PT975765E - Plant fatty acid epoxygenase genes and uses therefor - Google Patents

Description

EPOXYGENASE GENES OF VEGETABLE FATTY ACID AND ITS UTILIZATIONS "

FIELD OF THE INVENTION The present invention relates generally to novel genetic sequences encoding fatty acid epoxygenases. In particular, the present invention relates to genetic sequences encoding A12-epoxygenases fatty acid enzymes, as defined herein. More particularly, the present invention provides cDNAs and genomic gene sequences encoding plant fatty acid epoxygenases, preferably A12-epoxygenases of Crepis palaestin or Euphorbia lagascae. The genetic sequences of the present invention provide the means by which fatty acid metabolism can be altered or manipulated in organisms such as yeasts, molds, bacteria, insects, birds, mammals and plants, in particular to convert therein unsaturated fatty acids into epoxidized fatty acids. The invention extends to genetically modified oil accumulating organisms, transformed with the genetic sequences in question, and to the oils derived therefrom. The oils thus produced provide the means for the cost-effective raw material for use in the efficient production of coatings, resins, adhesives, plastics, surfactants and lubricants, among others.

Throughout this description and the claims that follow, unless the context requires otherwise, the word " comprises ", or variations, such as " or " comprising " will be understood to imply the inclusion of 1 a complete entity or a group of full entities referred to, but not the exclusion of any other complete entity or group of complete entities.

The bibliographic details of the publications referred to by the author in this description are gathered at the end of the description. Sequence identity numbers (SEQ ID Nos.) For the nucleotide and amino acid sequences referred to in the disclosure are set forth after the literature.

BACKGROUND OF THE INVENTION

There is considerable worldwide interest in the production of chemical raw materials, such as fatty acids, for industrial use from renewable plant sources rather than non-renewable petrochemicals. This concept has a wide acceptance by manufacturers and consumers based on the conservation of resources and provides a significant opportunity to develop new industrial crops for agriculture.

There is a diverse set of unusual fatty acids in nature and these have been well characterized (Badam & Patil, 1981 / Smith, 1970). Many of these unusual fatty acids have industrial potential and this has led to the interest in domestication of these species to allow agricultural production of particular fatty acids.

One class of fatty acids of particular interest are the epoxy fatty acids, consisting of an acyl chain wherein two adjacent carbon bonds are attached by an epoxy bridge. Due to their high reactivities, they have considerable application in the production of coatings, resins, glues, plastics, surfactants and lubricants. These fatty acids are currently produced by chemical epoxidation of vegetable oils, especially soybean oil and linseed oil, however this process produces mixtures of multiple and isomeric forms and involves significant processing costs.

Attempts are being made by others to develop some wild plants containing epoxy fatty acids (e.g., Euphorbia lagascae, Vernonia galamensis) in commercial sources of these oils. However, problems with agronomic aptitude and low yield potential severely limit the commercial utility of traditional approaches to plant breeding and breeding. The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating the efficiency of commercially important industrial processes by expressing genes isolated from a first organism or species in a second organism or species to thereby confer new phenotypes. More particularly, conventional industrial processes can be rendered more efficient or cost-effective, resulting in higher yields per unit cost, through the application of recombinant DNA techniques.

In addition, the appropriate choice of the host organism for the expression of a genetic sequence of interest provides the production of compounds which are not normally produced or synthesized by the host in high yield and purity.

However, in spite of the general effectiveness of recombinant DNA technology, the isolation of genetic sequences encoding enzymes important in fatty acid metabolism, in particular, the genes encoding the fatty acid A12-epoxygenases 3 enzymes responsible for the production of 12,13 epoxy-9,15-octadecenoic acid, among others, remains a major obstacle to the development of genetically engineered organisms that produce these fatty acids.

Until the present invention, only limited biochemical data exist indicating the nature of epoxygenases fatty acid enzymes, in particular A12-epoxygenases. However, in Euphorbia lagascae, the formation of 12,13-epoxy-9-octadecenoic acid (vernolic acid) from linoleic acid appears to be catalyzed by a cytochrome-P450-dependent A12-epoxygenase enzyme (Bafor et al., 1993; Blee et al., 1994). In addition, seed in developing flax plants has the ability to convert added vernolic acid to 12,13-epoxy-9,15-octadecadienoic acid through an endogenous Δ15 desaturase (Engeseth and Stymne, 1996). Epoxy fatty acids can also be produced by peroxide-dependent peroxygenase in plant tissues (Blee and Schuber, 1990).

In the work leading to the present invention, the inventors sought to isolate genetic sequences encoding genes that are important for the production of epoxy fatty acids, such as 12,13-epoxy-9-octadecenoic acid (vernolic acid) or 12,13- epoxy-9,15-octadecadienoic acid, and to transfer these genetic sequences into commercially productive oleaginous seed plants and / or other oil accumulating organisms.

SUMMARY OF THE INVENTION

One aspect of the invention provides an isolated nucleic acid molecule that encodes, or is complementary to, an isolated nucleic acid molecule encoding a fatty acid epoxygenase.

A second aspect of the invention provides an isolated nucleic acid molecule that hybridizes under conditions of at least low stringency with at least 20 contiguous nucleotides of SEQ ID Nos: 1 or 3 or 5 or 19 or 20, or a sequence thereof complementary.

A further aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that is at least 65% identical to SEQ ID NO: 1 or 3 or 5 or which is at least 75% identical to, at least 200 contiguous nucleotides in SEQ ID Nos: 19 or 20, or a complementary sequence thereof.

A further aspect of the invention provides a genetic construct comprising the above isolated nucleic acid molecule, either in the sense or antisense orientation, in operable connection with a promoter sequence.

A further aspect of the invention provides a method of altering the level of epoxy fatty acids in a cell, tissue, organ or organism, said method comprising the expression of a sense, antisense, ribozyme or co-suppression molecule comprising the molecule of the above isolated nucleic acid in said cell over a period of time and under conditions sufficient to increase or decrease the level of the epoxy fatty acids present therein.

A further aspect of the invention provides a method of producing an enzymatically active recombinant epoxygenase polypeptide in a cell, said method comprising expressing the above isolated nucleic acid molecule in said cell over a period of time and under conditions sufficient for epoxygenase so coded to be produced.

A further aspect of the invention provides a method of producing an enzymatically active recombinant epoxygenase polypeptide in a cell, said method comprising the steps of: (i) producing a genetic construct comprising the above isolated nucleic acid molecule operably placed under the controlling a promoter capable of conferring expression in said gene sequence in said cell, and optionally an expression enhancing element; (ii) transforming said genetic construct into said cell; and (iii) selecting transformants that express at a high level a functional epoxygenase encoded through the genetic sequence.

A still further aspect of the invention provides a method of producing an enzymatically active recombinant epoxygenase polypeptide in a transgenic plant comprising the steps of: (i) producing a genetic construct comprising the above isolated nucleic acid molecule operably placed under the control of a specific seed promoter and optionally an expression enhancing element, wherein said genetic sequence is also placed upstream of a transcription terminator sequence; (Ii) transforming said genetic construct into a cell or tissue of said plant; and (iii) selection of transformants expressing at a high seed level, a functional epoxygenase encoded through the genetic sequence.

A further aspect of the invention provides a recombinant epoxygenase polypeptide or a functional enzyme molecule.

A further aspect of the invention provides a recombinant epoxygenase comprising an amino acid sequence shown in any one of SEQ ID Nos: 2 or 4 or 6 or a homologue, analogue or derivative thereof that is at least about 50% identical thereto .

A still further aspect of the invention provides a method of producing an epoxygenated fatty acid in a cell, tissue, organ or organism, said method comprising incubating a cell, tissue, organ or organism which expresses an enzymatically active recombinant epoxygenase with a substrate of fatty acid and, preferably, an unsaturated fatty acid substrate, for a period of time and under conditions sufficient for at least one carbon bond, preferably a carbon double bond, of said substrate be converted into an epoxy group.

A further aspect of the invention provides an immunologically interacting molecule which binds to the recombinant epoxygenase polypeptide described herein, or a homologue, analogue or derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a linear representation of an expression plasmid comprising an epoxygenase structural gene operably placed under the control of the truncated napin promoter (FP1; right-hand dashed box) and placed upstream of the terminator sequence NOS (right-hand dotted box). The genetic sequence of epoxygenase is indicated by the rectangular white box on the right. The construct also comprises the NOS promoter (left-sided box) which directs the expression of the NPTII gene (left blank) and placed upstream of the NOS terminator (left dotted box). Also indicated are the left and right border sequences of the Ti plasmid of Agrobacterium tumefaciens. Figure 2 is a schematic representation showing the alignment of the amino acid sequences of the Crepis palaestin epoxygene polypeptide (Cpal2; SEQ ID NO: 2), an additional epoxygenase derived from Crepis sp., Different from C. palaestina, which produces a high levels of vernolic acid (CrepX; SEQ ID NO: 4), a partial amino acid sequence of an epoxygenase polypeptide derived from Vernonia galamensis (Vgall; SEQ ID NO: 6), the amino acid sequence of Δ12 (SEQ ID NO: 9), Brassica juncea (X91139; SEQ ID NO: 10), Glycine max (L43921; SEQ ID NO: 9), Crepis alpina acetylenase (Crepl; SEQ ID NO: 8), Δ12 desaturases of A. thaliana SEQ ID NO: 13), and the Δ12 hydroxylase of Ricinus communis (U22378; SEQ ID NO: 11), Solanum commersoni (X92847; SEQ ID NO: 12) and Glycine max (L43920; 14). Three histidine rich motifs that are conserved in non-heme mixed function monooxygenases are underlined. Figure 3 is a copy of a photographic representation of a Northern blot hybridization showing the specific seed expression of the Crepis palaestin epoxygenase gene exemplified through SEQ ID NO: 1. Northern blot analysis of total leaf RNA (lane 1) and developing seeds (lane 2) of Crepis palaestina. 15æg of total RNA was migrated on a Northern gel and transferred to Amersham Hybond N + membrane according to the manufacturer's instructions. The blot was hybridized at 60 ° C with a probe prepared from the 3 'untranslated region of SEQ ID NO: 1. The blot was washed twice in 2 x SSC (Sodium Citrate-NaCl buffer) at room temperature for 10 minutes, then in 0.1 x SSC at 60 ° C for 20 minutes. Figure 4 is a schematic representation showing the nucleotide sequence of the degenerate PCR primer (5 'to 3' direction) used to isolate the Euphorbia lagascae epoxygenase genes described herein. Figure 5 is a copy of a photographic representation of a RNA drop transfer hybridization showing the expression of the epoxygenase gene exemplified in SEQ ID NO: 3 in plants that produce vernolic acid compared to plants that do not produce vernolic acid. One pg of total RNA was isolated from the specified tissue and dripped onto Amersham Hybond N + membrane according to the manufacturer's instructions. The blot was hybridized at 42 ° C in 50% formamide with the 32 P-labeled probe prepared from SEQ ID NO: 3 for 16 hours. The blots were washed twice in 2 x SSC (Sodium Citrate-NaCl buffer) at room temperature, then in 0.5 x SSC at 55øC for 20 minutes. 9

Autoradiographies were obtained after an overnight exposure. Panel A shows total RNA of seed in development of Euphorbia lagascae (1), Euphorbia cyparissus (2), Vernonia galamensis (3), and flax (Linum usitatissimum) (4). Panel B shows total RNA of various tissues of Euphorbia lagascae, including seed in development (1), root (2) and leaf (3). Figure 6 is a schematic representation showing the subtractive hybridization method used to isolate the Euphorbia lagascae epoxygenase genes described herein. The + 6ADNc pool consisted predominantly of seed storage protein type sequences. A cluster of 15 of these sequences was biotinylated and further subtracted from + 6DNAc. HL = Long Hybridization - 20 h; HC = Short Hybridization - 3 h. Figure 7 is a copy of a photographic representation of a RNA drop transfer hybridization showing expression of the epoxygenase gene exemplified in SEQ ID NO: 20 in plants that produce vernolic acid compared to plants that do not produce vernolic acid. One pg of total RNA was isolated from the specified tissue and dripped onto Amersham Hybond N + membrane according to the manufacturer's instructions. The blot was hybridized at 42 ° C in 50% formamide with the 32 P-labeled probe prepared from SEQ ID NO: 20 for 16 hours. The blots were washed twice in 2 x SSC (Sodium Citrate-NaCl buffer) at room temperature, then in 0.5 x SSC at 55øC for 20 minutes. Autoradiographies were obtained after an overnight exposure. Panel A shows total RNA of developing seed of Euphorbia lagascae (1), Euphorbia 10 cyparissus (2), Vernonia galamensis (3), and flax (Linum usitatissimum) (4). Panel B shows total RNA of various tissues of Euphorbia lagascae, including seed in development (1), root (2) and leaf (3). Figure 8 is a schematic representation of a binary plasmid vector containing an expression cassette comprising the specific truncated napin (Napin) promoter and the nopaline synthase (NT) terminator, with a BamHI cloning site between the two, for in addition to the NPTII kanamycin resistance gene operably linked to the nopaline synthase (NP) and the nopaline synthase (NT) terminator sequences. The expression cassette is flanked by left-border (LB) and right-border (RB) T-DNA sequences. Figure 9 is a schematic representation of a binary plasmid vector containing an expression cassette comprising SEQ ID NO: 1 operably placed under the control of a seed specific truncated napin (Napin) promoter and upstream of the nopaline synthase terminator (NT), in addition to the kanamycin resistance gene NPTII operably linked to the nopaline synthase (NP) promoter and the nopaline synthase (NT) terminator sequences. The expression cassette is flanked by left (LB) and right (RB) T-DNA border sequences. To produce this construct, SEQ ID NO: 1 is introduced into the BamHI site of the binary vector shown in Figure 8. Figure 10 is a graphical representation of the gas chromatography profiles of fatty acid metal esters prepared from oilseeds of untransformed plants of Arabidopsis thaliana [panel (a)], or 11 plants of A. thaliana (transgenic line Cpal-17) which were transformed with SEQ ID NO: 1 using the genetic construct shown in Figure 9 [panels b) and (c)]. In panels (a) and (b), the fatty acid methyl esters were separated using packed column separation. In panel (c), the fatty acid methyl esters were separated using capillary column separation. The positions of elution of vernolic acid are indicated. Figure 11 is a graphical representation showing the joint distribution of pure plant epoxy fatty acids of Ti plants from Cpal2-transformed Arabidopsis thaliana plants as determined using gas chromatography. The levels of both vernolic acid (x-axis) and 12,13-epoxy-9,15-octadecadienoic acid (y-axis) were determined and plotted relative to each other. The data show a positive correlation between the levels of these fatty acids in transgenic plants. Figure 12 is a graphical representation showing the incorporation of 14C labeling in the chloroform phase obtained from lipid extraction of cotyledons from flax seed during feeding with labeled substrate. Symbols used; ♦, feed with [14C] oleic acid; , feeding with [14C] vernolic acid. Figure 13 is a graphical representation showing the incorporation of 14C labeling into phosphatidylcholine of linseed cotyledons during feeding with labeled substrate. Symbols used; ♦, feed with [14C] oleic acid; , feeding with [14C] vernolic acid. Figure 14 is a graphical representation showing the incorporation of 14C labeling into triacylglycerols of flax seed cotyledons during feeding with labeled substrate. Symbols used; ♦, feed with [14C] oleic acid; , feeding with [14C] vernolic acid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides an isolated nucleic acid molecule that encodes or is complementary to an isolated nucleic acid molecule encoding a fatty acid epoxygenase.

In which the isolated nucleic acid molecule of the invention encodes an enzyme that is involved in the direct epoxidation of arachidonic acid, and it is particularly preferred that the nucleic acid molecule in question is derived from a non-mammalian source.

As used herein, the term " derived from " shall be understood to indicate that a particular complete entity or complete entity group originated from the specified species but was not necessarily obtained directly from the specified source. The term " non-mammalian source " refers to any organism other than a mammal or a tissue or cell derived therefrom.

In the present context, the term " derived from a non-mammalian source " should be understood to indicate that a particular complete entity or a complete set of entities has been derived from bacteria, yeasts, birds, amphibians, reptiles, insects, plants, fungi, molds and algae or other non-mammals.

In a preferred embodiment of the present invention, the source organism is an organism having the genetic ability to synthesize epoxy fatty acids. More preferably, the source organism is a plant such as, but not limited to, Chrysanthemum spp., Crepis spp., Euphorbia spp. and Vernonia spp., among others.

Even more preferably, the source organism is selected from the list comprising Crepis biennis, Crepis aurea, Crepis conyzaefolia, Crepis intermédia, Crepis occidentalis, Crepis palaestina, Crepis vesicaria, Crepis xacintha, Euphorbia lagascae and Vernonia galamensis. No additional species are excluded.

In a particularly preferred embodiment of the present invention, the source organism is Crepis sp. which contains high levels of vernolic acid, such as Crepis palaestin, among others, or alternatively, Vernonia galamensis or Euphorbia lagascae.

Wherein the isolated nucleic acid molecule of the invention encodes an Δβ-epoxygenase or A9-epoxygenase enzyme or A12-epoxygenase or Al5-epoxygenase enzyme, or encodes at least one enzyme which is not involved in the direct epoxidation of arachidonic acid, and the nucleic acid molecule in question may be derived from any source producing said enzyme, including but not limited to yeast, mold, bacteria, insects, birds, mammals and plants. The nucleic acid molecule of the invention according to any of the foregoing embodiments may be DNA, such as a gene, cDNA molecule, RNA molecule or a synthetic oligonucleotide molecule, whether single-chain or double-stranded and independently of any characteristics of the secondary structure unless specifically mentioned.

Here reference to a " gene " is to be understood in its broader context and includes: (i) a classical genomic gene consisting of transcriptional and / or translation regulatory sequences and / or a coding region and / or untranslated sequences (ie, introns, 5 'and 3 'untranslated); or (ii) mRNA or cDNA corresponding to the coding regions (i.e., exons) and 5 'and 3' untranslated sequences of the gene. The term " gene " is also used to describe synthetic or fusion molecules encoding all or part of a functional product. The preferred epoxygenase genes of the present invention may be derived from a naturally occurring epoxygenase gene by conventional recombinant techniques. Generally, an epoxygenase gene can be mutagenized to produce single or multiple nucleotide substitutions, deletions and / or additions.

Nucleotide insert derivatives include 5 'and 3' terminal fusions as well as single and multiple nucleotide intra-sequence insertions. Nucleotide insert sequence variants are those in which one or more nucleotides are introduced at a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. 15

Deletion variants are characterized by the removal of one or more nucleotides from the sequence.

Nucleotide substitution variants are those wherein at least one nucleotide in the sequence has been removed and a different nucleotide introduced in its place. This replacement can be " silent " whereby the substitution does not change the amino acid defined by the codon. Alternatively, substituents are designed to change one amino acid to another similarly acting amino acid, or charge amino acid, polarity or similar hydrophobicity.

In the context of the present invention, the term " fatty acid epoxyigenase " should be understood to refer to any enzyme or functional equivalent or its enzymatically active derivatives which catalyze the biosynthesis of an epoxygenated fatty acid by conversion of a carbon bond of a fatty acid to an epoxy group and preferably by conversion of a carbon double bond of an unsaturated fatty acid to an epoxy group. Although not limiting the invention, a fatty acid epoxygenase can catalyze the biosynthesis of an epoxy fatty acid selected from the list comprising 12,13-epoxy-9-octadecenoic acid (vernolic acid), 12,13-epoxy-9,15- octadecadienoic acid, 15,16-epoxy-9,12-octadecadienoic acid, 9,10-epoxy-12-octadecenoic acid and 9,10-epoxy octadecanoic acid, among others. The term " epoxy ", " " epoxy group " and " epoxy residue " it will be known to those skilled in the art to refer to a three membered ring comprising two carbon atoms and one oxygen atom connected through single bonds as follows: 16 c

Accordingly, the term " epoxide " refers to compounds which comprise at least one epoxy group, as defined hereinbefore.

Those skilled in the art are aware that the fatty acid nomenclature is based on the length of the carbon chain and the position of the unsaturated carbon atoms within that carbon chain. Thus, fatty acids are designated using the shorthand notation:

Carbonototai: double bond binding of the double bond (Δ), in which the double bonds are cis, unless otherwise indicated. For example, palmitic acid (n-hexadecanoic acid) is a saturated fatty acid of 16 carbons (ie, 16: 0), oleic acid (octadecenoic acid) is an 18-carbon unsaturated fatty acid with a double bond between C- 9 and C-10 (ie, 18: 1Δ9) and linoleic acid (octadecadienoic acid) is an 18-carbon unsaturated fatty acid with two double bonds between C-9 and C-10 and between C-12 and C-13 ie, 18: 2 * 9'12).

However, in the present context an epoxygenase enzyme can catalyze the conversion of any carbon linkage to an epoxy group or alternatively, the conversion of any dipole into an unsaturated fatty acid substrate into an epoxy group. In this regard, it is well known to those skilled in the art that most mono-unsaturated fatty acids of higher organisms are 18-carbon unsaturated fatty acids (ie, 18: 1Δ9), while most polyunsaturated fatty acids 17 derived from organisms higher fatty acids are 18-carbon fatty acids with at least one of the double bonds therein located between C-9 and C-10. In addition, the bacteria also have C16 mono-unsaturated fatty acids. In addition, the epoxygenase of the present invention may act on more than one single fatty acid substrate molecule and accordingly the present invention is not limited by the nature of the substrate molecule on which the epoxygenase enzyme in question acts.

Preferably, the substrate molecule for the epoxygenase of the present invention is an unsaturated fatty acid containing at least one double bond.

In addition, the epoxygenases enzymes can act on any number of carbon atoms in any one substrate molecule. For example, they may be characterized as Δβ-epoxygenase, A9-epoxygenase, Δ12-epoxygenase or Î ± -epoxygenase enzymes, among others. Accordingly, the present invention is not limited by the position of the carbon atom on the substrate on which an epoxygenase enzyme can act.

As used herein, the term " Δβ-epoxygenase " should be understood to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ6 carbon bond of a fatty acid substrate into an Δ6 epoxy group and preferably catalyzes the conversion of the Δ6 double bond of at least one unsaturated fatty acid in an Δ6 epoxy group.

As used herein, the term " A9-epoxygenase " should be understood to refer to an epoxygenase enzyme which catalyzes the conversion of Δ9 carbon bond from a fatty acid substrate into an Δ9 epoxy group and preferably catalyzes the conversion of Δ9 double bond of at least one fatty acid unsaturated in an Δ9 epoxy group.

As used herein, the term " A12-epoxygenase " should be understood to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ12 carbon bond of a fatty acid substrate into an Δ12 epoxy group and preferably catalyzes the conversion of the Δ12 double bond of at least one unsaturated fatty acid in an Δ12 epoxy group.

As used herein, the term " al5-epoxygenase " should be understood to refer to an epoxygenase enzyme which catalyzes the conversion of the Δ15 carbon bond of a fatty acid substrate into an Δ15 epoxy group and preferably catalyzes the conversion of the Δ15 double bond of at least one unsaturated fatty acid in an Δ15 epoxy group. The present invention clearly extends to genetic sequences encoding all the epoxygenases enzymes listed above, among others.

In a preferred embodiment of the invention, the isolated nucleic acid molecule encodes a fatty acid epoxygenase enzyme which converts at least one carbon bond into palmitoleic acid (16: 1 to 9), oleic acid (18: 1Δ9), linoleic acid (18: 2Δ9'12), linolenic acid (18: 3Δ9'12'15), or arachidonic acid (20: 4a5'8'11: L4) in an epoxy bond. Preferably, the carbon bond is a carbon double bond.

More preferably, the isolated nucleic acid molecule of the invention encodes a fatty acid epoxygenase enzyme which converts at least one or both of the double bonds in linoleic acid to an epoxy group. According to this embodiment, an epoxygenase which converts both the double bonds Δ9 and Δ12 of linoleic acid into an epoxy group, can catalyze such conversions independently of each other, so that said epoxygenase is an A9-epoxygenase enzyme and / or al2-epoxygenase, as defined hereinbefore.

In an alternative preferred embodiment, the fatty acid epoxygenase of the present invention is an α2-epoxygenase, an Δ15-epoxygenase or an Δ9-epoxygenase, as defined hereinbefore.

More preferably, the fatty acid epoxyigenase of the invention is an Δ12-epoxygenase, as defined hereinbefore.

In a particularly preferred embodiment of the invention, there is provided an isolated nucleic acid molecule encoding the A12-epoxygenase linoleate, the enzyme which converts at least the Δ12 double bond of linoleic acid to a Δ12-εροχί group, thereby producing 12 , 13-epoxy-9-octadecenoic acid (vernolic acid).

Although not limiting the present invention, the preferred source of the A12-epoxygenase of the invention is a plant, in particular Crepis palaestin or Crepis sp. which is distinct from C. palaestina but contains high levels of vernolic acid, Vernonia galamensis or Euphorbia lagascae.

According to this embodiment, an Al2-epoxygenase can catalyze the conversion of palmitoleic acid to 9,10-epoxy-palmitic acid and / or the conversion of oleic acid to 9,10-epoxy stearic acid and / or conversion of linoleic acid in any one or more of 9,10-epoxy-12-octadecenoic acid or 12,13-epoxy-9-octadecenoic acid or 9,10,12,13-diepoxystearic acid and / or the conversion of linolenic acid to Any one or more of 9,10-epoxy-12,15-octadecadienoic acid or 12,13-epoxy-9,15-octadecadienoic acid or 15,16-epoxy-octadecadienoic acid or 9,10,12,13- diepoxy-15-octadecenoic acid or 9,10,15,16-diepoxy-12-octadecenoic acid or 12,13,15,16-diepoxy-9-octadecenoic acid or 9,10,12,13,15,16-triepoxy acid and / or the conversion of arachidonic acid to any one or more of 5,6-epoxy-8,11,14-tetracosatrienoic acid or 8,9-epoxy-5,11,14-tetracosatrienoic acid or 11,12 -epoxy-5,8,14-tetracatriatrienoic acid or 14.1 5-epoxy-5,8,11-tetracosatrienoic acid or OD KD LO 9-diepoxy-11,14-tetracosadienoic acid or 5,6,11,12-diepoxy-8,14-tetracosadienoic acid or 5,6,14,15 -depoxy-8,11-tetracosadienoic acid or 8,9,11,12-diepoxy-5,14-tetracosadienoic acid or 8,9,14,15-diepoxy-5,11-tetracosadienoic acid or 11,12,14,15 -depoxy-5,8-tetracosadienoic acid or 5,6,8,9,11,12-triepoxy-14-tetracosenoic acid or 5,6,8,9,14,15-triepoxy-11-tetracosenoic acid or 5,6 , 11,12,14,15-triepoxy-8-tetracosenoic acid or 8,9,11,12,14,15-triepoxy-5-tetracenoic acid, among others.

Those skilled in the art may be aware that not all substrates listed above may be derivable from a natural source, but nonetheless, they may be produced by chemical synthetic means. The conversion of both naturally occurring and chemically synthesized unsaturated fatty acids to epoxy fatty acids is within the scope of the present invention, the only requirement being that the nucleic acid molecule of the present invention, as described herein, encodes an enzyme or functional part of the which is capable of catalyzing said conversion.

According to the foregoing discussion, those skilled in the art will appreciate that a fatty acid epoxygenase enzyme may be a cytochrome 21 Ρ 450 dependent monooxygenase enzyme or a mixed function monooxygenase enzyme or alternatively a peroxide dependent peroxygen enzyme or similar enzyme , among others. However, the present invention is particularly directed to those epoxygenases enzymes which are mixed function monooxygenases and nucleic acid molecules encoding the same and their uses. Accordingly, it is particularly preferred that the nucleic acid molecule of the invention encodes a fatty acid epoxygenase which is a mixed function monooxygenase enzyme.

In the context of the present invention, the term " mixed function monooxygenase enzyme " should be understood to refer to any enzyme that catalyzes the epoxygenation of a carbon bond or carbon double bond in a fatty acid molecule, wherein said enzyme further comprises an amino acid sequence containing three histidine rich regions as follows: i) His- (Xaa) 3-4-His; (ii) His- (Xaa) 2-3-His-His; and (iii) His- (Xaa) 2-3-His-His, wherein His denotes histidine, Xaa designates any naturally occurring amino acid residue, as shown in Table 1, the (Xaa) 3-4 complete entity refers to to an amino acid sequence comprising three or four Xaa repeats, and the complete (Xaa) 2-3 refers to an amino acid sequence comprising two or three Xaa repeats. The term " mixed function monooxygenase enzyme " should be understood to refer to any enzyme comprising three of the histidine rich regions listed supra. 22

In the exemplification of the invention described herein, Applicants have demonstrated that the amino acid sequence of Crepis palaestin provided herein comprises an A12-epoxygenase enzyme which includes the characteristic amino acid sequence motifs of a monocyte fungus of mixed function as hereinbefore defined. The close identity of the amino acid sequence between the A12-epoxygenase enzyme of C. palaestina (SEQ ID NO: 2) and the amino acid sequences of polypeptides derived from a Crepis sp. and Vernonia galamensis as provided herein (SEQ ID Nos: 4 and 6), compared to the amino acid sequences of other mixed function monooxygenases, such as desaturases and hydroxylases, suggests that said amino acid sequences of Crepis sp. and V. galamensis are also fatty acid epoxygenases enzymes and may be A12-epoxygenase enzymes. In this regard, the amino acid sequence of Vernonia galamensis, exemplified herein, is a partial sequence comprising only a full histidine rich motif (ie, His-Arg-Asn-His-His) and a partial sequence of the first rich motif in histidine (ie, comprises the last two histidine residues of the His-Glu-Cys-Gly-His-His motif), because the corresponding nucleotide sequence encoding the same was amplified by polymerase chain reaction as a partial cDNA sequence, using a first primer for this first histidine rich motif and a second amplification primer designed for a region upstream of the third histidine rich motif (ie, His-Val-Met-His-His). In addition, the fact that the V. galamensis sequence was amplified using a specific primer for the first histidine rich motif indicates that the corresponding full size sequence would also comprise this motif. 23

Accordingly, in a particularly preferred embodiment, the nucleic acid molecule of the invention encodes a mixed function or a similar enzyme monooxygenase enzyme derived from Crepis spp., Including Crepes palaestina or alternatively, derived from Vernonia galamensis. According to this embodiment, it is still more preferred that the subject epoxygenase comprises at least one amino acid sequence containing three or more histidine rich regions as follows: (i) His-Glu-Cys-Gly- His-His (SEQ ID NO: 15); (ii) His-Arg-Asn-His-His (SEQ ID NO: 16); and (iii) His-Val-Met-His-His (SEQ ID NO: 17), or homologue, analogue or derivative thereof, wherein His designates histidine, Glu designates glutamate, Cys designates cysteine, Gly designates glycine, Arg refers to arginine, Asn designates asparagine, Val is valine, Met is methionine. The present invention clearly extends to epoxygenase genes derived from other species, including the epoxygenase genes derived from Chrysanthemum spp. and Euphorbia lagascae, among others.

In a preferred embodiment, although not limiting the present invention, the epoxygenase genes of other species which are encompassed by the present invention encode mixed function monooxygenase enzymes. The present invention further extends to isolated or recombinant polypeptides encoded by such genes and uses of said genes and polypeptides. The invention described in accordance with this embodiment does not encompass nucleic acid molecules encoding enzymatic activities other than epoxygenase activities as defined herein, in particular the A12-desaturase enzymes derived from Arabidopsis thaliana, Brassica juncea, Brassica napus or Glycine max, among others, which are known to contain similar histidine rich motifs.

In the present context, " homologues " of an amino acid sequence refers to those amino acid sequences or peptide sequences that are derived from polypeptides, enzymes or proteins of the present invention or alternatively substantially correspond to the amino acid sequences listed above, in spite of any substitutions, additions or deletions of naturally occurring amino acids therein.

For example, amino acids may be substituted for other amino acids having similar properties, for example, hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break α-helical structures or β-sheet structures, and so on. Alternatively, or in addition, the amino acids of a homologous amino acid sequence may be substituted for other amino acids having similar properties, for example, hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

The naturally occurring amino acid residues contemplated herein are described in Table 1.

A homologue of an amino acid sequence may be a synthetic peptide produced by any method known to those skilled in the art, such as by use of Fmoc chemistry. 25

Alternatively, a homologue of an amino acid sequence may be derived from a natural source, such as the same or another species such as the polypeptides, enzymes or proteins of the present invention. Preferred sources of amino acid sequence homologues listed above include any of the sources contemplated herein.

&Quot; analogs " of an amino acid sequence encompass those amino acid sequences which are substantially identical to the amino acid sequences listed above despite the occurrence there of any non-naturally occurring amino acid analogues therein.

The preferred non-naturally occurring amino acids contemplated herein are listed below in Table 2. The term " derived ", relative to an amino acid sequence, should be understood to refer to hereinafter amino acid sequence polypeptide mutants, fragments, or fusions listed above. Derivatives include modified amino acid sequences or peptides in which the ligands are linked to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules, such as radionuclides or fluorescent compounds. Also contemplated by the present invention are the glycosylated, fluorescent, acylated or alkylated forms of the subject peptides. In addition, the derivatives may comprise fragments or portions of an amino acid sequence disclosed herein and are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the sequences in question.

Methods for derivatizing peptides are well known in the art. 26

Substitutions encompass amino acid changes wherein one amino acid is substituted with a different naturally occurring or non-conventional amino acid residue. Such substitutions may be classified as " conservative ", in which case an amino acid residue is replaced with another naturally occurring amino acid of similar character, for example, Glye® Ala, Vale® Ilee® Leu, Asp > Glu , Lys > Arg, Asne < / RTI > Gln or Phe > Trp < - > Tyr.

Substitutions encompassed by the present invention may also be " non-conservative ", wherein an amino acid residue which is present in a repressor polypeptide is replaced with an amino acid having different properties, such as a naturally occurring amino acid from a different group ( eg, a charged or hydrophobic amino acid substituted with alanine), or alternatively, where a naturally occurring amino acid is substituted with an unconventional amino acid.

Amino acid substitutions are typically individual residues, but may be multiple residues, whether agglomerated or dispersed.

Amino acid deletions will generally be on the order of about 1-10 amino acid residues, while the insertions may be of any length. Deletions and insertions may be performed at the N-terminus, C-terminus or may be deletions or internal insertions. Generally, the insertions within the amino acid sequence will be smaller than amino- or carboxyl-terminal fusions and on the order of 1-4 amino acid residues. The present invention clearly extends to the subject isolated nucleic acid molecule when integrated into the genome of a cell as an addition to the endogenous cellular complement of epoxygenase genes. Alternatively, where the host cell does not normally encode enzymes required for epoxy fatty acid biosynthesis, the present invention extends to the subject isolated nucleic acid molecule when integrated into the genome of said cell as an addition to the endogenous cellular genome. TABLE 1

Amino Acid Three-letter abbreviation Symbol of a letter Alanine Ala Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F (continued)

Amino Acid Letter Abbreviation for a three letter symbol Prolina Pro P 28

Serine Ser S Threonine Thr T Tryptophan Trp Tyrosine Tyr Y Valine Val V Any amino acid as above Xaa X TABLE 2

Amino Acid Non-Conventional Code Amino Acid Amino Acid Amino Amino Amino Amino Amino Amino Amino Amino Amino Amino Amino Amino Amino Acid Mgabu LN-Methylarginine Amino Amino Acid Methyl Amino Acid Amino Amino Acid - LN-methylglutamine Nmgln carboxylate Norb LN-methylglutamic acid Nmglu cyclohexylalanine Chexa LN-methylhistidine Nmhis cyclopentylalanine Cpen LN-methylisoleucine Nmile D-alanine Dal LN-methyleucine Nmleu D-arginine Darg LN-methylisine Nmlys D-aspartic acid Dasp LN -methylmethionine Nmmet D-cysteine Dcys LN-methylnorleucine Nmnle D-glutamine Dgln LN-methylnorvaline Nmnva D-glutamic acid Dglu LN-methylornithine Nmorn (continued) Amino acid no Code Amino acid no Conventional conventional code D-histidine Dhis LN-methylphenylalanine Nmphe D-isoleucine Dile LN-methylproline Nmpro 29 D-leucine Dleu L- N-methylserine Nmser D-lysine Dlys LN-methylthreonine Nmthr D-methionine Dmet LN-methyltryptophan mtrp D-ornithine Dorn L-methyltyrosine Nmtyr D-phenylalanine Dphe LN-methylvaline Nmval D-proline Dpro L-methylethylglycine Nmetg D-serine Dser LN- methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr Î ± -methylaminoisobutyrate Maib D-valine Dvala-methyl-γ-aminobutyrate Mgabu Da-methylalanine Dmala a- methylcyclohexylalanine Mmaxa Da-methylarginine Dmax a-methylcyclopenten-alanine Mcpen Da-methylasparagine Dmasn α-methyl-α-naphthalalanine Manap Da-methylaspartate Dmasp α-methylpenicillamine Mpen Da-methylcysteine Dmcys N- (4-aminobutyl) glycine Nglu Da-methylglutamine Dmgln N- (2-aminoethyl) glycine Naeg Da-methylhistidine Dmhis N- (3-aminopropyl) glycine Norn Da-methylisoleucine Dmile N-amino-Î ± -methylbutyrate Nmaabu Da-methyleucine Dmleu α-napthylanine Anap Da-methylisine Dmlys N -benzylglycine Nphe D-methylmethioni (Carbamethylmethyl) glycine Nasm D-methylphenylalanine Dmp N- (2-carboxyethyl) glycine Nglu Da-methylproline Dmpro N- (carboxymethyl) glycine Nasp (continuation) Amino acid no Code Amino acid no Conventional conventional code Da-methylserine Dmser N-cyclobutylglycine Ncbut Da-methylthreonine Dmthr N-cycloheptylglycine Nchep Da-methyltryptophan Dmtrp N-cyclohexylglycine Nchex 30 Da-methyltyrosine Dmty N-cyclodecylglycine Ncdec Da-methylvaline Dmval N -cylododecylglycine Ncdod N-cycloalkylglycine Ncdn N-cyclooctylglycine Ncoct DN-methylarginine Dnmarg N-cyclopropylglycine Ncpro DN-methylasparagine Dnmasn N-cycloundecylglycine Ncund DN-methylaspartate Dnmasp N- (2,2-diphenylethyl) glycine Nbhm DN-methylcysteine Dnmcys N- 3,3-diphenylpropyl) glycine Nbhe DN-methylglutamine Dnmgln N- (3-guanidinopropyl) glycine Narg DN-methylglutamate Dnmglu N- (1-hydroxyethyl) glycine Nthr DN-methyl-h isethrin Nmis N- (hydroxyethyl) glycine Ndel N-methylisoleucine Dnmile N- (imidazolylethyl)) glycine Nhis DN-methyleucine Dnmleu N- (3-indolylethyl) glycine Nhtrp DN-methylisine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N N-methylcyclopentyl-alanine Nmcpen N-methylglycine Nala N-methylphenylalanine Dnmphe N-methylaminobutyrate Nmaib DN-methylproline Dnmpro N- (1-methylpropyl) glycine Nile DN-methylserine Dnmser N- (2-methylpropyl) glycine Nleu DN-methylthreonine Dnmthr DN-methyltryptophan Dnmtrp N- (1-methylethyl) glycine Nval Amino acid no Code Amino acid no Conventional conventional code DN-methyltyrosine Dnmtyr N-methyl napthalalanine Nmanap DN-methylvaline Dnmval N -methylpenicillamine Nmpen γ-aminobutyric acid Gabu N- (p-hydroxyphenyl) -glycine Nhtyr 31 Lt-butylglycine Tbug L-ethylglycine Etg L-homophenylalanine Hphe La-methylarginine Marg La-me Tylospartate Masp La-methylethyster Mcys La-methyl-histidine Mhis La-methylisoleucine Mile La-methyleucine Mleu La-methylmethionine Mmet La-methylnorvaline Mnva La-methylphenylalanine Mphe La-methylserine Mser La-methyltryptophan Mtrp La-methylvaline Mval N- (N- (2,2-diphenylethyl) carbamylmethyl) glycine Nnbhm 1-carboxy-1- (2,2-diphenylethylamino) cyclopropane Nmbc N- (thiomethyl) glycine Ncys penicillamine Pen La-methylalanine Mala La-methylasparagine Masn La- methyl-t-butylglycine methylethylmethylglycine methylethyl methylglycine Methylethylmethylglutamate Methylethylmethyl-homophenylalanine Mhphe N- (2-methylthioethyl) glycine Nmet La-methylisine Mlys La-methylnorleucine Mnle La-methylornithine Morn La-methylproline Mpro La-methylthreonine Mthr La -methylthiorosine Mtyr LN-methyl-homophenylalanine Nmhphe N- (N- (3,3-diphenylpropyl) -carbamylmethyl) glycine Nnbhe

A second aspect of the present invention provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID Nos: 1 or 3 or 5 or 19 or 20 or a complementary sequence thereof, or a homologue, analog thereof or derivative. 32

For the purposes of the nomenclature, the nucleotide sequence shown in SEQ ID NO: 1 is derived from Crepis palaestin and encodes the mixed function monooxygenase sequence or the mixed function monooxygenase type sequence shown in SEQ ID NO: 2. As exemplified herein, the amino acid sequence shown in SEQ ID NO: 2 has epoxygenase activity, more particularly Δ12-epoxygenase activity. The nucleotide sequence shown in SEQ ID NO: 3 corresponds to a cDNA derived from Crepis sp. different from C.

palaestin which contains high levels of vernolic acid. The amino acid sequence shown in SEQ ID NO: 4 corresponds to the amino acid sequence derived from the epoxygenase gene of

Crepis sp. provided in SEQ ID NO: 3. The nucleotide sequence shown in SEQ ID NO: 5 corresponds to amplified DNA derived from Vernonia galamensis using amplification primers derived from a consensus sequence of mixed function monooxygenases, including the sequences of the gene of epoxigenase from Crepis spp. of the invention. The amplified DNA comprises a partial sequence of the epoxygenase gene, which includes nucleotide sequences capable of encoding the His-Arg-Asn-His-His histidine rich motif which is characteristic of mixed function monooxygenases. The amino acid sequence shown in SEQ ID NO: 6 corresponds to the amino acid sequence derived from the epoxygenase gene of Vernonia galamensis provided in SEQ ID NO: 5. The nucleotide sequence shown in SEQ ID NO: 7 refers to partial sequence of a Crepis alpina acetylenase gene which has been used as a probe to isolate the nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 8 corresponds to the amino acid sequence derived from said partial sequence of the C. alpina acetylenase gene.

As used herein, the term " acetylenase " should be understood to refer to an enzyme which is capable of catalyzing the conversion of a carbon double bond to a fatty acid substrate molecule in a triple carbon bond or alternatively which is capable of catalyzing the formation of a triple carbon bond in a fatty acid molecule. The nucleotide sequence shown in SEQ ID NO: 18 corresponds to a degenerate amplification primer used to amplify putative sequences of the epoxygenase gene of

Euphorbia lagascae. In this regard, the nucleotide residues shown in SEQ ID NO: 18 are those recommended by

IUPAC-IUB Nomenclature Biochemical Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or

Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than

Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue. The nucleotide sequence shown in SEQ ID NO: 19 is derived from Euphorbia lagascae and encodes the cytochrome P-450 dependent monooxygenase sequence or the putative cytochrome P-450 dependent monooxygenase type sequence. The nucleotide sequence shown in SEQ ID NO: 20 is derived from Euphorbia lagascae and encodes a cytochrome P-450 dependent monooxygenase sequence or a putative cytochrome P-450 dependent monooxygenase type sequence. The present invention clearly extends to the genomic gene equivalents of the cDNA molecules exemplified in any one of SEQ ID Nos: 1, 3, 5, 19 or 20.

In a very particularly preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 19 or 20 or a genomic gene equivalent of said nucleotide sequence or a homologue, analogue or derivative thereof.

For the present purpose, " homologues " of a nucleotide sequence, is to be understood to refer to an isolated nucleic acid molecule that is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, despite occurrence within said sequence, of one or more more substitutions, insertions, deletions or nucleotide rearrangements. " Analogs " of a nucleotide sequence presented herein should be understood to refer to an isolated nucleic acid molecule that is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, despite the occurrence of any non-nucleotide constituents not usually present in said isolated nucleic acid molecule, for example, carbohydrates, radiochemicals, including radionucleotides, reporter molecules, such as but not limited to DIG, alkaline phosphatase or horseradish peroxidase, among others. 35 " Derivatives " of a nucleotide sequence presented herein should be understood to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a portion thereof.

Generally, homologs, analogs or derivatives of the nucleic acid molecule of the invention are produced by synthetic means or alternatively derived from naturally occurring sources. For example, the nucleotide sequence of the present invention may be mutagenized to produce single or multiple nucleotide substitutions, deletions and / or insertions as indicated supra.

In one embodiment of the invention, preferred homologues, analogs or derivatives of the nucleotide sequences set forth in any one of SEQ ID Nos: 1,3,5,19 or 20 or the complementary sequences thereof encode immunologically active or enzymatically active polypeptides.

As used herein, the term " immunologically active " should be understood to refer to the ability of a polypeptide molecule to induce an immune response in a mammal, in particular a sufficient immune response to produce an antibody molecule, such as, but not limited to, an IgM or IgG or serum molecule complete antibody containing said antibody molecule. The term " immunologically active " also extends to the ability of a polypeptide to induce a sufficient immune response for the production of monoclonal antibodies, synthetic Fab fragments of an antibody molecule, single chain antibody molecule or other immunoperative molecule. 36

As used herein, the term " enzymatically active " should be understood to refer to the ability of a polypeptide molecule to catalyze an enzymatic reaction, in particular an enzymatic reaction comprising the epoxygenation of a carbon bond in a fatty acid substrate molecule. More particularly, while not limiting the invention, the term " enzymatically active " may also refer to the ability of a polypeptide molecule to catalyze the epoxygenation of Δ-9 or Δ-12 in a fatty acid substrate molecule, such as linoleic acid or vernolic acid.

In an alternative embodiment, a preferred homologue, analogue or derivative of the nucleotide sequence set forth in any one of SEQ ID Nos: 1 or 3 or 5, or a complementary sequence thereof, comprises a nucleotide sequence that is at least 65 % identical to at least 20 contiguous nucleotides present therein, other than a nucleotide sequence encoding an acetylenase enzyme of Crepis sp.

More preferably, the percentage identity to any one of SEQ ID Nos: 1 or 3 or 5 is at least about 85%. Even more preferably, a homologue, analogue or derivative of SEQ ID Nos: 1 or 3 or 5 is at least about 90%, and even more preferably at least about 95% identical to , at least 100 or 250 or 500 or 1000 contiguous nucleotides present therein. The percent identity to SEQ ID NOS: 19 or 20, or their complementary sequences, is at least about 75%, relative to at least about 200 contiguous nucleotides, even more preferably at least, about 80%, still more preferably at least about 90%, and still more preferably at least about 95%, including at least about 99% identity. Nucleotide sequences 37 that are at least 65% relative to at least about 400 contiguous nucleotides in SEQ ID Nos: 19 or 20 are also within the scope of the invention.

Here reference to a percentage identity or percentage similarity between two or more nucleotides or amino acid sequences should be understood to refer to the number of identical or similar residues in an alignment of nucleotide or amino acid sequences as determined using any standard algorithm known from skilled in the art. In particular, nucleotide and / or amino acid sequence identities and similarities can be calculated using the Gap program, which uses the algorithm of Needleman and Wunsch (1970) to maximize the number of suitable residues and minimize the number of sequence gaps. The Gap program is part of the Sequence and Analysis Software Package of Computer Genetics Group Inc., University Research Park, Madison, Wisconsin, USA (Devereux et al., 1984).

In a further alternative embodiment, a preferred homologue, analogue or derivative of the nucleotide sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 19 or 20 or a complementary sequence thereof, hybridizes under conditions of at least low stringency with at least 20 contiguous nucleotides derived from said sequence.

More preferably, the hybridization stringency is at least moderate stringency, even more preferably at least high stringency.

For the purposes of setting the level of stringency, those skilled in the art will be aware that various different hybridization conditions may be utilized. For example, a low stringency may comprise hybridization and / or lavage performed in buffer 6 x SSC, 0.1% (w / v) SDS at 28 ° C. A moderate stringency may comprise hybridization and / or lavage performed in 2 x SSC buffer, 0.1% (w / v) SDS at a temperature in the range of 45øC to 65øC. A high stringency may comprise hybridization and / or washing performed in 0.1 x SSC buffer, 0.1% (w / v) SDS at a temperature of at least 65 ° C.

Generally, the stringency is increased by reducing the concentration of the SSC buffer, and / or increasing the concentration of SDS in the hybridization buffer or wash buffer and / or increasing the temperature at which hybridization and / or washing is performed. Conditions for hybridizations and washes are well understood by one skilled in the art. For the purposes of clarifying the parameters affecting the hybridization between nucleic acid molecules, reference may conveniently be made to pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is hereby incorporated by reference.

The isolated nucleic acid molecules disclosed herein may be used to isolate or identify homologs, analogs or derivatives thereof from other types of cells, tissues or organs, or from cells, tissues, organs of other species using any of a variety of means known to those skilled in the art.

For example, genomic DNA or mRNA or cDNA may be contacted under conditions of at least low stringency hybridization or equivalent with an effective hybridization amount of an isolated nucleic acid molecule comprising the nucleotide sequence shown in any one of SEQ ID Nos: 1, 3, 5, 19 or 20 or a complementary sequence thereof, or a functional part thereof, and the hybridization detected using detection means. 39

The detection means may be a reporter molecule capable of giving an identifiable signal (e.g., a radioisotope, such as 32 P or 35 S, or a biotinylated molecule) covalently linked to the isolated nucleic acid molecule of the invention.

In an alternative method, the detection means is any known format of the polymerase chain reaction (PCR). According to this method, degenerate pools of " starter molecules " of nucleic acid about 15-50 nucleotides in length based on the nucleotide sequences disclosed in SEQ ID Nos: 1, 3, 5, 19 or 20 or a complementary sequence thereof. Homologs, analogs or derivatives (ie, " template molecule ") are hybridized with two of said primer molecules, so that a first hybrid primer with a region in a template molecule chain and a second hybrid primer with a complementary sequence , wherein the first and second primers are not hybridized within the same or overlapping regions of the template molecule and wherein each primer is positioned in a 5 'to 3' orientation relative to the position where the other primer is hybridized to the opposite strand. Copies of the template nucleic acid molecule specific are enzymatically amplified in a polymerase chain reaction, a technique that is well known to a person skilled in the art.

The initiator molecules may comprise any naturally-occurring nucleotide residues (i.e., adenine, cytidine, guanine, thymidine) and / or comprise inosine or analogs or functional derivatives thereof, capable of being incorporated into a polynucleotide molecule. Nucleic acid primer molecules may also be contained in an aqueous mixture of other nucleic acid primer molecules or in substantially pure form. The detected sequence may be in a recombinant form, in a viral particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the related genetic sequence is from another plant species.

A third aspect of the present invention provides an isolated nucleic acid molecule encoding the amino acid sequence shown in any one of SEQ ID Nos: 2 or 4 or 6 or a homologue, analogue or derivative thereof.

In one embodiment contemplated herein, preferred homologues, analogs or derivatives of the amino acid sequences shown in SEQ ID Nos. 2, 4, or 6 are immunologically active or enzymatically active polypeptides as defined supra.

In an alternative embodiment of the invention, preferred homologues, analogs or derivatives of the amino acid sequence set forth in any one of SEQ ID Nos: 2, 4 or 6 comprise an amino acid sequence that is at least 60% identical to the same , and different from an acetylenase polypeptide of Crepis sp. More preferably, the homologues, analogs or derivatives of SEQ ID Nos: 2 or 4 or 6 which are encompassed by the present invention are at least about 85% identical, even more preferably at least about of 90% identical and even more preferably at least about 95% identical, and still more preferably at least about 99% -100% identical thereto.

Homologs, analogs or derivatives of any of SEQ ID Nos: 2 or 4 or 6 may further comprise a histidine rich region as defined supra. Even more preferably, the subject epoxygenase comprises at least one amino acid sequence containing three or more histidine rich regions as follows: (i) His-Glu-Cys-Gly-His-His (SEQ ID NO: ID No. 15); (ii) His-Arg-Asn-His-His (SEQ ID NO: 16); and (iii) His-Val-Met-His-His (SEQ ID NO: 17), or a homologue, analogue or derivative thereof. The invention described in accordance with this alternative embodiment does not encompass the A12-desaturases enzymes derived from Arabidopsis thaliana, Brassica juncea, Brassica napus or Glycine max, among others. The isolated nucleic acid molecule of the present invention is useful for the development of genetic constructs comprising a sense molecule, wherein said genetic constructs are designed for expression in a cell which does not normally express said nucleic acid molecule or overexpression of said molecule of nucleic acid in a cell which normally expresses said nucleic acid molecule.

Accordingly, a further aspect of the invention provides a genetic construct comprising a sense molecule that is operably linked to a promoter sequence. The term " sense molecule " as used herein should be understood to refer to an isolated nucleic acid molecule which encodes or is complementary to an isolated nucleic acid molecule encoding a fatty acid epoxygenase, wherein said nucleic acid molecule is provided in a format suitable for to produce a recombinant polypeptide when said sense molecule is introduced into a host cell by transfection or transformation.

Those skilled in the art will be aware that a genetic construct can be used to " transfect " a cell, which in this case is introduced into said cell without integration into the cell's genome. Alternatively, a genetic construct can be used to " transform " a cell, which in this case is stably integrated into the genome of said cell.

A sense molecule corresponding to a sequence of the fatty acid epoxygenase gene or a homologue, analogue or derivative thereof may be introduced into a cell using any known method for transfection or transformation of said cell. In which a cell is transformed by the genetic construct of the invention, an entire organism can be regenerated from a single transformed cell, using any method known to those skilled in the art.

Thus, the epoxygenase genes described herein can be used to develop individual cells or whole organisms which synthesize epoxy fatty acids not normally produced by wild or naturally occurring organisms belonging to the same genera or species as the genera or species from which they are derived the transfected or transformed cell, or to raise the levels of these fatty acids above the levels normally found in such wild or naturally occurring organisms.

In an alternative preferred embodiment, the isolated nucleic acid molecule of the invention is capable of reducing the level of epoxy fatty acids in a cell, when expressed therein, in the antisense orientation or as a ribozyme or co-suppression molecule , under the control of a suitable promoter sequence. Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar gene, is introduced into the cell. The present invention also extends to the use of co-suppression to inhibit the expression of an epoxygenase gene as described herein.

In the context of the present invention, an antisense molecule is an RNA molecule that is transcribed from the complementary strand of a nuclear gene, which is normally transcribed to produce a " sense " mRNA molecule " capable of being translated into a polypeptide. The antisense molecule is thus complementary to the sense mRNA, or a part thereof. While not limiting the mode of action of the antisense molecules of the present invention in any particular mechanism, the antisense RNA molecule has the ability to form a double stranded mRNA by base pairing with sense mRNA, which can prevent the translation of sense mRNA and subsequent synthesis of a polypeptide gene product.

Ribozymes are synthetic RNA molecules which comprise a hybridization region complementary to two regions, each of at least 5 contiguous nucleotide bases in the sense sense mRNA. In addition, the ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA. A complete description of ribozymes function is presented by Haseloff and Gerlach (1988) and contained in International Patent Application No. WO89 / 05852. The present invention extends to ribozymes which target a sense mRNA encoding an epoxygenase polypeptide described herein, thereby hybridizing to said sense mRNA and cleaving it, so as to be no longer capable of being translated into synthesize a functional polypeptide product.

According to this embodiment, the present invention provides a ribozyme or antisense molecule comprising a sequence of contiguous nucleotide bases which are capable of forming a hydrogen bonded complex with a sense mRNA, encoding an epoxygenase described herein to reduce the translation of said mRNA. Although preferred antisense and / or ribozyme molecules hybridize with at least about 10 to 20 nucleotides of the target molecule, the present invention extends to molecules capable of hybridizing with at least about 50-100 nucleotide bases in length, or a molecule capable of hybridizing to a full-size or substantially full-size epoxygenase mRNA. It is understood in the art that certain modifications, including nucleotide substitutions, among others, may be performed on the antisense and / or ribozyme molecules of the present invention without destroying the efficacy of said molecules in inhibiting the expression of the epoxygenase gene. It is thus within the scope of the present invention to include all variants, homologues, analogs or fragments of the nucleotide sequence of said gene encoding the same, the only requirement being that said variant of the nucleotide sequence, when transcribed , produces an antisense and / or ribozyme molecule which is capable of hybridizing to said sense mRNA molecule. The present invention extends to genetic constructs designed to facilitate the expression of a sense molecule, an antisense molecule, ribozyme molecule or co-suppression molecule which is capable of altering the level of epoxy fatty acids in a cell .

In a particularly preferred embodiment, the sense molecule, antisense molecule, ribozyme molecule, co-suppression molecule, or molecule directed to the gene that is capable of altering the epoxy fatty acid composition of a plant-derived cell or other organism, comprises a nucleotide sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 19 or 20, and more preferably in any one of SEQ ID Nos: 1 or 3 or 5 and, even more preferably in SEQ ID NO: 1 or a complementary, homologous, analogous or derivative thereof.

Those skilled in the art will also be aware that expression of a sense, antisense, ribozyme or co-suppression molecule may require that the nucleic acid molecule of the invention be put into operable linkage with a promoter sequence. The choice of the promoter for the present object may vary depending upon the level of expression of the required sense molecule and / or the species from which the host cell is derived and / or the tissue specificity or developmental specificity of the expression of the sense molecule which is required.

Here reference to a " promoter " should be understood in its broader context and includes the transcriptional regulatory sequences of a classical eukaryotic genomic gene, including the TATA box which is required for initiation of the rigorous transcription, with or without a sequence of the CCAAT box and additional regulatory elements (ie, upstream activating sequences, enhancers and silencers) that alter gene expression in response to developmental and / or external stimuli, or in a tissue-specific manner. In the context of the present invention, the term " promoter " also includes the transcriptional regulatory sequences of a classical prokaryotic gene, which in this case may include a -35 box sequence and / or box-10 transcriptional regulatory sequences.

In the present context, the term " promoter " is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of said sense molecule in a cell. Preferred promoters may contain additional copies of one or more specific regulatory elements to further enhance expression of the sense molecule and / or to alter the spatial expression and / or time expression of said sense molecule. For example, copper responsive regulatory elements may be placed adjacent to a heterologous promoter sequence that directs the expression of a sense molecule to impart copper-inducible expression.

By placing a sense, antisense, ribozyme or co-suppression molecule under the regulatory control of a promoter sequence, it means positioning said molecule so that the expression is controlled by the promoter sequence. A promoter is generally, but not necessarily, positioned upstream or downstream of a regulatory nucleic acid molecule. In addition, regulatory elements comprising a promoter are generally positioned within the 2 kb range of the transcription start site of the sense, antisense, ribozyme or co-suppression molecule, or chimeric gene, comprising the same. In constructing heterologous structural gene / promoter combinations it is generally preferred to position the promoter at a distance from the gene transcription start site which is approximately the same as the distance between that promoter and the control gene in its natural context, the gene from which the promoter is derived. It is known in the art that some variation in this distance can be accommodated without loss of promoter function. Likewise, the preferred positioning of a regulatory sequence element relative to a heterologous gene to be placed under its control is defined by positioning the element in its natural context, i. the genes from which the promoter is derived. Again, as is known in the art, some variation in this distance may also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include promoters derived from virus, yeast, fungal, bacterial, insect, bird, mammalian and plant genes that are capable of functioning in isolated cells or whole regenerated organisms. The promoter may regulate the expression of the sense, antisense, ribozyme or co-suppression molecule constitutively, or differentially, relative to the tissue in which the expression occurs, or, in the development phase in which expression occurs, or in response to external stimuli, such as physiological stresses, pathogens, or metal ions, among others.

Examples of promoters include the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Arabidopsis thaliana SSU gene promoter, seed-specific napin promoter, P32 promoter, BK5-T imm promoter, lac promoter, promoter tac promoters, lambda XL or XR phage promoters, CMV promoter (US Patent No. 5168062), T7 promoter, lacUV5 promoter, SV40 early promoter (U.S. Patent No. 5,116,827), late SV40 promoter (U.S. Patent No. 5,116,827), adenovirus, PIO baculovirus promoter or polyhedrin (U.S. Patent Nos. 5,264,041, 5,264,267, 5,266,317, 4,750,501 and 5,169,884), and the like. In addition to the specific promoters identified herein, cell promoters for so-called housekeeping genes are useful.

Preferred promoters according to this embodiment are those promoters which are capable of functioning in yeast, mold or plant cells. More preferably, promoters suitable for use according to this embodiment are capable of functioning in cells derived from oleaginous yeasts, oleaginous molds or oleaginous seed cultivars, such as linen sold under the trademark Linola® (referred to herein flax, safflower, soya, linseed, sesame, cottonseed, peanut, olive or palm oil, among others. Linola® is a registered trademark of the Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia.

In a more preferred embodiment, the promoter may be derived from a genomic clone encoding an epoxygenase enzyme, preferably derived from the genomic gene eigenvirus gene gene derived from Chrysanthemum spp. Crepis spp., Including C. palaestin or other Crepis sp., Euphorbia lagascae or Vernonia galamensis, which are referred to herein.

In a more preferred embodiment, the promoter can be derived from a highly expressed seed gene, such as the napin gene, among others. The genetic construct of the invention may further comprise a terminator sequence and be introduced into a suitable host cell where it is capable of being expressed to produce a recombinant polypeptide gene product or alternatively, a ribozyme or antisense molecule. The term " terminator " refers to a DNA sequence at the end of a transcription unit that signals the termination of transcription. Terminators are 3 'untranslated DNA sequences containing a polyadenylation signal that facilitates the addition of polyadenylate sequences to the 3' end of a primary transcript. Active terminators in cells derived from viruses, yeasts, molds, bacteria, insects, birds, mammals and plants are known and described in the literature. They can be isolated from bacteria, fungi, viruses, animals and / or plants.

Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, cauliflower mosaic virus (CaMV) 35S gene terminator, the gene terminator Zein mays, the Rubisco small subunit gene (SSU) terminator sequences, starch clover virus (SCSV) gene sequence terminators, any rho-independent E. coli terminator, among others.

Those skilled in the art will be aware of additional promoter sequences and terminator sequences that may be suitable for use in the practice of the invention. Such sequences can readily be used without any superfluous experimentation.

The genetic constructs of the invention may further comprise an origin of replication sequence which is required for replication in a specific cell type, for example, a bacterial cell, when said genetic construct is required to be maintained as an episomal genetic element (eg , plasmid or cosmid molecule) in said cell.

Preferred origins of replication include, but are not limited to, the origins of fl-ori and colE110 replication. The genetic construct may further comprise a selection marker gene (s) that is functional in a cell in which said genetic construct is introduced.

As used herein, the term " selection marker gene " includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and / or selection of cells that are transfected or transformed with a genetic construct of the invention or a derivative thereof.

Suitable selection marker genes contemplated herein include ampicillin resistance (Ampr), tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr), phosphinothricin resistance gene, neomycin phosphotransferase (nptll) gene, , hygromycin resistance gene, β-glucuronidase gene (GUS), chloramphenicol acetyltransferase (CAT) gene, and luciferase gene, among others.

A further aspect of the present invention provides a transfected or transformed whole cell, tissue, organ or organism expressing a recombinant epoxygenase polypeptide or a ribozyme, antisense or co-suppression molecule as described herein, or a homologue, analog or derivative.

Preferably, the isolated nucleic acid molecule is contained within a genetic construct as described herein. The genetic construct of the present invention may be introduced into a cell by various techniques known to those skilled in the art. The technique used may vary depending upon the successful techniques known for that particular organism.

Means for introducing recombinant DNA into bacterial cells, yeast cells, or cells or tissues of plants, insects, fungi (including mold), birds or mammals include, but are not limited to, transformation using CaCl 2 and its variations, in particular The method was described by Hanahan (1983), direct incorporation of DNA into protoplasts (Krens et al., 1982 / Paszkowski et al., 1984), PEG-mediated incorporation into protoplasts (Armstrong et al., 1990), microporticle bombardment, electroporation Fromm et al., 1985), microinjection of DNA (Crossway et al., 1986), bombardment of microparticles of tissue explants or cells (Christou et al., 1988; Sanford, 1988), vacuum infiltration of tissue with nucleic acid , or in the case of plants, Agrobacterium T-DNA mediated transfer to the plant tissue as essentially described by An et al. (1985), Herrera-Estrella et al. (1983a, 1983b, 1985).

For the bombardment of cell microparticles, a microparticle is propelled into a cell to produce a transformed cell. Any suitable ballistic cell transformation methodology and device may be used in carrying out the present invention. Exemplary devices and processes are disclosed by Stomp et al. (U.S. Patent No. 5122466) and Sanford and Wolf (U.S. Patent No. 4,945050). By using ballistic transformation processes, the genetic construct may incorporate a plasmid capable of replication in the cell to be transformed. 52

Examples of microparticles suitable for use in such systems include gold beads of 1 to 5 μm. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

In a particularly preferred embodiment, wherein the genetic construct comprises a " sense " molecule, it is particularly preferred that the recombinant epoxygenase polypeptide produced therein is enzymatically active.

Alternatively, an entire organism can be regenerated from the transformed cell, wherein the cell is derived from a multicellular organism and where relevant technology is available, according to procedures well known in the art.

Those skilled in the art will also be aware of methods for transforming, regenerating and propagating other cell types, such as those of fungi.

In the case of plants, plant tissue capable of subsequent clonal propagation may be transformed either by organogenesis or embryogenesis, with a genetic construct of the present invention and thereafter regenerated an entire plant. The particular tissue chosen will vary depending on the clonal propagation systems available, and more suitable, for the particular species to be transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (eg, apical meristem, axillary shoots and root meristems) and induced meristem tissue (eg, cotyledon meristem and hypocotyl meristem). The term " organogenesis " as used herein means a process by which shoots and roots are sequentially developed from meristematic centers. The term " embryogenesis " as used herein means a process whereby shoots and roots are co-developed in a concerted (not sequentially) form, either from somatic cells or gametes.

Transformed regenerated plants can be propagated through a variety of media, such as through clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be self-crosslinked to produce a second generation homozygous transformant (or T2), and the T2 plants propagated further through classical breeding techniques.

The regenerated transformed organisms contemplated herein may have a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed root grafted with an unprocessed shoot).

A further aspect of the invention provides a method of altering the level of epoxy fatty acids in a cell, tissue, organ or organism, said method comprising the expression of a sense, antisense, ribozyme or co-suppression molecule as described herein, in said cell for a period of time and under conditions sufficient to be increased or reduced the level of fatty acids present therein. 54

In a preferred embodiment, the method comprises the first additional step of transforming the cell, tissue, organ or organism to the sense, antisense, ribozyme or co-suppression molecule.

As discussed above, the isolated nucleic acid molecule may be contained within a genetic construct.

According to this embodiment, the cell, organ, tissue or organism in which the sense, anti-sense, ribozyme or co-suppression molecule in question is expressed can be derived from a bacterium, yeast, fungus (including a mold), insect, plant, bird or mammal.

Because a recombinant epoxygenase polypeptide can be produced in the regenerated as well as ex vivo transformant, an alternative preferred embodiment of the present invention provides a method of producing an enzymatically active recombinant epoxygenase polypeptide in a cell, said method comprising the steps of: (i) producing a genetic construct comprising the cDNA or the epoxygenase genomic genetic sequence of the invention operably placed under the control of a promoter capable of conferring expression on said gene sequence in said cell, and optionally an expression enhancing element; (ii) transforming said genetic construct into said cell; and (iii) selection of transformants expressing at a high level, the epoxigenase encoded by the genetic sequence.

A particularly preferred embodiment of the present invention provides a method of producing an enzymatically active recombinant epoxygenase polypeptide in a transgenic plant comprising the steps of: (i) producing a genetic construct comprising the cDNA or the epoxygenase genomic genetic sequence of the invention operatively placed under the control of a specific seed promoter, and optionally an expression enhancement element, wherein said gene sequence is also placed upstream of a transcription terminator sequence; (ii) transforming said genetic construct into a cell or tissue of said plant; and (iii) selection of the transformants expressing at a high seed level, the epoxigenase encoded by the genetic sequence.

In a more particularly preferred embodiment the plant is a kind of oleaginous seed which normally produces significant levels of linoleic acid, for example Linola® flax, oilseed rape, sunflower, safflower, soybean, linseed, sesame, cottonseed , peanuts, olives or olive oil, among others.

In an even more particularly preferred embodiment the plant is a kind of oleaginous seed which normally produces significant levels of linoleic acid, for example Linola®, sunflower or safflower, among others.

The enzymatically active recombinant epoxygenases described herein are particularly useful for the production of epoxygen fatty acids from unsaturated fatty acid substrates. The present invention particularly contemplates the production of specific epoxygen fatty acids in cells or regenerated transformed organisms that do not normally produce this specific epoxygen fatty acid.

Accordingly, a further aspect of the invention provides a method of producing an epoxygenated fatty acid in a cell, tissue, organ or organism, said method comprising incubating a cell, tissue, organ or organism expressing a recombinant epoxygenase enzymatically active compound of the present invention with a fatty acid substrate molecule, preferably an unsaturated fatty acid substrate molecule, for a period of time and under conditions sufficient for at least one carbon bond of said substrate to be converted into an epoxy group.

In an alternative embodiment, the method in question further comprises the first additional step of transforming or transfecting the cell, tissue, organ or organism with a nucleic acid molecule encoding said recombinant epoxygenase or a homologue, analogue or derivative thereof, such as described hereinbefore. As discussed above, the isolated nucleic acid molecule may be contained within a genetic construct.

According to this embodiment, the cell, organ, tissue or organism in which the subject epoxygenase is expressed is derived from bacterium, yeast, fungus (including a mold), insect, plant, bird or mammal. More preferably, the cell, organ, tissue or organism is derived from a yeast, plant or fungus, even more preferably from a yeast or oleaginous plant or fungus, or from an oil seed plant which is not normally expressed the recombinant epoxigenase of the invention. 57

Among the main economical oilseed plants, contemplated herein, the high linoleic genotypes of flax, sunflower, corn and safflower are preferred targets. Soybean and rape seed are alternate targets but are less suitable for maximum synthesis of fatty acid epoxy because of their lower levels of linoleic acid substrate and the presence of an active A15 desaturase competing with the epoxygenase for the substrate of linoleic acid.

An alternative embodiment is the transformation of Linola® (= linolenic acid flax) with the epoxygenase of the invention. Linola® linen normally contains about 70% linoleic acid, and very little (< 2%) is subsequently converted to linolenic acid by A15-desaturase (Green, 1986).

Preferred unsaturated fatty acid substrates contemplated herein include, but are not limited to, palmitoleic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid, among others.

In plant species which naturally contain high levels of vernolic acid, the β12-epoxyigenase present can be very efficient in the epoxidation of linoleic acid. Accordingly, the present invention particularly contemplates the expression of recombinant A12-epoxygenase derived from Euphorbia lagascae, Vernonia spp. and Crepis spp. at elevated levels in transgenic oilseeds during seed oil synthesis, to thereby produce high levels of vernolic acid.

Accordingly, linoleic acid is a particularly preferred substrate, in accordance with this embodiment of the invention. No additional substrates are excluded. 58

The products of the substrate molecules listed above are readily determined by those skilled in the art without undue experimentation. Particularly preferred epoxy fatty acids produced according to the present invention include 12,13-epoxy-9-octadecenoic acid (vernolic acid) and 12,13-epoxy-9,15-octadecadienoic acid, among others.

Conditions for incubating cells, organs, tissues or organisms expressing recombinant epoxygenase in the presence of the substrate molecule will vary depending on at least the incorporation of the substrate into the cell, tissue, organ or organism, and the affinity of epoxygenase to the substrate molecule in the particular environment selected. Optimal conditions may be readily determined by those skilled in the relevant art. The present invention clearly extends to the isolated oil containing the epoxy fatty acids, and / or to the isolated epoxy fatty acid itself, produced as described herein, and to any derivatives thereof, for example, coatings, resins, glues, plastics, surfactants and lubricants, among others.

Applicants have further shown that mixed function monooxygenases (MMOs) performing catalytic functions, such as desaturation, acetylenation, hydroxylation and / or epoxygenation, form a family of genes that share considerable nucleotide and amino acid sequence similarity. For example, desaturase, acetylenase, hydroxylase and / or epoxygenase enzymes which act on substrate molecules having a chain length and position of either (or) similar carbon double bond (s) (if present) are closely related to each other than enzymes acting on other substrates, and may be considered a " family ". 59

Without being bound by any theory or mode of action, sequence similarity between the members of any gene family is based on the identity of the substrate involved and the biochemical similarity of the reaction events occurring at the target carbon bond during the reaction suggesting that the divergent sequences within a family may comprise catalytic determinants or at least a functional part thereof which contributes to the specific catalytic properties of the family members.

An example of a family is the desaturase, acetylenase, hydroxylase and / or epoxygenase enzymes which catalyze the desaturation, acetylenation, hydroxylation and / or epoxygenation, respectively, of the Δ12 position of linoleic acid (hereinafter referred to as the " C18 family & ΜΜ0 "). The present inventors compared the nucleotide and amino acid sequences of members of the A12-MM0 C18 family to determine their divergent regions which potentially comprise the determinants of alternative catalytic functions at the Δ12 position (referred to hereinafter as " putative catalytic determinants ").

In addition, the presence of such families of fatty acid modifying MMOs is contemplated relative to other lengths of the fatty acid chain and double bond positions. For example, C18A15 desaturase is contemplated to belong to a family of related enzymes capable of desaturation, acetylenation, hydroxylation and / or epoxidation of the Δ15 position on C18 fatty acid substrates, the C18Δ15-ΜΜ0 family.

Through the production of synthetic genes in which these catalytic determinants have been exchanged (referred to as " domain exchange "), it is possible to convert genes encoding a catalytic function into them, encoding alternative catalytic functions. For example, the Δ 12 epoxygenase of the present invention can be converted to an Δ12 acetylenase by substituting portions of its C-terminal and N-terminal sequences with the equivalent domains of Δ12 acetylenase from Crepis alpina. Likewise, reverse domain exchange can also be performed.

As a further refinement, such changes in catalytic function may also be effected by making specific changes (eg, addition, substitution or deletion) only in those amino acids within each domain that are critical for determining the relevant catalytic function (such as directed mutagenesis).

Accordingly, a further aspect of the present invention contemplates a synthetic fatty acid gene comprising a nucleotide sequence derived from an epoxygenase gene as described herein, wherein said synthetic fatty acid gene encodes a polypeptide having epoxygenase activity or acetylenase or hydroxylase or desaturase, wherein said polypeptide comprises either an amino acid sequence that differs from an epoxygenase or acetylenase enzyme or naturally occurring hydroxylase or desaturase, or said polypeptide exhibits catalytic properties which are different from an enzyme epoxygenase or acetylenase or hydroxylase or naturally occurring desaturase, or said polypeptide comprises an amino acid sequence that is at least about 60% identical to a part of SEQ ID NO: 2 or 4 or 6 or homologue, analogue or derivative of said part.

Preferably, the synthetic fatty acid gene of the invention is derived from a Δ12 epoxygenase gene. 61

In one embodiment, the synthetic fatty acid gene of the invention encodes a fusion polypeptide in which the N-terminal and / or C-terminal amino acids of any one of SEQ ID Nos: 2 or 4 or 6 are screened, by amino acid sequences of a different member of the same family.

In a particularly preferred embodiment, the N-terminal and / or C-terminal amino acids of SEQ ID NO: 2 or 4 or 6 are replaced by corresponding regions of the acetylenase, desaturase or hydroxylase polypeptides shown in Figure 2. In a more preferred embodiment Preferably, at least about 30 amino acid residues of the N-terminal and / or C-terminus regions of any one of SEQ ID Nos: 2 or 4 or 6 are gratefully substituted by the corresponding regions of the acetylenase, desaturase or polypeptide polypeptides. hydroxylase compounds shown in Figure 2.

In an alternative embodiment, the synthetic fatty acid gene of the invention encodes a fusion polypeptide in which the N-terminal and / or C-terminal amino acids of a fatty acid acetylenase or fatty acid hydroxylase or fatty acid desaturase are substituted , by the N-terminal region and / or C-terminus of any one of SEQ ID Nos: 2 or 4 or 6.

In a particularly preferred embodiment, the N-terminal and / or C-terminal amino acids of a fatty acid acetylenase or fatty acid desaturase or fatty acid hydroxylase are gratefully substituted by the N-terminal and / or C-terminus region of any of SEQ ID NOS: 2 or 4 or 6. Still more preferably, the fatty acid acetylenase or fatty acid desaturase or fatty acid hydroxylase is selected from the list shown in Figure 2.

Yet still more preferably, at least about 30 amino acid residues of the N-terminal and / or C-terminal regions of a fatty acid acetylenase or fatty acid desaturase or fatty acid hydroxylase are replaced, in grate, by the N-terminal region and / or C-terminus of any one of SEQ ID Nos: 2 or 4 or 6.

Accordingly, the present invention extends to any variants of the epoxygenases enzymes referred to herein, wherein said variants are derived from an epoxygenase polypeptide as described herein and exhibit demonstrable acetylenase or hydroxylase or desaturase activity and comprise, or an amino acid sequence that differs from a naturally occurring acetylenase or hydroxylase or desaturase enzyme, or exhibit catalytic properties which are different from an naturally occurring acetylenase or hydroxylase or desaturase enzyme, or comprise an amino acid sequence which is at least one amino acid sequence, about 60% identical to any of SEQ ID Nos: 2 or 4 or 6.

As with other aspects of the invention, the variants described herein may be produced as recombinant polypeptides or in transgenic organisms once the synthetic genes concerned are introduced into a suitable host cell and expressed therein.

The recombinant polypeptides described herein or a homologue, analogue or derivative thereof may also be immunologically active molecules.

A further aspect of the present invention provides an immunologically interacting molecule which is capable of binding to a recombinant epoxygenase polypeptide of the invention. 63

Preferably, the recombinant epoxygenase polypeptide to which the immunologically interacting molecule is capable of binding comprises an amino acid sequence shown in any one of SEQ ID Nos: 2, 4 or 6, or a homologue, analogue or derivative thereof.

In one embodiment, the immunologically interacting molecule is an antibody molecule. The antibody molecule may be either monoclonal or polyclonal. Monoclonal or polyclonal antibodies may be selected from naturally occurring antibodies to an epitope, or peptide fragment, or synthetic epoxygenase peptide derived from a recombinant gene product or may be specifically produced against a recombinant epoxygenase or a homologue thereof, analogous or derivative.

Both the polyclonal and monoclonal antibodies are obtainable by immunization with a gene product, or epitope, or appropriate peptide fragment of a gene product. Alternatively, antibody fragments, such as Fab fragments, may be used. The present invention extends to recombinant and synthetic antibodies and to antibody hybrids. &Quot; synthetic antibody " is considered herein to include fragments and hybrids of antibodies

Antibodies contemplated herein can be used to identify genetic sequences expressing related epoxygenase polypeptides encompassed by the embodiments described herein. The only requirement for the successful detection of a related epoxygenase genetic sequence is that said genetic sequence is expressed to produce at least one epitope recognized by the antibody molecule. Preferably, for the purpose of obtaining expression to facilitate detection, the related genetic sequence is placed operably behind a promoter sequence, for example, the lac bacterial promoter. According to this preferred embodiment, the antibodies are used to detect the presence of a plasmid or bacteriophage which expresses the related epoxygenase. Accordingly, antibody molecules are also useful in purification of the plasmid or bacteriophage which expresses the related epoxygenase.

The subject antibody molecules may also be used to purify the recombinant epoxygenase of the invention or a naturally occurring equivalent or a homologue, analogue or derivative thereof. The present invention is further described by reference to the following non-limiting Examples. EXAMPLE 1

Characterization of epoxy fatty acids in Euphorbia lagascae and Crepis spp.

Seeds of the wild species of Euphorbia lagascae and various species of Crepis were screened by liquid gas chromatography for the presence of epoxy fatty acids.

As shown in Table 3, Euphorbia lagascae contain very high levels of the fatty acid epoxy vernolic acid in its seed oil. Crepis palaestin seeds have been shown to contain 61.4% by weight of vernolic acid and 0.71% by weight of the fatty acid acetylenic acid of crepeninic acid, of total fatty acids (Table 3). TABLE 3

Fatty acid composition of lipids derived from seeds of Crepis alpina, Crepis palaestina and Euphorbia lagascae Gordo Acid Distributes Crepis alpina. Relative (% Crepis palaestina by weight) to Euphorbia lagascae Palmitico 3, 9 5.1 4.3 Stearic 1,3 2.3 1.8 Oleic 1.8 6, 3 22.0 Linoleic 14.0 23.0 0 10.0 Crepeninic 75.0 0.7 0 Vernolic 0 61.4 58.0 Other 4.0 1.2 3 , 9 (continued) a Calculated from the% area of the total areas of the peaks integrated in the determination of gas liquid chromatography of methyl ester derivatives of the seed lipids EXAMPLE 2

Biochemical characterization of A12-epoxygenases linoleate in Euphorbia lagascae and Crepis palaestina 66 The enzyme linoleate A12-epoxygenase synthesizes vernolic acid from linoleic acid. The A12-epoxygenases linoleate derived from Euphorbia do and Crepis palaestina are located in the microsomes. Enzymes, at least of these species, may remain active in membrane fractions (microsomal) prepared from developing seeds.

The membrane preparations of Euphorbia lagascae and assays of their epoxygenase activities were performed as described by Bafor et al. (1993), with incubations containing NADPH, unless otherwise noted in Table 4. Lipid extraction, separation and methylation as well as separations by GLC and radio-GLC were performed essentially as described by Kohn et al. (1994) and Bafor et al. (1993).

The membrane preparations of Crepis alpina and Crepis palaestin were obtained as follows. The Crepis alpina and Crepis palaestina plants were grown in greenhouses and the seeds were harvested in the intermediate stage of development (17-20 days after flowering). Cotyledons were removed from their seed coatings by squeezing and homogenized with mortar and pestle in 0.1 M phosphate buffer, pH 7.2 containing 0.33 M sucrose, 4 mM NADH, 2 mM CoASH, 1 mg of bovine serum albumin / mL and 4000 units of catalase / mL. The homogenate was centrifuged for 10 minutes at 18,000 x g and the resulting supernatant centrifuged for 60 minutes at 150000 x g to obtain a microsomal precipitate.

Standard desaturase, acetylenase and epoxygenase assays with microspore membranes of the Crepis species were performed at 25 ° C with microsomal preparations equivalent to 0.2 mg of microsomal protein, resuspended in fresh homogenization buffer and 10 mmol either [1-14 C] 18: 1-CoA or [1-14 C] 18: 2-CoA 67 (specific activity of 85,000 dpm / nmol) in a total volume of 360 æL. When NADPH was used as a co-reducer, the membranes were resuspended in homogenization buffer, where NADH was replaced by NADPH. The biochemical characterization of the microsomal A12-epoxyigenase linoleate derived from Euphorbia lagascae and Crepis palaestina was performed and the data obtained were compared with the biochemical characteristics of the oleate A12-desaturase and A12-acetylenase linoleate enzymes derived from Crepis alpina microsomal preparations (Table 4) .

As shown in Table 4, Crepis palaestin A12-epoxygenase linoleate exhibits biochemical characteristics similar to those of the A12-acetylenase linoleate and Crepis alpina oleate A12-desaturase, with respect to the three enzymes requiring 02, to work equally well either with NADH or NADPH as co-reductors, and are inhibited by cyanide but not by carbon monoxide. In addition, none of these enzymes are inhibited by monoclonal antibodies against cytochrome P450 reductase.

The data in Table 4 suggest that Crepis palaestin A12-epoxygenase linoleate belongs to the same enzyme class as Crepis alpina A12-desaturase oleate and A12-acetylenase linoleate.

In contrast, E121-epoxygenase linoleate of Euphorbia lagascae requires NADPH as a co-reducer, is not inhibited by cyanide, but is inhibited by carbon monoxide (Table 4). In addition, we have discovered that Euphorbia lagascae Al2-epoxygenase linoleate is inhibited by monoclonal antibodies raised against a cytochrome P450 68 reductase enzyme. These data suggest that E121-epoxyigenase linoleate of Euphorbia lagascae belongs to the cytochrome P450 protein class and is therefore not biochemically related to Crepis palaestin A12-epoxygenase linoleate. TABLE 4

Comparison of the biochemical characteristics of the epoxygenases, acetylenases and desaturases derived from Crepis spp. and Euphorbia lagascae Treatment Enzymatic activity (% of control) Oleate A12-linoleate linoleate linoleate desaturase A12-A12- Δ12- of C. acetylenase epoxigenase alpha epoxigenase E. coli alpina palaestina lagascae carbon monoxide 85 84 88 3 (continuation)

Comparison of the biochemical characteristics of the epoxygenases, acetylenases and desaturases derived from Crepis spp. and Euphorbia lagascae

Treatment

Enzyme activity (% of control)

Oleate A12-linoleate linoleate linoleate desaturase A12-A12- Δ12- of C. acetylenase epoxigenase alpha epoxigenase of E. alpina C. alpina palaestina lagascae 69 antibodies Anti-P450 reductase (CsA5) 96 91 94 33 KCN 16 0 35 92 less NADH 100 plus NADPH 95 73 94 (control) minus NADPH 100 100 100 plus NADH (control) (control) (control) EXAMPLE 3

Strategy for Cloning of Crepis palaestin epoxygenase genes The cloning of the Crepis palaestina epoxygenase genes was based on the characteristics of the C. palaestina and C. alpina enzymes described in the preceding Examples.

In particular, poly (A) + RNA was isolated from developing seeds of Crepis palaestin using a QuickPrep Micro mRNA purification kit (Pharmacia

Biotechnology) and used to synthesize a d (T) oligosaccharide-initiated double stranded cDNA. The double stranded cDNA was ligated to EcoRI / NotI adapters (Pharmacia Biotechnology) and a cDNA library was constructed using the Gigapack ZAP-cDNA cloning kit (Stratagene).

Single-stranded cDNA was prepared from RNA derived from developing seeds of Crepis alpina, using standard procedures. A PCR fragment, designated as D12V (SEQ ID NO: 7), was obtained by amplification 70 of single stranded cDNA using primers derived from the deduced amino acid sequences of plant mixed function monooxygenases. The D12V fragment was subsequently randomly labeled and used to screen the above Crepis palaestin cDNA library on Amersham Hybond N + membrane filters as prescribed by the manufacturer using standard hybridization conditions. This approach resulted in the purification of a recombinant bacteriophage, designated Cpal2. The nucleotide sequence of the Cpal2 cDNA was determined and is shown in SEQ ID NO: 1. The Cpal2 cDNA appeared to be full-length. A schematic representation of an expression vector comprising the Cpal2 cDNA is shown in Figure 1. The genetic construct presented therein is designed for introduction into plant material for the production of a transgenic plant expressing the subject epoxygenase. Those skilled in the art will recognize that similar expression vectors can be produced without superfluous experimentation and used for the production of transgenic plants expressing any of the genetic sequences of the present invention by replacing the Cpβ2 cDNA with another structural gene sequence.

As shown in Figure 2, the nucleotide sequence of the Crepl cDNA encoded a polypeptide that was infrequently related to the amino acid level with at least one C. alpina acetylenase enzyme (Bafor et al., 1997)

International No. PCT / SE97 / 00247). The 1.4 kb insert of pCpal2 was sequenced (SEQ ID NO: 1) and shown to comprise an open reading frame encoding a polypeptide of 374 amino acids in length. The deduced amino acid sequence of Cpal2 showed 81% identity and 92% similarity to Crepis alpina A12-acetylenase and approximately 60% identity and 80% similarity to plant microsomal A12-desaturase proteins (Figure 2). However, the polypeptide encoded by Cpal2 comprised significant differences in the amino acid sequence compared to non-epoxygenetic enzymes. In particular, Cpal2 has a deletion of six contiguous amino acids in the 5 'terminal region compared to all microsomal Δ12 desaturases, and a deletion of two contiguous amino acids in the 3' terminal region compared to Crepl Δ12 acetylenase (Figure 2).

Although membrane bound fatty acid desaturase genes show limited sequence homologies, all contain three conserved histidine rich motif regions as follows: (i) His- (Xaa) 3-4-His; (ii) His- (Xaa) 2-3-His-His; and (iii) His- (Xaa) 2-3-His-His, wherein His denotes histidine, Xaa designates any naturally occurring amino acid residue as shown in Table 1, the complete (Xaa) 34 entity refers to an amino acid sequence comprising three or four Xaa repeats, and the complete (Xaa) 2-3 refers to an amino acid sequence comprising two or three Xaa repeats. It is suggested that these histidine rich regions are a part of the active site of the enzyme (Shanklin et al., 1994). The amino acid sequence encoded by the Cpal2 cDNA comprises three histidine rich motifs similar but not identical to the histidine rich motifs of the Al2-desaturases enzymes. These data suggest that the Cpal2 cDNA encodes an enzyme belonging to the mixed function monooxygenase class of the enzymes. Analysis of the fatty acids shown in Example 1 above indicated that vernolic acid was at least present in the seeds of Crepis palaestin. This enzyme may indeed be present exclusively in the seeds of C. palaestina. Expression of the Cpal2 gene was examined using the 3 'untranslated region of the Cpal2 cDNA clone as a hybridization probe in Northern blots of mRNA derived from developing seeds and C. palaestina leaves. As shown in Figure 3, the Cpal2 gene was highly expressed in developing seeds but no expression was detected in leaves. These data are consistent with the enzymatic activity profile of C. palaestina A12-epoxygenase linoleate in these tissues. EXAMPLE 4

Strategy for Cloning Euphorbia lagascae Epoxygenase Genes The cloning of Euphorbia lagascae epoxygenase genes was based on the characteristics of the E. lagascae enzymes as described in the preceding Examples.

In an approach performed to clone the Euphorbia lagascae epoxygenase genes, RNA was harvested from immature embryos of Euphorbia lagascae obtained in a phase of active synthesis of vernolic acid and used to construct a cDNA library. The cDNA library was constructed in the Lambda Zap II vector (Stratagene) as described in the preceding Example, except that the cDNA inserts were cloned in a directional manner at EcoRI-Xhol sites of the plasmid vector embedded in the lambda vector. The degenerate PCR primer shown in Figure 4 (SEQ ID NO: 18) was synthesized and used to amplify nucleotide sequences encoding P450 enzyme sequences from the Euphorbia lagascae cDNA library. For PCR amplification reactions, a 100æL fraction of the cDNA library was extracted with phenol [1: 1 (v / v)] and the DNA was precipitated by the addition of 2 volumes of ethanol and finally resuspended in 100æl of Water. A fraction (1æl) of the resuspended DNA was used as template in the PCR amplification reaction. PCR reactions were performed in 10 æl of TaqI polymerase buffer containing 200 æM of each dNTP, 10 æmol of the degenerate primer, 1 æmol of the T7 polymerase promoter primer and 0.4 æl of the Taq1 polymerase.

The amplification conditions were 2 min at 94 ° C, and five cycles, each cycle comprising 1 min at 48 ° C, followed by 2 min at 72 ° C, followed by 30 seconds at 93 ° C, then 28 cycles, each cycle comprising 30 seconds at 55Â ° C, followed by 90 seconds at 72Â ° C, followed by 30 seconds at 93Â ° C, and finally a cycle comprising 30 seconds at 55Â ° C, followed by 10 min at 72Â ° C, followed by 1 min at 25 ° C.

The PCR products were purified and digested using EcoRI and Xhol, and then subcloned into the Bluescript vector for sequence characterization. One of the PCR clones was found to encode a P450 sequence and was used as a probe to isolate a full-length cDNA clone. This nucleotide sequence is shown in SEQ ID NO: 19. SEQ ID NO: 19 had similarity to other members of the 2C family of P450 genes. In particular, SEQ ID NO: 19 shows an average of 40% identity with the human and mouse arachidonic epoxygenase sequences using the BLAST program.

Additionally, it has been shown that the transcript of SEQ ID NO: 19 is expressed on Euphorbia lagascae seeds but not on roots or leaves (Figure 5B). The transcript of SEQ ID NO: 19 was detected in the developing seeds of Vernonia galamensis but not those of E. ciparissis. or flax, two species that do not produce epoxy fatty acids (Figures 5A and 5B).

In an alternative approach to clone Euphorbia lagascae epoxygenase genes, a subtractive hybridization strategy was used to isolate genes that are specifically expressed in an organism that produces high levels of epoxy fatty acids.

In particular, the subtractive hybridization method described in Figure 6 was used to isolate epoxygenase genes that are specifically expressed in Euphorbia lagascae, which produces high levels of epoxy fatty acid, vernolic acid (Example 1), and not in the closely related species of Euphorbia ciparissus, which does not produce vernolic acid.

Accordingly, mRNA was isolated from developing embryos of Euphorbia lagascae at a stage where they actively synthesize vernolic acid, and used to produce the so-called " tester " cDNA. In addition, mRNA was isolated from developing embryos of E. ciparissis (at a similar stage of development relative to E. lagascae) and used to produce the so-called " dríver " cDNA. The subtractive hybridization process led to a library that was enriched for sequences expressed exclusively in Euphorbia lagascae. Clones of this library were sequenced and at least two sequences were identified as encoding P450 proteins based on similarity to other P450 sequences in the database. These two P450 PCR clones were used as probes to isolate the corresponding full-length cDNA clones from the above-referenced cDNA library.

One of the cDNAs isolated from P450, comprising the nucleotide sequence shown in SEQ ID NO: 20, appeared to be expressed in Euphorbia lagascae tissues (Figure 7B) and no homologous transcripts were detected in E. ciparrisus or flax seed tissue, two species that do not produce epoxy fatty acids. The deduced amino acid sequence of SEQ ID NO: 20 indicates that the cDNA clone is full size and encodes a P450 enzyme. These data suggest that the cDNA exemplified by SEQ ID NO: 20 may encode an expoxygenase, for example, to L12-epoxygenase linoleate which converts linoleic acid to vernolic acid. EXAMPLE 5

Demonstration of epoxygenase activity Confirmation that the cDNA clones exemplifying the invention encode epoxygenase activities was obtained by transforming Arabidopsis thaliana, which produces no epoxy fatty acids, in particular vernolic acid, with each individual candidate clone and examining the transformed tissue for the presence of epoxygen fatty acids which would otherwise not produce, or for hydroxy fatty acids which can be formed from the metabolism of an epoxygen fatty acid by the action of endogenous epoxide hydrolases (Blee and Schuber, 1990). The epoxygenase cDNA comprising SEQ ID NO: 1 was cloned into the Binary vector construct shown in Figure 8. Briefly, the DNA sequence was subcloned from plasmid pCpal2 (Figure 1) into the binary plasmid by digestion of pCpal2 with FcoRI and filling ends of the restriction fragment using the enzyme T4 DNA polymerase. The Binary vector (Figure 8) was linearized using BamRI and also filling ends using T4 DNA polymerase. For end-filling reactions, 1æg of cDNA or DNA insert from the linearized Binary vector was resuspended in 50æl of T4 DNA polymerase buffer (33mM Tris-acetate pH 7.9, 66mM potassium acetate, 10 mM magnesium acetate and 5 mM DDT) supplemented with 100 mM of each dNTP and 0.1 mg / ml BSA and 3 units of T4 DNA polymerase, and incubated for 6 min incubation at 37 ° C. The reaction was stopped by heating at 75 ° C for 10 min. Blunt-ended DNA and Binary vector DNA were ligated using T4 DNA ligase and standard binding conditions as recommended by Promega. Clones were selected in which the sequence of SEQ ID NO: 1 was introduced behind the napin promoter in the sense orientation, thus allowing expression of the epoxygenase polypeptide. The Binary plasmid harboring SEQ ID NO: 1, in the sense orientation, operably under control of the truncated napin promoter, is shown schematically in Figure 9. The Binary plasmid shown in Figure 9 was transformed into Agrobacterium AGLI strain using electroporation and used to transform Arabidopsis thaliana. Transgenic A. thaliana plants were obtained according to the method described by Valvekens et al. (1988) and Dolferus et al. (1994).

Transgenic plants and untransformed (i.e., control) plants were grown to maturity. One mature seed from each plant was analyzed for the composition in fatty acids by conventional techniques. Primary transformants (To) were established and the TI seed was harvested from each plant and analyzed for the fatty acid composition by gas chromatography. Twelve T0 plants were shown to contain vernolic acid in their TI seed lipids at concentrations ranging from 0.9% to 15.8% of the total fatty acids, while the non-transformed control plants did not contain any vernolic acid (Table 5 ). The highest expression plant line was Cpal-17, for which the GLC elution profiles (from packed column analysis and capillary column) are shown in Figure 10. The GLC elution profile from packed column for the untransformed control is also shown in Figure 10. TABLE 5

Levels of Vernolic Acid in transgenic lines of A. thaliana expressing SEQ ID NO: 1 Plant T0 No. Vernolic Acid (% by weight of total fatty acids of the seed) Cpal-4 1.4 Cpal-5 1.1 Cpal-8 2.7 Cpal-9 0.9 Cpal-13 0.9 78

Cpal-15 1.1 Cpal-17 15.8 Cpal-21 1.3 Cpal-23 1.4 Cpal-24 1.0 Cpal-25 CM V 1 Cpal-2 6 1.1 control line 0, 0 untransformed

Alternatively, or in addition, the putative fatty acid epoxygenase sequences described herein are each transformed into Linum usitatissimum (flax) and Arabidopsis thaliana under the control of the seed specific napin promoter. Flax and Arabidopsis thaliana transgenic plants are examined for the presence of epoxy fatty acids in developing seed oils. Previous work has shown that if epoxy fatty acids are supplied to developing flax embryos, they are incorporated into triglycerides (Example 10).

Alternatively, yeasts are also transformed with the epoxygenase clones of the invention and assayed for the production of epoxy fatty acids. EXAMPLE 6

Confirmation by Mass Spectroscopy of Arabidopsis Ti Seed Epoxy Fatty Acids Born in T0 Primary Transgenic Plants 79 Gas chromatography of methyl esters prepared from seed lipids from the TI seed of Cpal2-transformed Arabidopsis thaliana plants (Example 5) revealed the presence of two additional fatty acids compared to the untransformed controls. The first of these compounds had a retention time equivalent to that of a vernolic acid standard. The second compound had a longer retention time and was putatively identified as 12,13-epoxy-9,15-octadecadienoic acid, an expected derivative of vernolic acid, resulting from desulfurization at position A 15 by Arabidopsis thaliana A15-desaturase endogenous. Confirmation of the exact identity of the two peaks was obtained by mass spectroscopy of diols which were prepared from the fraction of epoxy fatty acid derived from plants transformed with Cpal2. The diols were further converted to trimethylsilyl ethers and analyzed by GC-MS DB23 on a fused silica capillary column (Hewlett-Packard 5890 II GC coupled to a Hewlett Packard 5989A MS working on electronic impact at 70eV15). The total ion chromatogram showed two peaks as follows: (i) The first elution peak had prominent mass ions 73, 172, 275 and 299, indicating that the epoxy group was positioned at C-12 of a C18 fatty acid and that a double bond has occurred between the epoxy group and the carboxyl terminus. This mass spectrum was the same as the spectrum of a trimethylsilyl ether derivative of diols prepared from pure vernolic acid (12,13-epoxy-9-octadecenoic acid); and (ii) The second elution peak had prominent mass ions 73, 171, 273 and 299, indicating the presence of two double bonds and a C-12 positioned epoxy group of a C18 fatty acid consistent with the mass for 12,13-epoxy-9,15-octadecadienoic acid. EXAMPLE 7

Fatty acid analysis of Cpal2 transgenic Arabidopsis plants Tl seed derived from transformed Arabidopsis thaliana plants expressing the Cpal2 cDNA clone under control of the napin promoter was germinated and T1 plants were established from five T0 lines (Nos. 4 , 8, 13, 17 & 21 in Table 5). The T2 seed was harvested from each T1 plant and analyzed for the fatty acid composition. The progeny of the transformants Nos. 4, 8, 13 and 21 (Table 5) were segregated as expected for the presence of vernolic acid, with those plants containing vernolic acid varying up to 3.1% (Table 6).

All Tl plants which contained vernolic acid (i.e., 18: 1 epoxy in Table 6) also contained 12,13-epoxy-9,15-octadecadienoic acid (i.e., 18: 2 epoxy in Table 6;

Figure 11), indicating that some of the vernolic acid synthesized by the Cpal2 epoxygenase was subsequently desaturated by the endogenous A15-desaturase. TABLE 6

Composition of pure seed fatty acids born in Ti plants derived from five primary Cpal2 transformants of Arabidopsis thaliana

Plant

Fatty acid 81 Acids Fatty non-fatty epoxy epoxy 16: 0 18: 0 18: 1 18: 2 20: 1 20: 1 22: 1 18: 1 18: 1 18-1: , 9 15.5 23.9 20.6 2.8 16.5 1.7 1.6 - - 4-2 7, 6 4.1 20.3 17.8 18.0 3.4 19.7 1 , 8 2.0 0.82 0.63 4-3 8.4 4.3 26.0 13.5 16.1 2.8 19.0 0 1.8 1.6 2.03 0.72 4-4 7, 6 4.0 25.2 14.3 16, 0 2.8 19, 8 2.1 1.7 1.99 0.92 4-5 7, 2 3.6 15.6 23.1 19, 9 3.1 19.7 1.6 2.1 - - 4-6 7, 0 3.7 19.2 17.8 18.4 3.2 20.3 1.9 2.1 0.87 0, 33 4-8 7, 4 3.9 16.0 23.6 20.1 3.1 18.7 1.6 1.8 - - 4-9 7, 6 4.0 24.8 13.4 15, 9 2.8 20.4 2.3 1.8 2.30 1.07 4-10 7, 6 4.2 24.0 13.5 16.2 3.1 20.4 1.9 1.8 1 , 97 0.83 4-11 7, 4 3.9 15.0 23.2 20.4 3.3 18.8 1.7 2.0 - - 4-12 8, 7 4.0 20.7 17 , 0 17.5 2.6 17.2 1.7 1.5 1.38 0.74 4-13 7, 2 4.1 21.9 16.4 17.7 7 3.2 21.0 1.7 1.9 1, 14 0.45 8-1 8, 1 3.9 26, 1 15.0 16, 0 2.6 19.5 2.0 1.6 1.79 0.82 (continued)

Composition of pure seed fatty acids born in Ti plants derived from five primary Cpal2 transformants of Arabidopsis thaliana

Plant Fatty Acid N ° Acids Fatty Epoxy Fatty Epoxy 16.0 18: 0 18: 1 18: 1 18: 3 20: 1 20: 1 22: 1 18: 1 18: 4.2 31.6 11.5 14.0 2.2 18.5 1.9 1.4 2.38 1.13 1 00 8.5 4.1 27.2 15.1 16.1 2.5 18.9 1.8 1.4 1.70 0.84 LO 1 00 9.1 4.2 27.7 14.7 16.2 2.4 18.3 1.7 1.5 1.70 0, 82 co 1 <9.8 4.0 26.0 17.2 17.2 2.3 16.9 1.6 1.2 1.36 0.71 8-7 10.0 3.5 15 , 2 25.3 22.3 2.3 14, 4 1.7 1.7 - - 00 1 00 8.4 4.3 32.2 10.7 13.3 2.5 20.3 1.6 1 , 5 1.92 0.82 1.00 9.8 3.6 15.9 25.3 22.0 2.4 14.5 1.6 1.3 - - 82 8-10 7.5 3.9 24 , 4 15.9 15.8 2.8 20.2 2.2 1.8 1.70 0.82 8-11 7.6 3.8 15.4 23.6 19.8 8 2.9 19.4 1.5 1.8 - - 8-12 9.4 4 3.7 24.2 16.7 16.7 2.2 17.6 6.9 1.2 1.46 0.65 8-13 10.3 4.3 25.3 17.1 17.9 2.2 16.0 1.8 1.3 1.48 0.73 13-1 7.0 4.3 33.3 8.1 11.22, 7 23.1 1.7 1.6 2.42 1.26 13-2 7.2 4.3 30.4 9.6 12.7 2.8 22.0 1.8 1.6 2.48 1 , 37 13-3 7.6 3.9 15.6 23.6 19.7 3.0 19.1 1.7 1.8 - - 13-4 7.7 4.0 15.2 22.5 19 , 3 3.1 18.0 1.6 1, 7 - - 13-5 8.0 4.2 16.3 22.2 17.5 4.4 19.4 2.0 2.0 - - 13-6 7.9 4.4 25.7 14.7 15.8 2.9 21.2 1.6 1.7 1.56 0.63 13-7 7.9 4.0 16.0 23.3 19.6 3.0 19.1 1.6 1, 8 - - 13-9 8.0 4.0 16, 1 23.6 20.0 2.9 18.7 1.6 1.6 - - 13-10 8.7 4.2 34.6 9.6 12.5 2.2 19.1 1.5 1.2 2.21 1.01 13-11 8.7 4.0 17.6 24.3 18.9 2.8 17.1 11.1 1, 4 - - 13-12 8.9 4.2 26.4 14.6 16.0 0 2.5 17.5 1.6 1.2 1.62 0.74 13-13 9.4 4.4, 9 14.4 15.3 2.5 18.9 1.5 1.4 1.30 0.77 (continued)

Composition of pure seed fatty acids born in Ti plants derived from five primary Cpal2 transformants of Arabidopsis thaliana Plant a Fatty acid non-epoxy Fatty acids epoxy 16.0 18: 0 18: 1 18: 3 20: 0 20 : 1 22: 0 22: 1 18: 1 18: 2 13-14 9.2 4.2 17.2 23.8 18.8 2.7 17.9 1.7 1.5 - - 13-15 8 , 4 4.2 19.7 20.9 18.6 2.7 17.7 7 1.4 1.5 0.40 0.16 13-16 8.2 4.3 23.0 17.1 17.3 2.8 19.3 1.5 1.5 0.97 0.42 13-17 8.3 4.1 15.7 23.9 19.9 2.8 17.6 1.6 1.6 - - 17-1 7.6 4.1 15.8 23.7 19.6 2.6 20.3 1.7 1.7 - - 17-2 8.3 4.1 16.4 24.4 20.1 2.3 16.8 8 1.5 1.4 - - 83 17-3 8.1 4.1 16.4 24.3 20.0 2.5 17.6 1.6 1.4 - 21-1 8 , 1 4.3 26.9 9 14.5 15.0 2.9 19.9 9 1.5 1.5 1, 64 0, 63 21-2 8.2 4.0 27.9 11.8 13.2 2.5 19.8 8 1.7 1.5 2, 18 0, 91 21-3 8.8 3.7 16.4 24.4 20.6 2.5 17.3 1.7 1.4-21 -4 7.9 3.9 19.6 19.8 8 17.8 2.7 18.7 1.7 1.7 0.66 0.46 21-5 7.2 4.2 26.5 12.9 14.4 3.0 21.5 0.9 1.8 1.78 0.84 21-6 8.3 4.2 27.4 13.9 15.4 2.6 19.9 1.7 1.5 1, 66 0, 65 21-7 7.2 4.2 26.8 13.5 13.4 3.0 21.9 1.7 1.8 1.74 0.80 21- 8 7.4 3.8 16.3 23.6 19.4 4 3.2 19.2 1.7 1.9 21-9 7.2 4.0 28.1 11.8 13.5 3.0 22 , 5 1.9 1.9 2, 15 1.05 21-10 7.2 4.2 26.1 13.8 14.6 3.0 22.3 1.7 1.8 1.64 0.82 21-11 7.1 4.2 29.2 11.5 12.7 3.0 22.5 1.8 1.8 2, 20 1.09 21-12 7.2 4.1 26.2 13, 6 14.2 3.1 22.4 1.8 1.9 1, 71 0.80 21-13 7.1 4.3 33.7 7.1 10.0 2.7 24.1 2.0 1 , 8 3, 05 1.47 21-14 7.4 3.7 16.9 9 21.9 19.6 13.1 19.2 1.8 2.0 0.29 vg 21-15 7.7 3, 6 15.6 24.3 20.2 2.9 18.1 1.8 1.8 EXAMPLE 8

Fatty Acid Analysis of Calla 2 Transgenic Linola Plants The above described binary plasmid construct comprising the Cpal2 cDNA clone (Figure 9) was transformed into Agrobacterium tumefaciens AGL1 strain, using electroporation. The transformed A. tumefaciens was used to infect explants of Linum usitatissimum var. Eyre as described by Lawrence et al. (1989), except that MS medium was used as the basal medium for root induction in the regenerated shoot material. 84

Two primary Linola transformants (TO plants) designated AP20 and AP21 were confirmed to be transgenic by PCR using primers directed against the Cpal2 gene and showing that these plants were resistant to kanamycin. Ten TI seeds from each plant were individually analyzed for the fatty acid composition using standard techniques.

As shown in Table 7, the AP20 seed secreted into 3 classes, comprised of three seeds without vernolic acid, two having more than 0.7% of vernolic acid, and five having intermediate levels (0.13-0.47 %) of vernolic acid.

Likewise, AP21 seeds secreted into 3 classes comprised of five seeds without vernolic acid, two having more than 0.25% vernolic acid and three having an intermediate level (0.09-0.14%) of (Table 8).

Thus, a total of twelve seeds containing vernolic acid were obtained. Eight of the twelve seeds of AP20 and AP21 containing vernolic acid also contained 12,13-epoxy-9,15-octadecadienoic acid

Composition of primary transformant fatty acids 10 individual Tl seeds of Linola Cpal2 ΆΡ20 Sement and Ti Fatty acids not epoxy Epoxy fatty acids 16: 0 18: 1 18: 1 18: 2 18: 3 20: 0 20: 1 22: 0 22: 1 18: 1 18: 2 1 6.4 3, 6 17.8 68.1 2.0 0.2 - 0.6 - 2 6, 0 3.5 25.4 60.8 1.4 0.2 0.2 0.70 0.23 3 6, 0 3.9 20.4 64.6 2.1 0.3 0.6 - 85 4 6.3 3.5 28.3 57 , 3 1.3 0.2 0.2 1.4 - 0.34 0.28 5 5.2 4.8 24.9 61.2 1.6 0.3 0.2 ο, - 0.37 - 6 5.8 4.1 23.3 63.1 1.9 0.2 0.2 0.2 - 0.47 - 7 5.9 4.3 21.7 64.1 2.2 0.2 0.2 0.2 - 0.13 0.12 8 5.9 3.3 22.3 65.2 2.0 0.2 0.2 ο, ι 0.2 - - 9 5.6 4.0 25.2 61.4 1.7 0.2 0.2 ο, ι - 0.84 - 10 6.2 4.4 27.4 57.9 1.7 0.2 0.2 0.2 - 0 , 54 - TABLE 8

Composition of primary transformant fatty acids 10 individual Tl seeds of Linola Cpal2 AP21 Sement and Ti Epoxy fatty acids Epoxy 16: 0 18: 0 18: 1 18: 2 18: 3 20: 0 20: 1 22: 0 22: 1 18: 1 18: 2 1 6.1 4.2 35.2 50.8 1.3 - - - 2.0 - - 2 5.7 5.0 32.9 53.3 1.4 0 , 2 0.2 0.2 0.14 0.21 3 5.9 4.0 35.1 50.8 1.3 0.2 0.2 0.1 1.5 - - 4 7.5 4, 1 38.8 45.5 1.2 0.2 0.3 - 1.7 - - 5 5.8 5.0 28.8 57.3 1.3 0.2 0.2 0.1 - 37 0.06 6 5.8 5.0 44, 1 41, 4 1.4 0.2 0.2 0.2 - - - 7 6.5 4.5 27.9 58.6 1.3, 2 0.1 0.1 - - - 8 6, 9 4, 6 37.6 48.1 1.2 - - - - 0.10 0.19 9 6.2 4.7 33.7 52.1 1 , 3 0.2 0.2 0.2 - 0.09 0.07 10 6.1 4.8 29.76 6.6 1.3 0.2 0.2 0.1 - 0.25 0, 04 86

Four TI plants were established from kanamycin resistant seedlings of AP20. All four plants were subsequently shown to produce vernolic acid in their T2 seed (Table 9). The levels of 18: 2 epoxy fatty acids were not analyzed in these T2 seeds. TABLE 9

Composition of progeny fatty acids from seeds Cpa.12 Linola T1 from ΆΡ20 T2 from T2E and T2 Fatty acids from epoxy 18: 0 22: 1 18: 1 A 3.4 3.0 27.4 65.5 0.6 na na na Na 0.06 B 3.5 3.1 30.2 62.6 0.6 na na na 0.07 C 3.6 2.7 33.3 59.86 na na na na 0.07 D 3.4 3.1 28.2 64.6 0.6 na na na na na = not analyzed EXAMPLE 9

Production of an oil rich in vernolic acid was achieved by transforming the epoxygenase gene described herein, in particular SEQ ID NO: 1, into Arabidopsis thaliana, as described in the foregoing Examples. As shown in Table 5, the A. thaliana transgenic line expressing SEQ ID NO: produces high levels of vernolic acid in its seeds relative to other fatty acids. In particular, in a transgenic line (Cpal-17), the produced vernolic acid is as much as 15.2% (w / w) of the total fatty acid content of the seed. The production of an oil rich in vernolic acid is also achieved by transformation of the epoxygenase gene described herein, in any one of SEQ ID Nos: 1, 3, 5, 19 or 20, and preferably any of SEQ ID NO: Nos: 1 or 3 or 5 in any oil accumulating organism which normally has very high levels of linoleic acid and minimum activities of other competitive enzymes capable of using linoleic acid as a substrate. The genetic sequences of the invention are placed operably under the control of a promoter which produces high level of oleaginous seed expression, for example, the seed specific napin promoter.

In an alternative approach to the transformation of A. thaliana, high linoleic genotypes of flax, sunflower, corn or safflower are transformed with the epoxygenase of the invention. Elevated levels of vernolic acid are produced by transgenic plants during seed oil synthesis when the epoxygenase gene is expressed at high levels.

Alternatively Linola® (= low linolenic acid) is transformed with the epoxygenase of the invention. Elevated levels of vernolic acid are produced by Linola® linseed transgenic plants during seed oil synthesis, when the epoxygenase gene is expressed at high levels.

In addition, applicants have shown that labeled vernolic acid supplied to developing flax seeds is not degraded but is incorporated into storage lipids at all three positions of the triglyceride molecule (see Example 10). Consistent with these data, high levels of vernolic acid synthesized by the introduced epoxygenase are readily deposited into the triglycerides of the seed oil of this species. EXAMPLE 10

Incorporation of oleic acid and vernolic acid in the lipids of developing linseed cotyledons

The prominent developing linseed cotyledons (six pairs in each incubation, duplicate incubation) at the intermediate seed development stage (20 days after flowering) were incubated with 10 nmol of the ammonium salts of either [1-14C] vernolic acid ( specific activity of 3000 dpm / nmol) or [1-114 C] oleic acid (specific activity of 5000 dpm / nmol) in 0.2 ml of phosphate buffer pH 7.2 for 30 min at 30 ° C. The cotyledons were then washed three times with 1 mL of distilled water and immediately extracted in an Ultra Turrax according to Bligh and Dyer (1959) or incubated further at 0.5 m. of 0.1 M phosphate buffer pH 7.2 for 90 or 270 min prior to extraction. A fraction of the lipids in the chloroform phase was methylated and separated on silica gel TLC plates in n-hexane / diethyl ether / acetic acid (85: 15: 1). The remainder of the lipids in the chloroform phase of each sample was applied to two separate silica gel TLC plates and the plates were developed in chloroform / methanol / acetic acid / water (85: 15: 10: 3.5 in vol) for separation of polar lipids and in n-hexane / diethyl ether / acetic acid (60: 40: 1.5) for separation of neutral lipids. The lipid areas with migration corresponding to authentic standards were removed and the radioactivity in each lipid was quantified by liquid scintillation counting. Recovery of the 14C labeling in the chloroform phase is described in Figure 12. [0009] More than half of the added radioactivity of both [14 C] oleic acid and [14 C] vernolic acid was incorporated by the cotyledons and recovered as lipophilic substances after 30 minute pulse marking. This amount remained virtually unchanged for the additional 270 min of incubation with both substrates. Separation of the radioactive methyl esters from the lipids showed that the majority of radioactivity (92%) of the [14C] vernolic acid provided to the experiments resided in compounds having the same migration as methyl veroleate indicating that the epoxy group remained intact on the linseed cotyledons at incubation time of 270 min.

About 28% of the [14C] vernolic acid supply activity that was present in the chloroform phase resided in phosphatidylcholine after 30 min and the radioactivity decreased to only 5% in 300 min incubation (Figure 13).

About 22% of the [14 C] oleic acid supply activity that was present in the chloroform phase resided in phosphatidylcholine after 30 min and the radioactivity decreased to about 11% at 300 min incubation (Figure 13).

About 32% of the [14 C] vernolic acid supply activity that was present in the chloroform stage resided 90 in triacylglycerols after 30 min and radioactivity increased to over 60% in 300 min incubation (Figure 14). The diacylglycerols contained about 24% of the activity in the [14 C] vernolic acid feed experiments and this amount remained practically constant during the incubation periods.

About 5% of the [14 C] oleic acid supply activity that was present in the chloroform phase resided in triacylglycerols after 30 min and radioactivity increased to 18% in 300 min incubation (Figure 14). The diacylglycerols contained about 19% of the activity after 30 min in the [14 C] oleic acid feed experiments and this amount remained practically constant during the incubation periods. The above experiment shows that flax cotyledons do not metabolize the epoxy group of vernolic acid to a great extent. Additionally, it shows that flax cotyledons have mechanisms to efficiently remove vernolic acid from membrane lipids and incorporate them into triacylglycerols. EXAMPLE 11

Cloning of A12-epoxygenase genes from other species containing epoxy acid

Al2-epoxygenase-Cp2 gene homologs are obtained from other species that are rich in epoxy fatty acids by cloning members of the gene family of A12 mixed function monooxygenases which are highly expressed in developing seeds and comparing their sequence of amino acids with 91 to those of known sequences of A12-desaturase and A12-epoxygenase. Such genes are cloned either by screening of seed cDNA libraries in development with gene based probes either in the Cpal2 gene (SEQ ID NO: 1) or in the D12V fragment (SEQ ID NO: 7), or by amplification of fragments of PCR using primers designed against conserved sequences of plant A12 mixed function monooxygenases, as described herein. The putative A12-epoxygenase sequences show superior overall sequence identity to the A12-epoxygenase sequences disclosed herein, than to the known A12-desaturase sequences.

In one example of this approach, a full length A12-epoxygenase sequence was obtained from a Crepis sp. unidentified strain containing high levels of vernolic acid in its seed oils and known to be non-crepis palaestin. Poly (A) + RNA was isolated from developing seeds of this Crepis sp. using a QuickPrep Micro mRNA purification kit (Pharmacia Biotechnology) and used to synthesize a d (T) oligosaccharide-initiated double strand cDNA. The thus obtained double-stranded cDNA was then ligated into EcoRI / Notl adapters (Pharmacia Biotechnology) and a cDNA library was constructed using the Gigapack ZAP-cDNA cloning kit (Stratagene). The cDNA library on Hybond N + membrane filters (Amersham) was screened with the randomly labeled D12V fragment (SEQ ID NO: 7) derived from Crepis alpina as prescribed by the manufacturer, using standard hybridization conditions. This resulted in the purification of a recombinant bacteriophage called CrepX. The nucleotide sequence of the CrepX cDNA was determined and is shown in SEQ ID NO: 3. The deduced amino acid sequence of CrepX (SEQ ID NO: 4) comprises a 374 92 amino acid protein having 97% identity with the A12-epoxygenase sequence Cpal2, but only 57% identity to the Δ12-desaturase sequence of Arabidopsis thaliana L26296. This clearly demonstrates the presence of one gene in another Crepis sp. which has a high content of vernolic acid, whose gene is highly homologous with the A12-epoxygenase gene Cpal2 and is not clearly a desaturase gene.

In a second example of this approach, a partial A12-epoxygenase-like sequence was obtained from the species containing Vernonia galamensis vernolic acid. First strand cDNA templates were prepared from total RNA isolated from developing seeds of V. galamensis, using standard procedures.

A PCR fragment (550 nucleotides in length), designated Vgall, was obtained by amplification of the single stranded cDNA using primers derived from the deduced amino acid sequence of plant mixed function monooxygenases. The nucleotide sequence of the amplified DNA was determined using standard procedures and is shown in SEQ ID NO: 5. Alignment of the deduced amino acid sequence of the Vgall PCR fragment (SEQ ID NO: 6) to the total sequence of

A12-epoxygenase Cpal2 and Arabidopsis thaliana A12-desaturase L26296 (Figure 2) demonstrates that the amplified Vgall sequence encodes an amino acid sequence that corresponds to the region covering amino acid residues 103-285 of the Cpβ2 polypeptide. Within this region, the Vgall sequence showed a higher amino acid identity with the Al2-epoxyigenase sequence Cpal2 (67%) than with the A. thaliana A12-desaturase sequence (60%), suggesting that the amplified DNA 93 corresponds to an epoxygenase sequence instead of desaturase.

Those skilled in the art will appreciate that the present invention is subject to variations and modifications other than those specifically disclosed herein. It is to be understood that the invention includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this disclosure, individually or collectively, and any and all combinations of any two or more of said steps or features.

REFERENCES 1. An et al. (1985) EMBO J. 4: 277-284. 2. Ausubel, F.M., Brent, R., Kingston, RE, Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338). 3. Badami, R.C., and Patil, K.B. (1981) Progress in Lipid Research, 19, 119-53. 4. Bafor, M., Smith, M.A., Jonsson, L., Stobart, K. and

Stymne, S. (1993) Arch. Biochem. Biophys. 303, 145-151. 5. Bafor, M., Banas, A., Wiberg, E., Lenman, M., Stahl, U. and

Stymne, S. (1997) In: Williams, J.P., Mobasher, K.U., Lem, N.W. (eds) Physiology, biochemistry and molecular biology of plant lipids. Kluwer Academic Publisher, Dordrecht. In print. 6. Blee and Schuber (1990) J. Biol. Chem. 265, 12887-12894. 7. Blee, E., Wilcox, A.L., Marnett, J. M., Schuber, F. (1993) J. Biol. Chem. 268, 1798-1715. 8. Blee, E., Stahl, S., Schuber, F. and Stymne, S. (1994) Biochem. Biophys. Comm. 197, 778-784. 9. Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 230, 379-288. 10. Bozak, K.R., Yu, H., Sirevag, R. and Christoffersen, R.E. (1990) Proc. Natl. Acad. Know. USA 87, 3904-3908. 11. Christou, P., McCabe, D.E., Swain, W.F. (1988). Plant Physiol 87, 671-674. 12. Crossway et al. (1986) Mol Gen Genet. 202, 179-185. 13. Devereux, J., Haeberli, P. and Smithies, O. (1984). Nucl. Acids Res. 12, 387-395. 14. Dolferus et al. Plant Physiol. (1994) 105, 1075-1087. 15. Engeseth, N. &amp; Stymne, S. (1996) Plant 198, 238-245 16. Fromm et al. (1985) Proc. Natl. Acad Sci. (USA) 82, 5824-5828. 17. Haseloff, J. and Gerlach, W.L. (1988). Nature 334, 586-594. 18. Herrera-Estrella et al. (1983a) Nature 303, 209-213. 19. Herrera-Estrella et al. (1983b) EMBO J. 2, 987-995. 95 20. Herrera-Estrella et al. (1985) In: Plant Genetic

Engineering, Cambridge University Press, NY, pp 63-93. 21. Kohn, G., Hartmann, E., Stymne, S. &amp; Beutelmann, P. (1994) J. Plant Physiol. 144, 265-271 22. Krens, F.A., Molendijk, L., Wullems, G.J.

Schilperoort, R.A. (1982). Nature 296, 72-74. 23. Lawrence, G.J., Ellis, J.G., Finnegan, E.J., Dennis, E.S. and Peacock, W. J. (1989) in: Breeding Research: The Key to Survival of the Earth (Iyama, S. and Takeda, G. eds) 6th International Congress of SABRAO. pp 535-538. 24. Lazo, G.R., Stein, P.A. and Ludwig, R.A. (1991).

Bio / technology 9, 963-967. 25. Needleman and Wunsch (1970) J. Mol. Biol. 48, 443-453. 26. Pazkowski et al. (1984) EMBO J. 3, 2717-2722. 27. Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986). Nucl. Acids Res. 14, 857-5868. 28. Sanger, F., Nicklin, S. and Coulson, A.R. (1977). Natl. Acad. Know. (USA) 72, 5463-5467. 29. Shanklin, J., Whittle, E. and Fox, B.G. (1994) Biochemistry 33, 12787-12794. 30. Valvekens et al. (1988) Proc. Natl Acad. Know. (USA) 85, 5536-5540. 96 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Commonwealth Scientific and Industrial Research Organization AND Sten Stymne

(ii) TITLE OF INVENTION: EPOXYGENASE GENES OF VEGETABLE FATTY ACID AND ITS UTILIZATIONS (iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: DAVIES COLISON CAVEARN

(B) STREET: 1 LITTLE COLINS STREET

(C) CITY: MELBOURNE

(D) STATE: VICTORIA

(E) COUNTRY: AUSTRALIA (F) POSTAL CODE: 3000 (v) COMPUTER READING FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM Compatible PC 97

(D) SOFTWARE: Patent In Release # 1.0, Version # 1.25 (vi) DATA OF THIS APPLICATION: (A) ORDER NUMBER: (B) ORDER DATE: ( vii) PREVIOUS ORDER DATA: (A) ORDER NUMBER: AU P06223 (B) ORDER DATE: APR 15, 1997 (vii) PREVIOUS ORDER DATA: (A) ORDER NUMBER: AU P06226 (B) DATE OF APPLICATION (Vii) PREVIOUS ORDERS: (A) APPLICATION NUMBER: US 60/043706 (B) ORDER DATE: APRIL 16, 1997 (vii) PREVIOUS ORDER DATA: (A) NUMBER OF ORDERS: ORDER FORM: US 60/050403 (B) ORDER DATE: JUN 20, 1997 (viii) INFORMATION BY THE MANDATORY / AGENT:

(A) NAME: HUGHES, DR. E JOHN L

(C) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: +61 3 9254 2777 (B) TELEFAX: +61 3 9254 2770 (C) TELEX: AA 31787 (C) 2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1358 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear ) TYPE OF MOLECULE: DNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 30..1151 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 99 53

GAGAAGTTGA CCATAAATCA TTTATCAAC ATG GGT GCC GGC GGT CGT GGT CGG Met Gly Ala Gly Gly Arg Gly Arg 1 ACA TCG GAA AAA TCG GTC ATG GAA CGT GTC TCA GTT GAT CCA GTA ACC Thr Ser Glu Lys Ser Vai Met Glu Arg Will Go Asp Pro Go Thr 10 15 20 TTC TCA CTG AGT GAA TTG AAG CAA GCA ATC CCT CCC CAT TGC TTC CAG 100

Phe Ser Leu Ser Glu Leu Lys Gin Ala Ile Pro Pro His Cys Phe Gin 25 30 35 40 AGA TCT GTA ATC CGC TCA TCT TAC TAT GTT GTT CAA GAT CTC ATT ATT 197 Arg Ser Vai Ile Arg Ser Ser Tyr Tyr Vai Vai Gin Asp Leu Ile Ile 45 50 55 GCC TAC ATC TTC TAC TTC CTT GCC AAC ACA TAT ATC CCT ACT CTT CCT 245 Wing Tyr Ile Phe Tyr Phe Leu Ala Asn Thr Tyr ile Pro Thr Leu Pro 60 65 70 ACT AGT CTA GCC TAC TTA GCT TGG CCC GTT TAC TGG TTC TGT CAA GCT 293 Thr Ser Leu Ala Tyr Leu Ala Trp Pro Vai Tyr Trp Phe Cys Gin Ala 75 80 85 AGC GTC CTC ACT GGC TTA TGG ATC CTC GGC CAC GAA TGT GGT CAC CAT 341 Ser Vai Leu Thr Gly Leu Trp Ile Leu Gly His Glu Cys Gly His His 90 * 95 100 GCC TTT AGC AAC TAC ACA TGG TTT GAC GAC ACT GTG GGC TTC ATC CTC 389 Ala Phe Ser Asn Tyr Thr Trp Phe Asp Asp Thr Val Gly Phe Ile Leu '105 110 115 120 CAC TCA TTT CTC CTC ACC CCG TAT TTC TCT TGG AAA TTC AGT CAC CGG 437 His Ser Phe Leu Leu Thr Pro Tyr P he Ser Trp Lys Phe Ser His Arg 125 130 135 AAT CAC CAT TCC AAC ACA AGT TCG ATT GAT AAC GAT GAA GTT TAC ATT 485 Asn His His Ser Asn Thr Ser Ser lie Asp Asn Asp Glu Val Tyr Ile 140 145 150 CCG AAA AGC AAG TCC AAA CTC GCG CGT ATC TAT AAA CTT CTT AAC AAC 533 Pro Lys Ser Lys Ser Lys Leu Ala Arg Ile Tyr Lys Leu Leu Asn Asn 155 160 165 CCA CCT GGT CGG CTG TTG GTT TTG ATT ATC ATG TTC ACC CTA GGA TTT 581 Pro Pro Gly Arg Leu Leu Leu Ile Ile Met Phe Thr Leu Gly Phe 170 175 180 CCT TTA TAC CTC TTG ACA AAT ATT TCC GGC AAG AAA TAC GAC AGT TTT 629 101

Pro Leu Tyr Leu Leu Thr Asn Ile Ser Gly Lys Lys 185 190 195

Tyr Asp Arg Phe 200 GCC AAC CAC TTC GAC CCC ATG AGT CCA ATT TTC AAA GAA CGT GAG CGG 677 Wing Asn His Phe Asp Pro Met Ser Pro Ile Phe Lys Glu Arg Glu Arg 205 210 215 TTT CAG GTC TTC CTT TCG GAT CTT GGT CTT CTT GCC GTG TTT TAT GGA 725 Phe Gin Go Phe Leu Ser Asp Leu Gly Leu Leu Ala Go Phe Tyr Gly 220 225 230 ATT AAA GTT GCT GTA GCA AAT AAA GGA GCT GCT TGG GTA GCG TGC ATG 773 Ile Lys Go Ala Go Ala Asn Lys Gly Ala Ala Trp Go Ala Cys Met 235 240 245 TAT GGA GTT CCG GTA TTA GGC GTA TTT ACC TTT TTC GAT GTG ATC ACC 821 Tyr Gly Vai Pro Go Leu Gly Go Phe Thr Phe Phe Asp Go Ile Thr 250 255 260 TTC TTG CAC CAC ACC CAT CAG TCG TCG CCT CAT TAT GAT TCA ACT GAA 869 Phe Leu His His Thr His His Ser Ser Pro His Tyr Asp Ser Thr Glu 265 270 275 280 TGG AAC TGG ATC AGA GGG GCC TTG TCA GCA ATC GAT AGG GAC TTT GGA 917 Trp Asn Trp Ile Arg Gly Ala Leu Ser Ala Ile Asp Arg Asp Phe Gly 285 290 295 TTC C TG AAT AGT GTT TTC CAT GAT GTT ACA CAC ACT CAT GTC ATG CAT 965 Phe Leu Asn Ser Go Phe His Asp Go His Thr His Go His Met His 300 305 310 CAT TTG TTT TCA TAC ATT CCA CAC TAT CAT GCA AAG GAG GCA AGG GAT 1013 His Leu Phe Ser Tyr Ile Pro His Tyr His Ala Lys Glu Ala Arg Asp 315 320 325 GCA ATC AAG CCA ATC TTG GGC GAC TTT TAT ATG ATC GAC AGG ACT CCA 1061 Ala Ile Lys Pro Ile Leu Gly Asp Phe Tyr Met Ile Asp Arg Thr Pro 330 335 340 ATT TTA AAA GCA ATG TGG AGA GAG GGC AGG GAG TGC ATG TAC ATC GAG 1109 102 1151

Ile Leu Lys Ala Met Trp Arg Glu Gly Arg Glu Cys Met Tyr Ile Glu 345 350 355 360

CCT GAT AGC AAG CTC AAA GGT GTT TAT TGG TAT CAT AAA TTG

Pro Asp Ser Lys Leu Lys Gly Vai Tyr Trp Tyr His Lys Leu 365 370 1211 1271 1331 1358

TGATCATATG CAAAATGCAC ATGCATTTTC AAACCCTCTA GTTACGTTTG TTCTATGTAT AATAAACCGC CGGTCCTTTG GTTGACTATG CCTAAGCCAG GCGAAACAGT TAAATAATAT CGGTATGATG TGTAATGAAA GTATGTGGTT GTCTGGTTTT GTTGCTATGA AAGAAAGTAT GTGGTTGTCG GTCAAAAAAA AAAAAAA (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 374 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Gly Ala Gly Gly Arg Gly Arg Thr Ser Glu Lys Ser Vai Met Glu 15 10 15

Arg Val Ser Go Asp Pro Go Thr Phe Ser Leu Ser Glu Leu Lys Gin 20 25 30

Ala Ile Pro Pro His Cys Phe Gin Arg Ser Val Ile Arg Ser Ser Tyr 35 40 45

Tyr Val Val Gin Asp Leu Ile Ile Ala Tyr Ile Phe Tyr Phe Leu Ala 50 55 60 103

Asn Thr Tyr Ile Pro Thr Leu Pro Thr Ser Leu Ala Tyr Leu Ala Trp 65 70 75 80

Pro Vai Tyr Trp Phe Cys Gin Ala Ser Vai Leu Thr Gly Leu Trp Ile 85 90 95

Leu Gly His Glu Cys Gly His His Ala Phe Ser Asn Tyr Thr Trp Phe 100 105 110

Asp Asp Thr Val Gly Phe Ile Leu His Ser Phe Leu Leu Thr Pro Tyr 115 120 125

Phe Ser Trp Lys Phe Ser His Arg Asn His His Ser Asn Thr Ser Ser 130 135 140

Ile Asp Asn Asp Glu Vai Tyr Ile Pro Lys Ser Lys Ser Lys Leu Ala 145 150 155 160

Arg Ile Tyr Lys Leu Leu Asn Asn Pro Pro Gly Arg Leu Leu Vai Leu 165 170 175

Ile Ile Met Phe Thr Leu Gly Phe Pro Leu Tyr Leu Leu Thr Asn Ile 1B0 185 190

Ser Gly Lys Lys Tyr Asp Arg Phe Ala Asn His Phe Asp Pro Met Ser 195 200 205

Pro Ile Phe Lys Glu Arg Glu Arg Phe Gin Phe Leu Ser Asp Leu 210 215 220

Gly Leu Leu Ala Go Phe Tyr Gly Ile Lys Go Wing Go Wing Asn Lys 225 230 235 240

Gly Ala Ala Trp Go Ala Cys Met Tyr Gly Go Pro Go Go Leu Gly Go 245 250 255

Phe Thr Phe Phe Asp Ile Ile Thr Phe Leu His His Thr His Gin Ser 260 265 270 104

Ser Pro His Tyr Asp Ser Thr Glu Trp Asn Trp Ile Arg Gly Ala Leu 275 280 285

Be Ala Ile Asp Arg Asp Phe Gly Phe Leu Asn Ser Vai Phe His Asp 290 295 300

Go Thr His Thr His Go His His His Leu Phe Ser Tyr Ile Pro His 305 310 315 320

Tyr His Ala Lys Glu Ala Arg Asp Ala Ile Lys Pro Ile Leu Gly Asp 325 330 335

Phe Tyr Met Ile Asp Arg Thr Pro Ile Leu Lys Ala Met Trp Arg Glu 340 345 350

Gly Arg Glu Cys Met Tyr Ile Glu Pro Asp Ser Lys Leu Lys Gly Vai 355 360 365

Tyr Trp Tyr His Lys Leu 370 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1312 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Crepis sp. 105

(ix) CHARACTERISTICS

(A) NAME / KEY: CDS (B) LOCATION: 26..1147 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGTTGACCAT AAATCATCTA TCAAC ATG GGT GCC GGC GGC CGT GGT CGG ACA 52 Met Gly Ala Gly Gly Arg Gly Arg Thr 1 5 TCG GAA AAG TCG GTC ATG GAA CGT GTC TCA GTT GAT CCA GTA ACC TTC 100 Ser Glu Lys Ser Vai Met Glu Arg Will Be Go Asp Pro Go Thr Phe 10 15 20 25 TCA CTG AGT GAT TTG AAG CAA GCA ATC CCT CCA CAT TGC TTC CAG CGA 148 Ser Leu Ser Asp Leu Lys Gin Ile Ile Pro Pro His Cys Phe Gin Arg 30 35 40 TCT GTC ATC CGT TCA TCT TAT TAC GTT GTT CAG GAT CTC ATA ATT GCC 196 Ser Vai Ile Arg Ser Ser Tyr Tyr Go Go Gin Asp Leu Ile Ile Ala 45 50 55 TAC ATC TTC TAC TTC CTT GCC AAC ACA TAT ATC CCT AAT CTC CCT CAT 244 Tyr Ile Phe Tyr Phe Leu Ala Asn Thr Tyr Ile Pro Asn Leu Pro His 60 65 70 CCT CTA GCC TAC TTA GCT TGG CCG CTT TAC TGG TTC TGT CAA GCT AGC 292 Pro Leu Ala Tyr Leu Ala Trp Pro Leu Tyr Trp Phe Cys Gin Ala Ser 75 80 85 GTC CTC ACT GGG TTA TGG ATC CTC GGC CAT GAA TGT GGT CAC CAT GCC 340 Go Leu Thr Gly Leu Trp Ile Leu Gly His Glu Cys Gly His His Ala 90 95 100 105 TAT AGC AAC TAC ACA TGG GTT GAC GAC ACT GTG GGC TTC ATC ATC CAT 388 Tyr Ser Asn Tyr Thr Trp Go Asp Asp Thr Go Gly Phe Ile Ile His 110 115 120 TCA TTT CTC CTC ACC CCG TAT TTC TCT TGG AAA TAC AGT CAC CGG AAT 436 Ser Phe Leu Leu Thr Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Asn 125 130 135 106 484 CAC CAT TCC AAC ACA AGT His His Ser Asn Thr Ser 140 AAA AGC AAG TCC AAA CTC Lys Ser Lys Ser Lys Leu 155 CCT GGT CGA CTG TTG GTT Pro Gly Arg Leu Leu Vai 170 175 TTA TAC CTC TTG ACA AAT Leu Tyr Leu Leu Thr Asn 190 AAC CAC TTC GAC CCC ATG Asn His Phe Asp Pro Met 205 CAG GTC TTC CTT TCG GAT Gin Phe Leu Ser Asp 220 AAA GTT GCT GTA GCA AAT Lys Go Ala Goa Wing Asn 235 GGA GTT CCG GTG CTA GGC Gly Go Pro Go Leu Gly 250 255 TTA CAC CAC ACC CAT CAG Leu His His Thr His Gin 270 AAC TGG ATC AGA GGG GCT Asn Trp Ile Arg Gly Ala 285 TCG ATT GAT AAC GAT GAA Ser Ile Asp Asn Asp Glu 145 AAG CGT ATC TAT AAA CTT Lys Arg Ile Tyr Lys Leu ile 165 TTG GTT ATC ATG TTC ACC Leu Ile Ile Phe Thr 180 ATT TCC GGC AAG AAA TAC Ile Ser Gly Lys Lys Tyr 195 AGT CCA ATT TTC AAA GAA Ser Pro Ile Phe Lys Glu 210 CTT GGT CTT CTT GCT GTG Leu Gly Leu Leu Ala Go 225 AAA GGA GCT GCT TGG GTG Lys Gly Ala Ala Trp Go 240 245 GTA TTT ACC TTT TTC GAT Go Phe Thr Phe Phe Asp 260 TCG TCG CCT CAT TAT GAT Ser Ser Pro His Tyr Asp 275 TTG TCA GCA ATC GAT AGN Leu Ser Ala Ile Asp Arg 290 GTT TAC ATT CCG is Tyr ile Pro 150 CTT AAC AAC CCA 532 Leu Asn Asn Pro CTA GGA TTT CCT 580 Leu Gly Phe Pro 185 GAT AGG TTT GCC 628 Asp Arg Phe Ala 200 CGT GAG CGG TTT 676 Arg Glu Arg Phe 215 TTT TAT GGA ATT 724 Phe Tyr Gly Ile 230 GCG TGC ATG TAT 772 Wing Cys Met Tyr GTG ATC ACG TTC 820 Vai Ile Thr Phe 265 TCA ACT GAA TGG 868 Ser Thr Glu Trp 280 GAC TTT GGG TTC 916 Asp Phe Gly Phe 295 107 CTG AAT AGT GTT TTC CAT GAT GTN ACA CAC ACT CAC GTC ATG CAT CAT 964 Leu Asn Ser Go To His His Asp Go His Thr His To His Met His His 300 305 310 TTG TTT TCA TAC ATT CCA CAC TAT CAT GCA AAG GAA GCA AGG GAT GCA 1012 Leu Phe Ser Tyr Ile Pro His Tyr His Ala Lys Glu Ala Arg Asp Ala 315 320 325 ATC AAA CCG ATC TTG GGC GAC TTT TAT ATG ATC GAT AGG ACT CCA ATT 1060 Xle Lys Pro Ile Leu Gly Asp Phe Tyr Met Ile Asp Arg Thr Pro Ile 330 335 340 345 TTA AAA GCA ATG TGG AGA GAG GGC AGG GAA TGC ATG TAC ATC GAG CCT 1108 Leu Lys Ala Met Trp Arg Glu Gly Arg Glu Cys Met Tyr Ile Glu Pro 350 355 360 GAT AGC AAG CTC AAA GGT GTT TAT TGG TAT CAT AAA TTG TGATCATATG 1157 Asp Ser Lys Leu Lys Gly Vai Tyr Trp Tyr His Lys Leu 365 370 CAAAATGCAC ATGCATTTTC AAACCCTCTA GTTACC TTTG TTCTATGTAT AATAAGACCG 1217 CCGGTCCTAT GGTTTTCTAT GCCTAAGCCA GGCGAAATAG TTAAATAATA TCGGTATGAT 1277 GTAATGAAAG TATGTGGTTG TCTAAAAAAA AAAAA 1312 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 374 amino acids (B) TYPE: amino acid (D ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 108

Met Gly Ala Gly Gly Arg Gly Arg Thr Ser Glu Lys Ser Vai Met Glu 15 10 15

Arg Will Go Asp Pro Will Thr Phe Be Leu Ser Asp Leu Lys Gin 20 25 30

Ala Ile Pro Pro His Cys Phe Gin Arg Ser Vai Ile Arg Ser Ser Tyr 35 40 45

Tyr Go Vai Gin Asp Leu Ile Ile Ala Tyr Ile Phe Tyr Phe Leu Ala 50 55 60

Asn Thr Tyr Ile Pro Asn Leu Pro His Pro Leu Ala Tyr Leu Ala Trp 65 70 75 80

Pro Leu Tyr Trp Phe Cys Gin Ala Ser Vai Leu Thr Gly Leu Trp Ile 85 90 95

Leu Gly His Glu Cys Gly His His Ala Tyr Ser Asn Tyr Thr Trp Val 100 105 110

Asp Asp Thr Val Gly Phe Ile Ile His Phe Leu Leu Thr Pro Tyr 115 120 125

Phe Ser Trp Lys Tyr Ser His Arg Asn His His Ser Asn Thr Ser Ser 130 135 140

Ile Asp Asn Asp Glu Vai Tyr Ile 145 150

Arg Ile Tyr Lys Leu Leu Asn Asn 165

Go Ile Met Phe Thr Leu Gly Phe 180

Ser Gly Lys Lys Tyr Asp Arg Phe 195 200

Pro Ile Phe Lys Glu Arg Glu Arg 210 215

Pro Lys Ser Lys Ser Lys Leu Lys 155 160

Pro Pro Gly Arg Leu Leu Vai Leu 170 175

Pro Leu Tyr Leu Leu Thr Asn Ile 185 190

Ala Asn His Phe Asp Pro Met Ser 205

Phe Gin Vai Phe Leu Ser Asp Leu 220 109

Gly Leu Leu Ala Go Phe Tyr Gly 225 230

Gly Ala Ala Trp Go Ala Cys Met 245

Phe Thr Phe Phe Asp Val Ile Thr 260

Ser Pro His Tyr Asp Ser Thr Glu 275 280

Ser Ala Ile Asp Arg Asp Phe Gly 290 295

Ile Lys Goes Ala Goes Ala Asn Lys 235 240

Tyr Gly Go Pro Go Go Leu Gly Go 250 255

Phe Leu His His Thr His Gin Ser 265 270

Trp Asn Trp Ile Arg Gly Ala Leu 285

Phe Leu Asn Ser Go Phe His Asp 300

Go Thr His Thr His Go His His His Leu Phe Ser Tyr Ile Pro His 305 310 315 320

Tyr His Ala Lys Glu Ala Arg Asp Ala Ile Lys Pro Ile Leu Gly Asp 325 330 335

Phe Tyr Met Ile Asp Arg Thr Pro Ile Leu Lys Ala Met Trp Arg Glu 340 345 350

Gly Arg Glu Cys Met Tyr Ile Glu Pro Asp Ser Lys Leu Lys Gly Vai 355 360 365

Tyr Trp Tyr His Lys Leu 370 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 550 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear 110 (ii) MOLECULE TYPE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Vernonia galamensis (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..549 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CAT CAC GCC TTC AGT GAC TAT CAA TGG ATA GAC GAC ACT GTG GGC TTC 48 His His Ala Phe Ser Asp Tyr Gin Trp Ile Asp Asp Thr Val Gly Phe 1 5 10 15 ATC CTT CAC TTT GCA CTC TTC ACC CCT TAT TTC TCT TGG AAA TAC AGT 96 Ile Leu His Phe Ala Leu Phe Thr Pro Tyr Phe Ser Trp Lys Tyr Ser 20 25 30 CAC CGT AAT CAC CAT GCC AAC ACA AAC TCT CTT GTA ACC GAT GAA GTA 144 His Arg Asn His His Wing Asn Thr Asn Ser Leu Go Thr Asp Glu Vai 35 40 45 TAC ATC CCT AAA GTT AAA TCC AAG GTC AAG ATT TAT TCC AAA ATC CTT 192 Tyr lie Pro Lys Go Lys Ser Lys Go Lys Ile Tyr Ser Lys Ile Leu 50 55 60 AAC AAC CCT CCT GGT CGC GTT TTC ACC TTG GCT TTC AGA TTG ATC GTG 240 Asn Asn Pro Pro Gly Arg Go Phe Thr Leu Ala Phe Arg Leu Ile Val 65 65 75 B0 GGT TTT CCT TTA TAC CTT TTC ACC AAT GTT TCA GGC AAG AAA TAC GAA 288 Gly Phe Pro Leu Tyr Leu Phe Thr Asn Will Be Gly Lys Lys Tyr Glu 85 90 95 CGT TTT GCC AAC CAT TTT GAT CCC ATG AGT CCC ATT TTC ACC GAG CGT 336 Arg Phe Ala Asn His Phe Asp Pro Met Ser Pro Ile Phe Thr Glu Arg 100 105 110 111 GAG CAT GTA CAA GTC TTG CTT TCT GAT TTT GGT CTC ATA GCA GTT GCT 384 Glu His Vai Gin Vai Leu Leu Ser Asp Phe Gly Leu Ile Ala Vai Ala 115 120 125 TAC GTG GTT CGT CAA GCT GTA CTG GCT GGA GGT GCT TGG GTG ATG 432 Tyr Go Go Arg Gin Ala Go Leu Ala Lys Gly Gly Ala Trp Go Met 130 135 140 TGC ATT TAC GGA GTT CCT GTG CTG GCC GTA AAC GCA TTC TTT GTT TTA 480 Cys Ile Tyr Gly Vai Pro Go Leu Ala Go Asn Ala Phe Phe Go Leu 145 150 155 160 ATC ACT TAT CTT CAC CAC ACG CAT CTC TCA CTG CCC CAC TAT GAT AGC 528 Ile Thr Tyr Leu His His Thr His Leu Ser Leu Pro His Tyr Asp Ser 1C5 170 175 TCA GAA TGG GAC TGG CTA CGA G 550 Ser Glu Trp Asp Trp Leu Arg 180 (2) INFORMATION FOR SEQ ID NO: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 183 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 112

His His Ala Phe Ser Asp Tyr Gin Trp Ile Asp Asp Thr Val Gly Phe 15 10 15

Ile Leu His Phe Ala Leu Phe Thr Pro Tyr Phe Ser Trp Lys Tyr Ser 20 25 30

His Arg Asn His His Wing Asn Thr Asn Ser Leu Go Thr Asp Glu Go 35 40 45

Tyr Ile Pro Lys Go Lys Ser Lys Go Lys Ile Tyr Ser Lys Ile Leu 50 55 60

Asn Asn Pro Pro Gly Arg Go Phe Thr Leu Ala Phe Arg Leu Ile Go 65 70 75 80

Gly Phe Pro Leu Tyr Leu Phe Thr Asn Will Be Gly Lys Lys Tyr Glu 85 90 95

Arg Phe Ala Asn His Phe Asp Pro 100

Glu His Vai Gin Vai Leu Leu Ser 115 120

Tyr Go Go Go Arg Gin Goes Leu 130 135

Cys Ile Tyr Gly Vai Pro Vai Leu 145 150

Ile Thr Tyr Leu His His Thr His 165

Met Ser Pro Ile Phe Thr Glu Arg 105 110

Asp Phe Gly Leu Ile Ala Goa Ala 125

Ala Lys Gly Gly Ala Trp Vai Met 140

Ala Vai Asn Ala Phe Phe Vai Leu 155 160

Leu Ser Leu Pro His Tyr Asp Ser 170 175

SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: 113 (A) LENGTH: 177 base pairs (B) TYPE: nucleic acid (C) STRAND TYPE (ix) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Crepis alpina (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 1..177 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAA TGC GGT CAC CAT GCC TTC AGC GAC TAC CAG TGG GTT GAC GAC AAT 48 Glu Cys Gly His His Ala Phe Ser Asp Tyr Gin Trp Val Asp Asp Asn 1 5 10 15 GTG GGC TTC ATC CTC CAC TCG TTT CTC ATG ACC CCG TAT TTC TCC TGG 96 Go Gly Phe Ile Leu His Ser Phe Leu Met Thr Pro Tyr Phe Ser Trp 20 25 30 AAA TAC AGC CAC CGG AAC CAC CAT GCC AAC ACA AAT TCG CTT GAC AAC 144 Lys Tyr Ser His Arg Asn His His Wing Asn Thr Asn Ser Leu Asp Asn 35 40 45 GAT GAA GTT TAC ATC CCC AAA AGC AAG GCC (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 amino acids (B) TYPE: amino acid (D) (SEQ ID NO: 8) ) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Glu Cys Gly His His Ala Phe Ser Asp Tyr Gin Trp Go Asp Asp Asn 15 10 15

Go Gly Phe Ile Leu His Phe Leu Met Thr Pro Tyr Phe Ser Trp 20 25 30

Lys Tyr Ser His Arg Asn His His Wing Asn Thr Asn Ser Leu Asp Asn 35 40 45

Asp Glu Vai Tyr Ile Pro Lys Ser Lys Ala Lys 50 55 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 383 amino acids (B) TYPE: amino acid (C) TYPE OF CHEMISTRY: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: 115 (A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Gly Ala Gly Gly Arg Met Pro Go Pro Thr Ser Ser Lys Lys Ser 15 10 15

Glu Thr Asp Thr Thr Lys Arg Val Pro Cys Glu Lys Pro Pro Phe Ser 20 25 30 Val Gly Asp Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45

Ile Pro Arg Ser Phe Ser Tyr Leu Ile Ser Asp Ile Ile Ile Ala Ser 50 55 60

Cys Phe Tyr Tyr Val Ala Thr Asn Tyr Phe Ser Leu Pro Pro Gin 65 70 75 80

Leu Ser Tyr Leu Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Cys Val 85 90 95

Leu Thr Gly Ile Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100 105 HO

Ser Asp Tyr Gin 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

Gin Lys Ser Ala Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 116

Gly Arg Ile Met Met Leu Thr Vai Gin Phe Go Leu Gly Trp Pro Leu 180 185 190

Tyr Leu Ala Phe Asn Will Be 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 Gin 210 215 220

Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Vai Cys Phe Gly Leu Tyr 225 230 235 240

Arg Tyr Ala Ala Ala Gin Gly Met Ala Ser Met Ile Cys Leu Tyr Gly 245 250 255

Go Pro Leu Leu Ile Go Asn Ala Phe Leu Go Leu Ile Thr Tyr Leu 260 265 270

Gin 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 Go Asp Arg Asp Tyr Gly Ile Leu 290 295 300

Asn Lys Will Phe His Asn Ile Thr Asp Thr His Will Ala His Hisu 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 Gin Phe Asp Gly Thr Pro Trp Tyr 340 345 350

Go Ala Met Tyr Arg Glu Ala Lys Glu Cys Ile Tyr Go Glu Pro Asp 355 360 365

(2) SEQUENCE CHARACTERISTICS: (A) LENGTH: 384 amino acids (B) TYPE: (a) LENGTH: 384 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Brassica juncea (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Gly Ala Gly Gly Arg Met Gin Will Be Pro Pro Lys Lys Ser 15 10 15

Glu Thr Asp Thr Leu Lys Arg Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30

Go Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys Arg Ser 35 40 45

Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Val Ile 50 55 60

Cys Phe Tyr Tyr Go Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 65 70 75 80

Leu Ser Tyr Go Ala Trp Pro Leu Tyr Trp Ala Cys Gin Gly Go Go 85 90 95 118

Leu Thr Gly Go Trp Go Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110

Ser Asp Tyr Gin Trp Leu Asp Asp Thr Go Gly Leu Ile Phe His Ser 115 120 125

Phe Leu Leu Go 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 Go Phe Go Pro Lys 145 150 155 160

Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175

Gly Arg Thr Vai Met Leu Thr Go Gin Phe Thr Leu Gly Trp Pro Leu 180 185 190

Tyr Trp Ala Phe Asn Will Be Gly Arg Pro Tyr Pro Glu Gly Phe Ala 195 200 205

Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220

Gin Ile Tyr Will Be Asp Ala Gly Ile Leu Ala Go Cys Tyr Gly Leu 225 230 235 240

Tyr Arg Tyr Ala Ala Ala Gin Gly Vai Ala Ser Met Vai Cys Leu Tyr 245 250 255

Gly Go Pro Leu Leu Ile Go Asn Ala Phe Leu Go Leu Ile Thr Tyr 260 265 270

Leu Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280 285

Asp Trp Leu Arg Gly Ala Leu Ala Thr Go Asp Arg Asp Tyr Gly Ile 290 295 300 119

Leu Asn Lys Will Phe His Asn Ile Thr Asp Thr His Will Ala His His 305 310 315 320

Leu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Vai Thr Lys Ala 325 330 335

Ile Lys Pro Ile Leu Gly Asp Tyr Tyr Gin Phe Asp Gly Thr Pro Trp 340 345 350

Go Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr Go Glu Pro 355 360 365

(A) LENGTH: 383 amino acids (B) TYPE: (a) LENGTH: 383 amino acids (B) TYPE: (a) LENGTH: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Glycine max (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 120

Met Gly Ala Gly Gly Arg Thr Asp Go Pro Pro Ala Asn Arg Lys Ser 1 S 10 15

Glu Go Asp Pro Leu Lys Arg Go Pro Phe Glu Lys Pro Gin Phe Ser 20 25 30

Leu Ser Gin Ile Lys Lys Ala Ile Pro Pro His Cys Phe Gin Arg Ser 35 40 45

Go Leu Arg Be Phe Be Tyr Go Go Tyr Asp Leu Thr Ile Ala Phe 50 55 60

Cys Leu Tyr Tyr Go Ala Thr His Tyr Phe His Leu Leu Pro Gly Pro 65 70 75 80

Leu Ser Phe Arg Gly Met Ala Ile Tyr Trp Ala Go Gin Gly Cys Ile 85 90 95

Leu Thr Gly Go Trp Go Ile Ala His Glu Cys Gly His His Ala Phe 100 105 110

Ser Asp Tyr Gin Leu Leu Asp Asp Ile Go Gly Leu Ile Leu His Ser 115 120 125

Ala Leu Leu Pro V 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 Go Phe Go Pro Lys 145 ISO 155 160

Gin Lys Ser Cys Ile Lys Trp Tyr Ser Lys Tyr Leu Asn Asn Pro Pro 165 170 175

Gly Arg Go Leu Thr Leu Ala Go Thr Leu Thr Leu Gly Trp Pro Leu 180 185 190

Tyr Leu Ala Leu Asn Will Be Gly Arg Pro Tyr Asp Arg Phe Ala Cys 195 200 205 121

His Tyr Asp Pro Tyr Gly Pro Ile Tyr Ser Asp Are Glu Arg Leu Gin 210 215 220

Ile Tyr Ile Ser Asp Ala Gly Vai Leu Ala Vai Vai Tyr Gly Leu Phe 225 230 235 240

Arg Leu Ala Met Ala Lys Gly Leu Ala Trp Vai Vai Cys Vai Tyr Gly 245 250 255

Go Pro Leu Leu Go Go Asn Gly Phe Leu Go Leu Ile Thr Phe Leu 260 265 270

Gin His Thr His Pro Ala Leu Pro His Tyr Thr Ser Ser Glu Trp Asp 275 280 285

Trp Leu Arg Gly Ala Leu Ala Thr Go Asp Arg Asp Tyr Gly Ile Leu 290 295 300

Asn Lys Will Phe His Asn Ile Thr Asp Thr His Will Ala His Hisu 305 310 315 320

Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala Thr Lys Ala Ile 325 330 335

Lys Pro Ile Leu Gly Glu Tyr Tyr Arg Phe Asp Glu Thr Pro Phe Vai 340 345 350

Lys Ala Met Trp Arg Glu Ala Arg Glu Cys Ile 'yr Vai Glu Pro Asp 355 360 365

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 383 amino acids (B) TYPE: amino acid (A) LENGTH: 383 amino acids 122 (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Solanum commersonii (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Gly Ala Gly Gly Arg Met Ser Ala Pro Asn Gly Glu Thr Glu Val 15 10 15

Lys Arg Asn Pro Leu Gin Lys Go Pro Thr Ser Lys Pro Pro Phe Thr 20 25 30

Go Gly Asp Ile Lys Lys Ala Ile Pro Pro His Cys Phe Gin Arg Ser 35 40 45

Leu Ile Arg Ser Phe Ser Tyr Will Go Tyr Asp Leu Ile Leu Will Be 50 55 60

Ile Met Tyr Tyr Go Ala Asn Thr Tyr Phe His Leu Leu Pro Ser Pro 65 70 75 80

Tyr Cys Tyr Ile Ala Trp Pro Ile Tyr Trp Ile Cys Gin Gly Cys Go 85 90 95

Cys Thr Gly Ile Trp Val Asn Ala His Glu Cys Gly His His Ala Phe 100 105 110

Ser Asp Tyr Gin Trp Go Asp Asp Thr Go Gly Leu Ile Leu His Ser 115 120 125

Ala Leu Leu Go Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 123

His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Go Phe Go Pro Lys 145 150 155 160

Pro Lys Ser Gin Leu Gly Trp Tyr Ser Lys Tyr Leu Asn Asn Pro Pro 165 170 175

Gly Arg Go Leu Ser Leu Thr Ile Thr Leu Thr Leu Gly Trp Pro Leu 180 185 190

Tyr Leu Ala Phe Asn Will Be Gly Arg Pro Tyr Asp Arg Phe Ala Cys 195 200 205

His Tyr Asp Pro Tyr Gly Pro Ile Tyr Asn Asn Arg Glu Arg Leu Gin 210 215 220

Ile Phe Ile Ser Asp Ala Gly Vai Leu Gly Vai Cys Tyr Leu Leu Tyr 225 230 235 240

Arg Ile Ala Leu Go Lys Gly Leu Ala Trp Leu Go Cys Go Tyr Gly 245 250 255

Go Pro Leu Leu Go Go Asn Gly Phe Leu Go Leu Ile Thr Tyr Leu 260 265 270

Gin His Thr His Pro Ser Leu Pro His Tyr Asp Ser Thr Glu Trp Asp 275 280 285

Trp Leu Arg Gly Ala Leu Ala Thr Cys Asp Arg Asp Tyr Gly Vai Leu 290 295 300

Asn Lys Will Phe His Asn Ile Thr Asp Thr His Will Will His Hisu 305 310 315 320

Phe Ser Thr Met Pro His Tyr Asn Ala Met Glu Ala Thr Lys Ala Go 325 330 335

Lys Pro Leu Leu Gly Asp Tyr Tyr Gin Phe Asp Gly Thr Pro Ile Tyr 340 345 350 124

Lys Glu Met Trp Arg Glu Ala Lys Glu Cys Leu Tyr Go Glu Lys Asp 355 360 365

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 387 amino acids (B) TYPE: amino acid (B) TYPE: amino acid (A) LENGTH: 387 amino acids (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Glycine max (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Gly Leu Ala Lys Glu Thr Thr Met Gly Gly Arg Gly Arg Val Ala 15 10 15

Lys Go Glu Go Gin Gly Lys Lys Pro Leu Ser Arg Go Pro Asn Thr 20 25 30

Lys Pro Pro Phe Thr Go Gly Gin Leu Lys Lys Ala Ile Pro Pro His 35 40 45 125

Cys Phe Gin Arg Ser Leu Leu Thr Ser Phe Ser Tyr Go Vai Tyr Asp 50 55 60

Leu Ser Phe Ala Phe Ile Phe Tyr 65 70 Leu Pro Gin Pro Phe Ser Leu Ile 85 Gin Gly Cys Leu Leu Thr Gly Val 100 His His Phe Ser Lys Tyr Gin 115 120 Thr Leu His Ser Thr Leu Leu Val 130 135

Ile Ala Thr Thr Tyr Phe His Leu 75 80

Ala Trp Pro Ile Tyr Trp Vai Leu 90 95

Trp Ile Ile His Glu Cys Gly 105 110

Trp Go Asp Asp Go Go Gly Leu 125

Pro Tyr Phe Ser Trp Lys Ile Ser 140

His Arg Arg His His Asn Thr Gly Ser Leu Asp Arg Asp Glu Val 145 150 155 160

Phe Go Pro Lys Pro Lys Ser Lys Go Ala Trp Phe Ser Lys Tyr Leu 165 170 175

Asn Asn Pro Leu Gly Arg Ala Will Be Leu Leu Will Thr Leu Thr Ile 180 185 190

Gly Trp Pro Met Tyr Leu Ala Phe Asn Will Be Gly Arg Pro Tyr Asp 195 200 205

Ser Phe Ala His His Tyr His Pro Tyr Ala Pro Ile Tyr Ser Asn Arg 210 215 220

Glu Arg Leu Leu Ile Tyr Will Be Asp Go Ala Leu Phe Be Go Thr 225 230 235 240

Tyr Ser Leu Tyr Arg Go Ala Thr Leu Lys Gly Leu Go Trp Leu Leu 245 250 255 126

Cys Go Tyr Gly Go Pro Leu Leu Ile Go Asn Gly Phe Leu Go Thr 260 265 270

Ile Thr Tyr Leu Gin His Thr His Phe Ala Leu Pro His Tyr Asp Ser 275 280 285

Ser Glu Trp Asp Trp Leu Lys Gly Ala Leu Ala Thr Met Asp Arg Asp 290 295 300

Tyr Gly Ile Leu Asn Lys Go Phe His His Ile Thr Asp Thr His Go 305 310 315 320

Ala His Hisu Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala 325 330 335

Thr Asn Ala Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gin Phe Asp Asp 340 345 350

Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Arg Glu Cys Leu Tyr 355 360 365

Go Glu Pro Asp Glu Gly Thr Ser Glu Lys Gly Vai Tyr Trp Tyr Arg 370 375 380

Asn Lys Tyr 385 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 387 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (ii) TYPE OF MOLECULE: protein 127 (vi) ORIGINAL SOURCE: (A) ORGANISM: Ricinus communis (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Gly Gly Gly Gly Arg Met Ser Thr Val Ile Ile Thr Ser Asn Asn Ser 15 10 15

Glu Lys Lys Gly Gly Ser Ser His Leu Lys Arg Ala Pro His Thr Lys 20 25 30

Pro Pro Phe Thr Leu Gly Asp Leu Lys Arg Ala Ile Pro Pro His Cys 35 40 45

Phe Glu Arg Ser Phe Go Arg Arg Ser Phe Ser Tyr Go Ala Tyr Asp Go 50 55 60

Cys Leu Ser Phe Leu Phe Tyr Ser Ile Ala Thr Asn Phe Phe Pro Tyr 65 70 75 80

Ile Ser Ser Pro Leu Ser Tyr Go 85

Gin Gly Cys Ile Leu Thr Gly Leu 100

His His Wing Phe Ser Glu Tyr Gin 115 120

Ile Goes His Being Lea Leu Leu Goes 130 135

His Arg Arg His His Be Asn Ile 145 150

Ala Trp Leu Vai Tyr Trp Leu Phe 90 95

Trp Ile Ile Gly His Glu Cys Gly 105 110

Leu Wing Asp Asp Ile Go Gly Leu 125

Pro Tyr Phe Ser Trp Lys Tyr Ser 140

Gly Ser Leu Glu Arg Asp Glu Val 155 160

Phe Vai Pro Lys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 165 170 175 128

Asn Asn Pro Pro Gly Arg Vai Leu Thr Leu Ala Ala Thr Leu Leu Leu 180 185 190

Gly Trp Pro Leu Tyr Leu Ala Phe Asn Will Be Gly Arg Pro Tyr Asp 195 200 205

Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 210 215 220

Glu Arg Leu Gin Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr 225 230 235 240

Phe Vai Leu Tyr Gin Ala Thr Met Ala Lys Gly Leu Ala Trp Vai Met 245 250 255

Arg Ile Tyr Gly Go Pro Leu Leu Ile Go Asn Cys Phe Leu Vai Met 260 265 270

Ile Thr Tyr Leu Gin His Thr His Pro Ala Ile Pro Arg Tyr Gly Ser 275 280 285

Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Vai Thr Vai Asp Arg Asp 290 295 300

Tyr Gly Go Leu Asn Lys Go Phe His Asn Ile Ala Asp Thr His Go 305 310 315 320

Ala His Hisu Phe Ala Thr Go Pro His Tyr His Ala Met Glu Ala 325 330 335

Thr Lys Ala Ile Lys Pro Ile Met Gly Glu Tyr Tyr Arg Tyr Asp Gly 340 345 350

Thr Pro Phe Tyr Lys Ala Leu Trp Arg Glu Ala Lys Glu Cys Leu Phe 355 360 365 129

Go Glu Pro Asp Glu Gly Ala Pro Thr Gin Gly Go Phe Trp Tyr Arg 370 375 380

Asn Lys Tyr 385 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (ii) TYPE OF MOLECULE: peptide (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) STRANDEDNESS: (A) LENGTH: 5 amino acids ) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 130 (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) TYPE OF FRAGMENT: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17

SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear 131 (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TGGAATTCCY TBMGNNNNYT SGGNHTBGG 29 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: 1610 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (vi) ORIGINAL SOURCE: (A) ORGANISM : Euforbia lagascae (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 8..1546 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGATACA ATG AAC ACT AAG GAG AAG AAG AAG AAG AAC AGG GTT TCT AAC 49

Met Asn Thr Lys Glu Lys Lys Lys Lys Asn Arg Will Be Asn 15 10 ATG TCT ATT CTT CTT TGC TTC CTT TGC CTT CTT CCA GTT TTC CTT GTT 97

Met Ser Ile Leu Leu Cys Phe Leu Cys Leu Leu Pro Go Phe Leu Go 15 20 25 30 132 145 TCT CTT TCT ATT CTT TCT AAG AGG CTT AAG Ser Leu Ser Ile Leu Ser Lys Arg Leu Lys 35 40 CCA CCA GGA CCA AAG ACT CTT CCA ATT Pro Pro Gly Pro Lys Thr Leu Pro Ile Ile 50 55 AGG CAA GAT CCA GAG GCT TCT CTT TCT CAA Arg Gin Asp Pro Glu Ala Ser Leu Ser Gin 65 70 CCA GTT GTT CAT TGC GAG AAG CTT GAG TCT Pro Go Glycine Glycerol Glycerol Glycerol Glycogen Glycerol Glycogen Glycogen Glycogen Glycogen Glycogen Glycerol Glycogen Glycogen Glycogen Glycogen Glycogen Glycogen Glycogen Glycogen Glycogen Gly Gly Gly Gly Lys Ser Pro 115 120 ACT GGA GGA TAT GGA CTT GAG AGG AAG Thr Gly Gly Tyr Gly Leu Glu Arg Ser Lys 130 135 AAG GAG ACT TGG TCT GCT TTC AGG CAA TAT Lys Glu Thr Trp Ser Ala Phe Arg Gin Tyr 145 150 GGA ATG GGA GGA AGG TCT TTC GAG CTT ATG Gly Met Gly Gly Arg Ser Phe Glu Leu Met 160 165 TGC CTT GTT GAT GGA TAT GTT TCT AGG AAG Cys Leu Vai Asp Gly Tyr Will Be Arg Lys 175 180 CCA TCT AAG TGG AAG CTT Pro Ser Lys Trp Lys Leu 45 GGA AAC CTT CAA GAT GAG 193 Gly Asn Leu Gin Asp Glu 60 GGA CAT ATT GCT AGG GGA 241 Gly His Ile Ala Arg Gly 75 TTC GGA ACT CAA CCA ACT 289 Phe Gly Thr Gin Pro Thr 90 GCT CTT CTT CAT GGA GCT 337 Leu Leu His Gly Ala 105 110 CCA AAC GAT GCT TGG GAT 385 Pro Asn Asp Ala Trp Asp 125 AAC GAG AGG TGG AAG GAG 433 Asn Glu Arg Trp Lys Glu 140 AGG ACT CTT AGG GCT TTC 481 Arg Thr Leu Arg Ala Phe 155 AGG TGG CAA GAG GCT CAT 529 Arg Trp Gin Glu Ala His 170 GCT TCT GGA ACT GAT CCA 577 Ala Ser Gly Thr Asp Pro 185 190 133 625 ACT AAG GAT CTT GAG GAT TCT AGG TTC AAC ATT ATT ATG GGA GCT.ACT Thr Lys Asp Leu Glu Asp Ser Arg Phe Asn Ile Ile Met Gly Ala Thr 195 200 205 TTC AAC CAA GGA CTT GAT TAT AAG ATT AAG ACT TTC CTT GAT AGG CAT 673 Phe Asn Gin Gly Leu Asp Tyr Lys Ile Lys Thr Phe Leu Asp Arg His 210 215 220 GAG AGG AGG AAC TTC CAA TTC AAC AAT GTT GAT GCT GTT TAT CAT CAA 721 Glu Arg Arg Asn Phe Gin Phe Asn Asn Asp Ala Vai Tyr His Gin 225 230 235 ATG AAG GAT GCT GAG AGG GGA TTC GTT GAT TCT AGG GGA TGG CAA GAT 769 Met Lys Asp Ala Glu Arg Gly Phe Go Asp Ser Arg Gly Trp Gin Asp 240 245 250 GAG TTC GGA ATT GCT CTT CAA CAA GTT GTT GCT CAA ATT CTT GAT AAG 817 Glu Phe Gly Ile Ala Leu Gin Gin Go Vai Ala Gin Ile Leu Asp Lys 255 260 265 270 CCA CTT GAT CAT CAA AAG GCT CTT GAG AGG TGG CAA CCA AGG GAT TCT 865 Pro Leu Asp His Gin Lys Ala Leu Glu Arg Trp Gin Pro Arg Asp Ser 275 280 285 CTT AAC CAT TTC ATT GGA GCT AGG GAT GAT GAG ATG GTT CAA ATT AAG 913 Leu Asn His Phe Ile Gly Ala Arg Asp Asp Glu Met Vai Gin Ile Lys 290 295 300 TAT TGT TGC AAG GAT GCT CTT AGG ATG TTC GAT ACT GGA ATT CTT 961 Tyr Asp Phe Cys Lys Asp Ala Leu Arg Met Phe Asp Thr Gly .I Leu 305 310 315 GCT GCT GAT CTT CAA TCT TCT ACT TCT TCT ATT AGG TGG GAG CCA ATT 1009 Ala Ala Asp Leu Gin Ser Ser Thr Ser Ser Arg Ile Trp Glu Pro Ile 320 325 330 GTT GTT ATG CTT CAA GCT GAG GTT AAG GGA GAG ATT TGC GAG GAG CTT 1057 Vai Vai Met Leu Gin Ala Glu Vai Lys Gly Glu Ile Cys Glu Glu Leu 335 340 345 350 134 GAT AGG GTT ATT GCT AGG CAT CAA AGG CCA TCT ATG AAG GAT AAG ATG 1105 Asp Arg Ile Ile Ala Arg His Gin Arg Pro Ser Met Lys Asp Lys Met 355 360 365 GTT AAG AGG TAT ACT GCT GCT GTT GTT TGC GAG CTT GAT AGG TAT GCT 1153 Go Lys Arg Tyr Thr Ala Ala Goa Cys Glu Leu Asp Arg Tyr Ala 370 375 380 AAG CTT CTT CCA TCT TCT CTT AGG TGC GTT GCT GCT GAT GAG TGG AAG 1201 Lys Leu Leu Pro Ser Ser Leu Arg Cys Go Ala Ala Asp Glu Trp Lys 385 390 395 TTC AGG GAG TAT CTT ATT CCA GTT GGA ATG ACT GTT GGA AAC CTT AAG 1249 Phe Arg Glu Tyr Leu Ile Pro Go Gly Met Thr Go Gly Asn Leu Lys 400 405 410 ACT ACT GTT ATG CTT GAT CAA AAG GA T CCA GTT GAT CCA GAG CTT TTC 1297 Thr Thr Go Met Leu Asp Gin Lys Asp Pro Go Asp Pro Glu Leu Phe 415 420 425 430 GAT GGA ATG TAT GGA CTT GAT GCT GAG GTT CAT TTC GAT AAG ACT GAT 1345 Asp Gly Met Tyr Gly Leu Asp Ala Glu His Phe Asp Lys Thr Asp 435 440 445 AGG TTC ATG CCA CCA TTC TCT GCT GGG AGG ATT GCC TGC GCT GGA CAA 1393 Arg Phe Met Pro Pro Phe Ser Ala Gly Arg Ile Ala Cys Ala Gly Gin 450 455 460 CTT CTT GCT GCT GCT CTT TTC CTT TTC TTC TGT ACT ATT GCT GAT 1441 Leu Leu Wing Ala Tyr Glu Leu Phe Leu Phe Phe Trp Thr Ile Ala Asp 465 470 475 GTT TCA CAA ATT TTC TCT CTT GCT CAA TTC AAG GAG GGA CAT TGC ACT 1489 Go Phe Gin Ile Phe Ser Leu Ala Gin Phe Lys Glu Gly His Cys Thr 480 485 490 GCT GTT ACT CTT ATT ATT GAT TGC CTT GCT GTT AGG TAT GAT CTT TGC 1537 Ala Vai Thr Leu Ile Ile Asp Cys Leu Ala Go Arg Tyr Asp Leu Cys 495 500 505 510 135 1586 1610 CTT GCT AGG TAGGGACCTT TACCGTTTGT GTGA CCGTGT CAATGCTTGC Leu Ala Arg AATGGGCTTT TAATAATATT ATTA (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1698 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (ix) CHARACTERISTICS:

(A) NAME / KEY: CDS (B) LOCATION: 8..1504 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GAGAACA ATG GCA CAA TTC GGC ACG AGG GAA ATT CTA GTC TCA CTC TTT 49 Met Ala Gin Phe Gly Thr Arg Glu Ile Leu Will Be Leu Phe 1 5 10 CTC TTT CTA ATA CTA ATA AAG TTC ACA TTT TTA AAA CTC AAA ACC CCC 97 Leu Phe Leu Ile Leu Ile Lys Phe Thr Phe Leu Lys Leu Lys Thr Pro 15 20 25 30 CAA AAC CTC CCC CCA TCA CCA CCA TCT TTT CCA ATC ACC GGC CAT CTC 145 Gin Asn Leu Pro Pro Ser Pro Pro Ser Phe Pro Ile Thr Gly His Leu 35 40 45 136 193 CAT CTC CTA AAA CAA CCA ATC CAC AGA ACT CTC CAC CAA ATC GCC ACC HiS Leu Leu Lys Gin Pro Ile His Arg Thr Leu His Gin Ile Ala Thr 50 55 60 AAG TAC GGG GAC ATC TTA TTC CTC CGA TTC GGA ACA CGA AAA GTC CTA Lys Tyr Gly Asp Ile Leu Phe Leu Arg Phe Gly Thr Arg Lys Vai Leu 65 70 75 GTC ATC TCC TCT CTC CCC GCC GTA CAA GAA TGT TTC ACT ATA AAC GAC Go Ile Ser Ser Leu Pro Ala Go Gin Glu Cys Phe Thr Ile Asn Asp 80 B5 90 ATC ATT TTC GCT AAC CGC CCA ACA ATT CTC GCC GGG AAG CAC CTC AAT Ile Ile Phe Ala Asn Arg Pro Thr Ile Leu Ala Gly Lys His Leu Asn 95 100 105 110 TAC AAT TCC ACC ACC ATG GGA TTC GCC TCC TAT GGC GAT CAC TGG CGT 385 Tyr Asn Ser Thr Met Gly Phe Ala Ser Tyr Gly Asp His Trp Arg 115 120 125 CAT CTC CGA CGA CTC ACA ACA ATT GAG CTC TTC TCT GCA AAT CGT GTT 433 His Leu Arg Arg Leu Thr Thr Ile Glu Leu Phe Ser Ala Asn Arg Val 130 135 140 GCC ATG TTT TCC GGG TTC CGG GCC GAT GAA AGA ACA GCT TTT TAT CAA 481 Ala Met Phe Ser Gly Phe Arg Ala Asp Glu Ser Thr Ala Phe Tyr Gin 145 150 155 ACA GTT GTT CCA GGA AAT CGG GAT TCG GGA AAG ATA GTA ACT TTG ACA 529 Thr Go Go Pro Gly Asn Arg Asp Ser Gly Lys Ile Go Thr Leu Thr 160 165 170 TCG AAA CTG ATG GAG CTT ACA CTG AAT AAC ATA ATG AGA ATG GCT GCC 577 Ser Lys Leu Met Glu Leu Thr Leu Asn Asn Ile Met Arg Met Ala Ala 175 180 185 190 GG A AAA CGG TTT TAC GGG AAA GAA GTG AAG GAT GAA GGT GAG TTG 625 Gly Lys Arg Phe Tyr Gly Lys Glu Vai Lys Asp Glu Glu Gly Glu Leu 195 200 205 137 673 TTG CAG GAT CTT ATG AAG AAA ATG GAG GCG CTC CGG GGG AAT TCA ACG Leu Gin Asp Leu Met Lys Lys Met Glu Ala Leu Arg Gly Asn Ser Thr 210 215 220 GTG AAA CGA GAT TAT TTT CCA GTA TTG CAG TGG ATT GAT ΤΑΓ CAG GGA ΤΑΓ CAG GGA V Lys Arg Asp Tyr Phe Pro V Leu Gin Trp Ile Asp Tyr Gin Gly 225 230 235 GTA AAG AAG AAG ATG AGG AAC CTG ATG AAG AAA ATG GAC GGG TTC TTG Go Lys Lys Lys Met Arg Asn Leu Met Lys Lys Met Asp Gly Phe Leu 240 245 250 CAA AAT CTC ATT GAT GAA CAC CGA AAC ACG ACG TTG TGG ATC AAT CAA Gin Asn Leu Ile Asp Glu His Arg Asn Thr Thr Leu Trp Ile Asn Gin 255 260 265 270 721 769 817 GTT CGA GCA ACT CGG ACA AAA AGA GGA ACT TGG ACA CTG GTA GAT GTT Go Arg Ala Thr Arg Thr Lys Arg Gly Thr Trp Thr Leu Va Asp Va 275 2Θ0 285 ATG TTG AAT CTT AAA AAG ACA CAA CCT GAC TTC TAC ACT GAT CTA ACT Met Leu Asn Leu Lys Lys Thr Gin Pro Asp Phe Tyr Thr Asp Leu Thr 290 295 300 ATC AAA GGT GTC ATT CAG ACA ACA CTG ACT GCA GGA TCT CAA ACG TCA Ile Lys Gly Ile Ile Gin Thr Thr Leu Thr Ala Gly Ser Gin Thr Ser 305 310 315 GCA GTT ACA CTA GAA TGG GCG CTG TCA CTT CTT CTC AAC CAT CCT CAA Ala Vai Thr Leu Glu Trp Ala Leu Ser Leu Leu Leu Asu His Pro Gin 320 325 330 GTA ATG CAC AAA GCT TAT GCC GAA ATA GAG GCG ATT GTC GGG ACC AAC Go Met His Lys Ala Tyr Ala Glu Ile Glu Ala Ile Go Gly Thr Asn 335 340 345 350 CGC TTA TTA AAC GAA GCC GAC TTA CCA CAT CTA AGC TAT TTA CAA AAC Arg Leu Leu Asn Glu Ala Asp Leu Pro His Leu Ser Tyr Leu Gin Asn 355 360 365 865 913 961 1009 1057 138 1105 1153 ATA ATC ACC GAG ACA TTT CGA CTC TTC CCA CCA GTA CCA CTT TTA CTA Ile Ile Thr Glu Thr Phe Arg Leu Phe Pro Pro Go Pro Leu Leu Leu 370 375 380 ccc CAT AAA TCA TCA GCA GAT TGC ATA GTT TCC GGG TTT CAC ATA CCA 1201 Pro His Lys Ser Ser Al Asp Cys Ile Will Be Gly Phe His Ile Pro 385 390 395 CGG GGC ACA ATG TTG CTA GTG AAC ACA TGG AGC ATG AAT AGA AAT CCA 1249 Arg Gly Thr Met Leu Leu Will Asn Thr Trp Ser Met Asn Arg Asn Pro 400 405 410 AGA TTA TGG AAG GAA CCA GAG AAA TTC ATA CCA GAA AGA TTT GAA GGA 1297 Arg Leu Trp Lys Glu Pro Glu Lys Phe Ile Pro Glu Arg Phe Glu Gly 415 420 425 430 GGA GAA AAT ACT GAA GGG TGT AAC TAT AAA TTG CTT CCT TTC GGT GCA 1345 Gly Glu Asn Thr Glu Gly Cys Asn Tyr Lys Leu Leu Pro Phe Gly Ala 435 440 445 GGA AGG CGG GCT TGT CCG GGG GCC GGT GTG GCG AAA CGA ATG GTA GGA 1393 Gly Arg Arg Ala Cys Pro Gly Ala Gly Va Ala Lys Arg Met Val Gly 450 455 460 CTC ACT TTA GGT GCA TTG ATT CAG TGT TTT GAG TGG GAA AGA ATT GGG 1441 Leu Thr Leu Gly Ala Leu Ile Cys Phe Glu Trp Glu Arg Ile Gly 465 470 4 5 GAA GAA GAA ATA GAT TTG AGT GAA GGA ACA GGT CTT ACT ATG CCA AAA 1489 Glu Glu Glu Ile Asp Leu Ser Glu G ly Thr Gly Leu Thr Met Pro Lys 480 485 490 GAT TTC CTT TGG AAG TAATATGCAA ACCTCGGCAA AACATGATTA ACTTTCTTTC 1544

Asp Phe Leu Trp Lys 495 TACATTGTTA TAAAAGGTGG GTTTCTTTGC AGGTGCCAAC CCTAATTCAA ATATCGCATT 1604 TTTTCCCTGC AACCCAGCTG CTAACCAAAT ATCACTGTTT CTCATTATTC CTTATATAAA 1664 139

ACCTTAAAGC ACTATTTGCC TCCTAAAAAA AAAA 1698

Lisbon, 14 December 2006

Claims (34)

An isolated nucleic acid molecule encoding or complementary to an isolated nucleic acid molecule encoding an epoxygenase which is a mixed function monooxygenase polypeptide which is capable of catalyzing the epoxygenation of a carbon bond in a fatty acid molecule , wherein said epoxygenase comprises an amino acid sequence having the three histidine rich motifs: (i) His- (Xaa) 3-4-His; (ii) His- (Xaa) 2-3-His-His / e (iii) His- (Xaa) 2-3 His-His. An isolated nucleic acid molecule according to claim 1, wherein the carbon bond is a double bond on an unsaturated fatty acid molecule. An isolated nucleic acid molecule according to claims 1 or 2, wherein said epoxygenase is an Δβ-epoxygenase enzyme, an A9-epoxygenase enzyme, an A12-epoxygenase enzyme or an Al5-epoxygenase. An isolated nucleic acid molecule according to any one of claims 1 to 3, derived from a plant. The isolated nucleic acid molecule according to claim 4, wherein the plant is selected from the list comprising Crepis spp. Euphorbia spp., Chrysanthemum spp. and Vernonia spp. 1 The isolated nucleic acid molecule according to claim 4, wherein the plant produces high levels of vernolic acid. The isolated nucleic acid molecule according to claim 5, wherein the plant is selected from the list comprising Crepis biennis, Crepis aurea, Crepis conyzaefolia, Crepis intermédia, Crepis occidentalis, Crepis palaestina, Crepis vesicaria, Crepis xacintha and Vernonia galamensis. An isolated nucleic acid molecule according to any one of claims 1 to 7, comprising a nucleotide sequence that is at least about 65% identical to any one of SEQ ID Nos: 1, 3 or 5 or a sequence thereof. sequence. An isolated nucleic acid molecule according to any one of claims 1 to 8 capable of hybridizing under at least moderate stringency conditions to at least 20 contiguous nucleotides contained within any of SEQ ID Nos: 1,3 or 5 or a complementary sequence thereof. An isolated nucleic acid molecule according to any one of claims 1 to 9, comprising the nucleotide sequence shown in SEQ ID Nos: 1, 3 or 5, or a complementary nucleotide sequence thereof, or at least 20 contiguous nucleotides the same. A genetic construct comprising the isolated nucleic acid molecule according to any one of claims 1 to 10, operably linked to a promoter sequence, wherein said nucleic acid molecule is capable of being transcribed in the sense or anti-sense orientation -sense relative to the in vivo transcription direction of a naturally occurring mixed function epoxygenase monooxygenase gene. A method of altering the level of epoxy fatty acids in a cell, tissue, organ, non-human organism, or microorganism, said method comprising introducing a sense, antisense, ribozyme or co-suppression molecule comprising the nucleic acid according to any one of claims 1 to 10 in a cell, tissue, organ or a non-human organism and the incubation of said cell for a period of time and under conditions sufficient to occur the expression of said sense molecule, sense, ribozyme or co-suppression. A method according to claim 12, wherein the step of introducing the sense, antisense, ribozyme or co-suppression molecule comprises the stable transformation of the non-human cell, tissue, organ or organism to the sense molecule, antisense, ribozyme or co-suppression. A method of producing a recombinantly enzyme-linked mixed function epoxygenase polypeptide in a cell, said method comprising culturing a cell comprising the isolated nucleic acid molecule according to any one of claims 1 to 10 for a period of time and under conditions sufficient for expression to occur. A method according to claim 14, comprising the further first step of transforming the cell with the isolated nucleic acid molecule. 3 A method of producing a recombinantly enzyme-linked mixed function epoxygenase polypeptide in a cell, said method comprising the steps of: (i) producing a genetic construct comprising the isolated nucleic acid molecule according to any one of claims 1 to 10, operably placed under the control of a promoter capable of conferring expression on said gene sequence in said cell, and optionally an expression enhancing element; (ii) transforming said genetic construct into said cell; and (iii) selection of transformants expressing a functional epoxygenase encoded by the genetic sequence at a high level. A method of producing a recombinantly enzyme-linked mixed function epoxygenase polypeptide in a transgenic plant comprising the steps of: (i) producing a genetic construct comprising the isolated nucleic acid molecule according to any one of claims 1 to 10 operably placed under the control of a specific seed promoter and optionally an expression enhancement element, wherein said gene sequence is also placed upstream of a transcription terminator sequence; (ii) transforming said genetic construct into a cell or tissue of said plant; and (iii) selection of transformants that express a functional epoxigenase encoded by the genetic sequence at a high seed level. A method according to claim 17, wherein the plant is a kind of oleaginous seed which normally produces high levels of linoleic acid. A method according to claim 17 or 18, wherein the plant is selected from the list comprising Linola® flax, oilseed rape, sunflower, safflower, soybean, flaxseed, sesame, cottonseed, peanut, olive or palm- of-olive oil. A recombinant mixed function epoxygenase monooxygenase polypeptide produced according to the method according to any one of claims 14 to 19. A recombinant mixed function epoxygenase monooxygenase polypeptide according to claim 20, which comprises an amino acid sequence shown in any one of SEQ ID Nos: 2, 4 or 6 or which is at least about 50% identical to it. A method of producing an epoxygenated fatty acid in a cell, tissue, organ, non-human organism, or microorganism, said method comprising incubating a non-human cell, tissue, organ or organism which expresses the recombinant polypeptide according to the invention. claims 20 or 21 with a fatty acid substrate for a period of time and under conditions sufficient for at least one carbon bond of said substrate to be converted into an epoxy group. 5 A method according to claim 22, wherein the fatty acid substrate is an unsaturated fatty acid and the carbon bonding of said substrate which is epoxygenated is a carbon double bond. A method according to claims 22 or 23, wherein the fatty acid substrate is selected from the list comprising palmitoleic acid, oleic acid, linoleic acid, linolenic acid, 9,15-octadecadienoic acid and arachidonic acid. A method according to any one of claims 22 to 24, wherein the carbon bond which is epoxygen is a Δ 6 carbon bond or a Δ 9 carbon bond or a Δ 12 carbon bond or a Δ 15 carbon bond. A method according to any one of claims 22 to 25, wherein the epoxygenated fatty acid which is produced is vernolic acid. A method according to any one of claims 22 to 26, comprising the first further step of transforming or transfecting the non-human cell, tissue, organ or organism with a nucleic acid molecule encoding recombinant mixed-function epoxygenase monooxygenase. A method according to any one of claims 22 to 27, wherein the cell, organ, tissue or non-human organism in which the recombinant mixed function epoxygenase monooxygenase is expressed is derived from bacteria, yeast, fungus, mold, insect, plant, bird or mammal. 6 A method according to claim 28, wherein the yeast, plant, fungus or mold is a yeast, plant, fungus or oil mold. A method according to claim 29, wherein the plant is an oleaginous seed plant which does not normally express the recombinant mixed function epoxygenase monooxygenase at a high level. A method according to claim 30, wherein the oleaginous seed plant is selected from the list comprising Linola® flax, oleaginous seed rape, sunflower, safflower, soybean, flaxseed, sesame, cottonseed, peanut, olive or palm of olive oil. A plant transformed with the isolated nucleic acid molecule according to any one of claims 1 to 10 or a derivative cell, tissue or organ thereof or the progeny of said plant which also comprises said nucleic acid molecule. A transformed plant which is capable of expressing the recombinant polypeptide according to claim 20 or 21 or a derivative cell, tissue or organ thereof or the progeny of said plant which is also capable of expressing said recombinant polypeptide. A plant or a cell, tissue or organ thereof or the progeny thereof according to claim 32 or 33, which is or is derived from Linola® flax, oilseed rape, sunflower, safflower, soybean, flaxseed, sesame, cotton seed, peanut, olive or olive oil. The plant or a cell, tissue or organ thereof derived therefrom or its progeny according to claim 32 or 33, which is or is derived from Arabidopsis thaliana or Linum usitatissimum. Lisbon, December 14, 2006