgacgtcca ttaacgtaaa gctc
242221DNAArtificial SequencePrimer CaFAE1 stop 22ttaggaccga
ccgttttggg c 212326DNAArtificial SequencePrimer AtKCS F
23gggtggctct tcgcaatgtc gagccc 262425DNAArtificial SequencePrimer
CsFAE1 5 prime RACE 24gaggcttttc cggcaagtaa cgccg
252524DNAArtificial SequencePrimer AtKCS cons F 25ggtatgaatt
ggcttacacg gaag 242624DNAArtificial SequencePrimer CsKCSA F
26tatgaattgg cttacacgga agcc 242729DNAArtificial SequencePrimer
CsFAE1A R2 27tatattgcca atataagtat taaaggtcc 292824DNAArtificial
SequencePrimer AtKCS cons F 28ggtatgaatt ggcttacacg gaag
242929DNAArtificial SequencePrimer CsFAE1B R 29tatattgcca
atataagtat taaaggtcc 293024DNAArtificial SequencePrimer AtKCS cons
F 30ggtatgaatt ggcttacacg gaag 243124DNAArtificial SequencePrimer
CsFAE1C R 31ggtagagatc gtttgtggta agcg 243222DNAArtificial
SequencePrimer CsFAD2 start 32atgggtgcag gtggaagaat gc
223330DNAArtificial SequencePrimer CsFAD2 stop 33tcataactta
ttgttgtacc agtacacacc 303424DNAArtificial SequencePrimer CsFAE1
start 34atgacgtccg ttaacgcaaa gctc 243524DNAArtificial
SequencePrimer CsFAE1 stop 35ttaggaccga ccgtttttga catg
243624DNAArtificial SequencePrimer CsACT For 36acaatttccc
gctctgctgt tgtg 243724DNAArtificial SequencePrimer CsACT Rev
37agggtttctc tcttccacat gcca 243826DNAArtificial SequencePrimer
CsACT probe 38tgtttcaaac gctctatccc tcgctc 263923DNAArtificial
SequencePrimer CsFAD2 A For1 39ctgcgagaaa ccaccgttca ccc
234026DNAArtificial SequencePrimer CsFAD2 all Rev 40cacgagtagt
caacgaggta aaccgg 264129DNAArtificial SequencePrimer CsFAD2 all
probe 41ccacttctat tcccatctcc aacacaacc 294226DNAArtificial
SequencePrimer CsFAE1 all For 42aacctttgct tgtttccgtt aacggc
264326DNAArtificial SequencePrimer CsFAE1 all Rev 43cacgagtagt
caacgaggta aaccgg 264429DNAArtificial SequencePrimer CsFAE1 all
probe 44ccacttctat tcccatctcc aacacaacc 29451152DNACapsella rubella
45atgggtgcag gtggaagaat gccggttcct tcttcttcca agaagtcgga aaccaatgcc
60atcaagcgtg tgccgtgcga gaaaccgcct ttcacggttg gagatctcaa gaaagcaatc
120ccaccgcagt gtttcaaacg ctctatccct cgctctttct cctaccttat
cactgacatc 180attattgcct cctgcttcta ctacgtcgcc accaattact
tctctctcct ccctcagcct 240ctctcttact ttgcttggcc cctctactgg
gcctgtcagg gctgtgtcct taccggtgtc 300tgggtcattg cccacgaatg
cggtcaccat gctttcagcg actaccaatg gctggatgac 360acagttggtc
ttatcttcca ctccttcctc ctcgtccctt acttctcctg gaagtacagt
420catcgccgcc accattccaa cacgggatct cttgaaaggg atgaagtatt
tgttccaaag 480cagaaatccg ctatcaagtg gtacggcaaa taccttaaca
acccttttgg acgtatcatg 540atgttaaccg tccagtttgt cctcggatgg
cccttgtact tggcctttaa cgtctcaggc 600agaccttacg atgggttcgc
ttgccatttc
ttccccaacg ctcccatcta caacgaccgc 660gaacgccttc agatctatat
ctcggatgcc ggtattctag cagtctgtta tggtctttac 720cgttacgctg
ctgcacaagg aatggcctcg atgatctgcc tctacggagt accacttctg
780atagtgaacg cgttccttgt cttgatcaca tacttgcagc acactcatcc
tgcgttgcct 840cactacgatt catccgagtg ggattggctt aggggagctt
tggctaccgt agacagagac 900tatggaatct tgaacaaggt gttccacaac
atcacggaca cacatgtggc tcatcatctg 960ttctcgacaa tgcctcatta
caacgcgatg gaagctacaa aggcgataaa gcctatactc 1020ggagactatt
accagtttga tggaacaccg tggtatgtgg cgatgtatag ggaggcaaag
1080gagtgtatct atgtagaacc ggacagggaa ggtgacaaga aaggtgtgta
ctggtacaac 1140aataagttat ga 1152461152DNAArabidopsis lyrata
46atgggtgcag gtggaagaat gccggttcct acttcttcca agaaatcgga aaccgacacc
60ataaagcgtg tgccgtgcga gaaaccgcct ttctcggtgg gagatctgaa gaaagcaatc
120ccccagcatt gtttcaaacg ctcaatccct cgctctttct cctaccttat
cggtgacatc 180ataattgcct catgcttcta ctacgttgcc accaattact
tctctctcct acctcagcct 240ctctcttact tggcttggcc actctattgg
gcctgtcaag gctgtgtcct aactggtgtc 300tgggtcatag cccacgaatg
cggtcaccac gcattcagcg actaccaatg gctggatgac 360acagtcggtc
ttatcttcca ttccttcctc ctcgtccctt acttctcctg gaagtatagt
420catcgccgtc accattccaa cacgggatcc ctcgaaagag atgaagtatt
tgtcccaaaa 480cagaaatccg caatcaagtg gtacggcaaa tacctcaaca
accctcttgg acgcatcatg 540atgttaaccg tccagtttgt cctcgggtgg
cccttgtact tagcctttaa cgtttcgggc 600agaccgtatg acgggttcgc
ttgccatttc ttccccaacg ctcccatcta caatgaccgc 660gaacgcctcc
agatatacct ctcggatgcg ggtattctag ccgtctgttt tggtctttac
720cgttacgccg ctgcacaagg aatggcctct atgatctgcc tctacggagt
accgcttctg 780atagtgaatg cgttcctcgt cttgatcact tacttgcagc
acactcaccc ctccttgcct 840cactacgatt catcagagtg ggactggctc
aggggagctt tggctaccgt agacagagac 900tatggaatct tgaacaaggt
gttccacaac attacagaca cacacgtggc acatcacctg 960ttctcgacaa
tgccgcatta taacgcaatg gaagctacaa aggcgataaa gccaatactg
1020ggagactatt accagttcga tggaacaccg tggtatgtgg cgatgtatag
ggaggcaaag 1080gagtgtatct acgtagaacc ggacagggaa ggtgacaaga
aaggtgtgta ctggtacaac 1140aataagttat ga 1152471518DNAArabidopsis
lyrata 47atgacgtccg ttaacgcaaa gctcctttac cattacgtct taaccaactt
tttcaacctc 60tgtttgttcc cgttaacggc gttcgtcgcc ggaaaagcct ctcggcatac
cacaaacgat 120ctccacaact tcttttccta tctccaacac aaccttataa
ccgtagctat actctttgct 180ttcactgtct ttggttttgt tctctacatg
gtaacccgac ccaaaccggt ttatctcgtt 240gactactcgt gctaccttcc
accaccgcat ctcagagcca gtgtctccag agtcatggat 300gttttctatc
aaataagaaa agctgatact tcacggaacg tggcatgcga tgatccgtcc
360tcgcttgatt tcctgaggaa gattcaagag cgttcaggtc taggtgatga
gacctacggt 420cccgagggac tccttcacgt gcccccacgg aagacttttg
cagcggcacg tgaagagaca 480gagcaggtta tcattggtgc gctcgaaaat
ctattccaga acaccaaagt taaccctaga 540gagattggta tacttgtggt
gaactcaagc atgtttaatc caactccttc gctttccgcg 600atggtcgtta
atactttcaa gctccgaagc aacatcaaaa gctttaatct tggaggaatg
660ggttgtagtg ctggtgttat cgccattgat cttgcgaagg acttgttgca
tgttcataaa 720aacacttatg ctcttgtggt gagcactgag aacatcactc
aagggattta tgctggcgaa 780aatagatcaa tgatggttag caattgcttg
ttccgtgttg gtggggccgc gattttgctc 840tccaacaagc ctagagatcg
gagacggtcc aagtacaagc tagctcacac ggttcgaacc 900catacgggag
ctgatgacaa gtcttttcga tgtgtgcaac aagaagacga tgagagtggt
960aaaatcggtg tttgtctgtc aaaggacata accaatgttg cggggacaac
gctaaagaaa 1020aacatagcaa cattgggtcc gttgattctt cctttaagcg
agaaatttct ttttttcgtt 1080accttcgtcg ccaagaaact tttaaaggac
agaatcaagc attactatgt cccggatttt 1140aagcttgcta ttgaccattt
ctgtattcat gccggaggca gagccgtgat cgatgagcta 1200gagaagagct
taggactatc accgatcgac gtggaggcat ctagatcaac gttacataga
1260tttgggaata cgtcgtctag ctcaatttgg tatgaattgg catacataga
ggcaaaagga 1320agaatgaaga aagggaataa agcttggcag attgctttag
gatcagggtt taagtgtaat 1380agtgcggttt gggtggctct acgcaatgtc
aagccttcgg caaatagtcc ttggcaacat 1440tgcatcgata gatatccggc
taaaattgat tctgatttgt caaagtcaga gactcatgtc 1500aaaaacggtc ggtcctaa
1518481155DNACamelina hispida 48atgggtgcag gtggaagaat gccagttcct
tcttcttctt ccaagaaatc ggaaaccaat 60gccataaagc gtgtgccctg cgagaaaccg
ccgttcacgg ttggagaact gaagaaagca 120atcccaccgc attgtttcaa
acgctctatc cctcgctctt tctcctacct tatcactgac 180atcattattg
cctcctgctt ctactacgtc gccaccaatt acttctctct cctccctcag
240cctctctctt acttggcttg gcctctctat tgggcctgtc aaggctgtgt
cctaaccggt 300gtctgggtca tagcccatga atgcggtcac cacgcattca
gcgactacca atggctcgat 360gacacagttg gtcttatctt ccattccttc
cttctcgtcc cttacttctc ctggaagtac 420agtcatcgcc gtcaccattc
caacacagga tctcttgaaa gagatgaagt atttgtccca 480aagcagaaat
cagctatcaa gtggtatggc aaatacctca acaaccctcc tggacgcatc
540atgatgttaa ccgtccagtt tgtcctcggg tggcccttgt acttggcctt
taacgtctcg 600ggcagaccat acgacgggtt cgcttgccat ttcttcccca
acgctcccat ctacaacgac 660cgtgaacgcc tccagatata tctctctgat
gctggtattc tagcagtctg ttttgggctt 720taccgttatg ccgctgcaca
aggattggct tcgatgatct gcctctacgg agtaccgctt 780ctgatagtga
acgcgttcct cgtcttgatc acttacttgc agcacactca tcctgcgttg
840cctcactacg attcatccga gtgggattgg cttaggggag ctttggctac
cgtagacaga 900gactatggaa tcttgaacaa ggtgttccac aacatcacgg
acacacatgt ggctcatcat 960ctgttctcga caatgccgca ttataatgcg
atggaagcta caaaggcgat aaagccaata 1020ctcggtgact actaccagtt
cgacggaaca ccgtggtatg tggcgatgta tagggaggca 1080aaggagtgta
tctatgtaga accggacagg gaaggtgaca agaaaggtgt gtactggtac
1140aacaataagt tatga 1155491518DNACamelina hispida 49atgacgtccg
ttaacgcaaa gctcctttac cattacgtcc taaccaactt tttcaacctt 60tgcttgtttc
cgttaacggc gttacttgtc ggaaaagtct ctcggcttac cgcaaacgat
120ctctaccact tctatttcca tctccaacac aatctcataa ccgttattct
actctttgct 180ttcaccactt ttggtttggt tctctacatt gtaacccggc
ccaaaccggt ttacctcgtt 240gactactcgt gctaccttcc accaccgcat
ctcaaagtta gtgtttccaa agtcatggat 300attttctacc aaataagaaa
agctgatact tcacggaacg tggcatgcga tgatccatcc 360tcgcttgatt
tcctgaggaa gattcaagaa cgttcaggtc taggtgatga aacctacagt
420cccccgggac tcattcacgt gccaccacaa aagacctttg cagcgtcacg
tgaagagaca 480gagcaggtaa tcatcggtgc gctagaaaag ctattcgaga
acactaaagt taaccctaga 540gatattggta tacttgtggt caactcaagc
atgtttaatc caactccttc gttatccgct 600atggtcgtaa atactttcaa
gcttcgaagc aacatcaaaa gctttagtct cggaggaatg 660ggttgtagtg
ctggtgttat cgccattgat cttgcaaagg acttgttgca tgttcataaa
720aacacttatg cacttgtggt gagcactgag aacatcactc acggcattta
ttctggcgaa 780aatagatcca tgatggttag taattgcttg ttccgtgttg
gtggggcagc gattttgctc 840tccaacaagc caggagatcg tagacggtcc
aagtacaagc tatgtcacac cgttcgaacg 900cataccggag ctgatgacaa
gtcttttcga tgtgtgcaac aaggagacga tgagagcggt 960aaaattggag
tttgtctgtc aaaggacata acagttgttg cggggacagc gcttaagaaa
1020aacatagcaa cgttaggtcc attgattctt cctttaagcg aaaagtttct
gtttttagta 1080accttcatcg ccaagaaagt tttgaaggac aagatcaaga
actattacgt cccggatttc 1140aagcttgcta ttgaccactt ctgtattcat
gcgggaggca gagccgtgat cgatgtgctt 1200gagaagagct taggactatc
tccaatcgat gtggaggcat ctagatcaac gttacataga 1260tttgggaata
cttcgtctag ctcaatttgg tatgaattgg catacataga agcaaaagga
1320aggatgaaga aagggaatag agcttggcag attgctttag ggtcagggtt
taagtgtaac 1380agtgcggttt gggtggctct atgcaatgtc aaggcttcgg
cgaatagtcc ttgggagcat 1440tgcatcgata gatatccggt tcaaattgat
tctgattcat caaaatcaga gactcatgtc 1500aaaaacggtc ggtcctaa
1518501518DNACamelina hispida 50atgacgtccg ttaacgcaaa gctcctttac
cattacgtcc taaccaactt tttcaacctt 60tgcttgtttc cgttaacggc gttacttgcc
ggaaaagcct ctaggcttac cacaaacgat 120ctctaccact tctattccca
tctccaacac aaccttgtaa ccgtaatttt actctttgct 180ttcacctctt
tcggtttggt tctctacatt gtaacccggc ccaaaccggt ttacctcgtt
240gactactcgt gctaccttcc accaccgcac ctcaaagtta gtgtttccaa
ggtcatggat 300attttctacc aaatcagaaa agctgatact tcacgaaacg
tggcatgcga tgatccatcc 360tcgcttgatt tcttgaggaa gattcaagaa
cgttcaggtc taggtgatga aacctacagt 420cccccgggac tcattaacgt
gcccccacaa aagacctttg cagcttcacg tgaagagaca 480gagcaggtaa
tcatcggtgc gctagaaaag ctattcgaga acaccaaagt taaccctaga
540gagattggta tacttgtggt gaactcaagc atgtttaatc caactccttc
gctatctgcg 600atggtcgtta acactttcaa gctccgaagc aacatcaaaa
gcttaagtct cggaggaatg 660ggttgtagtg ctggtgtcat cgccattgat
cttgcaaagg acttgttgca tgttcataaa 720aacacttatg ctcttgtggt
gagcactgag aacatcactc aaggcatcta tgctggcgaa 780aatagatcca
tgatggttag caattgcttg ttccgtgttg gtggcgcagc gattttgctc
840tccaacaagg cgggagatcg gagacggtct aagtacaagc tgtgtcacac
tgttcgaacg 900cataccggag ctgatgacaa gtcttttcga tgtgtgcaac
aaggagacga tgagagcggt 960aaaattggag tttgtctgtc aaaggacata
accgttgttg cggggacagc gcttaagaaa 1020aacatagcaa cgttgggtcc
gttgattctt cctttaagcg aaaagtttct gtttttagta 1080accttcatcg
ccaagaaact tttgaaggac aagatcaagc actattacgt ccccgatttc
1140aagcttgcta ttgaccattt ctgtattcat gcgggaggca gagccgtaat
cgatgttctt 1200gagaagagct taggactatc tccaatagat gtggaggcct
ctagatcaac gttacataga 1260tttgggaata cttcgtctag ctcaatttgg
tatgaattgg catacataga agcaaaagga 1320aggatgaaga aagggaatag
agcttggcag attgctttag ggtcagggtt taagtgtaac 1380agtgcggttt
gggtggctct atgcaatgtc aaggcttcgg cgaatagtcc ttgggaacat
1440tgcatcgata gatatccggt tcaaattgat tctgattcat caaaatcaga
gactcatgtc 1500aaaaacggtc ggtcctaa 1518511158DNACamelina laxa
51atgggtgcag gtggaagaat gccagttcct tcttcttctt cttccaagaa atctgaaacc
60gatgccataa agcgtgtgcc ctgcgagaaa ccgccgttca cgcttggaga actgaagaaa
120gcaatccccc cgcagtgttt caaacgctct atccctcgct ctttctccta
ccttatcact 180gacatcattg ttgcctcctg cttctactac gtcgccacca
attacttctc tctcctccct 240cagcctctct cttacttggc ttggcctctc
tactgggcct gtcaaggctg tgtcctaacc 300ggtgtctggg tcatagctca
cgaatgcggt caccacgcat tcagcgacta ccaatggctt 360gatgacacag
ttggtcttat cttccattcc ttccttctcg tcccttactt ctcctggaag
420tacagtcatc gtcgtcacca ttccaacaca ggatctctcg aaagagatga
agtatttgtc 480ccaaagcaga aatcagctat caagtggtat ggcaaatacc
tcaacaaccc tcctggacgc 540atcatgatgt taaccgtcca gtttgtcctc
gggtggccct tgtacttggc ctttaacgtc 600tcgggcagac cgtacgacgg
gttcgcttgc catttcttcc ccaacgctcc catctacaac 660gaccgcgaac
gcctccagat atatctctct gatgccggta tcctagcagt ctgttttggg
720ctttaccgtt acgttgctgc acaaggaatg gcctcgatga tctgcctcta
cggagtaccg 780cttctgatag tgaacgcgtt cctcgtcttg atcacttact
tgcagcacac tcatcctgcg 840ttgcctcact acgattcatc cgagtgggat
tggcttaggg gagctttggc taccgttgac 900agagactatg gaatcttgaa
caaggtgttc cacaacatca cggacacaca tgtggctcat 960catctgttct
cgacaatgcc acattataat gcgatggaag ctacaaaggc gataaagcca
1020atactcggtg actactacca gttcgacgga acaccgtggt atgtggcgat
gtatagggag 1080gcaaaggagt gtatctatgt agaaccggac agagaaggtg
acaagaaagg tgtgtactgg 1140tacaacaata agttatga 1158521518DNACamelina
laxa 52atgacgtccg ttaacgcaaa gctcctttac cattacgtcc taaccaactt
tttcaacctt 60tgcttgtttc cgttaacggc gttacttgtc ggaaaagtct ctcggcttac
cgcaaacgat 120ctctaccact tctatttcca tctccaacac aatctcataa
ccgttattct actctttgct 180ttcaccactt ttggtttggt tctctacatt
gtaacccggc ccaaaccggt ttacctcgtt 240gactactcgt gctaccttcc
accaccgcat ctcaaagtta gtgtttccaa agtcatggat 300attttctacc
aaataagaaa agctgatact tcacggaacg tggcatgcga tgatccatcc
360tcgcttgatt tcctgaggaa gattcaagaa cgttcaggtc taggtgatga
aacctacagt 420cccccgggac tcattcacgt gccaccacaa aagacctttg
cagcgtcacg tgaagagaca 480gagcaggtaa tcatcggtgc gctagaaaag
ctattcgaga acactaaagt taaccctaga 540gatattggta tacttgtggt
caactcaagc atgtttaatc caactccttc gttatccgct 600atggtcgtaa
atactttcaa gcttcgaagc aacatcaaaa gctttagtct cggaggaatg
660ggttgtagtg ctggtgttat cgccattgat cttgcaaaag acttgttgca
tgttcataaa 720aacacttatg cacttgtggt gagcactgag aacatcactc
acggcattta tgctggcgaa 780aatagatcca tgatggttag caattgcttg
ttccgtgttg gtggggcggc gattttgctc 840tccaacaagc cgggagatcg
gagacgggcc aagtacaagc tatgtcacac tgttcgaacg 900cataccggag
ctgatgacaa gtcttttcga tgtgtgcaac aaggagatga tgagagcggt
960aaaatcggag tttgtctgtc aaaggacata accgctgttg cggggacagc
gcttaagaaa 1020aacatagcaa cgttaggtcc gttgattctt cctttaagcg
aaaaatttct gtttttagta 1080accttcatcg ccaagaaact tttgaaggac
aagatcaagc actattacgt cccggatttc 1140aaggttgcta ttgaccattt
ctgtattcat gccggaggta gagccgtgat cgatgtgctt 1200gagaagagct
taggactatc tccaatcgat gtcgaggcat cgagatcaac gttacacaga
1260tttgggaata cttcgtctag ctcaatttgg tatgaattgg catacataga
agcaaaagga 1320aggatgaaga aagggaataa agcttggcaa attgctttag
ggtcagggtt taagtgtaac 1380agtgcggttt gggtggctct atgcaatgtc
aaggcttcgg cgaatagtcc ttgggaacat 1440tgcatcgata gatatccggt
tcaaatagat tttgattcat caaaatcaga cactcatgtc 1500aaaaacggtc ggtcctaa
1518531518DNACamelina laxa 53atgacgtccg ttaacgcaaa gctcctttac
cattacgtcc taaccaactt tttcaacctt 60tgcttgtttc cgttaacggc gttacttgcc
ggaaaagcct ctaggcttac cacaaacgat 120ctctaccact tcaattccca
tctccaacac aacattgtaa cggttgtttt actctttgct 180tttaccgctt
tcggtttggt tctttacgtt gtaacccggc ccaaaccggt ttacctcgtt
240gactactcgt gctaccttcc accaccgcat ctcaaagtta gtgtttccaa
ggtcatggat 300attttctacc aaataagaaa agctgatacc acacgaaacg
tggcatgcga tgatccatcc 360tcgcttgatt tcctgaggaa gattcaagaa
cgttcaggtc taggtgatga aacctacagt 420ccccagggac tcattaacgt
gcccccacaa aagacctttg cagcttcacg tgaagagaca 480gagcaggtaa
tcatcggtgc gctagaaaag ctattcgaga acaccaaagt taaccctaga
540gagattggta tacttgtggt gaactcaagc atgtttaatc caactccttc
gctatctgcg 600atggtcgtta acactttcaa gctccgaagc aatatcaaaa
gctttagtct cggaggaatg 660ggttgtagtg ctggtgtcat cgccattgat
cttgcaaaag acttgttgca tgttcataaa 720aacacttatg cacttgtggt
gagcactgag aacatcactc aaggcattta tgctggcgaa 780aatagatcca
tgatggttag caattgcttg ttccgtgttg gtggggcggc gattttgctc
840tccaacaagc cgggagatcg gagacgggcc aagtacaagc tatgtcacac
tgttcgaacg 900cataccggag ctgatgacaa gtcttttcga tgtgtgcaac
aaggagatga tgagagcggt 960aaaatcggag tttgtctgtc aaaggacata
accgctgttg cggggacagc gcttaagaaa 1020aacatagcaa cgttaggtcc
gttgattctt cctttaagcg aaaaatttct gtttttagta 1080accttcatcg
ccaagaaact tttgaaggac aagatcaagc actattacgt cccggatttc
1140aaggttgcta ttgaccattt ctgtattcat gccggaggta gagccgtgat
cgatgtgctt 1200gagaagagct taggactatc tccaatcgat gtcgaggcat
cgagatcaac gttacacaga 1260tttgggaata cttcgtctag ctcaatttgg
tatgaattgg catacataga agcaaaagga 1320aggatgaaga aagggaataa
agcttggcaa attgctttag ggtcagggtt taagtgtaac 1380agtgcggttt
gggtggctct atgcaatgtc aaggcttcgg cgaatagtcc ttgggaacat
1440tgcatcgata gatatccggt tcaaatagat tttgattcat caaaatcaga
cactcatgtc 1500aaaaacggtc ggtcctaa 1518541155DNACamelina microcarpa
54atgggtgcag gtggaagaat gccagttcct tcttcttctt ccaagaaatc tgaaaccgat
60gccataaagc gtgtgccctg cgagaaacca ccgttcacgc tgggagatct gaagaaagca
120atcccaccgc agtgtttcaa acgctctatc cctcgctctt tctcctacct
tatcactgac 180atcattattg cctcctgctt ctactacgtc gccaccaatt
acttttctct cctccctcag 240cctctctctt acttggcttg gcccctctat
tgggcttgtc aaggctgtgt cctaaccggt 300gtctgggtca tagcccacga
atgcggtcac cacgcattca gcgactacca gtggctcgat 360gacacagtcg
gtcttatctt ccattccttc cttctcgtcc cttacttctc ctggaagtac
420agtcatcgcc gtcaccattc caacacagga tccctcgaaa gagatgaagt
atttgtccca 480aagcagaagt ccgctatcaa gtggtatggc aaatacctca
acaaccctgc tggacgcatc 540atgatgttga ccgtccagtt tgtcctcggg
tggcccttgt acttggcctt taacgtctcc 600ggcagaccat acgacgggtt
cgcttgccat ttcttcccca acgctcccat ctacaacgac 660cgtgaacgcc
tccagatata tctctctgat gccggtattc tagcagtctg ttttgggctt
720taccgttacg ccgctgcaca aggattggcc tcgatgatct gcctctacgg
agtaccactt 780ctgatagtga acgcgttcct cgtcttgatc acttacttgc
agcacactca tcctgcgttg 840cctcactacg attcatccga gtgggattgg
cttaggggag ctttggctac cgtagacaga 900gactatggaa tcttgaataa
ggtgttccac aacatcacgg acacacatgt ggctcatcat 960ctgttctcga
caatgccgca ttataatgcg atggaagcta caaaggcgat aaagccaata
1020ctcggtgact attaccagtt cgacggaaca ccatggtatg tggccatgta
tagggaggca 1080aaggagtgta tctatgtaga accggacagg gaaggtgaca
agaaaggtgt gtactggtac 1140aacaataagt tatga 1155551158DNACamelina
microcarpa 55atgggtgcag gtggaagaat gccggttcct tcttcttctt cttccaagaa
atcagaaacc 60gatgccatga agcgtgtgcc ctgcgagaaa ccaccgttca cgctgggaga
attgaagaaa 120gcgatcccac cgcagtgttt caaacgctct atccctcgct
ctttctccta ccttatcact 180gacatcattg ttgcctcctg cttctactac
gtcgccacca attacttctc tctcctccct 240cagcctctct cttacttggc
ttggcccctc tattgggcct gtcaaggctg tgtcctaacc 300ggtgtctggg
tcatagccca cgaatgcggt caccacgcat tcagtgacta ccaatggctt
360gatgacacag ttggtcttat cttccattcc ttccttctcg taccttactt
ctcctggaag 420tacagtcatc gccgtcacca ttccaacaca ggatctctcg
aaagagatga agtatttgtc 480ccaaagcaga agtccgctat caagtggtat
ggcaaatacc tcaacaaccc tcctggacgc 540atcatgatgt taaccgtcca
gtttgtcctc gggtggccct tgtacttggc ctttaacgtc 600tcgggcagac
catacgacgg gttcgcttgc catttctttc ccaacgctcc catctacaac
660gaccgcgaac gcctccagat atatctctct gatgccggta ttctagcagt
ctgttttggg 720ctttaccgtt acgcagctgc gcaaggaatg gcctcgatga
tctgcctcta cggagtaccc 780cttctgatag tgaacgcgtt cctcgtcttg
atcacttact tgcagcacac tcaccctgcg 840ttgcctcact acgattcgtc
cgagtgggat tggcttaggg gagctttggc taccgtagac 900agagactacg
gaatcttgaa caaggtgttc cataacatca cggacacaca tgtggctcat
960catctgttct cgacaatgcc acattataat gctatggaag cgacaaaggc
gataaagcca 1020atactcggtg actactacca gttcgacgga acaccgtggt
atgtggccat gtatagggag 1080gcaaaggaat gtatctatgt agaaccggac
agggaaggtg acaagaaagg tgtgtactgg 1140tacaacaata agttatga
1158561155DNACamelina microcarpa 56atgggtgcag gtggaagaat gccggttcct
tcttcttctt ccaagaaatc agaaaccgat 60gccataaagc gtgtgccttg cgagaaaccg
ccgttcacac tgggagaatt gaagaaagcg 120atcccaccgc agtgtttcaa
acgctctatc cctcgctctt tctcctacct tatcactgac 180atcattgttg
cctcctgctt ctactacgtc gccaccaatt acttctctct cctccctcag
240cctctctctt acttggcttg gcccctctat tgggcttgtc aaggctgtgt
cctaaccggt 300gtctgggtca tagcccacga atgcggtcac cacgcattca
gcgactacca atggcttgat 360gacacagttg gtcttatctt ccattccttc
cttctcgtcc cttacttctc ctggaagtac 420agtcatcgcc gtcaccattc
caacacagga tctctcgaaa gagatgaagt atttgtccca 480aagcagaagt
ccgctatcaa gtggtatggc aaatacctca acaaccctgc tggacgcatc
540atgatgttaa ccgtccagtt tgtcctcggg tggcccttgt acttggcctt
taacgtctcg 600ggcagaccat acgatgggtt cgcttgccat ttcttcccca
acgctcccat ctacaacgac 660cgtgaacgcc tccagatata tctctctgat
gccggtattc tagcagtctg ttttgggctt 720taccgttacg ccgctgcaca
aggattggcc tcgatgatct gcctctacgg agtaccactt 780ctgatagtaa
acgcgttcct cgtcttgatc acttacttgc agcacactca tcctgcgttg
840cctcactacg attcatccga gtgggattgg cttaggggag ctttggctac
cgtagacaga 900gactatggaa tcttgaacaa ggtgttccac aacatcacgg
acacacatgt ggctcatcat 960ctgttttcga caatgccgca ttataatgcg
atggaagcta caaaggcgat aaagccaata 1020ctcggtgact attaccagtt
cgacggaaca ccatggtatg tggccatgta tagggaggca 1080aaggagtgta
tctatgtaga accggacagg gaaggtgaca agaaaggtgt gtactggtac
1140aacaataagt tatga 1155571518DNACamelina microcarpa 57atgacgtccg
ttaacgcaaa gctcctttac cattacgtcc taaccaactt tttcaacctt 60tgcttgtttc
cgttaacggc gttacttgcc ggaaaagcct ctaggcttac ctcaaacgat
120ctctaccact tctattccca tctccaacac aaccttataa ccgtaatttt
actctttgct 180ttcaccgctt tcggtttggt tctctacatt gtaacccggc
ccaaaccggt ttacctcgtt 240gactactcgt gctaccttcc accaccgcat
ctcaaagtta gtgtttccaa ggcgatggat 300attttctacc aaataagaaa
agctgatacc tcacggaacg tggcatgcga tgatccatcc 360tcgcttgatt
tcctgaggaa gattcaagaa cgttcaggtc taggtgatga aacctacagt
420ccccagggac tcattaacgt gcccccacga aagacctttg cagcttcacg
tgaagagaca 480gagcaggtaa tcatcggtgc gctagataag ctattcgaga
ataccaaagt taaccctaga 540gaaattggta tacttgtggt caactcaagc
atgtttaatc caactccttc gctatctgcg 600atggtcgtta atactttcaa
gcttcgaagc aacatcaaaa gctttagtct cggaggaatg 660ggttgtagtg
ctggtgtcat cgccattgat cttgcaaagg acttgttgca tgttcataaa
720aacacttatg cacttgtggt gagcactgag aacatcactc aaggcattta
tgctggcgaa 780aatagatcca tgatggttag caattgcttg ttccgtgttg
gtggcgcagc gattttgctc 840tccaacaagc caggagatcg gagacggtcc
aagtacaagt tatgtcatac tgttcgaacg 900cataccggag ctgatgacat
gtcttttcga tgtgtgcaac aaggagacga tgagagcggt 960aaaatcggag
tttgtctgtc aaaggacata accgttgttg cggggatagc gcttaagaaa
1020aacatagcaa cgttgggtcc gttgattctt cctttaaggg aaaaatttct
gtttttagta 1080accttcatcg ccaagaaact tttgaaggac aagatcaagc
actattacgt cccggatttc 1140aagcttgcta ttgaccattt ctgtattcat
gcgggaggca gagccgtgat cgatgtgctt 1200gagaagagct taggactatc
tccaatcgat gtggaggcat ctagatcaac gttacacaga 1260tttgggaata
cttcgtctag ctcaatttgg tatgaattgg catacataga agcaaaagga
1320aggatgaaga aagggaatag agcttggcag attgctttag ggtcaggatt
taagtgtaac 1380agtgcggttt gggtggctct atgcaatgtc aaggcttcgg
cgaatagtcc ttgggaacat 1440tgcatcgata gatatccggt tcaaattgat
tctggttcat caaaatcaga tactcatgtc 1500aaaaacggtc ggtcctaa
1518581518DNACamelina microcarpa 58atgacgtccg ttaacgcaaa gctcctttac
cattacgttc taaccaactt tttcaacctt 60tgcttgtttc cgttaacggc gttacttgcc
ggaaaagcct ctaagcttac agcaaacgat 120ctctaccact tctattccca
tctccaacac aaccttataa ccgtaatttt actctttgct 180ttcaccgctt
tcggtttggt tctctacatt gtaacccggc ccaaaccggt ttacctcgtt
240gactactcgt gctaccttcc accaccgcat ctcaaagtta gtgtttccaa
ggcgatggat 300attttctacc aaataagaaa agctgatacc tcacggaacg
tggcatgcga tgatccatcc 360tcgcttgatt tcctgaggaa gattcaagaa
cgttcaggtc taggtgatga tacgtacagt 420ccccagggac tcattaacgt
gcccccacaa aagacctttg cagcttcacg tgaagagaca 480gagcaggtaa
tcatcggtgc gctagaaaag ctattcgaga acaccaaagt aaaccctaga
540gagattggta tacttgtggt gaactcaagc atgtttaatc caactccttc
gctatctgcg 600atggtcgtta acactttcaa gctccgaagc aacatcaaaa
gctttagtct cggaggaatg 660ggttgtagtg ctggtgttat cgccattgat
cttgcaaagg acttgttgca tgttcataaa 720aacacttatg cacttgtggt
gagcactgag aacatcactc aaggcattta tgctggcgaa 780aacagatcca
tgatggttag caattgcttg tttcgtgttg gtggggcagc gattttgctc
840tccaacaaac tgggagatcg gagacggtcc aagtacaagc tatgtcatac
tgttcgaacg 900cataccggag ctgatgacaa gtcttttcga tgtgtgcaac
aaggagacga tgagggcggt 960aaaatcggag tttgtctgtc aaaggacata
accgttgttg cggggacagc gcttaagaaa 1020aacatagcaa cgttgggtcc
gttgattctt cctttaagcg aaaagtttct gtttttagtt 1080accttcatcg
ccaagaaact tttgaaggac aagatcaagc actgttacgt cccggatttc
1140aagcttgcta tcgaccattt ctgtattcat gcgggaggca gagccgtgat
cgatgtgctt 1200gagaagagct taggactatc gccaatcgat gtggaggcat
ctagatcaac gttacataga 1260tttgggaata cttcgtctag ctcaatttgg
tatgaattgg catacataga agcaaaagga 1320aggatgaaga aagggaatag
agcttggcag attgctttag ggtcagggtt taagtgtaac 1380agtgcggttt
gggtggctct atgcaatgtc aaggcttcgg cgaatagtcc ttgggaagat
1440tgcatcgata gatatccggt tcaaattgat tctgattcat caaaatcaga
gactcatgtc 1500aaaaacggtc ggtcctaa 1518591518DNACamelina microcarpa
59atgacgtccg ttaacgcaaa gctcctttac cattacgttc taaccaactt tttcaacctt
60tgcttgtttc cgttaacggc gttacttgcc ggaaaagcct ctaagcttac cgcaaacgat
120ctctaccact tctattccca tctccaacac aaccttataa ccgtaatttt
actctttgct 180ttcaccgctt tcggtttggt tctctacatt gtaacccggg
ccaaaccggt ttacctcgtt 240gactactcgt gctaccttcc accaccgcat
ctcaaagtta gtgtttccaa ggtgatggat 300attttctacc aaataagaaa
agctgatacc tcacggaacg tggcatgcga tgatccatcc 360tcgcttgatt
tcctgaggaa gattcaagaa cgttcaggtc taggtgatga aacctacggt
420ccccaaggac tcattaatgt tccaccacaa aagacctttg cagcgtcacg
tgaagagaca 480gagcaggtaa tcatcggtgc gctagaaaag ctattcgaga
acactaaagt taaccctaga 540gagattggta tacttgtggt gaactcaagc
atgtttaatc caactccttc gctatccgcg 600atggtcgtta atactttcaa
gctccgaagc aacatcaaaa gctttagtct cggaggaatg 660ggttgtagtg
ctggtgttat cgccattgat cttgcaaagg acttgttgca tgttcataaa
720aacacttatg cacttgtggt gagcacagag aacatcactc aaggcattta
tgctggcgaa 780aatagatcca tgatggttag caattgcttg ttccgtgttg
gtggggcagc gattttgctc 840tccaacaagc cgggagatgg gagacggtcc
aagtacaagc tatgtcatac tgttcgaaca 900cataccggag ctgatgacaa
gtcttttcga tgtgtgcaac aaggagacga tgagagcggt 960aaaatcggag
tatgtctgtc aaaggacata accgttgttg cggggacagc gcttaagaaa
1020aacatagcaa cgttgggtcc gttgattctt cctttaagcg aaaagtttct
ctttttagtt 1080accttcatcg ccaagaaact tttgaaggac aagatcaagc
actattacgt cccggatttc 1140aagcttgcta ttgaccattt ctgtattcat
gccggaggca gagccgtaat cgatgtgctt 1200gagaagagct taggactatc
tccaatcgat gtggaggcat ctagatcaac gttacataga 1260tttgggaata
cttcgtctag ctcaatttgg tatgaattgg catacataga agcaaaagga
1320aggatgaaga aagggaatag agcttggcag attgctttag ggtcagggtt
taagtgtaac 1380agtgcggttt gggtggctct atgcaatgtc aaggcttcgg
cgaatagtcc ttgggaacat 1440tgcatcgata gatatccggt tcaaattgat
tctgattcat caaaattaga gactcatgtc 1500aaaaacggtc ggtcctaa
1518601155DNACamelina rumelica 60atgggtgcag gtggaagaat gccagttcct
tcttcttctt ccaagaaatc tgaaaccgat 60gccataaagc gtgtgccctg cgagaaaccg
ccgttcacgg ttggagaact gaagaaagca 120atcccaccgc attgtttcaa
acgctctatc cctcgctctt tctcctacct tatcactgac 180atcattgttg
cctcctgctt ctactacgtc gccaccaatt acttctctct cctccctcag
240cctctctctt acttggcttg gcctctctat tgggcctgtc aaggctgtgt
cctaaccggt 300gtctgggtca tagcccacga atgcggtcac cacgcattca
gcgactacca atggcttgat 360gacacagttg gtcttatctt ccattccttc
cttctcgtcc cttacttctc ctggaagtac 420agtcatcgcc gtcaccattc
caacacagga tctctcgaaa gagatgaagt atttgtccca 480aagcagaaat
cagctatcaa gtggtatggc aaatacctca acaaccctcc tggacgcatc
540atgatgttga ccgtccagtt tgtcctcggg tggcccttgt acttggcctt
taacgtctcg 600ggcagaccat acgacgggtt cgcttgccat ttcttcccca
acgctcccat ctacaacgac 660cgtgaacgcc tccagatata tctctccgat
gccggtattc tagcagtctg ttttgggctt 720taccgttacg cagctgcaca
aggaatggcc tcgatgatct gcctctacgg agtaccactt 780ctgatagtga
acgcgttcct cgtcttgatc acttacttgc agcacactca tcctgcgttg
840cctcactacg attcatccga gtgggattgg cttaggggag ctttggctac
cgtagacaga 900gactatggaa tcttgaacaa ggtgttccac aacatcacgg
acacacatgt ggctcatcat 960ctgttctcga caatgccgca ttataatgcg
atggaagcga cgaaggcgat aaagccaata 1020ctcggtgact actaccagtt
cgacggaaca ccgtggtatg tggcgatgta tagggaggca 1080aaggaatgta
tctatgtaga accggacagg gaaggtgaca agaaaggtgt gtactggtac
1140aacaataagt tatga 1155611155DNACamelina rumelica 61atgggtgcag
gtggaagaat gccggttcct tcttcttctt ccaagaaatc tgaaaccgat 60gccatgaagc
gtgtgccctg cgagaaacca ccgttcacgc tgggagaact gaagaaagca
120atcccaccgc agtgtttcaa acgctctatc cctcgctctt tctcctacct
tatcactgac 180atcattgttg cctcctgttt ctactacgtc gccaccaatt
tcttctctct cctccctcag 240cctctctctt acttggcttg gcctctctat
tgggcttgtc aaggctgtgt cctaaccggt 300gtctgggtca tagctcacga
atgcggtcac cacgcattca gtgactacca atggcttgat 360gacacagttg
gtcttatctt ccattccttc cttctcgtcc cttacttctc ctggaagtac
420agtcatcgcc gtcaccattc caacacagga tctctcgaaa gagatgaagt
atttgtccca 480aagcagaagt ccgctatcaa gtggtatggc aaatacctca
acaaccctcc tggacgcatc 540atgatgttaa ccgtccagtt tgtcctcggg
tggcccttgt acttggcctt taacgtctcg 600ggcagaccat acgacgggtt
cgcttgccat ttcttcccca acgctcccat ctacaacgac 660cgcgaacgcc
tccagatata tctctctgat gccggtattc tagcagtctg ttttgggctt
720taccgttacg cagctgcaca aggaatggcc tcgatgatct gcctctacgg
agtaccactt 780ctgatagtga acgcgttcct cgtcttgatc acttacttgc
agcacactca ccctgcgttg 840cctcactacg attcgtccga gtgggattgg
cttaggggag ctttggctac cgtagacaga 900gactacggaa tcttgaacaa
ggtgttccat aacatcacgg acacacatgt ggctcatcat 960ctgttctcga
caatgccaca ttataatgct atggaagcga caaaggcgat aaagccaata
1020ctaggtgact actaccagtt cgacggaaca ccgtggtatg tggccatgta
tagggaggca 1080aaggaatgta tctatgtaga accggacagg gaaggtgaca
agaaaggtgt gtactggtac 1140aacaataagt tatga 1155621518DNACamelina
rumelica 62atgacgtccg ttaacgcaaa gctcctttac cattacgtcc taaccaactt
tttcaacctt 60tgcttgtttc cgttaacggc gttacttgcc ggaaaagcct ctaggcttac
cacaaacgat 120ctctaccact tctattccca tctccaacac aacctcataa
ccgttattct actctttgct 180tttaccgctt ttggtttggt tctctacatt
gtaacccggc ccaaaccggt ttacctcgtt 240gactactcgt gctaccttcc
accaccgcat ctcaaagtta ctgtttccaa ggtcatggat 300attttctacc
aaataagaaa agctgatact tcacggaacg tggcatgcga tgatccatcc
360tcgcttgatt tcctgaggaa gattcaagaa cgttcaggtc taggtgatga
aacctacagt 420cccccgggac tcattaacgt gcccccacaa aagacctttg
cagcttcacg tgaagagaca 480gagcaggtaa tcatcggtgc gctagaaaag
ctattcgaga acaccaaagt taaccctaga 540gagattggta tacttgtggt
gaactcaagc atgtttaatc caactccttc gctatccgct 600atggtcgtta
acactttcaa gctccgaagc aatatcaaaa gctttagtct tggaggaatg
660ggttgtagtg caggtgttat cgccattgat cttgcaaagg acttgttgca
tgttcataaa 720aacacttatg cacttgtggt gagcactgag aacatcactc
aaggcattta tgctggcgaa 780aatagatcca tgatggttag caattgcttg
ttccgtgttg gtggggcagc cattttgctc 840tccaacaagc caggagatcg
gagacggtcc aagtaccagc tatgtcatac tgttcgaaca 900cataccggag
ctgatgacag gtcttttcga tgtgtgcaac aaggagacga tgagagcggt
960aaaatcggag tttgtctgtc aaaggacata accgctgttg cggggacagc
gcttaagaaa 1020aacatagcaa cgttgggtcc attgattctt cctttaagcg
aaaagtttct gtttttagta 1080accttcatcg ccaagaaact tttgaagaac
aagatcaagc actattacgt cccggatttc 1140aagcttgcta ttgaccattt
ctgtattcat gccggaggca gagccgtaat cgatgtgctt 1200gagaagagct
taggactatc gccaatcgat gtggaagcat ctagatcaac gttacataga
1260tttgggaata cttcgtctag ctcaatttgg tatgaattgg catacataga
agcaaaagga 1320aggatgaaga aagggaatag agcttggcag attgctttag
ggtcagggtt taagtgtaac 1380agtgcggttt gggtggctct atgcaatgtc
aaggcttcgg cgaatagtcc ttgggaacat 1440tgcatcgata gatatccggt
tcaacttaat tctgattcat caaaatcaga gactcatgtc 1500aaaaacggtc ggtcctaa
1518631518DNACamelina rumelica 63atgacgtccg ttaacgcaaa gctcctttac
cattacgttc taaccaactt tttcaacctt 60tgcttgtttc cgttaacggc gttacttgcc
ggaaaagcct ctaagcttac cgcaaacgat 120ctctaccact tctattccca
tctccaacac aaccttataa ccgtaatttt actctttgct 180ttcacctctt
tcggtttggt tctctacatt gtaacccggc ccaaaccggt ttacctcgtt
240gattactcgt gctaccttcc accaccgcat ctcaaagtta gtgtttccaa
ggtgatggat 300attttctacc aaataagaaa agctgatacc tcacgaaacg
tggcatgcga taatccatcc 360tcgcttgatt tcctgaggaa gattcaagaa
cgttcaggtc taggtgatga aacctacagt 420ccccaaggac tcattaatgt
tccaccacaa aagacctttg cagcgtcacg tgaagagaca 480gagcaggtaa
tcatcggtgc gctagaaaag ctattcgaga acactaaagt tagccctaga
540gagattggta tacttgtggt gaactcaagc atgtttaatc caactccttc
gctatccgcg 600atggtcgtta atactttcaa gctccgaagc aacatcaaaa
gctttagtct cggaggaatg 660ggttgtagtg ctggtgttat cgccattgat
cttgcaaagg acttgttgca tgttcataaa 720aacacttatg cacttgtggt
gagcactgaa aacatcactc aaggcattta tgctggcgaa 780aatagatcca
tgatggttag caattgcttg ttccgtgttg gtggcgcagc gattttgctc
840tccaacaagc cgggagatcg gagacggtcc aagtacaagc tatgtcatac
tgttcgaaca 900cataccggag ctgacgacaa gtcttttcga tgtgtgcaac
aaggagacga tgagagcggt 960aaaatcggag tatgtctgtc aaaggacata
accgttgttg cggggatagc gcttaagaaa 1020aacatagcaa cgttgggtcc
gctgattctt cctttaagcg aaaaatttct gtttttagta 1080agcttcatcg
ccaagaaact tttgaaggac aagatcaagc actattacgt cccggatttc
1140aagcttgcta ttgatcattt ctgtattcat gccggaggca gagccgtaat
cgatgtgctt 1200gagaagagct taggactatc tccaatcgat gtggaggcat
ctagatcaac gttacataga 1260tttgggaata cttcgtctag ctcaatttgg
tatgaattgg catacacaga agcaaaagga 1320aggatgaaga aagggaatag
agcttggcag attgctttag ggtcagggtt taagtgtaac 1380agtgcggttt
gggtggctct atgcaatgtc aaggcttcgg cgaatagtcc ttgggaacat
1440tgcatcgata gatatccggt taaaattgat tctgattcat caaaatcaga
gactcatgtc 1500aaaaacggtc ggtcctaa 151864383PRTCapsella rubella
64Met Gly Ala Gly Gly Arg Met Pro Val Pro Ser Ser Ser Lys Lys Ser 1
5 10 15 Glu Thr Asn Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro Phe
Thr 20 25 30 Val Gly Asp Leu Lys Lys Ala Ile Pro Pro Gln Cys Phe
Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp
Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Asn Tyr
Phe Ser Leu Leu Pro Gln Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro
Leu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp
Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr
Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe
Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135
140 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys
145 150 155 160 Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn
Asn Pro Phe 165 170 175 Gly Arg Ile Met Met Leu Thr Val Gln Phe Val
Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn Val Ser Gly Arg
Pro Tyr Asp Gly Phe Ala Cys 195 200 205 His Phe Phe Pro Asn Ala Pro
Ile Tyr Asn Asp Arg Glu Arg Leu Gln 210 215 220 Ile Tyr Ile Ser Asp
Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu Tyr 225 230 235 240 Arg Tyr
Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu Tyr Gly 245 250 255
Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr Leu 260
265 270 Gln His Thr His Pro Ala Leu Pro His Tyr Asp Ser Ser Glu Trp
Asp 275 280 285 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr
Gly Ile Leu 290 295 300 Asn Lys Val Phe His Asn Ile Thr Asp Thr His
Val Ala His His Leu 305 310 315 320 Phe Ser Thr Met Pro His Tyr Asn
Ala Met Glu Ala Thr Lys Ala Ile 325 330 335 Lys Pro Ile Leu Gly Asp
Tyr Tyr Gln Phe Asp Gly Thr Pro Trp Tyr 340 345 350 Val Ala Met Tyr
Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro Asp 355 360 365 Arg Glu
Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380
65383PRTArabidopsis lyrata 65 Met Gly Ala Gly Gly Arg Met Pro Val
Pro Thr Ser Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg
Val Pro Cys Glu Lys Pro Pro Phe Ser 20 25 30 Val Gly Asp Leu Lys
Lys Ala Ile Pro Gln His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg
Ser Phe Ser Tyr Leu Ile Gly Asp Ile Ile Ile Ala Ser 50 55 60 Cys
Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln Pro 65 70
75 80 Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys
Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His
His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly
Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp
Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu
Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Gln Lys Ser Ala
Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg
Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro Leu 180 185 190
Tyr Leu Ala Phe Asn Val Ser Gly
Arg Pro Tyr Asp Gly Phe Ala Cys 195 200 205 His Phe Phe Pro Asn Ala
Pro Ile Tyr Asn Asp Arg Glu Arg Leu Gln 210 215 220 Ile Tyr Leu Ser
Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu Tyr 225 230 235 240 Arg
Tyr Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu Tyr Gly 245 250
255 Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr Leu
260 265 270 Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu
Trp Asp 275 280 285 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp
Tyr Gly Ile Leu 290 295 300 Asn Lys Val Phe His Asn Ile Thr Asp Thr
His Val Ala His His Leu 305 310 315 320 Phe Ser Thr Met Pro His Tyr
Asn Ala Met Glu Ala Thr Lys Ala Ile 325 330 335 Lys Pro Ile Leu Gly
Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Trp Tyr 340 345 350 Val Ala Met
Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro Asp 355 360 365 Arg
Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380
66505PRTArabidopsis lyrata 66Met Thr Ser Val Asn Ala Lys Leu Leu
Tyr His Tyr Val Leu Thr Asn 1 5 10 15 Phe Phe Asn Leu Cys Leu Phe
Pro Leu Thr Ala Phe Val Ala Gly Lys 20 25 30 Ala Ser Arg His Thr
Thr Asn Asp Leu His Asn Phe Phe Ser Tyr Leu 35 40 45 Gln His Asn
Leu Ile Thr Val Ala Ile Leu Phe Ala Phe Thr Val Phe 50 55 60 Gly
Phe Val Leu Tyr Met Val Thr Arg Pro Lys Pro Val Tyr Leu Val 65 70
75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro Pro His Leu Arg Ala Ser Val
Ser 85 90 95 Arg Val Met Asp Val Phe Tyr Gln Ile Arg Lys Ala Asp
Thr Ser Arg 100 105 110 Asn Val Ala Cys Asp Asp Pro Ser Ser Leu Asp
Phe Leu Arg Lys Ile 115 120 125 Gln Glu Arg Ser Gly Leu Gly Asp Glu
Thr Tyr Gly Pro Glu Gly Leu 130 135 140 Leu His Val Pro Pro Arg Lys
Thr Phe Ala Ala Ala Arg Glu Glu Thr 145 150 155 160 Glu Gln Val Ile
Ile Gly Ala Leu Glu Asn Leu Phe Gln Asn Thr Lys 165 170 175 Val Asn
Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met Phe 180 185 190
Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys Leu 195
200 205 Arg Ser Asn Ile Lys Ser Phe Asn Leu Gly Gly Met Gly Cys Ser
Ala 210 215 220 Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His
Val His Lys 225 230 235 240 Asn Thr Tyr Ala Leu Val Val Ser Thr Glu
Asn Ile Thr Gln Gly Ile 245 250 255 Tyr Ala Gly Glu Asn Arg Ser Met
Met Val Ser Asn Cys Leu Phe Arg 260 265 270 Val Gly Gly Ala Ala Ile
Leu Leu Ser Asn Lys Pro Arg Asp Arg Arg 275 280 285 Arg Ser Lys Tyr
Lys Leu Ala His Thr Val Arg Thr His Thr Gly Ala 290 295 300 Asp Asp
Lys Ser Phe Arg Cys Val Gln Gln Glu Asp Asp Glu Ser Gly 305 310 315
320 Lys Ile Gly Val Cys Leu Ser Lys Asp Ile Thr Asn Val Ala Gly Thr
325 330 335 Thr Leu Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu
Pro Leu 340 345 350 Ser Glu Lys Phe Leu Phe Phe Val Thr Phe Val Ala
Lys Lys Leu Leu 355 360 365 Lys Asp Arg Ile Lys His Tyr Tyr Val Pro
Asp Phe Lys Leu Ala Ile 370 375 380 Asp His Phe Cys Ile His Ala Gly
Gly Arg Ala Val Ile Asp Glu Leu 385 390 395 400 Glu Lys Ser Leu Gly
Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser 405 410 415 Thr Leu His
Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu 420 425 430 Leu
Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Lys Ala 435 440
445 Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp
450 455 460 Val Ala Leu Arg Asn Val Lys Pro Ser Ala Asn Ser Pro Trp
Gln His 465 470 475 480 Cys Ile Asp Arg Tyr Pro Ala Lys Ile Asp Ser
Asp Leu Ser Lys Ser 485 490 495 Glu Thr His Val Lys Asn Gly Arg Ser
500 505 67384PRTCamelina hispida 67Met Gly Ala Gly Gly Arg Met Pro
Val Pro Ser Ser Ser Ser Lys Lys 1 5 10 15 Ser Glu Thr Asn Ala Ile
Lys Arg Val Pro Cys Glu Lys Pro Pro Phe 20 25 30 Thr Val Gly Glu
Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg 35 40 45 Ser Ile
Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile Ile Ala 50 55 60
Ser Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln 65
70 75 80 Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln
Gly Cys 85 90 95 Val Leu Thr Gly Val Trp Val Ile Ala His Glu Cys
Gly His His Ala 100 105 110 Phe Ser Asp Tyr Gln Trp Leu Asp Asp Thr
Val Gly Leu Ile Phe His 115 120 125 Ser Phe Leu Leu Val Pro Tyr Phe
Ser Trp Lys Tyr Ser His Arg Arg 130 135 140 His His Ser Asn Thr Gly
Ser Leu Glu Arg Asp Glu Val Phe Val Pro 145 150 155 160 Lys Gln Lys
Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro 165 170 175 Pro
Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro 180 185
190 Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala
195 200 205 Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu
Arg Leu 210 215 220 Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val
Cys Phe Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Ala Gln Gly Leu
Ala Ser Met Ile Cys Leu Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val
Asn Ala Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr His
Pro Ala Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu
Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305 310
315 320 Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys
Ala 325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly
Thr Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys
Ile Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp Lys Lys Gly Val
Tyr Trp Tyr Asn Asn Lys Leu 370 375 380 68505PRTCamelina hispida
68Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Val Gly
Lys 20 25 30 Val Ser Arg Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr
Phe His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe
Ala Phe Thr Thr Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg
Pro Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Pro Gly Leu 130 135
140 Ile His Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Asp Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr His Gly Ile 245 250 255
Tyr Ser Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg
Arg 275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Val Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Val Leu 355 360 365 Lys Asp
Lys Ile Lys Asn Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Ser Asp Ser Ser Lys Ser 485 490 495 Glu
Thr His Val Lys Asn Gly Arg Ser 500 505 69505PRTCamelina hispida
69Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly
Lys 20 25 30 Ala Ser Arg Leu Thr Thr Asn Asp Leu Tyr His Phe Tyr
Ser His Leu 35 40 45 Gln His Asn Leu Val Thr Val Ile Leu Leu Phe
Ala Phe Thr Ser Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg
Pro Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Pro Gly Leu 130 135
140 Ile Asn Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Leu
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255
Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Ala Gly Asp Arg
Arg 275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Val Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp
Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Ser Asp Ser Ser Lys Ser 485 490 495 Glu
Thr His Val Lys Asn Gly Arg Ser 500 505 70385PRTCamelina laxa 70Met
Gly Ala Gly Gly Arg Met Pro Val Pro Ser Ser Ser Ser Ser Lys 1 5 10
15 Lys Ser Glu Thr Asp Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro
20 25 30 Phe Thr Leu Gly Glu Leu Lys Lys Ala Ile Pro Pro Gln Cys
Phe Lys 35 40 45 Arg Ser Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr
Asp Ile Ile Val 50 55 60 Ala Ser Cys Phe Tyr Tyr Val Ala Thr Asn
Tyr Phe Ser Leu Leu Pro 65 70 75 80 Gln Pro Leu Ser Tyr Leu Ala Trp
Pro Leu Tyr Trp Ala Cys Gln Gly 85 90 95 Cys Val Leu Thr Gly Val
Trp Val Ile Ala His Glu Cys Gly His His 100 105 110 Ala Phe Ser Asp
Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe 115 120 125 His Ser
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg 130 135 140
Arg His His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val 145
150 155 160 Pro Lys Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu
Asn Asn 165 170 175 Pro Pro Gly Arg Ile Met Met Leu Thr Val Gln Phe
Val Leu Gly Trp 180 185 190 Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly
Arg Pro Tyr Asp Gly Phe 195
200 205 Ala Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu
Arg 210 215 220 Leu Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val
Cys Phe Gly 225 230 235 240 Leu Tyr Arg Tyr Val Ala Ala Gln Gly Met
Ala Ser Met Ile Cys Leu 245 250 255 Tyr Gly Val Pro Leu Leu Ile Val
Asn Ala Phe Leu Val Leu Ile Thr 260 265 270 Tyr Leu Gln His Thr His
Pro Ala Leu Pro His Tyr Asp Ser Ser Glu 275 280 285 Trp Asp Trp Leu
Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly 290 295 300 Ile Leu
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His 305 310 315
320 His Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys
325 330 335 Ala Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly
Thr Pro 340 345 350 Trp Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys
Ile Tyr Val Glu 355 360 365 Pro Asp Arg Glu Gly Asp Lys Lys Gly Val
Tyr Trp Tyr Asn Asn Lys 370 375 380 Leu 385 71505PRTCamelina laxa
71Met Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1
5 10 15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Val Gly
Lys 20 25 30 Val Ser Arg Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr
Phe His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe
Ala Phe Thr Thr Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg
Pro Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro
Pro Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile
Phe Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala
Cys Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln
Glu Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Pro Gly Leu 130 135
140 Ile His Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr
145 150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu
Asn Thr Lys 165 170 175 Val Asn Pro Arg Asp Ile Gly Ile Leu Val Val
Asn Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met
Val Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe
Ser Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile
Asp Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr
Tyr Ala Leu Val Val Ser Thr Glu Asn Ile Thr His Gly Ile 245 250 255
Tyr Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260
265 270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg
Arg 275 280 285 Arg Ala Lys Tyr Lys Leu Cys His Thr Val Arg Thr His
Thr Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly
Asp Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys
Asp Ile Thr Ala Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile
Ala Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe
Leu Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp
Lys Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Val Ala Ile 370 375 380
Asp His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385
390 395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser
Arg Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser
Ile Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met
Lys Lys Gly Asn Lys Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly
Phe Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val
Lys Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp
Arg Tyr Pro Val Gln Ile Asp Phe Asp Ser Ser Lys Ser 485 490 495 Asp
Thr His Val Lys Asn Gly Arg Ser 500 505 72505PRTCamelina laxa 72Met
Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1 5 10
15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly Lys
20 25 30 Ala Ser Arg Leu Thr Thr Asn Asp Leu Tyr His Phe Asn Ser
His Leu 35 40 45 Gln His Asn Ile Val Thr Val Val Leu Leu Phe Ala
Phe Thr Ala Phe 50 55 60 Gly Leu Val Leu Tyr Val Val Thr Arg Pro
Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro
Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile Phe
Tyr Gln Ile Arg Lys Ala Asp Thr Thr Arg 100 105 110 Asn Val Ala Cys
Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln Glu
Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Gln Gly Leu 130 135 140
Ile Asn Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr 145
150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu Asn
Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn
Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val
Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe Ser
Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile Asp
Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr Tyr
Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255 Tyr
Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260 265
270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg Arg
275 280 285 Arg Ala Lys Tyr Lys Leu Cys His Thr Val Arg Thr His Thr
Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly Asp
Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys Asp
Ile Thr Ala Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile Ala
Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe Leu
Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp Lys
Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Val Ala Ile 370 375 380 Asp
His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385 390
395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg
Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile
Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys
Lys Gly Asn Lys Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly Phe
Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val Lys
Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp Arg
Tyr Pro Val Gln Ile Asp Phe Asp Ser Ser Lys Ser 485 490 495 Asp Thr
His Val Lys Asn Gly Arg Ser 500 505 73384PRTCamelina microcarpa
73Met Gly Ala Gly Gly Arg Met Pro Val Pro Ser Ser Ser Ser Lys Lys 1
5 10 15 Ser Glu Thr Asp Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro
Phe 20 25 30 Thr Leu Gly Asp Leu Lys Lys Ala Ile Pro Pro Gln Cys
Phe Lys Arg 35 40 45 Ser Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr
Asp Ile Ile Ile Ala 50 55 60 Ser Cys Phe Tyr Tyr Val Ala Thr Asn
Tyr Phe Ser Leu Leu Pro Gln 65 70 75 80 Pro Leu Ser Tyr Leu Ala Trp
Pro Leu Tyr Trp Ala Cys Gln Gly Cys 85 90 95 Val Leu Thr Gly Val
Trp Val Ile Ala His Glu Cys Gly His His Ala 100 105 110 Phe Ser Asp
Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His 115 120 125 Ser
Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg 130 135
140 His His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro
145 150 155 160 Lys Gln Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu
Asn Asn Pro 165 170 175 Ala Gly Arg Ile Met Met Leu Thr Val Gln Phe
Val Leu Gly Trp Pro 180 185 190 Leu Tyr Leu Ala Phe Asn Val Ser Gly
Arg Pro Tyr Asp Gly Phe Ala 195 200 205 Cys His Phe Phe Pro Asn Ala
Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr Leu Ser
Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu 225 230 235 240 Tyr Arg
Tyr Ala Ala Ala Gln Gly Leu Ala Ser Met Ile Cys Leu Tyr 245 250 255
Gly Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile Thr Tyr 260
265 270 Leu Gln His Thr His Pro Ala Leu Pro His Tyr Asp Ser Ser Glu
Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp
Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr
His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His Tyr
Asn Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly
Asp Tyr Tyr Gln Phe Asp Gly Thr Pro Trp 340 345 350 Tyr Val Ala Met
Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg
Glu Gly Asp Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380
74385PRTCamelina microcarpa 74Met Gly Ala Gly Gly Arg Met Pro Val
Pro Ser Ser Ser Ser Ser Lys 1 5 10 15 Lys Ser Glu Thr Asp Ala Met
Lys Arg Val Pro Cys Glu Lys Pro Pro 20 25 30 Phe Thr Leu Gly Glu
Leu Lys Lys Ala Ile Pro Pro Gln Cys Phe Lys 35 40 45 Arg Ser Ile
Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile Val 50 55 60 Ala
Ser Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro 65 70
75 80 Gln Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln
Gly 85 90 95 Cys Val Leu Thr Gly Val Trp Val Ile Ala His Glu Cys
Gly His His 100 105 110 Ala Phe Ser Asp Tyr Gln Trp Leu Asp Asp Thr
Val Gly Leu Ile Phe 115 120 125 His Ser Phe Leu Leu Val Pro Tyr Phe
Ser Trp Lys Tyr Ser His Arg 130 135 140 Arg His His Ser Asn Thr Gly
Ser Leu Glu Arg Asp Glu Val Phe Val 145 150 155 160 Pro Lys Gln Lys
Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn 165 170 175 Pro Pro
Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp 180 185 190
Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe 195
200 205 Ala Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu
Arg 210 215 220 Leu Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val
Cys Phe Gly 225 230 235 240 Leu Tyr Arg Tyr Ala Ala Ala Gln Gly Met
Ala Ser Met Ile Cys Leu 245 250 255 Tyr Gly Val Pro Leu Leu Ile Val
Asn Ala Phe Leu Val Leu Ile Thr 260 265 270 Tyr Leu Gln His Thr His
Pro Ala Leu Pro His Tyr Asp Ser Ser Glu 275 280 285 Trp Asp Trp Leu
Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly 290 295 300 Ile Leu
Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His 305 310 315
320 His Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys
325 330 335 Ala Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly
Thr Pro 340 345 350 Trp Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys
Ile Tyr Val Glu 355 360 365 Pro Asp Arg Glu Gly Asp Lys Lys Gly Val
Tyr Trp Tyr Asn Asn Lys 370 375 380 Leu 385 75384PRTCamelina
microcarpa 75Met Gly Ala Gly Gly Arg Met Pro Val Pro Ser Ser Ser
Ser Lys Lys 1 5 10 15 Ser Glu Thr Asp Ala Ile Lys Arg Val Pro Cys
Glu Lys Pro Pro Phe 20 25 30 Thr Leu Gly Glu Leu Lys Lys Ala Ile
Pro Pro Gln Cys Phe Lys Arg 35 40 45 Ser Ile Pro Arg Ser Phe Ser
Tyr Leu Ile Thr Asp Ile Ile Val Ala 50 55 60 Ser Cys Phe Tyr Tyr
Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro Gln 65 70 75 80 Pro Leu Ser
Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys 85 90 95 Val
Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala 100 105
110 Phe Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His
115 120 125 Ser Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His
Arg Arg 130 135 140 His His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu
Val Phe Val Pro 145 150 155 160 Lys Gln Lys Ser Ala Ile Lys Trp Tyr
Gly Lys Tyr Leu Asn Asn Pro 165 170 175 Ala Gly Arg Ile Met Met Leu
Thr Val Gln Phe Val Leu Gly Trp Pro 180 185 190 Leu Tyr Leu Ala Phe
Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala 195 200 205 Cys His Phe
Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln
Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu 225 230
235 240 Tyr Arg Tyr Ala Ala Ala Gln Gly Leu Ala Ser Met Ile Cys Leu
Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu
Ile Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ala Leu Pro His Tyr
Asp Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr
Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn
Ile Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr
Met Pro His Tyr Asn Ala Met
Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr
Gln Phe Asp Gly Thr Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu
Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp
Lys Lys Gly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380
76505PRTCamelina microcarpa 76Met Thr Ser Val Asn Ala Lys Leu Leu
Tyr His Tyr Val Leu Thr Asn 1 5 10 15 Phe Phe Asn Leu Cys Leu Phe
Pro Leu Thr Ala Leu Leu Ala Gly Lys 20 25 30 Ala Ser Arg Leu Thr
Ser Asn Asp Leu Tyr His Phe Tyr Ser His Leu 35 40 45 Gln His Asn
Leu Ile Thr Val Ile Leu Leu Phe Ala Phe Thr Ala Phe 50 55 60 Gly
Leu Val Leu Tyr Ile Val Thr Arg Pro Lys Pro Val Tyr Leu Val 65 70
75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro Pro His Leu Lys Val Ser Val
Ser 85 90 95 Lys Ala Met Asp Ile Phe Tyr Gln Ile Arg Lys Ala Asp
Thr Ser Arg 100 105 110 Asn Val Ala Cys Asp Asp Pro Ser Ser Leu Asp
Phe Leu Arg Lys Ile 115 120 125 Gln Glu Arg Ser Gly Leu Gly Asp Glu
Thr Tyr Ser Pro Gln Gly Leu 130 135 140 Ile Asn Val Pro Pro Arg Lys
Thr Phe Ala Ala Ser Arg Glu Glu Thr 145 150 155 160 Glu Gln Val Ile
Ile Gly Ala Leu Asp Lys Leu Phe Glu Asn Thr Lys 165 170 175 Val Asn
Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met Phe 180 185 190
Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys Leu 195
200 205 Arg Ser Asn Ile Lys Ser Phe Ser Leu Gly Gly Met Gly Cys Ser
Ala 210 215 220 Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu His
Val His Lys 225 230 235 240 Asn Thr Tyr Ala Leu Val Val Ser Thr Glu
Asn Ile Thr Gln Gly Ile 245 250 255 Tyr Ala Gly Glu Asn Arg Ser Met
Met Val Ser Asn Cys Leu Phe Arg 260 265 270 Val Gly Gly Ala Ala Ile
Leu Leu Ser Asn Lys Pro Gly Asp Arg Arg 275 280 285 Arg Ser Lys Tyr
Lys Leu Cys His Thr Val Arg Thr His Thr Gly Ala 290 295 300 Asp Asp
Met Ser Phe Arg Cys Val Gln Gln Gly Asp Asp Glu Ser Gly 305 310 315
320 Lys Ile Gly Val Cys Leu Ser Lys Asp Ile Thr Val Val Ala Gly Ile
325 330 335 Ala Leu Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile Leu
Pro Leu 340 345 350 Arg Glu Lys Phe Leu Phe Leu Val Thr Phe Ile Ala
Lys Lys Leu Leu 355 360 365 Lys Asp Lys Ile Lys His Tyr Tyr Val Pro
Asp Phe Lys Leu Ala Ile 370 375 380 Asp His Phe Cys Ile His Ala Gly
Gly Arg Ala Val Ile Asp Val Leu 385 390 395 400 Glu Lys Ser Leu Gly
Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser 405 410 415 Thr Leu His
Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu 420 425 430 Leu
Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Arg Ala 435 440
445 Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val Trp
450 455 460 Val Ala Leu Cys Asn Val Lys Ala Ser Ala Asn Ser Pro Trp
Glu His 465 470 475 480 Cys Ile Asp Arg Tyr Pro Val Gln Ile Asp Ser
Gly Ser Ser Lys Ser 485 490 495 Asp Thr His Val Lys Asn Gly Arg Ser
500 505 77505PRTCamelina microcarpa 77Met Thr Ser Val Asn Ala Lys
Leu Leu Tyr His Tyr Val Leu Thr Asn 1 5 10 15 Phe Phe Asn Leu Cys
Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly Lys 20 25 30 Ala Ser Lys
Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr Ser His Leu 35 40 45 Gln
His Asn Leu Ile Thr Val Ile Leu Leu Phe Ala Phe Thr Ala Phe 50 55
60 Gly Leu Val Leu Tyr Ile Val Thr Arg Pro Lys Pro Val Tyr Leu Val
65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro Pro His Leu Lys Val Ser
Val Ser 85 90 95 Lys Ala Met Asp Ile Phe Tyr Gln Ile Arg Lys Ala
Asp Thr Ser Arg 100 105 110 Asn Val Ala Cys Asp Asp Pro Ser Ser Leu
Asp Phe Leu Arg Lys Ile 115 120 125 Gln Glu Arg Ser Gly Leu Gly Asp
Asp Thr Tyr Ser Pro Gln Gly Leu 130 135 140 Ile Asn Val Pro Pro Gln
Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr 145 150 155 160 Glu Gln Val
Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu Asn Thr Lys 165 170 175 Val
Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met Phe 180 185
190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys Leu
195 200 205 Arg Ser Asn Ile Lys Ser Phe Ser Leu Gly Gly Met Gly Cys
Ser Ala 210 215 220 Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu Leu
His Val His Lys 225 230 235 240 Asn Thr Tyr Ala Leu Val Val Ser Thr
Glu Asn Ile Thr Gln Gly Ile 245 250 255 Tyr Ala Gly Glu Asn Arg Ser
Met Met Val Ser Asn Cys Leu Phe Arg 260 265 270 Val Gly Gly Ala Ala
Ile Leu Leu Ser Asn Lys Leu Gly Asp Arg Arg 275 280 285 Arg Ser Lys
Tyr Lys Leu Cys His Thr Val Arg Thr His Thr Gly Ala 290 295 300 Asp
Asp Lys Ser Phe Arg Cys Val Gln Gln Gly Asp Asp Glu Gly Gly 305 310
315 320 Lys Ile Gly Val Cys Leu Ser Lys Asp Ile Thr Val Val Ala Gly
Thr 325 330 335 Ala Leu Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu Ile
Leu Pro Leu 340 345 350 Ser Glu Lys Phe Leu Phe Leu Val Thr Phe Ile
Ala Lys Lys Leu Leu 355 360 365 Lys Asp Lys Ile Lys His Cys Tyr Val
Pro Asp Phe Lys Leu Ala Ile 370 375 380 Asp His Phe Cys Ile His Ala
Gly Gly Arg Ala Val Ile Asp Val Leu 385 390 395 400 Glu Lys Ser Leu
Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser 405 410 415 Thr Leu
His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu 420 425 430
Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Arg Ala 435
440 445 Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala Val
Trp 450 455 460 Val Ala Leu Cys Asn Val Lys Ala Ser Ala Asn Ser Pro
Trp Glu Asp 465 470 475 480 Cys Ile Asp Arg Tyr Pro Val Gln Ile Asp
Ser Asp Ser Ser Lys Ser 485 490 495 Glu Thr His Val Lys Asn Gly Arg
Ser 500 505 78505PRTCamelina microcarpa 78Met Thr Ser Val Asn Ala
Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1 5 10 15 Phe Phe Asn Leu
Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly Lys 20 25 30 Ala Ser
Lys Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr Ser His Leu 35 40 45
Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe Ala Phe Thr Ala Phe 50
55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg Ala Lys Pro Val Tyr Leu
Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro Pro His Leu Lys Val
Ser Val Ser 85 90 95 Lys Val Met Asp Ile Phe Tyr Gln Ile Arg Lys
Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala Cys Asp Asp Pro Ser Ser
Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln Glu Arg Ser Gly Leu Gly
Asp Glu Thr Tyr Gly Pro Gln Gly Leu 130 135 140 Ile Asn Val Pro Pro
Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr 145 150 155 160 Glu Gln
Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu Asn Thr Lys 165 170 175
Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn Ser Ser Met Phe 180
185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val Val Asn Thr Phe Lys
Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe Ser Leu Gly Gly Met Gly
Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile Asp Leu Ala Lys Asp Leu
Leu His Val His Lys 225 230 235 240 Asn Thr Tyr Ala Leu Val Val Ser
Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255 Tyr Ala Gly Glu Asn Arg
Ser Met Met Val Ser Asn Cys Leu Phe Arg 260 265 270 Val Gly Gly Ala
Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Gly Arg 275 280 285 Arg Ser
Lys Tyr Lys Leu Cys His Thr Val Arg Thr His Thr Gly Ala 290 295 300
Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly Asp Asp Glu Ser Gly 305
310 315 320 Lys Ile Gly Val Cys Leu Ser Lys Asp Ile Thr Val Val Ala
Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile Ala Thr Leu Gly Pro Leu
Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe Leu Phe Leu Val Thr Phe
Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp Lys Ile Lys His Tyr Tyr
Val Pro Asp Phe Lys Leu Ala Ile 370 375 380 Asp His Phe Cys Ile His
Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385 390 395 400 Glu Lys Ser
Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg Ser 405 410 415 Thr
Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile Trp Tyr Glu 420 425
430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys Lys Gly Asn Arg Ala
435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly Phe Lys Cys Asn Ser Ala
Val Trp 450 455 460 Val Ala Leu Cys Asn Val Lys Ala Ser Ala Asn Ser
Pro Trp Glu His 465 470 475 480 Cys Ile Asp Arg Tyr Pro Val Gln Ile
Asp Ser Asp Ser Ser Lys Leu 485 490 495 Glu Thr His Val Lys Asn Gly
Arg Ser 500 505 79384PRTCamelina rumelica 79Met Gly Ala Gly Gly Arg
Met Pro Val Pro Ser Ser Ser Ser Lys Lys 1 5 10 15 Ser Glu Thr Asp
Ala Ile Lys Arg Val Pro Cys Glu Lys Pro Pro Phe 20 25 30 Thr Val
Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg 35 40 45
Ser Ile Pro Arg Ser Phe Ser Tyr Leu Ile Thr Asp Ile Ile Val Ala 50
55 60 Ser Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Leu Pro
Gln 65 70 75 80 Pro Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys
Gln Gly Cys 85 90 95 Val Leu Thr Gly Val Trp Val Ile Ala His Glu
Cys Gly His His Ala 100 105 110 Phe Ser Asp Tyr Gln Trp Leu Asp Asp
Thr Val Gly Leu Ile Phe His 115 120 125 Ser Phe Leu Leu Val Pro Tyr
Phe Ser Trp Lys Tyr Ser His Arg Arg 130 135 140 His His Ser Asn Thr
Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro 145 150 155 160 Lys Gln
Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro 165 170 175
Pro Gly Arg Ile Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro 180
185 190 Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe
Ala 195 200 205 Cys His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg
Glu Arg Leu 210 215 220 Gln Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala
Val Cys Phe Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Ala Gln Gly
Met Ala Ser Met Ile Cys Leu Tyr 245 250 255 Gly Val Pro Leu Leu Ile
Val Asn Ala Phe Leu Val Leu Ile Thr Tyr 260 265 270 Leu Gln His Thr
His Pro Ala Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285 Asp Trp
Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290 295 300
Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His 305
310 315 320 Leu Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr
Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp
Gly Thr Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu
Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp Lys Lys Gly
Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380 80384PRTCamelina
rumelica 80 Met Gly Ala Gly Gly Arg Met Pro Val Pro Ser Ser Ser Ser
Lys Lys 1 5 10 15 Ser Glu Thr Asp Ala Met Lys Arg Val Pro Cys Glu
Lys Pro Pro Phe 20 25 30 Thr Leu Gly Glu Leu Lys Lys Ala Ile Pro
Pro Gln Cys Phe Lys Arg 35 40 45 Ser Ile Pro Arg Ser Phe Ser Tyr
Leu Ile Thr Asp Ile Ile Val Ala 50 55 60 Ser Cys Phe Tyr Tyr Val
Ala Thr Asn Phe Phe Ser Leu Leu Pro Gln 65 70 75 80 Pro Leu Ser Tyr
Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys 85 90 95 Val Leu
Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala 100 105 110
Phe Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His 115
120 125 Ser Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg
Arg 130 135 140 His His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val
Phe Val Pro 145 150 155 160 Lys Gln Lys Ser Ala Ile Lys Trp Tyr Gly
Lys Tyr Leu Asn Asn Pro 165 170 175 Pro Gly Arg Ile Met Met Leu Thr
Val Gln Phe Val Leu Gly Trp Pro 180 185 190 Leu Tyr Leu Ala Phe Asn
Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala 195 200 205 Cys His Phe Phe
Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile
Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu 225 230 235
240 Tyr Arg Tyr Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu Tyr
245 250 255 Gly Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile
Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ala Leu Pro His Tyr Asp
Ser Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val
Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile
Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met
Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala
325 330 335 Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gln Phe Asp Gly Thr
Pro Trp 340 345 350 Tyr Val Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile
Tyr Val Glu Pro 355 360 365 Asp Arg Glu Gly Asp Lys Lys Gly Val Tyr
Trp Tyr Asn Asn Lys Leu 370 375 380 81505PRTCamelina rumelica 81Met
Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1 5 10
15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly Lys
20 25 30 Ala Ser Arg Leu Thr Thr Asn Asp Leu Tyr His Phe Tyr Ser
His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe Ala
Phe Thr Ala Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg Pro
Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro
Pro His Leu Lys Val Thr Val Ser 85 90 95 Lys Val Met Asp Ile Phe
Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala Cys
Asp Asp Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln Glu
Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Pro Gly Leu 130 135 140
Ile Asn Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr 145
150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu Asn
Thr Lys 165 170 175 Val Asn Pro Arg Glu Ile Gly Ile Leu Val Val Asn
Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val
Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe Ser
Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile Asp
Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr Tyr
Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255 Tyr
Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260 265
270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg Arg
275 280 285 Arg Ser Lys Tyr Gln Leu Cys His Thr Val Arg Thr His Thr
Gly Ala 290 295 300 Asp Asp Arg Ser Phe Arg Cys Val Gln Gln Gly Asp
Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys Asp
Ile Thr Ala Val Ala Gly Thr 325 330 335 Ala Leu Lys Lys Asn Ile Ala
Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe Leu
Phe Leu Val Thr Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asn Lys
Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380 Asp
His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385 390
395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg
Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile
Trp Tyr Glu 420 425 430 Leu Ala Tyr Ile Glu Ala Lys Gly Arg Met Lys
Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly Phe
Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val Lys
Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp Arg
Tyr Pro Val Gln Leu Asn Ser Asp Ser Ser Lys Ser 485 490 495 Glu Thr
His Val Lys Asn Gly Arg Ser 500 505 82505PRTCamelina rumelica 82Met
Thr Ser Val Asn Ala Lys Leu Leu Tyr His Tyr Val Leu Thr Asn 1 5 10
15 Phe Phe Asn Leu Cys Leu Phe Pro Leu Thr Ala Leu Leu Ala Gly Lys
20 25 30 Ala Ser Lys Leu Thr Ala Asn Asp Leu Tyr His Phe Tyr Ser
His Leu 35 40 45 Gln His Asn Leu Ile Thr Val Ile Leu Leu Phe Ala
Phe Thr Ser Phe 50 55 60 Gly Leu Val Leu Tyr Ile Val Thr Arg Pro
Lys Pro Val Tyr Leu Val 65 70 75 80 Asp Tyr Ser Cys Tyr Leu Pro Pro
Pro His Leu Lys Val Ser Val Ser 85 90 95 Lys Val Met Asp Ile Phe
Tyr Gln Ile Arg Lys Ala Asp Thr Ser Arg 100 105 110 Asn Val Ala Cys
Asp Asn Pro Ser Ser Leu Asp Phe Leu Arg Lys Ile 115 120 125 Gln Glu
Arg Ser Gly Leu Gly Asp Glu Thr Tyr Ser Pro Gln Gly Leu 130 135 140
Ile Asn Val Pro Pro Gln Lys Thr Phe Ala Ala Ser Arg Glu Glu Thr 145
150 155 160 Glu Gln Val Ile Ile Gly Ala Leu Glu Lys Leu Phe Glu Asn
Thr Lys 165 170 175 Val Ser Pro Arg Glu Ile Gly Ile Leu Val Val Asn
Ser Ser Met Phe 180 185 190 Asn Pro Thr Pro Ser Leu Ser Ala Met Val
Val Asn Thr Phe Lys Leu 195 200 205 Arg Ser Asn Ile Lys Ser Phe Ser
Leu Gly Gly Met Gly Cys Ser Ala 210 215 220 Gly Val Ile Ala Ile Asp
Leu Ala Lys Asp Leu Leu His Val His Lys 225 230 235 240 Asn Thr Tyr
Ala Leu Val Val Ser Thr Glu Asn Ile Thr Gln Gly Ile 245 250 255 Tyr
Ala Gly Glu Asn Arg Ser Met Met Val Ser Asn Cys Leu Phe Arg 260 265
270 Val Gly Gly Ala Ala Ile Leu Leu Ser Asn Lys Pro Gly Asp Arg Arg
275 280 285 Arg Ser Lys Tyr Lys Leu Cys His Thr Val Arg Thr His Thr
Gly Ala 290 295 300 Asp Asp Lys Ser Phe Arg Cys Val Gln Gln Gly Asp
Asp Glu Ser Gly 305 310 315 320 Lys Ile Gly Val Cys Leu Ser Lys Asp
Ile Thr Val Val Ala Gly Ile 325 330 335 Ala Leu Lys Lys Asn Ile Ala
Thr Leu Gly Pro Leu Ile Leu Pro Leu 340 345 350 Ser Glu Lys Phe Leu
Phe Leu Val Ser Phe Ile Ala Lys Lys Leu Leu 355 360 365 Lys Asp Lys
Ile Lys His Tyr Tyr Val Pro Asp Phe Lys Leu Ala Ile 370 375 380 Asp
His Phe Cys Ile His Ala Gly Gly Arg Ala Val Ile Asp Val Leu 385 390
395 400 Glu Lys Ser Leu Gly Leu Ser Pro Ile Asp Val Glu Ala Ser Arg
Ser 405 410 415 Thr Leu His Arg Phe Gly Asn Thr Ser Ser Ser Ser Ile
Trp Tyr Glu 420 425 430 Leu Ala Tyr Thr Glu Ala Lys Gly Arg Met Lys
Lys Gly Asn Arg Ala 435 440 445 Trp Gln Ile Ala Leu Gly Ser Gly Phe
Lys Cys Asn Ser Ala Val Trp 450 455 460 Val Ala Leu Cys Asn Val Lys
Ala Ser Ala Asn Ser Pro Trp Glu His 465 470 475 480 Cys Ile Asp Arg
Tyr Pro Val Lys Ile Asp Ser Asp Ser Ser Lys Ser 485 490 495 Glu Thr
His Val Lys Asn Gly Arg Ser 500 505
* * * * *
References