Note: Descriptions are shown in the official language in which they were submitted.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
FLAX PROMOTERS FOR MANIPULATING GENE EXPRESSION
TECHNICAL FIELD
This invention relates to gene promoters useful
for the genetic manipulation of plants. More
particularly, the invention relates to gene promoters
isolated from flax useful, for example, for
manipulating the expression of indigenous genes or
transgenes in flax and other plants to modify
l0 endogenous characteristics or to introduce new ones.
BACKGROUND ART
Flax (hinum usitatissimum) is the second most
important oilseed crop in Canada and an important crop
worldwide. Unfortunately, the use of flax seed oil is
limited by the narrow range of natural fatty acids
present in it. Therefore, there is a need to create
new cultivars with a wider range of fatty acid
composition to supplement the existing food and
confections markets (Rowland et al., 1995 - please
refer to the "References" section below for full
reference identification details). Also, there is a
commercial interest in using flax as a vehicle for
biofarming of pharmaceutical-related products by
molecular genetic manipulation of appropriate
transgenes (Moloney and van Rooijen, 1996). A need for
flax varieties tolerant to various abiotic and biotic
stresses has also been recognized (Rowland et al.,
1995). For example, herbicide-tolerant flax varieties
would be very useful in crop rotation programs. There
is always, of course, a need for promoters useful for
expressing foreign genes in various other plants.
Molecular genetic manipulation of flax seed
composition or other characteristics, such as stress
tolerance, can be achieved by expressing appropriate
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
2
transgenes using seed-specific or constitutive gene
promoters. While a cDNA sequence corresponding to a
flax gene has been reported (Singh et al., 1994), no
promoter has yet been characterized from flax. There
is, therefore, a need to identify and isolate one or
more genes and promoters from flax to facilitate
genetic manipulation of the flax plant and other
plants.
DISCLOSURE OF INVENTION
An object of the invention is to identify and
isolate one or more genes and promoter sequences from
flax and to utilize such sequences in the genetic
manipulation of plants.
Another object of the invention is to provide a
vector containing a promoter sequence from flax for
introducing an indigenous gene or a transgene into flax
or other plants.
Another object of the invention is to provide a
method of modifiying flax and other plants to change
characteristics thereof.
Stated in general terms, the present invention is
based on the isolation, purification and
characterization by the inventors of the present
invention of two genes from flax and two promoters from
those genes. The sequences obtained are used for
regulating the expression of a heterologous gene
(foreign, reporter or transgene) in flax and other
plant species. This can result in flax plants having
different range of fatty acids than natural flax and
can result in the development of transgenic plants
suitable for the production of specific products or
having new and useful characteristics. Such plants and
products are of commercial and industrial interest.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
3
According to one aspect of the present invention,
there is provided isolated and purified
deoxyribonucleic acid of SEQ ID N0:1 or SEQ ID N0:2.
These sequences relate to the novel flax genes isolated
and characterized by the inventors of the present
invention.
These identified and isolated genes are useful in
themselves for making antisense or sense constructs
based on the derived sequences. Both types of contruct
l0 can be used to reduce the levels of similar mRNA during
expression of the natural genes. This would result in
an increase in 18:0 fatty acid in membrane or storage
lipids in flax and other plant species. Sense
constructs may also be used in enhancing the levels of
mRNA. Such enhancement will result in the increase of
16:1 or 18:1 fatty acids in membranes or storage lipids
in.flax and other plant species. Such plants will be
of increased commercial interest and value.
Thus, according to another aspect of the
invention, there is provided a method of changing fatty
acids of membrane and storage lipids of plants,
characterized by making an antisense or sense construct
based on SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3 or SEQ
ID N0:4, ligating the constuct into a plant
transformation vector, using the vector to transform
the genome of a plant or plant seed, and then growing
the plant or plant seed and extracting membrane or
storage lipids from the plants.
. According to another aspect of the invention,
there is provided isolated and purified
deoxyribonucleic acid of SEQ ID N0:3 or SEQ ID N0:4
(deposited as plasmids ATCC 98193 and 98192,
respectively, see details below). These are the
promoters that are useful for enhancing or enabling the
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
4
expression of genes introduced into flax or other
plants.
According to another aspect of the invention,
there is provided a gene expression cassette comprising
a sequence according to SEQ ID N0:1, SEQ ID N0:2, SEQ
ID N0:3 or SEQ ID N0:4. The gene expression cassette
is useful in itself as this part of the plasmids
mentioned above can be used to construct other plasmid
suitable to transform other plant species.
l0 According to yet another aspect of the invention,
there is provided a vector for introduction of a gene
into a plant cell, the vector comprising a promotor of
SEQ ID N0:3 or SEQ ID N0:4.
The invention also relates to transgenic plants
and plant seeds having a genome containing an
introduced promoter sequence of SEQ ID N0:3 or SEQ ID
N0:4 regulating the expression of an introduced gene,
and a method of producing such plants and plant seeds.
The invention also relates to substantially
homologous DNA sequences (e.g. greater than or equal to
40o homology, more preferably greater than or equal to
70o homology) isolated and/or characterized by known
methods using the sequence information of SEQ ID NO:1,
SEQ ID N0:2, SEQ ID N0:3 or SEQ ID N0:4, and to parts
of reduced length of promoter sequences SEQ ID N0:3 or
SEQ ID N0:4 that are still able to function as
promoters of gene expression. It will be appreciated
by persons skilled in the art that small changes in the
identities of nucleotides in a specific promoter
sequence may result in reduced or enhanced
effectiveness of the promoters and that partial
promoter sequences often work as effectively as the
full length versions. The ways in which promoter
sequences can be varied or shortened are well known to
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
persons skilled in the art, as are ways of testing the
effectiveness of promoters. All such variations of the
promoters are therefore claimed as part of the present
invention.
5 It should be noted that.the term "promoter" in
this disclosure includes the core promoter elements
(TATA box and initiator) and upstream regulatory
elements (enhancers)(Datla et al., 1997).
As will be appreciated from the description above,
the promoters of the invention are beneficial in
manipulating the expression of genes in flax and other
crops.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows genomic DNA sequence of the SAD1 [SEQ
ID N0:1; identified in Fig. 1 as LUSAD1.SEQ] and SAD2
[SEQ ID N0:2; identified in Fig. 1 as LUSAD2.SEQ] genes
and the corresponding SAD cDNA sequence [SEQ ID N0:5;
identified in Fig. 1 as LUCDNA]. Nucleotides (nt) are
represented by capital letters. Nucleotides different
from the cDNA sequence are shaded, including those of
introns. Differences between SAD1 and SAD2 are shown
in shaded lower case letters. Gaps in the sequences are
presented by dashes. The start and stop codons on the
cDNA sequence are boxed.
Fig. 2A is a partial restriction map of the SADl
gene, and Fig. 2B shows the result of a DNA blot
analysis identifying the regulatory sequences of SAD1
and SAD2.
Fig. 3 shows an outline of the scheme employed to
isolate the promoter regions of the two SAD genes.
Position and direction of the primers used in IPCR are
indicated by arrowheads. Various abbreviations are as
follows: E, exon; I, Intron; RE, 5'- regulatory
elements (promoters); and UT, untranslated regions.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
6
Fig. 4 discloses nucleotide sequences [SEQ ID N0:3
(SAD1) and SEQ ID N0:4 (SAD2)] of the 5'- regulatory
regions of the two SAD genes. Homologous nt are
represented by a dash (-), gaps by a dot (.), and
additions by lower case letters. A putative
transcriptional site is indicated by +l, and a TATA box
is overlined. Key restriction sites are also shown.
Fig. 5 shows salient features of the plasmids
CDC214 and pCDC220. Various abbreviations are as
follows: flax promoter I, SAD1 gene promoter; flax
promoter II, SAD2 gene promoter; GUS (uidA), gene for
(3-glucuronidase enzyme; nos-T, transcriptional
terminator of the nopaline synthase gene; nptll,
neomycin phospho-transferase expression cassette. The
arrowheads indicate the direction of transcription.
Key restriction sites are shown. Regions outside the
left and right border (LB and RB) are that of a
previously described binary plant transformation
vector, pRD410 (Datla et al., 1992).
Fig. 6 shows the expression of a heterologous gene
(uidA) by the two SAD gene promoters in various tissues
of flax. Different tissues are abbreviated as YL+A,
young leaves and apices; ML, mature leaves: S, stems;
R, roots; B, buds; 1/2 OF, half open flower; F1,
Flower; and MS, seeds at about mid-development. Data
presented are from one generation of two plants
transformed with a tandem 35s promoter (2x35s), two
generations of two plants transformed with pCDC214
(SAD1), and one generation of two plants transformed
with pCDC220(SAD2).
Fig. 7 shows the expression of a heterologous
gene(uidA) by the two SAD gene promoters during flax
seed development and in relation to,fatty acid and
protein biosyntheses. For GUS assays, data represent
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
7
one generation of two plants transformed with a tandem
35s promoter (2x35s), two generations of two plants
transformed with pCDC214(SAD1), and two generations of
a plant transformed with pCDC220 (SAD2). For fatty
acids, three individual embryos of var. McGregor were
analyzed. For protein content, data are from two
transgenic plants transformed with pCDC214 and 220.
Fig. 8 shows the expression of a heterologous
gene(uidA) by the two SAD gene promoters in tobacco
leaves and mid-developmental seeds. Data represent 5
to 8 transgenic plants transformed with pCDC214 (SADl),
pCDC220 (SAD2), pRD410 (35s), and pRD420 (uidA alone).
Fig. 9 shows the expression of a heterologous
gene(uidA) by the two SAD gene promoters during tobacco
seed development. Various developmental stages of
tobacco seeds were identified according to de Silva et
al. (1992) and are abbreviated as W, white; LB, light
brown; B, brown; DB, dark brown; and M, mature. Data
represent 5 to 8 transgenic plants transformed with
pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and
pRD420 (uidA alone).
Fig. 10 shows the expression of a heterologous
gene(uidA) by the two SAD gene promoters in canola
leaves and mature seeds. Data represent 2 to 5 plants
transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410
(35s), and untransformed plants (UT).
BEST MODES FOR CARRYING OUT THE INVENTION
In flax, endogenous SAD activity can be detected
from about 10 days after pollination (dap) to seed
maturity, suggesting a promoter of this gene would be
useful in manipulating gene expression during seed
development. Moreover, SAD has been found to be the
key enzyme in manipulating the levels of saturated
fatty acids in rapeseed and soybean triacylglycerols
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
8
(Knutzon et al., 1992; see Topfer et al., 1995).
During studies carried out by the inventors aimed at
diversifying flax as a crop, it was discovered that
there are two SAD genes in flax. The isolation,
purification and characterization of these genes and
their promoters is disclosed below, as well as the
expression capabilities of the promoters in flax and
other plant species.
The promoters developed according to the present
invention can be used to modify an endogenous
characteristic of flax or another plant species, or to
to add a new characteristic. An example of a
modification of an endogenous characteristic of flax
is, for example, the alteration of levels of different
types of fatty acids in the seed oils. The
introduction of a new characteristic is, for example,
the production of a thermoplastic polymer in plants
that normally do not produce thermoplastics. While it
is normally easy to detect added characteristics, it is
sometimes difficult to detect altered characteristics
because of natural variation of characteristics in
plants. The alterations can, however, be detected by
comparing the average characteristics of a
statistically significant number of the plants under
examination with a statistically significant number of
genomically-unmodified plants of the same genotype,
grown under identical environmental conditions at the
same time. If there is an appreciable difference in
the measured characteristic, then it can be said that
there has been an alteration of that characteristic and
that the alteration is a result of the genomic-
modification.
In the case of an added characteristic, again the
comparison can be made with genomically-unmodified
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
9
plants of the same genotype, again grown under
identical environmental conditions at the same time.
The promoters of the present invention belong to a
two-member gene family encoding the enzyme D9
desaturase (Stearoyl-acyl carrier protein desaturase;
SAD; EC 1.14.99.6). Stearoyl-acyl carrier protein
desaturase is the first enzyme in the fatty acid
desaturation pathway, and it catalyzes the conversion
of stearoyl-ACP(18:0-ACP) to oleoyl-ACP(18:1~9-ACP).
The promoters were isolated using the inverse
polymerase chain reaction (IPCR) technique. They are
capable of expressing a foreign gene, e.g. uidA (which
encodes(3-glucuronidase: GUS), in various tissues with
high level of expression in seeds.
In developing seeds, both promoters showed a
similar temporal expression pattern for uidA (measured
as GUS activity). The GUS activity could be detected
as early as 4 dap in developing seeds and in desiccated
seeds (~50 dap) of transgenic flax. In developing
seeds, the ability of the promoters to effect uidA gene
expression correlated well with both fatty acid and
protein biosyntheses and the maximum activity of GUS
preceded the maximal accumulation of fatty acids and
proteins.
The promoters of the invention are useful in
manipulating transgene expression in a variety of
tissues including seeds. Some of the products which
are possible using these promoters include, but are not
limited to, the following: plants with enhanced
herbicide, pest, pathogen, and stress resistance;
plants containing oil, protein, and carbohydrate of
altered composition and content; plants with reduced
anti-nutritional substances: plants producing
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
pharmaceutical compounds such as antibodies,
neuropeptides, recombinant proteins, and biodegradable
thermoplastics (Bennett, 1993; Moloney and van
Rooijen, 1996; Datla et al., 1997).
5 The effectiveness of the promoters of the present
invention is predictable from the effectiveness of
known promoters. For example, it is well established
that promoters such as cauliflower mosaic virus (CaMV)
are capable of expressing a wide variety of genes in a
10 wide varity of plant species. Napin promoter (from
rapeseed) has been used to express a variety of genes
in canola/rapeseed (Knutzon et al., 1992; Jones et al.,
1995; Dahesh et al., 1996). Phaseolin gene promoter
(from bean) has also been used to express several genes
in rapeseed (Ritz et al., 1995). The (3-conglycinin
promoter (from soyabean) has been used to express genes
not only in soyabean but also in Petunia (Kinney, 1997;
Chen et al., 1986).
Moreover, by testing the promoters in two very
diverse plant species, as will become apparent from the
experimental detail below, the inventors have
demonstrated that the promoters would function in other
diverse plant species as well.
Further demonstration of this principle can be
obtained from Chen ZL, Schuler MA, Beachy RN. 1986;
Dehesh K, Jones A, Knutzon DS, Voelker TA. 1996; Hitz
WD, Mauvis CJ, Ripp KG, Reiter RJ, DeBonte L, Chen, Z.
1995; Jones A, Davies HM, Voelker TA. 1995; Kinney, AJ.
1997; and Knutzon et al., 1992.
It is believed that the present invention can now
best be described by presenting experimental details
forming a specific illustration. It should be kept in
mind, however, that the present invention is not
limited to these details.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
EXPERIMENTAL DETAILS
Molecular Biological Techniques
Isolation of plasmid DNA, restriction digestion,
modification and ligation of DNA, PCR, gel
electrophoresis, and transformation and culture of E.
coli strains were carried out according to standard
procedures (Sambrook et al., 1989). Nucleotide
sequencing was performed using double stranded plasmid
DNA by the dideoxy chain termination method (Sanger et
al . , 1977 ) using a Taq DYEDEOXYTM terminator cycle
sequencing kit (available from Applied Biosystems) and
an Applied Biosystems Model 370A Sequencer (available
from Applied Biosystems). The oligodeoxy-
ribonucleotides used in nucleotide sequencing, and PCR
techniques were synthesised using a phosphoramidate
synthesis procedure in a Biosearch 8750 DNA synthesizer
(New Brunswick Scientific Co.), and purified by HPLC-
based protocols (Gait, 1984). IPCR was done according
to Ochman et al. (1993) and Warner et al. (1993).
Plant DNA was extracted using the protocol of
Dellaporta et al. (Dellaporta et al., 1983) except that
RNA was removed by adding 100~,g of RNAase B (Sigma)
followed by incubation at 65°C for 20 min. The DNA was
extracted once with an equal volume of
phenol: chloroform (1:1, v/v) and once with an equal
volume of chloroform:isoamyl alcohol (24:1, v/v). Five
~g of DNA was digested with the appropriate restriction
enzyme, fractionated on a 0.8% agarose gel, and
pressure-blotted onto Hybond-NTM nylon membranes
(Amersham) using the PosiBlotTM apparatus (Stratagene)
after depurination, denaturation and neutralization of
the DNA (Sambrook et al., 1989). The blotting solution
contained 0.02 M NaOH and 1 M NH9-acetate. The DNA was
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
12
immobilized on the membrane by baking the membranes at
80°C for 1 h.
A radioactive probe for identifying promoters was
prepared by annealing 10 ng of oligo-29A and 30A (Table
1 below) and then filling in the ends using the Klenow
fragment of DNA polymerase and random primer kit
solutions (GIBCO BRL).
Table 1
l0 Nucleotide sequence of various
oligonucleotides (OL) used
OL-24 (-) 5'-GAAI3mATGCCATCAT-
ACTCCAATCAT-3' [SEQ ID N0:6]
OL-25 (+) 5'-GAAIZOCCTTCAACAAC-
AATGGCTCTC-3' [SEQ ID N0:7]
OL-29A (+) 5'-izoCCTTCAACAACAATGGCTCTCAAGC-
TCAACCCAGTCACCACCTT-3' [SEQ ID N0:8]
OL-30A (-) 5'-is9GGAGAAGTTGTTGAGGGAGCGTGTT-
GAAGGGAAGGTGGTGACTGGGTTGA-3' [SEQ ID N0:9]
OL-39 (-) 5'-zs3TTGGTGGAGGTGGAACTGAA-3' [SEQ ID N0:10]
OL-110 (+) 5'-zs3AGCTAAAGAAGTCACATGGAC-3' [SEQ ID NO:11]
NOTE: The number in subscript corresponds to the nucleotide
residue in the SAD cDNA sequence (Singh et al., 1994). + and -
indicate coding and non-coding strand.
The sequence of oligo-29A corresponded to nt 120-
163 of SAD cDNA (reported by Singh et al., 1994). The
sequence of oligo-30A corresponded to nt 145-194. In
this way, radioactive probe fragments spanning 75 bps
in the 5' end region of SAD cDNA were obtained.
Prehybridization was done at 65°C for 3 h in 5x
SSPE, 5x Denhardts solution, 0.5o SDS, and 500~g of
CA 02268745 1999-04-15
l3
Salmon sperm DNA (amersham) . Hybridizat,i on s~ras done at
55C for h. The membrane was washed at room
18
temperaturein 2x SSPE and 0.1~ SDS for 15 a~d 5
min
and then 50 C in lx SSPE and 0.1% for 10 min.
at SDS
At this point the membrane was free of backgrou~:d
signal. Autoradiograms were obtained by expcsing t:e
membranes for variable lengths of tire to Kodak X-
OMATT'"' AR films wi th intensifying screens at -70°C.
Reporter Gene Constructs
l0 A 1.747 kb DNA fragment containing only t::e
regulatory region and a part of the untranslated region
of the SAD1 gene ~,~as amplified by PCR and cloned into
the pCRII vector (Invitrogen Corp). The same f=agr.:en~
was retrieved as an EccRI fragment from the pCR=I
i5 vector and subseauently cloned into pBluescrioW"' =_
(Stratagene) to gain some cloning sites. The __'_a-%a:'
5' - regulatory regi on, approximately 1 . 257 '.{a, ..~ _:~.e
SAD2 gene was PCR-amplified but using the piv ~'~a
polymerase (Stratagene) , and cloned into an cc?.'~ ==to
20 of the pBluescrip~. II SK vector.
The SAD1 and SAD2 gene 5' regulatory eleme.~.ts r~==a
cloned into pRD420 as a SalI-Sc~aI fragment i:: -= J.. _
the uidA. The plasmid pRD420 was obtained f=o~
R.S.S. Datla, NRC Plant Biotechnology Instit~.:te, _=
25 Gymnasium Place, Saskatoon, Saskatchewan, Carat , ~,'i
OW9 (Dada et al., 1992). The resulting const=v~~~
were labeled as pCDC214 and pCDC220. These con~_=v=~s
were deposited on October 3, 1996 (tested for -%'_~t___ty
on October 9, 1996, deposit receipt dated October -
30 1996) under the terms of the Budapest Treaty at _.._
American Type Culture Collection, 10801 Univers_=~:
Blvd., Manassas VA 20110-2209, USA, under depos:= ~-:s.
ATCC 98193 and 98192, respectively. The plasmi~s
CA 02268745 1999-04-15
~a
CDC214 and 220 were transferred directly to
Agrobacterium strain GV3101 containing helper plasmid
pMP90 (Koncz and Sc'nel 1 , 196) using a freeze-taw
method of transformation (An et al., 1983).
Plant Transformation
Flax seeds were surface sterilized by stirring in
70 o ethanol for ? minutes, followed by three i0 :,li.~.uta
washes in 0.5=~ sodium hypochlorite (freshly uilute~
from the commercial product), and 5 rinses i:~ steri'_e
l0 distilled wa~ar. Seeds were germinated on basal medium
consisting of Murashige and Skoog (MS) major and minor
salts and Gamborg vitamins (Sigma 0404), 3° sucrose and
0.8~ agar. '_'he per: of the medium was adjusted to 5.8
before autoclaving. About 10 surface-sterilizes seeds
l5 were placed in each 100x15 mm plate. The p1_tes were
sealed with paraf_lm and placed in the dark at 22°C =or
5 to 7 days.
Derivatives of Agrobacteria tumefaciens stra'_~.
GV3101/pMP90 carrning pCDC214 and pCDC220 were :rown c::
20 solidified 2x YT :~.edi um (Sambrook et a1. 1 989)
supplemented vita. 50 ug/m1 kanamycin and 50 ug/~!'
gentamycin sulfate. Single colonies trom ~ to .. aay-
old culture plates were used to inoculate 10 m_ '__~wid
2x YT medium containing antibiotics as above an2~~ uM
?5 acetosyringone. Cul Lures were grown at 28°C wi=_~
rotary agitation nor about 24 hours. Prior to
inoculation of flax tissues, the cell concentra=_o:, of
the suspension was adjusted to 1x109 cells/ml.
The following methods for obtaining transfor:~ed .lax
30 callus were modified from Mlynarova et al. (19:s?.
Hypocotyls of 5-7 day aseptic flax seedlings were cut
into segments 3-4 mm long. To avoid dehydratic::, _'.'.~.e
segments were maintained in a small volume of l:~u_d
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
basal medium until all the hypocotyls were cut. The
hypocotyl segments were immersed in bacterial
suspension (1x109 cells/ml) for 30 minutes with
occasional swirling. The suspension was removed by
5 aspiration and the hypocotyl segments were transferred
to sterile filter paper to remove excess liquid. The
segments were placed on agar-solidified (0.80) basal
medium supplemented with 4.44~,M 6-benzylaminopurine
and 0.54~.M naphthaleneacetic acid (MSD4x2 medium;
l0 Basiran et al., 1987). Maltose (30) replaced sucrose
as the carbohydrate source. About 25 explants were
placed in each 100x15 mm Petri dish and maintained at
22-24°C, with a 16 h photoperiod and photon density of
approximately 50 ~,mol/m2/s. After 2 days the segments
15 were transferred to the same medium supplemented with
100~g/ml kanamycin for selection of transformed cells
and 200~,g/ml cefotaxime to eliminate Agrobacteria.
The explants were maintained under the same growth
conditions for 3 weeks. As a control, non-inoculated
segments were treated in the same way.
Green callus formed at the cut ends of most of the
inoculated hypocotyl segments, whereas little or no
callus appeared on non-inoculated segments and they
were completely bleached after 3 weeks on the selection
medium. Callus was excised and transferred to basal
medium (3o maltose) supplemented with S~,M zeatin and
antibiotics as above. Shoots regenerated from some of
the calli within 3-4 weeks.
When the shoots had elongated to 0.5 to 1.0 cm, they
were removed from the callus and placed in capped glass
tubes (100x25 mm) containing 8 ml rooting medium: 1/2
strength MS salts, 3o sucrose, 0.1 ~,M IAA, 0.8o agar,
pH 5.8, and 30~,g/ml kanamycin for selection of
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
16
transformed shoots. The shoots were maintained under
low light (<25~.mo1/m2/s) for 5-8 days by which time
some of the shoots had roots about 2-3 mm long. The
plantlets were transferred to pots in the growth
chamber within 10-14 days, when roots had elongated to
about 2 cm and the shoots were 3-5 cm tall. Transgenic
plants were grown under 18 h of light (300-500
~.mol/m2/s) and day/night temperature of 20/17°C. The
plants were fertilized just before flowering with a
solution containing 27 g of 15N:30P:15K supplemented
with 0.9 g CuS04 in 9 liters of water.
Transformation of canola and tobacco were performed
according to Moloney et al. (1989) and Horsch et al.
(1985), respectively.
Tissue Samplin
Various tissues and developing seeds at different
stage of development were harvested and immediately
frozen in liquid N2 and stored at -80°C until analyzed.
In progeny generations, these tissues were combined
from a total of 8 plants .
Fluorimetric GUS Enzyme Assay
Fluorimetric GUS assay was done essentially
according to Jefferson (1987). The assays were done in
a micro well titer plate and fluorescence of the
reactions was measured by CytoFluorTM II multi-well
fluorescence plate reader (PerSeptive Biosystems}.
Determination of Fatty Acid and Protein Content in
Seeds
The fatty acid content of seeds of different ages
was determined by fatty acid methyl ester analysis of
seed homogenates as described previously (Taylor et
al., 1992).
The same protein extracts which were used for GUS
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
17
assays were used far protein estimation. Protein
concentration was determined using a modified Bradford
assay method (Bio-Rad protein assay) and BSA as the
standard.
RESULTS
Isolation and characterization of the two SAD genes
The inventors of the present invention have found
that three lines of evidence prove there are two SAD
genes in flax, namely: the amplification of two
different sized DNA fragments by PCR, the results of
restriction analysis of cloned PCR products, and the
results of DNA blot analysis of flax genomic DNA.
The genomic sequences of the two SAD genes were
amplified by PCR. Several oligonucleotide primers were
synthesized based on the nucleotide sequence of the
published SAD cDNA sequence (Singh et al., 1994).
These primers were used in all possible combinations
with flax genomic DNA as the template to amplify
different segments of SAD genes. The molecular size of
the PCR products was determined by agarose gel
electrophoresis: in most reactions two products of very
similar molecular size were detected, suggesting the
possibility of two SAD genes in flax. Amplification
with oligo-25 and 24 (Table 1) yielded a fragment of
about 2.6 kb. This fragment contained the whole SAD
gene as determined by sequence data.
The amplified SAD gene fragments were cloned into
pCRII vector (Invitrogen Corp.). The identity of the
amplified gene products was confirmed by comparison of
their nucleotide sequences with the SAD cDNA sequence
(Singh et al., 1994). Sequence analyses indicated that
the SAD1 and SAD2 genes have 97.2% similarity with each
other in the coding region and 96.2% and 93.7% with the
published flax cDNA sequence, respectively (Fig. 1).
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
18
It is clear that the mRNA for SAD cDNA, reported by
Singh et al. (1994), was transcribed from the SAD1
gene. Some general features of the flax SAD genes have
been deduced from sequence analysis. As expected on
the basis of the cDNA sequence, the coding region of
the gene is 1192 bps. This consists of three exons
interrupted by two introns of approximately 0.6 to 0.7
kb. Exon 1 consists of 123 bp, whereas exons 2 and 3
are 507 by and 561 by long, respectively.
Verification for the presence of two SAD genes in
flax comes from the analyses of two independent clones,
each containing the full length gene. Although the
nucleotide sequences of the coding regions are almost
identical, there are several base changes. One of
these has altered a restriction enzyme site, NcoI,
resulting in the observation that the two clones have
different restriction digestion patterns. The two
clones also differ significantly in their intron
sequences (Fig. 1). The different intron sequences are
presumably responsible for the slight difference in the
molecular size of the two PCR products generated by the
same primer combination.
Identification of SAD Gene Promoter Sequences in Flax
Genome
Genomic DNA was extracted from 7-10 days old
seedlings of flax var. McGregor (obtained from Dr. G.
Rowland, Crop Development Centre, 51 Campus Dr.,
Saskatoon, Saskatchewan S7N 5A8), digested with
restriction enzyme, BamHI, BclI, BglII, NdeI or SstI,
gel-fractionated and blotted onto nylon membrane for
probing. These restriction enzymes would cut within
the flax SAD genomic sequence as indicated in Figure 2A
and elsewhere in the flax genome. When the DNA blot
was hybridized with the probe, DNA fragments containing
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
19
the 5'- upstream region and a part of the 5'-
untranslated and coding region of the SAD gene were
expected to hybridize (Figure 2A).
The result of one such experiment is shown in Figure
2B. In each lane, two different size fragments
hybridized with the probe indicating the existence of
two SAD genes in flax. Singh et al. (1994) have shown
only one SAD gene in flax. Since both the genes might
be active, the inventors decided to isolate the 5'
regulatory DNA sequences of both SAD genes.
Isolation and Characterization of Promoter Elements
5'- regulatory DNA sequences of the two SAD genes
were amplified using the IPCR technique.
DNA blot analysis of the flax genome indicated that
the two fragments obtained from the digestion of flax
DNA with the restriction enzyme SstI would contain
about 1.7 and 1.2 kb of 5' flanking regions of the SADl
and SAD2 gene, respectively (Figs. 2B, 3 and 4). These
fragments are expected to contain sufficient 5'-
regulatory elements required for gene expression. SstI
was used to cut the flax genomic DNA, and the
circularized DNA template required for IPCR was
prepared. An outline of the promoter isolation scheme
is shown in Fig. 3, and is believed to be self-
explanatory.
Flax genomic DNA was digested with the restriction
enzyme SstI and gel fractionated. DNA fragments were
isolated from a region of the agarose gel where the two
promoter fragments that hybridized with the SAD probe
were expected (Fig. 2B and 3). These DNA fragments
were ligated at a concentration favoring the
circularization of single DNA molecules (Ochman et al.,
1993; Warner et al., 1993). The circularized DNA was
then used as a template in the IPCR with two primers
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/008I2
(oligo-39 and oligo 110; Table 1). The orientation of
the each member of the primer set used in the IPCR is
opposite to that normally used in a regular PCR (Fig.
3). Two distinct fragments of the expected sizes, 2.2
5 kb and 1.7 kb, were amplified using IPCR. The
untranslated region and parts of the exon 1 and exon 2
constituted the additional approximately 0.5 kb
(Fig.3). The two fragments could also be digested with
SstI indicating the authenticity of the PCR product.
10 The two DNA fragments were cloned in the pCRII
vector (Invitrogen Corp.) and sequenced. The DNA
sequence of the 5'- regulatory regions of the two SAD
genes was compiled and compared (Fig. 4). The two SAD
promoters are quite homologous. A large deletion of
15 368 by in the SAD2 gene promoter (corresponding to nt
759 to 391 in the SAD1 promoter) is very conspicuous.
There are a few short deletions, some substitutions and
minor gaps in both the promoters. Based on the
sequence data, 3'- regions of these DNA fragments were
20 matched with the 5'- coding regions of the two SAD
genes, and thereby assigned the promoters to their
respective SAD genes.
Expression of the~3-qlucuronidase Gene by Flax
Promoters in Transgenic Plants
The ability of a promoter to regulate expression of
a gene spatially and temporally can be demonstrated by
using it to express a heterologus gene. To achieve
this here, first, reporter gene constructs were made by
fusing the promoter of the SAD1 or SAD2 gene with the
uidA gene (Fig. 5). These expression constructs were
then used to transform flax, canola and tobacco, and
independent transgenic plants of these species were
obtained.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
21
Different tissues were sampled and assayed for GUS
activity to determine spatial or tissue-specific
expression. Developing seeds were also collected at
various stages of development to analyse the temporal
expression pattern of the two promoters during seed
development.
These promoters were capable of expressing the uidA
gene in various tissues, with high level of expression
in seeds (Fig. 6). In developing seeds, both the
l0 promoters showed similar temporal expression patterns
for GUS (Fig. 7). The GUS activity could be detected
as early as 4 dap in developing seeds and in desiccated
seeds (approximately 50 dap) of transgenic flax with
higher activities around mid-development (14 to 28
dap ) .
In tobacco, GUS activity in leaf was insignificant
with both the promoters whereas in seeds GUS activity
could be detected easily (Fig. 8). In developing
tobacco seeds, GUS activity was highest at about mid-
development (Fig. 9). In canola, GUS activity could be
detected easily in both leaves and seeds (Fig. 10).
Utility of the Flax Promoters in Regulating Gene
Expression
The utility of the flax promoters disclosed here is
demonstrated by comparing their effect on uidA gene
expression with both lipid and protein biosynthesis in
developing flax seeds. In developing seeds, uidA
expression correlated well with both fatty acid and
protein biosynthesis (Fig. 7). Tn seeds, maximum
expression of the uidA gene controlled by the SAD gene
promoters preceded the maximum accumulation of fatty
acids and proteins. Also, in tobacco the temporal
pattern of uidA gene expression correlated well with
the lipid biosynthesis (de Silva et al., 1992).
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
22
Therefore, these promoters are useful in manipulating
gene expression in seeds. Since these promoters are
also active in other tissues they are useful in
manipulating gene expression in a variety of tissues.
Utility of SAD Genes
The utility of the genes can be demonstrated by
carrying out the following predictive experiments
(similar experiments have been reported in Knutzen et
al., 1992; Topfer et al., 1995). Firstly, antisense or
sense constructs are made using the disclosed or other
promoters. For example, these genes or their parts can
be ligated into a SmaI restriction site of pCDC 214 or
220 (Fig. 5) or any other convenient cloning site of
another plant transformation vector. These recombinant
plasmids can then be mobilized, for example, into an
Agrobacterium strain which can then be used to
transform a variety of plant species. Any changes in
fatty acids of membrane and storage lipids can be
evaluated by routine methods described in this
application.
Both type of constructs are expected to reduce the
levels of similar mRNA during expression of the natural
genes resulting in an increase of 18:0 fatty acid in
membrane or storage lipids. Sense constructs can also
be used in enhancing the levels of mRNA. Such
enhancement will likely result in the increase of 16:1
or 18:1 fatty acids in membranes or storage lipids of
plants. Such plants will be of increased commercial
interest and value.
It will be appreciated by persons skilled in the art
that various modifications and alterations may be made
to the present invention without departing from the
general scope of the invention as defined by the
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/008I2
23
following claims. All such variations and
modifications should be considered part of this
invention.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
24
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Ravinder Kumar Jain
(B) STREET: 2913 Irvine Avenue
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7J 2A9
(A) NAME: Roberta Gail Thompson
(B) STREET: 117 Capilano Court
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7K 4B9
(A) NAME: David Charles Taylor
(B) STREET: 622 Wollaston Bay
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7J 9C3
(A) NAME: Gordon Grant Rowland
(B) STREET: 213 Lake Crescent
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7H 3A1
(A) NAME: Alan Gordon McHUghen
(B) STREET: 35 Cathedral Bluffs Road
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7P lAl
(A) NAME: Samuel Leonard MacKenzie
(B) STREET: 17 Cambridge Crescent
(C) CITY: Saskatoon
(D) STATE: Saskatchewan
(E) COUNTRY: Canada
(F) POSTAL CODE (ZIP): S7H 3P9
(ii) TITLE OF INVENTION: Flax Promoters For Manipulating Gene
Expression
CA 02268745 1999-04-15
WO 98118948 PCT/CA97/00812
(iii) NUMBER OF SEQUENCES: 11
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/029,916
(B) FILING DATE: 30-OCT-1996
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2701 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(B) STRAIN: McGregor
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0: 1:
ACAACCATTC AATTCAAAAGTTCCATTTCC TCATCTGCCTTACCCATAAA60
TTTTTCCAAT
TCTCGACGGA CACCAAAAAATTGCCCCCAA ACAACAGCGCAGAAAAACCT120
CTCAGCCAGC
TCAACAACAA TGGCTCTCAAGTCACCACCT TCCCTTCAACACGCTCCCTC180
GCTCAACCCA
AACAACTTCT CCTCCAGATCTTTCTCATGG CTGCTTCCACTTTCAATTCC240
TCCTCGCACC
ACCTCCACCA AGTAAGCATCCGGAATCTCC GCCGATTTCTTTTAAGCGAT300
TCCTCCTCCT
TGATCGTAGA TAAATTTGTCCGTTCATCAA AATCTGCACGGTTCGTTTCT360
GGTTGCTTAC
TCTTCTGCGC CTAGATTGCAGTTCGCTTTC CGATTTGACTGACCGACATA920
TTATGTCATT
AATCAATTCC TTTGTGTTTCGTTTTGCGCT GTAATTGATT~GTCAGTGTTT980
ACGATTCTGG
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
26
GCACAGGTTT CCCCTTCTCC TCCTCCGTCC ATCAAATGCA TGTTATTACC ATTTCAATTT 540
CAGTTTCCTT CTCTGAAATA TCCGTCTCTG GGAAAATAAG TCTCTGTATC TACTATCCTA 600
TCAGCTTGTT TAGGAGAGGT TCGATATTCG TTTACATAAA CCAATTGGCT TACAGTCCTT 660
GAACGTTCTA AATGTTGGTC GCGGTGATAA TAGGTTCTCA AAAGAGGTTT GTCTATGTTG 720
TTTGGCAAAA TCTTGTTTCT GTGAATCATG TTTAAGGTCC TTGGAAGAAT GACTAATGAG 780
CTATGACATG ATTACGACGT AGTAGTTATT GAACTGCTGA TAATTCAATA TAGGGGTAAC 890
TTTGTTGATT GTTTGGTCAC AGGGAGGCTG AGAAGCTAAA GAAGTCACAT GGACCACCAA 900
AAGAGGTGCA TATGCAAGTG ACCCATTCCA TGCCCCCACA GAAGCTGGAG ATATTTAAGT 960
CTCTGGAAGG TTGGGCTGAG GATGTTCTAT TACCGCACCT GAAGCCAGTT GAGAAATGCT 1020
GGCAGCCACA GGATTTCCTG CCCGAACCTG AGTCGGATGG GTTCGAGGAG CAAGTGAAGG 1080
AGCTCAGGGC AAGGGCCAAA GAACTGCCCG ATGACTATTT TGTTGTGCTG GTTGGGGATA 1190
TGATCACCGA AGAAGCTCTG CCGACTTACC AGACAATGCT CAACACCCTT GACGGGGTGA 1200
GGGACGAGAC TGGAGCCAGC CTTACGCCGT GGGCAATCTG GACAAGGGCG TGGACCGCTG 1260
AAGAGAATAG GCACGGTGAC CTTCTCAACA AGTATCTATA CCTCTCTGGA AGGGTGGACA 1320
TGAGGCAAAT TGAAAAGACC ATTCAGTATC TCATCGGCTC TGGAATGGTA TGTAATCACA 1380
TACTTCATCC TTTTCTATTA ATCTTTGGGT GAACAAAATT CACTACACTG GTAGCAGCTG 1440
AAACTTTAGA TGATTTTTTT TACTGCCTAG CTTCTATGAA ACAAAACCAC GTAAGTCAAA 1500
TAGGGTTGAC AATGAGTTCA AGTGGCAAAA TTTTTCTTAT ATACCAACTT CGAACCACTT 1560
TATATGACAT ACCAACTCCT AGTTCGGTTA AAATTCCTCC GTCGAAGATA TAATACTTGG 1620
ATTGGTTAAA TGAATTGTGA AAGGATACAC GTGATGTGGT CTGGAATTAA TTTGTTTGAA 1680
TGATCAGTTG GGTTCGGGGC GACAACTGTG AACTGGAACC ACCCTAAGTA AATTTTCTTT 1790
CTGTCCTACA AATTTGAGGT TCTCCTTGAT CACCTTAGTC CATCTTAGGT TTGCCCGTTA 1800
GTAAGATCTG CATTTAGCAG TTTGTCCTGG TATCTGATAT CACTAGTATC TTTGTTTGAT 1860
TCCCTAGCAT CTCTGAAACC ATCGGACAAG TAGGTGGTTT AGGACAAATT TGGTTCATTG 1920
CGGCATTTTT TGTTTGTATC GCCGTATCAT CTGGAAGAAG CAGACAGTTT TGCAAAGTGG 1980
CATCAAGCTC AAGAAAGCAA CGGCTAGAAG AAGTTCTACA TCTGATGCTT TCCTTTTGTT 2040
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
27
TCTTTGTGTG CTTTTTGGAC TTTGTTCTTTGATCCAAGAT CCAAAAACAG2100
TTTCCTGTAG
AAAACAACCC CTACCTCGGT TTCATCTACAAGAGAGGGCA ACGTTCATCT2160
CCTCATTCCA
CCCACGGAAA CACAGCCAGA CTCGCCAAGGCATGAAGCTG GCGCAGATCT2220
ACCATGGGGA
GCGGGATCAT CGCAGCAGAC GAGAAACGGCATACACCAAG ATCGTCGAGA2280
ACGAAACCGC
AGCTCTTCGA GATCGACCCT GACGGTACAGGGCGGACATG ATGAGGAAGA2340
TGCTGGCACT
AGATATCGAT GCCCGCCCAC TTGATGTACGCGACAACCTC TTCGACAATT2400
ATGGAGAAGA
ACTCGTCAGT CGCTCAACGC ATCGGGGTGTGGATTATGCC GATATCCTGG2460
ATACTGCCAA
AGTTCCTGGT GGGGAGGTGG AAAGTGGATGGCTTTCCGGG GAAGGGAACA2520
CTTTTACGGG
AAGCTCAGGA TTTTGTCTGC GGGCTTCCTGAAAGTTGGAG GAGAGGGCTG2580
CGAGGATTCG
CGGGGAGGGC AAAGCAAACG TCGAAATCTGCTGGATCTTC AGCAGAGAAT2680
TCCCGTTCAG
TGGTACTCTA ATGGAGTTTG CTTGAGAGTTATGATTGGAG TATGATGGCA2700
AGAGTGTGGA
T
2701
(2) INFORMATION FOR SEQ ID
NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2705 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: l.lnear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(B) STRAIN: Mc6regor
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
ACAACCATTC AATTCAATAT CTCACATTCA AGTTTTTCCA ACTTCCATTT CCTCATCTGC 60
CTTACCCATA AATCTCGACA CCAAAACACT CAGCCAGCTT CGTCCCAAAC AACGCAGAAA 120
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
28
AACCTTCAAC AACAATGGCT CTCAAGCTCA ACCCAGTCAC 180
CACCTTCCCT TCGACCCGCT
CCCTCAACAA CTTCTCCTCC AGATCTCCTC GCACCTTTCT 240
CATGGCTGCT TCCACTTTCA
ATTCCACTTC CACCAAGTAA GTTCCCGTCA CCATCTCCTC 300
TTCCTCGGAA TCTCCGCCGT
TTCATTTAAG CGATTGATCG TAGAAAATCT GTCGGTTGCT 360
TAGCGTTCAT TCAAATCTGC
GCGGTTCGTT TCTTTTTCTT TCTTCAGACT GCATCATCTG 420
CATTATGTTA TTGTTCGTTT
CCGATTTGAC TAACCTACAT AATCAATTCC TTTGTGTTTC 480
ACGAGTCTGG ATTTTGCGCT
GTAATTGATT GTCAGTGTTT GGACAGGTTT CCATTTCTCC 590
ACCTCCGTCC ATCAAATGCA
TGTTATTACC TACCAATTTC AGCGTCTTTC TCTGGAAATT 600
TCTGTCTCTG TATCTACTAT
CCTATTAGCT TGTTTGAGAG AGGTTCAATA TTGGTTTGCA 660
TGAACCAAGT GGCTTACAAT
CCTTCAACGT TCTAAATGTT GGTCGCAGTA ACAATAGGTT 720
CTCAAAAGAG GTTTTTCTAT
GTTGTTTGGC AAAATCTTGT TTCTGTGAAT CATGTTAAGG 780
TCCTGGGAAG AATGATTAAT
GAGCTATGAC ATGATTAAGG CGTAGTAGTT ATTGAACTGC 890
TGATAATTCA ATATAGGGGT
AACTTTGTTG GTTGTTTGGT GACAGGGAGG CTGAGAAGCT 900
AAAGAAGTCA CATGGACCAC
CAAAAGAGGT GCATATGCAA GTGACCCATT CCATGCCCCC 960
ACAGAAGCTG GAGATCTTTA
AGTCCCTTGA AGGTTGGGCA GAGGACGTTC TGTTGCCGCA 1020
CCTGAAGCCG GTTGAGAAAT
GCTGGCAGCC ACAAGATTTC CTGCCCGAAC CCGAGTCGGA 1080
TGGGTTCGAG GAGCAAGTGA
AGGAGCTCAG GGCAAGGGCT AAAGAACTCC CCGATGACTA 1140
TTTTGTTGTG CTGGTTGGGG
ATATGATCAC CGAAGAAGCT CTACCGACTT ACCAGACAAT 1200
GCTCAACACC CTTGACGGGG
TGAGGGACGA GACTGGAGCC AGCCTTACGC CGTGGGCAAT 1260
CTGGACAAGG GCGTGGACCG
CTGAAGAGAA TAGGCACGGT GACCTTCTCA ACAAGTATCT 1320
TTACCTCTCT GGAAGGGTGG
ACATGAGGCA AATTGAAAAG ACCATTCAGT ATCTCATCGG 1380
CTCTGGAATG GTATATACTC
ACATCCTATC TGCCCCTTTA TCCTTTTCCA TTAATCTTTG 1440
ATTGAACAAA ATTCAATAAA
CTGGTAGCTG AAACTTTAGA TGATTTGTTA CTGCCTAGCT 1500
TCTATGAGAA AACCACTGAA
GTCAAATAGG TTTGACAATG GGTTTAAATG GAAAAAGTTT 1560
CATATACCAT CTTCCATCTA
TTTTATATGA CATACCAACT TCTACTTTGG AGAAAATTCG 1620
CCGTGGATAA TCATATTATT
GAAGATATAG TACTTAGTAG ATTGGTTAGA TGAACTGTTA 1680
AACAATACAT GTGATGTCGT
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
29
GTGCAATTAA TTTGTGTAAA TGATTAGCTGGACAAATGTG AACTGGAACC1790
GGTTCGGGAC
CTAGTAAACT ATGAATTGAG GTTGTCCTTCTCTGTCCTGG GTCTGTTTGC1800
ATCACTTTAT
CTGTTTGCAA GATCTGCATG TAGCAGTTTGTGCTACCAGT GGTATCTTTG1860
TCCTGGTATT
TTTGATTCCC TAGCATCTCT GAAAACATCGCTGGTTAGGA CAAATTTGGT1920
GACCAAGTAT
TCATTGCGGC ATTTTTTGTT TGTATCGCTGAAGAGCAGAC AGTTTTGCAA1980
TATCGTCTGG
AGTGGCATCA AGCTCAAGAA AGCAACGGCTCTACATCTGA TGCGTTCCTT2040
AGAAGAAGTT
TTGTTTCTTT GTGTGCTTTT TGGACTTTGTTGTAGGATCC AAGATCCAAA2100
TCTTTTTGCC
AACAGAAAAC AACCCCTACC TCGGTTTCATTTCCAAGAGA GGGCAACGTT2160
CTACACCCCA
CATCTCCCAC GGAAATACGG CCAGACTCGCGGGGACATGA AGCTGGCGCA2220
CAAGGACCAC
GATCTGCGGG ATCATCGCAG CAGACGAGAAACAGCATACA CCAAGATCGT2280
GCGGCACGAA
CGAGAAGCTC TTCGAGATCG ACCCTGACGGGCTCTGGCGG ACATGATGAG2340
TACAGTGTTG
GAAGAAGATA TCGATGCCCG CCCACTTGATGAAGACGACA ACCTCTTCGA2900
GTACGATGGA
CAATTACTCG TCGGTCGCTC AACGCATCGGGCCAAGGATT ATGCTGATAT2960
GGTGTATACT
CCTGGAGTTC CTGGTGGGGA GGTGGAAAGTACGGGACTTT CCGGGGAAGG2520
GGATGCTTTT
GAACAAAGCT CAGGAGTTTG TCTGTGGGCTATTCGAAAAT TGGAGGAGAG2580
TCCAGCGAGG
GGCTGCGGGG AGGGCAAAGC AAACGTCGAATTCAGCTGGA TCTTCAGCAG2690
ATCTGTCCCA
AGAATTGGTA CTCTAATGGA GTTTGCCCGATGGAATGATT GGAGTATGAT2700
GAGTTGAGTG
GGCAT
2705
(2) INFORMATION FOR SEQ ID
NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1693 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(B) STRAIN: McGregor
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID NO:
3:
GAGCTCTCAATGTAGTAACACAAAGCCTTC TGTCTTCTTT CAATGCTAGA60
CTGTAACGTT
ACTTGTCTTCTTATAACTGTTTGTTTGCTT CTTCAGCTAA GGATGGAGCC120
TGTTGGAGAA
ACGGAGATCCCGGTAAAGCAAAGGATGGAT CGAGAGGAGA AGAGAACATG180
CGGTGGCTCG
GAAGCATTGCACAGAGCCGTCACGTTGGAA GTGCCTCATT GTCTCGGTAT240
CGCAGGCCCC
GGAACATTTGGTGGTGGTGAGGTTGAAGAA GAGGAGAAAG TCATCATCTA300
ATGCCGTAGT
CTGGGATGGATTGATCCGGCCAGCATGTTC TCCTCCCGAA CCCTATTGAT360
ATCGACCTGT
GACAATGTAACATCAATGTCAATCTCTGCA GATATCTGTT CATGATTCTT420
AGGATCAGGT
TTTTGGTTGATTCTTGTGAATGTGTAACAT TGATGTAAGC TGTAATATCT480
TATTTGTTGT
GATTTTGTTGTTGCTTTGATCAATCAAATA AATCTCGTTC TAAGCCTCTT540
AACGCGATCA
TCATATTCATTTTGACGACTATGTATAGTC GTACAAACTA TAATCTACAT600
TTCGGTTAAC
CAAGTCGGAATTAGCTAGACATTGTCAAGG AGGAGGAAAA ATTGGATGAG660
TATCAAGAAA
GAAATCATACACCCAATTCTGAAGCTGATT CTTCATCTAT TTCGACTTTT720
GATTTCGAGT
TTTGAGTCTCAACTGTGATTTCGAGTTTCG ACTTGATTTG ATTCGAAATT780
GCTCTTTGAT
AAATGCCTCCAAAGTGCTCTCTACTTGCGG TTGGCCTGGT ATCATTGAAT840
TCAATGGCGA
GACAGAACTAGACAGCTACCAGGTGCAAAA AACATTTGTT TGCATTAATG900
AATGTCTTCT
TCCATGTTTTCTGCATTTTAATCTTTCCCC AAACACCTAA CATTGATCCT960
TATATAGCTT
CCTCTCACGGTTGCAGATCTCGTTGCTGAT AACACATACA ACTCTAAAAC1020
TGGCTACAAG
GGTTCAAAGTGAAATTGTTTTGGTGGTAGA GTTGTGTGTT AAAGTTCTGG1080
TGGTGACTCG
ATTCGAATCCAGCATTCCCCACAAAATAGA CACCAACGTA ACCGTCTTCT1140
GTGTTTATTT
ATCTTGTATTGACCGAGAGTTACGATATAC TCCGACAAAA TCCACATCAT1200
AAAGACATCT
CAAATGGATCCGTAGTTAGTGCAGTGGCTC GATTAACATA GGAAAAAATT1250
AATGAAAAAA
TGCCTGAAATCGATGCTCAAAACAAGTAGA AATTCATTCA GACAAACACG1320
AACATATTTA
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
31
ATCATTTAGC ATCATCAAAT TAATAACAAG AGCAAACAAT 1380
AAAGCACATA GCAAAACATA
CAATAGTCGT CTTGCAATGT CATATGATAA TAAGCCAGTG 1940
AAACCATGAA GCCCAAGTGA
AGTGGTCAAG TGGGAGCTGA AAGCTTCCGA ACCCAAGCCC 1500
CCGCTACCGG GTTAGGACAT
ACGACACGCG ACATGCTACG AAACTTAAAA ATCGGTCACG 1560
CAGTTAATGG AACAAATGAA
ACGCAACGAC TATTAAGTGA CCATTTTGCA GAAATGATAT 1620
GAAAAAGTGA CCATTTAGAC
AAATGAGCAA AGAAAATACA AGTGGCGAGT GCTGACATAA 1680
TAAACCGAAT GCAGGCGTTA
CCATCCAATT TTA 1693
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1191 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(B) STRAIN: McGregor
(xi) SEQUENCE
DESCRIPTION:
SEQ ID NO:
4:
GAGCTCTCAA CAAACTCTTTTTTTTCCATA ACGTTGAATGTTAGAACTTT60
TGTAGTAACA
GTCTTTTTAT TTCATGAAGCTGATCAGCTG ATGTTGGAGAAGGATGGAGC120
AACTGTTTCT
CACGGAGATT AAAGGATGGAACGAGAGGAG ACGGTGACTCGAGAGTACAG180
CCTGAAAAGC
GGAAGCATTG TCACGCTTGCAGTGCCTCAT TCAGAGTTCTTGTCTCGGTA240
CACAGAGCTG
TGGAACATTT ACGTTGAAGAAGAGGAAGAA AGATGCTATGGTTCATCATC300
AGTGGCGGTG
TAGTGGGAAG GCCGGCATGTTCTCCTCCCG AAATCGGGCCGTCCCAATTG360
GATTGATCCA
ATGACAATGT TCAATCTCTGCAGATTTTTG TTAGCAGCAG~GTCATGATTC920
AACATCAATG
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97100812
32
TTTTTTGGTT GATTCTTGTG AATGTAAGCT ATTTGTTGTT GTAATATATG CATTGATTGT 9BC
GATTTTGTTT TAGCTTTGAT CAATGAAATA AATCTCGTTC AACCCAACCA TCAGGCTCTT 59C
TCATATTCAT TTTGACGACT ATATATACAT AATCGTACAA ACTATTCGGT TAACTAATCT 600
ACAGAAAGTC GGAGTTAGCT AGAGATTGTC AAGGAGGAGG AGATCATACA CCTAATTTTG 660
AAGCTGATTC TTCATCTATG ATTTCGAGTT TTGACTTGAT TTGGCTCTTC GATATTCGAA 720
ATTAAATGCC TCAATGCCTC CAAAGTGCTC TCTACTTGCG GGTGGACCTA CAAAACTAGG 780
CAAACAGGTG CAAAAAACAT GTGTTTACAC GTCCATGTTA TCTTGCATTG GCCCATGTTT 890
TCTGCATTGT AAATCTTTCC CCAAACACAT AGTTAGACGA AGTCGATAAT CTAGCACCAT 900
CAAATCAATA ACACGAGCAA ATAATAAAGT AAATAGTGAA ACCATGAAGC CTAATTGGTC 960
GAGTGGAGCT GAAAGCTTTC ATCGGTATCG AACCCAACCC CCCCTGCTAC GAAACTTAAA 1020
AATGGGTTAC GCTATTACAC TCGATAGAAC TGATGAAACG CAACGATTGT TAAGTAACCA 1080
TTTTGCAGAA ACGATAATTG ACAAGTGACC ATTTGGATAA ATGACCAGGG AAAATACAAG 1190
TGGCGAGTGC TGACATAATA AACCGAATGC GGGCGTTACC ATCCAATTTT A 1191
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1371 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv} ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CGACAACCAT TCAATTCAAA AGTTTTTCCA ATTTCCATTT CCTCATCTGC CTTACCCATA 60
AATCTCGACG GACACCAAAA AACTCAGCCA GCTTGCCCCC AAACAACAGC GCAGAAAAAC 120
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
33
CTTCAACAAC CTTCCCTTCA ACACGCTCCC180
AATGGCTCTC
AAGCTCAACC
CAGTCACCAC
TCAACAACTTCTCCTCCAGA TCTCCTCGCA GGCTGCTTCC ACTTTCAGTT240
CCTTTCTCAT
CCACCTCCACCAAGGAGGCT GAAGCTAAAG GACCACCAAA AGAGGTGCAT300
AAGTCACATG
ATGCAAGTGACCCATTCCAT GCCCCCACAG GATATTTAAG TCTCTGGGAA360
GAAGCTGGGA
GGTTGGGGCTGAGGGATGTT CTTATTTCGC AGTTGAGAAA TGCTGGCAGC920
ACCTGAAGCC
CACAGGATTTCCTGCCCGAA CCTGAGTCGG GGAGCAAGTG AAGGAGCTCA480
ATGGGTTCGA
GGGCAAGGGCCAAAGAACTG CCCGATGACT GCTGGTTGGG GATATGATCA540
ATTTTGTTGT
CCGAAGAAGCTCTGCCGACT TACCAGACAA CCTTGACGGG GTGAGGGACG600
TGCTCAACAC
AGACTGGAGCCAGCCTTACG CCGTGGGCAA GGCGTGGACC GCTGAAGAGA660
TCTGGACAAG
ATAGGCACGGTGACCTTCTC AACAAGTATC TGGAAGGGTG GACATGAGGC720
TATACCTCTC
AAATTGAAAAGACCATTCAG TATCTCATCG GGATCCAAAA ACAGAAAACA780
GCTCTGGAAT
ACCCCTACCTCGGTTTCATC TACACCTCAT GGCAACGTTC ATCTCCCACG890
TCCAAGAGAG
GAAACACAGCCAGACTCGCC AAGGACCATG GCTGGCGCAG ATCTGCGGGA900
GGGACATGAA
TCATCGCAGCAGACGAGAAA CGGCACGAAA CAAGATCGTC GAGAAGCTCT96D
CCGCATACAC
TCGAGATCGACCCTGACGGT ACAGTGCTGG CATGATGAGG AAGAAGATAT1020
CACTGGCGGA
CGATGCCCGCCCACTTGATG TACGATGGAG CCTCTTCGAC AATTACTCGT1080
AAGACGACAA
CAGTCGCTCAACGCATCGGG GATACTGCCA CGATATCCTG GAGTTCCTGG1190
AGGATTATGC
TGGGGAGGTGGAAAGTGGAT GCTTTTACGG GGAAGGGAAC AAAGCTCAGG1200
GGCTTTCCGG
ATTTTGTCTGCGGGCTTCCT GCGAGGATTC GGAGAGGGCT GCGGGGAGGG1260
GAAAGTTGGA
CAAAGCAAACGTCGAAATCT GTCCCGTTCA CAGCAGAGAA TTGGTACTCT1320
GCTGGATCTT
AATGGAGTTTGCTTGAGAGT AGAGTGTGGA TATGATGGCA T 1371
ATGATTGGAG
(2)
INFORMATION
FOR
SEQ
ID
NO:
6:
(i)
SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 29 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
cDNA
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
34
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAAATGCCAT CATACTCCAA TCAT 24
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
GAACCTTCAA CAACAATGGC TCTC 29
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97100812
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CCTTCAACAA CAATGGCTCT CAAGCTCAAC CCAGTCACCA CCTT gq
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GGAGAAGTTG TTGAGGGAGC GTGTTGAAGG GAAGGTGGTG ACTGGGTTGA 50
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
36
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
TTGGTGGAGG TGGAACTGAA 20
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Linum usitatissimum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AGCTAAAGAA GTCACATGGA C 21
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
37
REFERENCES OF INTEREST TO THE PRESENT INVENTION
l.An,G., Ebert, P. R., Mitra, A., and Ha, S. B. 1988.
Binary vectors, in: S. B. Gelvin, R. A. Schilperoort
and D. P. S. Verma (Eds.), Plant Molecular Biology
Manual, Kluwer Academic. Pp A3:1-19.
2.Basiran N., Armitage, P., Scott, R. J., and Draper,
J. 1987. Genetic transformation of flax (Linum
usitatissimum) by Agrobacteria tumefaciens .
regeneration of transformed shoots via a callus
phase. Plant Cell Reports 6:396-399.
3.Bennett, J. 1993. Genes for crop improvement. In
Genetic Engineering, Vol. 15. J. K. Setlow (ed.).
Plenum Press, NY. Pp.165-189.
4.Chen ZL, Schuler MA, Beachy RN. 1986. Functional
analysis of regulatory elements in a plant embryo-
specific gene. Proc Natl Acad Sci U S A 1986 83:85~0-
8564.
5.Datla, R.S.S., Hammerlindl, J.K., Panchuck, B.,
Pelcher, L.E., and Keller, W. 1992. Modified binary
plant transformation vectors with the wild-type gene
encoding NPTII. Gene 211:383-384.
6.Datla, R., Anderson, J. W., and Selvaraj, G. 1997.
Plant promoters for transgene expression. Biotech.
Ann. Rev. (In press).
7.Dehesh K, Jones A, Knutzon DS, Voelker TA. 1990.
Production of high levels of 8:0 and 10:0 fatty acids
in transgenic canola by overexpression of Ch FatB2, a
thioesterase cDNA from Cuphea hookeriana. Plant J
9:167-172.
8. Dellaporta, S.L., Wood, J., and Hicks J.B. 1983. A
plant DNA mini preparation: version II. Plant Mol.
Biol. Rep. 1:19-21.
9.de Silva, J., Robinson, S. J., and Safford, R. 1992.
The isolation and functional characterization of a B.
napus acyl carrier protein 5' flanking region
involved in the regulation of seed storage lipid
synthesis. Plant Mol. Biol. 18:1163-1172.
lO.Gait, M. J. 1989. Oligonucleotide,Synthesis-A
Practical Approach. IRL Press, Oxford.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
38
ll.Hitz WD, Mauvis CJ, Ripp KG, Reiter RJ, DeBonte L,
Chen, Z. 1995. The use of cloned rapeseed genes for
cytoplasmic fatty acid desaturases and the plastid
acyl-ACP thioesterase to alter relative levels of
polyunsaturated and saturated fatty acids in rapeseed
oil. Proc. 9th International Cambridge Rapeseed
Congress, UK. Pp 47D-472.
12. Horsch, R. B. , Fry, J. E. , Hoffmann, N. L. ,
Eichholtz, D., Rogers S. G., and Fraley R. T. 1985. A
simple and general method for transferring genes into
plants. Science 227:1229-1231.
13.Jefferson, R. A. 1987. Assaying chimeric genes in
plants: the GUS gene fusion system. Plant Mol. Biol.
Rep. 5:387-405.
l4.Jones A, Davies HM, Voelker TA. 1995. Palmitoyl-aryl
carrier protein (ACP) thioesterase and the
evolutionary origin of plant acyl-ACP thioesterases.
Plant Cell 7:359-371.
l5.Kinney, AJ. 1997. Development of genetically
engineered oilseeds. in: JP Williams, MU Khan,and NW
Lem(Eds.), Physiology, Biochemistry, and Molecular
Biology of Plant Lipids, Kluwer Academic Publ. Pp
298-300.
l6.Knutzon, D. S., Thompson, G. A., Radke, S. E.,
Johnson, W. B., Knauf, V. C., and Kridl, J. C. 1992.
Modification of Brassica seed oil by antisense
expression of a stearoyl-acyl carrier protein
desaturase gene. Proc. Natl. Acad. Sci. USA. 89:2624-
2628.
l7.Koncz, C., and Schell, J. 1986. The promoter of TL-
DNA gene 5 controls the tissue-specific expression of
chimeric genes carried by a novel type of
Agrobacterium binary vector. Mol. Gen. Genet.
204:383-396.
l8.Mlynarova, L., Bauer, M., Nap, J.-P., and Pretova,
A. 1994. High efficiency Agrobacterium-mediated gene
transfer to flax. Plant Cell Reports 13:282-285.
l9.Moloney, M. M. and van Rooijen, G. J. H. (1996)
Recombinant proteins via oleosin partitioning. Inform
7:107-113.
CA 02268745 1999-04-15
WO 98/18948 PCT/CA97/00812
39
20.Moloney, M. M., Walker, J. M., and Sharma K. K.
1989. High efficiency transformation of Brassica
napus using Agrobacterium vectors. Plant Cell Rep.
8:238-242.
2l.Ochman, H., Ayala, F. J., and Hartl, D. L. 1993. Use
of polymerase chain reaction to amplify segments
outside boundaries of known sequences. Meth. Enzymol.
218:309-321.
22.Rowland, G. G., McHughen, A., Gusta, L. V., Bhatty,
R. S., MacKenzie, S. L., and Taylor, D. C. 1995. The
application of chemical mutagenesis and biotechnology
to the modification of linseed. Euphytica 85:317-321.
23.Sanger, F., Nickler, S., and Coulson, A. R. 1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA. 74:5463-5467.
24.Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989.
Molecular Cloning: A Laboratory Manual, 2nd ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor,
NY.
25.Singh, S., McKinney, S., and Green, A. 1994.
Sequence of a cDNA from Linum usitatissimum encoding
the stearoyl-aryl carrier protein desaturase. Plant
Physiol. 104:1075.
26.Taylor, D. C., Barton, D. L., Rioux, K. P., Reed, D.
W., Underhill, E. W., MacKenzie, S. L., Pomeroy, M.
K., and Weber, N. (1992). Biosynthesis of acyl lipids
containing very-long chain fatty acids in
microspore-derived and zygotic embryos of Brassica
napus L, cv. Reston. Plant Physiol. 99:1609-1618.
27.Topfer, R., Martini, N., and Schell, J. 1995.
Modification of plant lipid synthesis. Science
268:681-686.
28.Warner, S. A. J., Scott, R., and Draper, J. 1993.
Isolation of an asparagus intracellular PR gene
(AoPR1) wound-responsive promoter by the inverse
polymerase chain reaction and its characterization in
transgenic tobacco. Plant J. 3:191-201.
