AU731298B2 - Genes for microsomal delta-12 fatty acid desaturases and related enzymes from plants - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

ORIGINAL

Name of Applicant: Address for Service: TO BE COMPLETED BY APPLICANT E. I. DU PONT DE NEMOURS AND COMPANY CALLINAN LAWRIE, 711 High Street, Kew, 3101, Victoria, Australia "GENES FOR MICROSOMAL DELTA-12 FATTY ACID DESATURASES AND RELATED ENZYMES FROM PLANTS Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me:- 1 '0 WO 94/11516 PCF/US93/09997 1

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GENES'FOR MICROSOMAL DELTA-12 FATTY ACID DESATURASES JIND RELATED ENZYMES FROM PLANTS FIELD OF'THE INVENTION The invention relates to the preparation and use of nucleic acid fragments encoding fatty acid desaturase enzymes to modify plant lipid composition. Chimeric genes incorporating such nucleic acid fragments and suitable regulatory sequences may be used to create transgenic plants with altered levels of unsaturated fatty acids.

BACKROUND OF THE INVENTION Plant lipids.have a variety of industrial and nutritional uses and are central to plant membrane function and climatic adaptation. These lipids represent a vast array of chemical structures, and these structures determine the physiological and industrialproperties of the lipid. Many of these structures o* result either directly or indirectly from metabolic 20 processes that alter the degree of unsaturation of the lipid. Different metabolic regimes in different plants produce these altered lipids, and either domestication of exotic plant species or modification of agronomically "**adapted species is usually required to economically produce large amounts of the .desired lipid..

Plant lipids find their major use as edible oils in 00* the form of triacylglycerols. The specific performance and health attributes of edible oils are determined largely by their fatty acid composition. Most vegetable oils derived from commercial plant varieties are composed primarily of palmitic stearic (18:0), oleic linoleic (18:2) and linolenic (18:3) acids. Palmitic and stearic acids are, respectively, 16- and 18-carbon-long, saturated fatty acids. Oleic, linoleic, and linolenic acids are 18-carbon-long, I I fl.

WO 94/11516 PCF/US93/09987 2 unsaturated fatty acids containing one, two, and three double bonds, respectively. Oleic acid is referred to as a mono-unsaturated fatty acid, while linoleic and linolenic acids are referred to as poly-unsaturated fatty acids. The relative amounts of saturated and unsaturated fatty acids in commonly used, edible vegetable oils are summarized below (Table 1): 2AELLI Percentages of Saturated and Unsaturated Fatty Acids in the Oils of Releeted OIL Crop unstuatd unsaturated Can6% 58% 36% 24% 61% *Cor 13% 25% 62% Pea.ut. 18% 48% 34% 9% 13% 78% .Snflow. 9% 41% 51% 30% 19% 51% Many recent research efforts have examined the role that saturated and unsaturated fatty acids play in reducing the risk of coronary heart disease. In the past, it was believed that mono-unsaturates, in contrast to saturates and poly-unsaturates, had no effect on serum cholesterol and coronary heart disease risk.

Several recent human clinical studies suggest that diets high in mono-unsaturated fat and low in saturated fat may reduce the "bad" (low-density lipoprotein) cholesterol while maintaining the "good" (high-density lipoprotein) cholesterol (Mattson et al., Journal of Lipid Research (1985) 26:194-202).

A vegetable oil low in total saturates and high in mono-unsaturates would provide *significant health

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WO 94/11516 PCF/US93/09987 3 benefits to consumers as well as economic benefits to oil processors. As an example, canola oil is considered a very healthy oil. However, in use, the high level of poly-unsaturated fatty acids in canola oil renders the.

oil unstable, easily oxidized, and susceptible to development of disagreeable odors and flavors (Gailliard, 1980, Vol. 4, pp. 85-116 In: Stumpf, P. K., Ed., The Biochemistry of Plants, Academic Press, New York). The levels of poly-unsaturates may be reduced by hydrogenation, but the expense of this process and the concomitant production of nutritionally questionable trans isomers of the remaining unsaturated fatty acids reduces the overall desirability of the hydrogenated oil (Mensink et al., New England J. Medicine (1990) N323: 15 439-445). Similar problems exist with soybean and corn oils.

For specialized uses, high levels of polyunsaturates can be desirable. Linoleate and linolenate are essential fatty acids in human diets, and an edible 20 oil high in these fatty acids can be used for nutritional supplements, for example in baby foods.

::Mutation-breeding programs have met with some success in altering the levels of poly-unsaturated fatty acid levels found in the edible oils of agronomic species. Examples of commercially grown varieties are high oleic sunflower and low linolenic flax (Knowles, (1980) pp. 35-38 In:- Applewhite, T. Ed., World Conference on Biotechnology for the Fats and Oils Industry Proceedings, American Oil Chemists' Society).

Similar commercial progress with the other plants shown in Table 1 has been largely elusive due to the difficult nature of the procedure and the pleiotropic effects of the mutational regime on plant hardiness and yield potential.

(U i WO 94/11516 PCr/US93/09987 4 The biosynthesis of the major plant lipids has been the focus of much research (Browse et al., Ann. Rev.

Plant Physiol. Mol. Biol. (1991) 42:467-506). These studies show that, with the notable exception of the soluble stearoyl-acyl carrier protein desaturase, the controlling steps in the production of unsaturated fatty acids are largely catalyzed by membrane-associated fatty acid desaturases. Desaturation reactions occur in plastids and in the endoplasmic reticulum using a variety of substrates including galactolipids, sulfolipids, and phospholipids. Genetic and physiological analyses of Arabidapsis thaliana nuclear mutants defective in various fatty acid desaturation reactions indicates that most of these reactions are catalyzed by 15 enzymes encoded at single genetic loci in the plant.

The analyses show further that the different defects in fatty acid desaturation can have profound and different effects on the ultra-structural morphology, cold sensitivity, and photosynthetic capacity of the plants 20 (Ohlrogge, et al., Biochim. Biophys. Acta (1991) 1082:1-26). However, biochemical characterization of the desaturase reactions has been meager. The instability of the enzymes and the intractability of their proper assay has largely limited researchers to investigations of enzyme activities in crude membrane preparations. These investigations have, however, demonstrated the role of delta-12 desaturase and desaturase activities in the production of linoleate and linolenate from 2 -oleoyl-phosphatidylcholine and 2 -linoleoyl-phosphatidylcholine, respectively (Wang et al., Plant Physiol. Biochem.

(1988) 26:777-792). Thus, modification of the activities of these enzymes represents an attractive target for altering the levels of lipid unsaturation by genetic engineering.

WO 94/11516 PCrIUS93/0997 Nucleotide sequences encoding microsomal delta-9 stearoyl-coenzyme-A desaturases from yeast, rat, and mice have been described (Stukey, et al., J. Biol.

*Chem. (1990) 2 *65:20144-20149; Thiede, et al., J. Biol.

Chem. (1986) 261:13230-13235; Kaestnerr et al., J. Biol.

Chem. (1989) 264:14755-1476). Nucleotide sequences encoding soluble delta-9 stearoyl-acyl carrier protein desaturases from higherplants have also been described (Thompson# et al., Proc. Natl. Acad Sci. U.S.A. (1991) 88:2578-2582; Shanklin et al., Proc. Natl. Acad. Sci.

USA (1991) 88:2510-2514). A nucleotide sequence from coriander plant encoding a soluble fatty acid desaturase, whose deduced amino acid sequence is highly identical to thto the stearoyl-acyl carrier protein 15 desaturase and which is responsible for introducing the :double bond in petroselinic fatty acid (18:1, 6c), 'has also been described [Cahoon, et. al. (1992) Proc. Natl.

Acad. Sci. U.S.A. 89:11184-11188J. Two fatty acid ***.desaturase genes from the cyanobacterium, Svnehocgstfs PCC6803, have been described: one encodes a fatty acid desaturase, designated des A, that catalyzes the conversion of oleic acid at the sn-l position of galactolipids to linoleic acid (Wada, et al., Nature (1990) 347:200-203]3: another encodes a delta-6 fatty a cid desaturase that catalyzes the conversion of linoleic acid at the sn-i position' of galactolipids to y-linolenic acid (18:2, 6c,9c) (WO 93067121. Nucleotide sequences encoding higher plant membraae-bound microsomal and plastid delta-15 fatty acid desaturases have also been described (NO 93112453; Arondel, V. et.

al. (1992) Science 258:1353-1355). There is no report of the isolation of higher plant genes encoding fatty acid desaturases other than the soluble delta-6 and delta-9 desaturases and the membrane-bound (microsomal and plastid) delta-15 desaturases. While there is WO 94/11516 PC/US93/09987 6 extensive amino acid sequence identity between the soluble desaturases and significant amino acid sequence identity between the higher plant microsomal and plastid desaturases, there is no significant homology between the soluble and the membrane-bound desaturases.

Sequence-dependent protocols based on the sequences encoding delta-15 desaturases have been unsuccessful in cloning sequences for microsomal delta-12 desaturase.

For example, nucleotide sequences of microsomal or plastid delta-15 desaturases as hybridization probes have been unsuccessful in isolating a plant microsomal delta-12 desaturase clone. Furthermore, while we have used a set of degenerate oligomers made to a stretch of 12 amino acids, which is identical in all plant 15 desaturases and highly conserved (10/12) in the cyanobacterial des A desaturase, as a hybridization probe to isolate a higher plant nucleotide sequence encoding plastid delta-12 fatty acid desaturase, this method has been unsuccessful in' isolating the microsomal "20 delta-12 desaturase cDNAs. Furthermore, there has been no success in isolating the microsomal delta-12 desaturase by using the polymerase chain reaction products derived from plant DNA, plant RNA or plant cDNA library using PCR primers made to stretches of amino acids that are conserved between the higher plant delta-15 and des A desaturases. Thus, there are no teachings which enable the isolation of plant microsomal delta-12 fatty acid desaturases or plant fatty acid desaturase-related enzymes. Furthermore, there is no evidence for a method to control the the level of delta-12 fatty acid desaturation or hydroxlylation in plants using nucleic acids encoding delta-12 fatty acid desaturases or hydroxylases.

The biosynthesis of the minor plant lipids has been less well studied. While hundreds of different fatty 0 1I WO 94/11516 PCTUS93/09987 7 acids have been found, many from the plant kingdom, only a tiny fraction of:all plants have been surveyed for their lipid content (Gunstone, et al., Eds., (1986) The Lipids Handbook, Chapman and Hall Ltd., Cambridge).

Accordingly, little is known about the biosynthesis of these unusual fatty acids and fatty acid derivatives.

Interesting chemical features found in such fatty acids include, for example, allenic and conjugated double bonds, acetylenic bonds, trana double bonds, multiple double bonds, and single double bonds in a wide number of positions and configurations along the fatty acid chain. Similarly, many of the structural modifications found in unusual lipids hydroxylation, epoxidation, cyclization, etc.) are probably produced 15 via further metabolism following chemical activation of the fatty acid by desaturation or they involve a chemical reaction that is mechanistically similar to.

desaturation. Many of these fatty acids and derivatives *having such features within their structure could prove commercially useful if an agronomically viable species could be induced to synthesize them by introduction of a gene encoding the appropriate desaturase. Of particular interest are vegetable oils rich in 12-hydroxyoctadeca- 9-enoic acid (ricinoleic acid). Ricinoleic acid and its derivatives are widely used in the manufacture of S. lubricants, polymers, cosmetics, coatings and pharmaceuticals see Gunstone, et al., Eds., (1986) The Lipids Handbook, Chapman and Hall Ltd., Cambridge). The only commercial source of ricinoleic acid is castor oil and 100% of the castor oil used by the U.S. is derived from beans grown elsewhere in the world, mainly Brazil. Ricinoleic acid in castor beans is synthesized by the addition of an hydroxyl group at the delta-12 position of oleic acid (Galliard Stumpf (1966) J. Biol. Chem. 241: 5806-5812). This reaction ft WO 94/11516 PCI/US93/09987 8 resembles the initial reaction in a possible mechanism for the desaturation of oleate at the delta-12 position to linoleate since dehydration of 12-hydroxyoctadeca-9enoic acid, by an enzyme activity analogous to the hydroxydecanoyl dehydrase of E. cali (Cronan et al.

(1988) J. Biol. Chem. 263:4641-4646), would result in the formation of linoleic acid. Evidence for the hydroxylation reaction being part of a general mechanism of enzyme-catalyzed desaturation in eukaryotes has been obtained by substituting a sulfur atom in the place of carbon at the delta-9 position of stearic acid. When incubated with yeast cell extracts the thiostearate was converted to a 9-sulfoxide (Buist et al. (1987) Tetrahedron Letters 28:857-860). This sulfoxidation was 15 specific for sulfur at the delta-9 position and did not occur in a yeast delta-9-desaturase deficient mutant (Buist Marecak (1991) Tetrahedron Letters 32:891-894).

The 9-sulfoxide is the sulfur analogue of 9-hydroxyoctadecastearate, the proposed intermediate of stearate 20 desaturation.

Hydroxylation of oleic acid to ricinoleic acid in castor bean cells, like microsomal desaturation of oleate in plants, occurs at the delta-12 position of the fatty acid at the sn-2 position of phosphatidylcholine in microsomes (Bafor et al. (1991) Plant Physiol 280:507-514). Furthermore, castor oleate delta-12 hydroxylation and plant oleate microsomal delta-12 desaturation are both inhibited by iron chelators and require molecular oxygen (Moreau Stumpf (1981) Plant Physiology 67:672-676; Somerville, C. (1992) MSU-DOE Plant Research Laboratory Annual Report]. These biochemical similarities in conjunction with the observation that antibodies raised against cytochrome completely inhibit the activities of both oleate delta-12 desaturation in safflower microsomes and oleate 1 11 (L WO94/11516 PC/US93/09987 9 delta-12 hydroxylase in castor microsomes [Somerville, C. (1992) MSU-DOE Plant Research Laboratory Annual Report] comprise strong evidence that the hydroxylase and the desaturase are functionally related. It seems reasonable to assume, therefore, that the nucleotide sequence encoding a plant delta-12 desaturase would be useful in cloning the oleate hydroxylase gene from castor by sequence-dependent protocols. For example, by screening a castor DNA library with oligomers based on amino acid regions conserved between delta-12 desaturases, or regions conserved between delta-12 and other desaturases, or with oligomers based on amino acids conserved between delta-12 desaturases and known membrane-associated hydroxylases.. It would be more 15 efficient to isolate the castor. oleate hydroxylase cDNA by combining the sequence dependent protocols with a "differential" library approach. One example of such a difference library would be based on different stages of castor seed development, since ricinoleic acid is not synthesized by very young castor seeds (less than 12 DAP, corresponding to stage I and stage II seeds in the scheme of Greenwood Bewley, Can. J. Bot. (1982) S60:1751-1760), in the 20 days following these early stages the relative ricinoleate content increases from 0% to almost 90% of total seed fatty acids .(James et al.

Biochem. J. (1965) 95:448-452, Canvin. Can. J. Biochem.

Physiol. (1963) 41:1879-1885). Thus it would be possible to make a cDNA "difference" library made from mRNA present in a stage when ricinoleic acid was being synthesized at a high rate but from which mRNA present in earlier stages was removed. For the earlier stage mRNA, a stage such as stage II (10 DAP) when ricinoleic acid is not being made but when other unsaturated fatty acids are, would be appropriate. The construction of libraries containing only differentially expressed genes WO 94/11516 PCT/US93/09987 is well known in the art (Sargent. Meth. Enzymol. (1987) 152:423-432). Assembly of the free ricinoleic acid, via ricinoleoyl-CoA, into triacylglycerol is readily catalyzed by canola and safflower seed microsomes (Bafor et al., Biochem J. (1991) 280:507-514, Wiberg et al.

International Symposium on the MetaboliSm, Strucure Function of Plant Lipids (1992), Jerba, Tunisia) and ricinoleic acid is removed from phosphatidylcholine by. a lipase common to all oilseeds investigated. Thus, expression of the castor bean oleate hydroxylase gene in oil crops, such as canola seeds and soybeans, would be expected to result in an oil rich in triglycerides containing ricinoleic acid.

SUMMARY OF THE INVENTION 15 Applicants have discovered a means to control the nature and levels'of unsaturated fatty acids in plants.

Nucleic acid fragments from cDNAs or genes encoding fatty acid desaturases are used to create chimeric genes. The chimeric genes may be used to transform 20 various plants to modify the fatty acid composition of the plant or the oil produced by the plant. More specifically, one embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50%, 60%, 90% or greater respectively to the polypeptide encoded by SEQ ID NOS:1, 3, 5, 7, 9, 11, or Most specifically, the invention pertains to a gene sequence for plant microsomal delta-12 fatty acid desaturase or desaturase-related enzyme. The plant in this embodiment may more specifically be soybean, oilseed Brassica species, Arabidopsis thaliana, castor, and corn.

Another embodiment of this invention involves the use of these nucleic acid fragments in sequence- I ft WO 94/11516 PCT/US93/09987 11 dependent protocols. Examples include use of the fragments as hybridization probes to isolate nucleotide sequences encoding other fatty acid desaturases or fatty acid desaturase-related enzymes. A related embodiment involves using the disclosed sequences for amplification of RNA or DNA fragments encoding other fatty acid desaturases or fatty acid desaturase-related enzymes.

Another aspect of this invention involves chimeric genes capable of modifying the fatty acid composition in the seed of a transformed plant, the gene comprising nucleic acid fragments related as defined to SEQ ID NOS:1, 3, 5, 7, 9, or 15 encoding fatty acid desaturases or SEQ ID NOS:11 encoding a desaturase or desaturaserelated enzyme operably-linked in suitable orientation 15 to suitable regulatory sequences. Preferred are those chimeric genes which incorporate nucleic acid fragments encoding microsomal delta-12 fatty acid desaturase or desaturase-related enzymes.

Yet another embodiment of the invention involves a 20 method of producing seed oil containing altered levels of unsaturated fatty acids comprising: transforming a plant cell with a chimeric gene described above; growing sexually mature plants from the transformed plant cells of step screening progeny seeds from the sexually mature plants of step (bj for the •desired levels of unsaturated fatty acids, and processing the progeny seed of step to obtain seed oil containing altered levels of the unsaturated fatty acids. Preferred plant cells and oils are derived from soybean, rapeseed, sunflower, cotton, cocoa, peanut, safflower, coconut, flax, oil palm, and corn.

Preferred methods of transforming such plant cells would include the use of Ti and Ri plasmids of Agrobectgrium, electroporation, and high-velocity ballistic bombardment.

I WO 94/11516 PCT/US93/09987 12 The invention also is embodied in a method of RFLP breeding to obtain'altered levels of oleic acids in the seed oil of oil producing plant.species. This method involves making a cross between two varieties of oil producing plant species differing in the oleic acid trait; making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross; and hybridizing the Southern blot with the radiolabelled nucleic acid fragments encoding the fatty acid desaturases or desaturase-related enzymes.

The invention is also embodied in a method of RFLP mapping that uses the isolated microsomal delta-12 desaturase cDNA or related genomic fragments described 15 herein.

The invention is also embodied in plants capable of producing altered levels of fatty acid desaturase by virtue of containing the chimeric genes described herein. Further, the invention is embodied by seed oil o* 20 obtained from such plants.

BRIEF DESCRTPTION OF THE SEODENCE DESCRIPTIONS The invention can be more fully understood from the following detailed description and the Sequence Descriptions which form a part of this application. The 25 Sequence Descriptions contain the three letter codes for amino acids as defined in 37 C.F.R. 1.822 which are incorporated herein by reference.

SEQ ID NO:1 shows the 5' to 3' nucleotide sequence of 1372 base pairs of the Arabidopin thaliana cDNA which encodes microsomal delta-12 desaturase.

Nucleotides 93-95 and nucleotides 1242-1244 are, respectively, the putative initiati6n codon and the termination codon of the open reading frame (nucleotides 93-1244). Nucleotides 1-92 and 1245-1372 are, respectively, the 5' and 3' untranslated nucleotides.

WO 94/11516 PCr/US93/0997 13 SEQ ID NO: 2 -is the 383 amino acid protein sequence deduced from thie open reading frame (nucleotides 93-1244 in SEQ ID 140:1.

SEQ ID 140:3 shows the 5' to 3' nucleotide sequence of 1394 base pairs of the Brassiain nap=a~ CDNA which encodes microsomal delta-12 desaturase in plaamid pCF2-165d. Nucleotides 99 to 101 and nucleotides 1248 to 1250 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 99 to 1250). Nuclebotides 1 to 98 and 1251 to 1:394 are, respectively, the 5' and 3' untranslated nucleotides.

SEQ ID BOA: is the 383 amino acid protein sequenbe deduced from the open reading frame (nucleotides 99 to 1250) in SEQ ID 140:3.

SEQ ID N40:5 shows the 58 to 3' nucleotide sequence of 1369 base pairs of soybean (IOlJnn mi=) cDKA which *.*encodes microsomal delta-12 desaturase in plasmid pS.F2-169K. Nucleotides 108 to 110 and nucleotides 1245 to 1247 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 108 to 1247) N4ucleotides 1 to 107 and 1248 to 1369 are, respectively, the 5' and 3' :..untranslated nucleotides..

SEQ ID NO: 6 is the 381 amino acid protein sequence deduced f rom the open reading frame (nucleotides 113 to 1258) in SEQ ID 140:5.

SEQ ID 140:7 shows the 5' to 3' nucleotide sequence of 1790 base pairs of corn (Z=a mana) cDN& which encodes microsomal delta-12 devaturase in plasmid pFad24l.

Nucleotides 165 to 167 and nucleotides 1326 to 1328 are, respectively, the putative initiation codon and the termination codon or the open reading f rame (nucleotides 164 to 1328). Nucleotides 1 to 163 and 1329 to 1790 WO 94/11516 PCI'11S9310997 14 are, respectively; the 5' and 3' untranslated nucleotides.

SEQ ID 110:8 is the 387 amino acid protein sequence deduced from the open reading frame (nucleotides 164 to.

1328) in SEQ ID 110:7.

SEQ ID 110:9 shows the V to 3' nucleotide sequence of 673 base pairs of castor (ile-niza nnminjna) incomplete cDNA which encodes part of a microsomal delta-12 desaturase in pla szdd p1RF2-1C. The sequence encodes an open reading frame from base 1 to base 673.

SEQ ID 110:10 is the 219 amino acid protein sequence deduced from the open reading frame (nucleot ides 1 to 657) in SEQ ID 110:9.

SEQ ID 110:11 shows the 5' to 3' nucleotide sequence of 1369 base pairs of castor (Ric±nna em-nat) cDNA which encodes part of a microsomal delta-12 desaturase or desaturase-related enzyme in plasmid pRF197C-42.

***Nucleotides 184 to 186 and nucleotides 1340 to 1342 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 184 to 1347) Nucleotides 1 to 183. and 1348 to 1369 are, respectively, the 5' and 3' untranslated nucleotides.

SEQ ID 110:12 is the 387 amino acid protein sequence deduced from the open reading frame (nucleotides 184 to 1342) in SEQ ID 110:11.

SEQ ID NO0:13 is the sequence of a set of 64-fold degenerate 26 nucleotide-long oligomers, designated 1153, made to conserved amino acids 101-109 of SEQ ID 110:2, designed to be used as* sense primers in PCR to isolate novel sequences encoding microsomal delta-12 desaturases or desaturase-like enzymes.

SEQ ID NO0:14 is the sequence of a set of 64-fold degenerate and 26 nucleotide-long oligomers, designated 1159, which is made to conserved amino acids 313-321 of

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WO 94/11516 PCF/US93/09987 SEQ ID NO:2 and designed to be used as antisense primers in PCR to isolate novel sequences encoding microsomal delta-12 desaturases or desaturase-like enzymes.

SEQ ID NO:15 shows the 5' to 3' nucleotide sequence of 2973 bp of Arabidopsis thaliana genomic fragment containing the microsomal delta-12 desaturase gene contained in plasmid pAGF2-6. Its nucleotides 433 and .2938 correspond to the start and end, respectively, of SEQ ID NO:1. Its nucleotides 521 to 1654 are the 1134 bp intron.

SEQ ID NO:16 is the sequence of a set of 256-fold degenerate and 25 nucleotide-long oligomers, designated which is made to conserved amino acids 318-326 of S" SEQ ID NO:2 and designed to be used as antisense primers 15 in PCR to isolate novel sequences encoding microsomal delta-12 desaturases or desaturase-like enzymes.

SEQ ID NO:17 is the sequence of a set of 128-fold degenerate and 25 nucleotide-long oligomers, designated which is made to conserved amino acids 318-326 of .20 SEQ ID NO:2 and designed to be used as antisense primers in PCR to isolate novel sequences encoding microsomal delta-12 desaturases or desaturase-like enzymes.

DETAIL.ED DESCRIPTION OF TfE INVENTION Applicants have isolated nucleic acid fragments 25 that encode plant fatty acid desaturases and that are useful in modifying fatty acid composition in oilproducing species by genetic transformation.

Thus, transfer of the nucleic acid fragments of the invention or a part thereof that encodes a functional enzyme, along with suitable regulatory sequences that direct the transcription of their nRNA, into a living cell will result in the production or over-production of plant fatty acid desaturases and will result in increased levels of unsaturated fatty acids in cellular lipids, including triacylglycerols.

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WO 94/11516 PC/US93/09987 16 Transfer of the nucleic acid fragments of the invention or a part thereof, along with suitable regulatory sequences that direct the transcription of their antisense RNA, into plants will result in the inhibition of expression of the endogenous fatty acid desaturase that is substantially homologous with the transferred nucleic acid fragment and will result in decreased levels of unsaturated fatty acids in cellular lipids, including triacylglycerols.

Transfer of the nucleic acid fragments of the invention or a part thereof, along with suitable regulatory sequences that. direct the transcription of their mRNA, into plants may result in'inhibition by cosuppression of the expression of the endogenous fatty 15 acid desaturase gene that is substantially homologous with the transferred nucleic acid fragment and may result in decreased levels of unsaturated fatty acids in cellular lipids, including triacylglycerols.

The nucleic acid fragments of the invention can also be used as restriction fragment length polymorphism (RFLP) markers in plant genetic mapping and plant breeding programs.

The nucleic acid fragments of the invention or oligomers derived therefrom can also be used to isolate other related fatty acid desaturase genes using DNA, RNA, or a library of cloned nucleotide sequences from the same or different species by well known sequencedependent protocols, including, for example, methods of nucleic acid hybridization and amplification by the polymerase chain reaction.

Definitions In the context of this disclosure, a number of terms shall be used. Fatty acids are specified by the number of carbon atoms and the number and position of the double bond: the numbers before and after the colon WO 94/11516 PC/US93/09987 17 refer to the chain length and the number of double bonds, respectively.- The number following the fatty acid designation indicates the position of the double bond from the carboxyl end of the fatty acid with the affix for the cis-configuration of the double bond.

For example, palmitic acid stearic acid (18:0), oleic acid (18:1,9c), petroselinic acid (18:1, 6c), linoleic acid (18:2,9c,12c), 7-linolenic acid (18:3, 6c,9c,12c) and a-linolenic acid (18:3, 9c,12c,15c).

Unless otherwise specified 18:1, 18:2 and 18:3 refer to oleic, linoleic and linolenic fatty acids. Ricinoleic acid refers to an 18 carbon fatty acid with a cis-9 double bond and a 12-hydroxyl group. The term "fatty acid desaturase" used herein refers to an enzyme which 15 catalyzes the breakage of a carbon-hydrogen bond and the introduction of a carbon-carbon double bond into a fatty acid molecule. The fatty acid may be free or esterified to another molecule including, but not limited to, acylcarrier protein, coenzyme A, sterols and the glycerol moiety of glycerolipids. The term "glycerolipid desaturases" used herein refers to a subset of the fatty acid desaturases that act on fatty acyl moieties esterified to a glycerol backbone. "Delta-12 desaturase" refers to a fatty acid desaturase that .25 catalyzes the formation of a double bond between carbon positions 6 and 7 (numbered from the methyl end), those that correspond to carbon positions 12 and 13 .(numbered from the carbonyl carbon) of an 18 carbon-long.

fatty acyl chain. "Delta-15 desaturase" refers to a fatty acid desaturase that catalyzes the formation of a double bond between carbon positions 3 and 4 (numbered from the methyl end), those that correspond to carbon positions 15 and 16 (numbered from the carbonyl carbon) of an 18 carbon-long fatty acyl chain. Examples of fatty acid desaturases include, but are not limited D 1L WO 94/11516 PCr/US93/09987 18 to, the microsomal delta-12 and delta-15 desaturases that act on phosphatidylcholine lipid substrates; the chloroplastic or plastid delta-12 and desaturases that act on phosphatidyl glycerol and galactolipids; and other desaturases that act on such fatty acid substrates such as phospholipids, galactolipids, and sulfolipids. "Microsomal desaturase" refers to the cytoplasmic location of the enzyme, while "chloroplast desaturase" and "plastid desaturase" refer to the plastid location of the enzyme. These fatty acid desaturases may be found in a variety of organisms including, but not limited to, higher plants, diatoms, and various eukaryotic and prokaryotic microorganisms as fungi and photosynthetic bacteria and algae.

The term "homologous fatty acid desaturases" refers to fatty acid desaturases that catalyze the same desaturation on the same lipid substrate. Thus, microsomal delta-15 desaturases, even from'different plant species, are homologous fatty acid desaturases.

The term "heterologous fatty acid desaturases" refers to fatty acid desaturases that catalyze desaturations at different positions and/or on different lipid substrates. Thus, for example, microsomal delta-12 and desaturases, which act on phosphatidylcholine lipids, are heterologous fatty acid desaturases, even when from the same plant. Similarly, microsomal delta-15 desaturase, which acts on phosphatidylcholine lipids, and chloroplast delta-15 desaturase, which acts on galactolipids, are heterologous fatty acid desaturases, even when from the same plant. It should be noted that these fatty acid desaturases have never been isolated and characterized as proteins.

Accordingly, the terms such as "delta-12 desaturase" and desaturase" are used as a convenience to describe the proteins encoded by nucleic acid fragments WO 94/11516 PCr/US93/09987 19 that have been isolated based on the phenotypic effects caused by their disruption. They do not imply any catalytic mechanism. For example, delta-12 desaturase refers to the enzyme that catalyzes the formation of a double bond between carbons 12 and 13 of an 18 carbon fatty acid irrespective of whether it "counts" the carbons from the methyl, carboxyl end, or the first double bond. The term "fatty acid desaturase-related enzyme" refers to enzymes whose catalytic product may not be a carbon-carbon double bond but whose mechanism of action is similar to that of a fatty acid desaturase (that is, catalysis of the displacement of a carbonhydrogen bond of a. fatty acid chain to form a fattyhydroxyacyl intermediate or end-product). Examples 15 include delta-12 hydroxylase which means a delta-12 *fatty acid hydroxylase or the oleate hydroxylase responsible for the synthesis of ricinoleic acid from.

oleic acid.

The term "nucleic acid" refers to a large molecule 20 which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, a phosphate and either a purine or pyrimidine. A "nucleic acid fragment" is a fraction of a given nucleic acid molecule. In higher plants, deoxyribonucleic acid (DNA) 25 is the genetic material while ribonucleic acid (RNA) is involved in the transfer of the information in DNA into proteins. A "genome" is the entire body of genetic material contained in each cell of an organism. The term "nucleotide sequence" refers to the sequence of DNA or RNA polymers, which can be single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The term "oligomer" refers to short nucleotide sequences, usually up to 100 bases long. As used herein, the term "homologous to" refers D t WO 94/11516 PCr/US93/09987 to the relatedness between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Biggins, Eds. (1985) Nucleic Acid Hybridisation,

IRL

Press, Oxford, or by the comparison of sequence similarity between two nucleic acids or proteins, such as by the method of Needleman et al. Mol. Biol.

(1970) 48:443-453). As used herein, "substantially homologous" refers to nucleotide sequences that have more than 90% overall identity at the nucleotide level with the coding region of the claimed sequence, such as 15 genes and pseudo-genes corresponding to the coding regions. The nucleic acid fragments described herein .include molecules which comprise possible variations,.

both man-made and natural, such as but not limited to those that involve base changes that do not cause a change in an encoded amino acid, or which involve base changes that alter an amino acid but do not affect the functional properties of the protein encoded by the DNA sequence, those derived from deletions, rearrangements, amplifications, random or controlled 25 mutagenesis of the nucleic acid fragment, and even occasional nucleotide sequencing errors.

"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding non-coding) and following (3' non-coding) the coding region. "Fatty acid desaturase gene" refers to a nucleic acid fragment that expresses a protein with fatty acid desaturase activity. "Native" gene refers to an isolated gene with its own regulatory sequences as found in nature. "Chimeric gene" refers to a gene that comprises heterogeneous regulatory and WO 94/11516 PCT/US93/09987 21 coding sequences not found in nature. "Endogenous" gene refers to the native gene normally found in its natural location in the genome and is not isolated. A "foreign" gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.

"Pseudo-gene" refers to a genomic nucleotide sequence that does not encode a functional enzyme.

"Coding sequence" refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", lacking an intron or it may include one or more introns bounded by appropriate splice junctions.

An "intron" is a nucleotide sequence that is transcribed in the primary transcript but that is removed through 15 cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a *protein.

"Initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides in a coding 20 sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation). "Open reading frame" refers to the coding sequence uninterrupted by introns between initiation and termination codons that encodes an amino acid sequence.

S 25 "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription -of a DNA sequence. When the RNA transcript is a perfect complementary copy of the:DNA sequence, it is referred to as the .primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.

"Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a double-stranded DNA that is complementary to and derived from mRNA. "Sense" RNA V WO 94/11516 PCT/US93/09987 22 refers to RNA transcript that includes the mRNA.

"Antisense RNA" refers to a RNA transcript that is complementary to all or part of.a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or. translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. "Ribozyme" refers to a catalytic RNA and includes sequence-specific endoribonucleases.

As used herein, Usuitable regulatory sequences" o.refer to nucleotide sequences in native or chimeric genes that are located upstream within, and/or downstream to the nucleic acid fragments of the invention, which control the expression of the nucleic 20 acid fragments of the invention. The term "expression", as used herein, refers to the transcription and stable accumulation of the sense (mRNA) or the antisense RNA derived from the nucleic acid fragment(s) of the invention that, in conjunction with the protein 25 apparatus of the cell, results in altered levels of the fatty acid desaturase(s). Expression or overexpression of the gene involves transcription of the gene and translation of the mRNA into precursor or mature fatty acid desaturase proteins. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.

"Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.

"Cosuppression" refers to the expression of a foreign WO 94/11516. PC/US93/09987 23 gene which has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene. "Altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or.proportions that differ from that of normal or non-transformed organisms.

"Promoter" refers to a DNA sequence in a gene, usually upstream to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. In artificial DNA constructs promoters can also be used to transcribe antisense RNA. Promoters may also contain DNA sequences that are involved in the binding of 15 protein factors which control the effectiveness of o* *transcription initiation in response to physiological or developmental conditions. It may also contain enhancer oelements. An "enhancer" is a DNA sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter. "Constitutive promoters" refers to those that direct gene expression in all tissues and at all times. "Tissue-specific" or "development-specific" promoters as referred to herein are those that direct gene expression almost exclusively in specific tissues, such as leaves or seeds, or at specific development stages in a tissue, such as in early or late embryogenesis, respectively.

The non-coding sequences" refers to the DNA sequence portion of a gene that contains a polyadenylation signal and any other regulatory signal capable of affecting mRNA processing or gene expression.

The polyadenylation signal is usually characterized by WO 94/11516 PCIT/US93/09987 24 affecting the addition of polyadenylic acid tracts to the 3' end of the ZmRNA precursor.

"Transformation" herein refers to the transfer of a foreign gene into the genome of a host organism and its genetically astable inheritance. "Restriction fragment length polymorphism" (RPLP) refers to different sized restriction fragment lengths due to altered nucleotide sequences in or around variant forms of genes.

"Moleular breeding" refers to the use of DNA-based diagnostics, such as RFLP, RAPDs, and PCR in breeding.

"Fertile" refers to plants that are able to propagate sexually.

"Plants" refer to photosynthetic organisms, both eukaryotic and prokaryotic, whereas the term "Higher plants" refers to eukaryotic plants. "Oil-producing species" herein refers to plant species which produce.

and store triacylglycerol In specific organs, primarily in seeds. Such species include soybean. (Glycine I=x), rapeseed and canola (including Alrassica nap=i, caznesi4, sunflower (Bnllahthusa An=u), cotton ~(G.aaxniu b.±xmztlim), ICorn (2=maaj cocoa (Thngbxgziw safflower (fCarxjhamuna Znt.iu), oil palm *(Elapi gUinPnnai), coconut palm (Cgj= nmafgra), flax (Lizwim uflitatinimm), castor (B±-±num a moma) and peanut (Arnech4iq hy~gnexJ. The group also includes nonagronomic species which are useful in developing appropriate expression vectors such as tobacco, rapid cycling xAraasoa, species, and kbdla thAllaA, and wild species which may be a source of unique fatty acids.

"mSequence-dependent protocols" refer to techniques that rely on a nucleotide sequence for their utility.

Examples of sequence-dependent protocols include, but are not limited to, the methods of nucleic acid and -oligorner hybridization and methods of DNA and RNA WO 94/11516 PCr/IS93/O9M8 amplification such as are exemplified in various uses of the polymerase chain reaction (PCR).

various solutions used in the experimental manipulations are referred to by their cmmn names such as "SSCI, OSSPE", "Denhardt'a solution", etc. The composition of these solutions may be. found by reference to Appendix B of Sam brook, et al. (Molecular Cloning, A Laboratory manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).' T-inMA Hutagang-Rn anlIIn~i~t nr ofAn Arnhldgtpni Mtnt Dafact1ve In in T-DNAk mutagenesis (Feldmann, et Science (1989) 243:1351-1354), the integration of T-DNA in the genome can interrupt normal expression of the gene at or near the site of the integration. If the resultant' mutant phenotype can be detected and shown genetically be tightly linked to the T-DNA insertion, then the "tagged" mutant locus and its wild type counterpart can 20 be readily isolated by molecular cloning by one skilled in the art.

Arghtdia thalinna seeds were transformed by Agrnbaeteriuu tizmfarimnS C58Clrif strain harboring the **avirulent Ti-plasmid pGV3850::pAK1003 that has the T-DNA.

region between the left and right ?-DNA borders replaced by the origin of replication region and anpicillin resistance gene of plasmid pBfl322, a bacterial kanamycin .resistance gene, and a plant kanamycin rebistance gene (Feldmann, et al., Mol. Gen. Genetics (1987) 208:1-9).

Plants from the treated seeds were self-fertilized andthe resultant progeny seeds, germinated in the presence of kanamycin, were self-fertilized to give rise to a population, designated T3, that was segregating for T-DNA insertions. T3 seeds from approximately 1700 T2 plants were germinated and grown under controlled WO 94/11516 PCT/US93/09987 26 environment. One leaf from each of ten T3 plants of each line were pooled and analyzed for fatty acid composition. One line, designated 658, showed an incresed level of oleic acid Analysis of twelve individual T3 seeds of line 658 identified two seeds that contained greater than 36% oleic acid while the remaining seeds contained 12-221 oleic acid. The mutant phenotype of increased level of oleic acid in leaf and seed tissues of line 658 and its segregation in individual T3 seeds suggested that line 658 harbors a mutation that affects desaturation of oleic acid to linoleic acid in both leaf and seed tissues. When approximately 200 T3 seeds of line 658 were tested for their ability to germinate in the presence of kanamycin, 15 four kanamycin-sensitive seeds were identified, suggesting multiple, possibly three, T-DNA inserts in the original T2 line. When progeny seeds of 100 individual T3 plants were analyzed for fatty acid composition and their ability to germinate on kanamycin, one plant, designated 658-75, was identified whose progeny segregated 7 wild type:2 mutant for the increased oleic acid and 28 sensitive:60 resistant for kanamycin resistance. Approximately 400 T4 progeny seeds of derivative line 658-75 were grown and their leaves analyzed for fatty acid composition. Ninety one of these seedlings were identified as homozygous for the mutant (high oleic acid) phenotype. Eighty-three of these homozygous plants were tested for the presence of nopaline, another marker for T-DNA, and all of them were nopaline positive. On the basis of these genetic studies it was concluded that the mutation in microsomal delta-12 desaturation was linked to the T-DNA.

41

V

WO 94/11516 PC/JS93/O9987 27 I~oltionof rablop~i 6A-75 t~bnaomi DNA- Cnntaining the niarupted Cgnts roentr^11irng In order to isolate the gene controlling microsomal 5 delta-12 desaturation from wild-type 1=xid~aL a T-DNA-plant DNA "un~ction" fragment containinig a T-DRA border integrated into the host plant DNA was isolated from the homozygous mutant plants of the 658-75 line of Araihida3nig. For this, genornic DNA from the mutant plant was isolated and completely digested by either Bamn KI or Sal I restriction enzymes. In'each case, one of the resultant fragments was expected to contain the origin of replication and ampicillin-resistance gene of pBR322 as well as the left T-DNA-plauat DNA junction fragment. Such fragments were rescued as plasmids by ligating the digested genomic DNA fragments at a dilute concentration to facilitate self-ligation and then using the ligated fragments to transform enU. cells. While no ampicillin-resistant colony was obtained from the plasmid rescue of Sal I-digested plant genomic DNA, a t Cu:. single ampicillin-resistant colony was obtained from the plasmid rescue of Earn HI-digested plant genomic DNA.

The plasmid obtained from this transformant was designated p658-1; Restriction analysis of plasmid p 6 58-1 with Barn HI, Sal I and Eco RI restriction enzymes strongly suggested that it contained the expected.

14.2 kb portion of the T-DNA (containing pBR322 sequences) and a putative plant DNA/left T-DNA border fragment in a 1.6 kB Eco RI-Dam HI fragment. The 1.6 kb Eco RI-Barn HI fragment was subcloned into p~luescript SK (Stratagenel by standard cloning procedures described in Sambrook et al., (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press) and the resultant plasmid, designated pS1658.

11 .1 WO 94/11516 PCr/US93/O997 28 Tgolation of Kieronal D)ltx-!12 pe-gatturm-g rnNA ndr GeneA f rom WI Id A-rahi dgas4 R The 1.6 kb Eco RI-Ban HI fragment, which contained the putative plant DNA flanking T-VNA, in plasmid p658-i was isolated and used as a radiolabeled hybridization probe to screen a cONA library made to polyA* mp.NA from the above-ground parts of Ah a~aiAL thaliann plants, which varied in size from those that had Just opened their primary leaves to plants which bad bolted and were flowering [Elledge et al. (1991) Pz-oc. Kati. Acad Sci.

USA 88:.1731-1735). The cDNA insertsin the library were' made into an Xho I site flanked by Eco RI sites in Dose*:lambda Yes vector [Elledge et al. (1991) Proc.* Nati.

Acad Sci. USA t8:1731-1735). Of the several positivelyhybridizing plaques, four were subjected to plaque purification. P1&smids were excised from the purified phages by site-specific recombination using the cre-lox Dos recombination system in*K. noli stanrN3 Elledge et al. (1991) Proc. NatI. Acad 'Sci. USA 88:1731-1735].

20 The four excised plasmids were digested by Eco RI restriction enzyme and shown to contain cDNA inserts ranging in size between 1 kB and 1.5 kE. Partial nucleotide sequence determination and restrictionlenzyme mapping of all four cDNAs revealed their common 25 "identity.

The partial nucleotide sequences of two CDNAs, 0 designated pSF2b and p92103, containing inserts of ca.

1.2 kB and ca. 1.4 kB, respectively, were determined.

The composite sequence derived from these plasmids is shown as SEQ ID NO:1 and is expected to be contained completely in plasmnid p92103. SEQ ID 110:1 shows the 51 to 3' nucleotide sequence of 1372 base pairs of the Arabidnpnin cDNA which encodes microsomal delta-12 fatty acid desaturase. Nucleotides 93-95 are the putative initiation codon of the open reading frame (nucleotides II .I WO 94/11516 PCT/US93/09987 29 93-1244), (identified by comparison of other plant delta-12 desaturases in this application). Nucleotides 1242-1244 are the termination codon. Nucleotides 1 to 92 and 1245-1372 are the 5' and 3' untranslated nucleotides, respectively. The 383 amino acid protein sequence in SEQ ID NO:2 is that deduced from the open reading frame and has an estimated molecular weight of 44 kD.

The gene corresponding to SEQ ID N0:1 was isolated by screening an Arabidopia genomic DNA library using radiolabeled pSF2b cDNA insert, purifying the positively-hybridizing plaque, and subcloning a 6 kB ind III insert fragment from the phage DNA in pBluescript vector. The sequence of 2973 nucleotides of 15 the gene is shown in SEQ ID NO:15. Comparison of the sequences of the gene (SEQ ID NO:15) and the cDNA (SEQ ID NO:1) revealed the presence of a single intron of 1134 bp at a position between nucleotide positions 88 and 89 of the cDNA, which is 4 nucleotides 5' to the initiation codon.

The 1.6 kB Eco RI-Bam HI genomic border fragment insert in pS1658 was also partially sequenced from the Barn HI and Eco RI ends. Comparison of the nucleotide sequences of the gene (SEQ ID NO:15), the cDNA (SEQ ID 25 NO:1), the border fragment, and the published sequence of the left end of T-DNA (Yadav et al., (1982) Proc.

Natl. Acad. Sci. 79:6322-6326) revealed that a) the sequence of the first 451 nucleotides of the border fragment from the Barn HI end is collinear with that of nucleotides 539 (Bam HI site) to 89 of the cDNA, b) from the Eco RI end, the border fragment is collinear from nucleotides 1 to 61 with that of the left end of T-DNA (except for a deletion of 9 contiguous nucleotides at position 42 in the border fragment), and is collinear from nucleotides 57 to 104 with that of nucleotides WO 94/11516 PCT/UIS93/09M8 4 1-88 of the cDNA, and c) the sequence divergences between the border fragment and the .cDNA are due to the presence of the intron in the border fragment. These results show that the T-DNA disrupted the microsomal delta-12 desaturase gene in the transcribed region between the promoter and the codingq region and 5' to the intron in the untranslated sequence.

A phage DNA containing Azblican inicrosomal delta-12 desaturase gene was used as a RFLP marker on a Southern blot containing genomic DNA from several progeny of Arabidg~gja thAlivina (ecotype Wassileskija and marker line 11100 ecotype Lman desberg background) digested with Kind 111. This mapped the microsomal delta-12 desaturase gene 1.3.6 CM proximal to locus 15 c3838, 9.2 cM distal to locus 1At228, and 4.9 cM proximal to Fad D locus on chromosome 3 (Koorneef, M. et al., (1993) in Genetic Maps, Ed. O'Brien, S. J7.; Yadav .9 .9.et al. (1993) Plant-Physiology 1DU:467-476). This position corresponds closely to previously suggested locus for microsomal delta-12 desaturation (Fad 2).

[Hugly, S. et al., (1991) Heredity 82:43211.

The open reading frames in SEQ ID 110:1 and in sequences encoding Aki hj~ iA micjros omal delta-iS desaturase (IW0 9311245], Arabaida99ia plastid desaturase (NO0 9311245], and cyanobacterial-desaturase, des A, [Wada, et al., Nature (1990) 347:200-203; Genbank ID:CSDESA; Gen~ank Accession No:X535081 as well as their deduced amino acid sequences were compared by the method of Needleman et al. 1401. Biol. (1970) 48:443-453] using gap weight and gap length weight values of 5.0 and 0.3, respectively, for the nucleotide sequences and and 0.1, respectively, for protein sequences. The overall identities are summarized in Table 2.

WO 94/11516 PCrTUS93/09987 31 TABLE 2 Percent Identity Between Different Fatty Acid Desaturases at the Nucleotide and Amino Acid LeveIl A3 ad dea A a2 nucleotide 48(8 gaps) 46(6 gaps) 43(10 gaps) amino acid 39(9 gaps) 34(8 gaps) 24(10 gaps) a3 nucleotide 65(1 gap) 43(9 gaps) amino acid 65(2 gaps) 26(11 gaps) ad nucleotide 43(9 gaps) amino acid 26(11 gaps) a2, a3, ad, and des A refer, respectively, to SEQ ID NO:1/2, Arabidopsi microsomal delta-15 desaturase,- Arabidopai plastid delta-15 desaturase, and cyanobacterial desaturase, des A. The percent 5 identities in each comparison are shown at both the nucleotide and amino acid levels; the number of gaps imposed by the comparisons are shown in brackets following the percent identities. As expected on the basis of unsuccessful attempts in using delta-15 fatty acid nucleotide sequences as hybridization probes to isolate nucleotide sequences encoding microsomal delta-12 fatty acid desaturase, the overall homology at the nucleotide level between microsomal delta-12 fatty acid desaturase (SEQ ID NO:1) and the nucleotide 15 sequences encoding the other three desaturases is poor (ranging between 43% and .At the amino acid level too, the microsomal delta-12 fatty acid desaturase (SEQ ID NO:2) is poorly related to cyanobacterial des A (less than 24% identity) and-the plant delta-15 desaturases (less than 39% identity).

While the overall relatedness between the deduced amino acid sequence of the said invention and the published fatty acid desaturases is limited, more significant identities are observed in shorter stretches WO 94/11516 PCF/US93/0997 32 of amino acid sequaences in the above comparisons These results confirmed that the T-DNA in line 658-75 had interrupted the normal expression of a fatty acid desaturase gene. Based on the fatty acid phenotype of homozygous mutant line .658-75, Applicants concluded that SEQ ID NO:1 encoded the delta-12 desaturase. Further, Applicants concluded that it was the microsomal delta-12 desaturaae, and not the chioroplastic delta-12 desaturase, since: a) the mutant phenotype was expressed strongly in the. seed but expressed poorly, if at ail, in the leaf of line 658-75, and b) the delta-!12 desaturase polypeptide, by-comparison to the microsomal and plastid delta-iS desaturase polypeptides (NO 93112453, did not have an N-terminal extension of a 15 transit peptide expected for a nuclear-encoded plastid desaturase.

Plasmid. p92103 was deposited on October 16, 1992.

.*.with the American Type Culture Collection of Rockville, Maryland under the provisions of the Budapest Treaty and bears accession number ATCC 69095.

EXprasminn Of Mirosnomal nlta 2Fat Acid nesaturagp In ArahjdpnRgi FAd2-1 Muant To omIumnt tq Mutation.

Tn Delta-i2 Fatty Aid De-aturation To confirm the identity of SEQ ID NO:2 ltkableld-g microsomal delta-12 fatty acid desaturase cDNA) a chimeric gene comprising of SEQ.ID NO:1 was transformed into an Arhf~i mutant affected in microsomal delta-12 fatty acid denaturation. For this, the ca.

1.4 kb Eco RI fragment containing the cDNA (SEQ) ID NO: 1) was isolated from plaszidd p92103 and sub-cloned in PGA748 vector [An et. al. (1988) Binary Vectors. In: Plant Molecular Biology Manual. Eds Gelvin, S. B. et al.

Kluwer Academic Press], which was previously linearized with Eco RI restriction enzyme. In one of the resultant binary plasmid, designated pqA-Fad2, the CDNA was placed WO 94/11516 PCr/US93/09997 33 in the sense orientation behind the CaMV 35S promotor of the vector to provide, cons titut ive expression.

Binary vector pGA-Fad2 Was transformed by the freeze/thaw method (Holsters et al. (1978) Mal. Gen.

Genet. .163:181-1871 into h&grgbar_+Ar±im Xn~ai strain R1000, carrying the Ri plasmid pRiA4b from 1~rnbnqerIuiw rbi [Moore et al., (1979) Plasmld 2:617-6261 -to result in. transformants R2OOO/pGA-Fad2.

Agrobacterium strains R1000 and RlOOO/pGh-Fad2 were used to transform Aat~a mutant fad.2-1 [Miquel, MI.

a Browse, J. (1992) Journal of Biological Chemistry 267:1502-1509] and strain R1000 was used to transform wild type Ar~h±doiz.. Young bolts of plants'were sterilized and cut so that a single node was present in :1::15 each explant. Explants were inoculated by Agrobacteria and incubated at 25*C in the dark on drug-free MS minimal organics medium with 30 gIL sucrose (Gibco) After four days, the explants were transferred to fresh MS medium containing 500 mg/L cefotaxime and 250 mg/mi carbenicillin for the counterselection of Agrnbar_+erinm.

*After 5 days, hairy roots derived from R1OOO/pGA-Fad2 transformation were excised and transferred to the same.

medium containing 50 mg/mi kanamycin. Fatty acid methyl esters were prepared from 5-10 mm of the roots essentially as described by Browse et al.,.(Anal.

Biochem. (1986) 152:141-145) except that 2.5% 12S04 in methanol was used as the methylation reagent and samples were heated for 1.5 h at 80*C to effect the methanolysis of the seed txiglycerides. The results are shown in Table 3. Root samples 41 to 46, 48 to 51, 58, and 59 are derived from transformation of fad2-1 plants with R1000/pGA Fad2; root samples 52, 53, and 57 were derived from transformation of fad2-1 plants with R1000 and serve as controls; root sample 60 is derived from WO 94/11516 WO 9411516PCr/UIS93/09M8 34 transformation of widld type Arahipjj1i with R1000 and also serves as a control.

Fatty acid Composition in Tranagenic ArabidosaLa fad2-1 Hairy Roots Transformed with A~nratorum R10QO/or.A--Fxd2 41 24.4 1.6 1.7 5.0 29.4 33.8 42 25.6 3.7 1.3 20.0 22.0 27.5 43 23.6 1.6 7.2 27.6 36.1 44 24.4 1.3 4.6 16.0 18.1 33.6 20.7 8.1 44.7 11.8 14.8 20.1 1.8 7.5 33.7 36.0 48 26.1 2.9 2.1l 9.5 17.6 33.4 .:49 30.8 1.0 2.4 8.7 18.7 31.1 19.8 1.9 3.3 27.7 21.8 24.4 :51 20.9 1.1 5.0 13.7 25.0 32.1 58 23.5 0.3 1.4 3.6 22.1 45.9 *59 22.6 0.6 1.4 2.8 29.9 40.4 52, cont. 12.3 2.6 64.2 4.6 16.4 53, cont. 20.3 9.1 2.2 55.2 1.7 9.2 57, cont. 10.4 2.4 0.7 65.9 3.8 12.7 6 0,wT 23.0 1.7 0.8 6:.0 35.0 31.8 5 These results show that expression of a±~a microsomal delta-12 desaturase in a mutant lacking delta-12 desaturation can result in partial to complete complementat ion of the mutant. The decrease in oleic acid levels in tranagenic roots is accompanied by increases in the levels of both 18:2 and 18:3. Thus, overexpression of this gene in other oil crops, especially canola, which is a close relative of Arhldn~nj and which naturally has high levels of 18:1 in seeds, is also expected to result in higher levels of 18:2, which in conjunction with WO 94/11516 PCr/US93/0998 the overexpression of the microsomal delta-15 fatty acid desaturase will result in very high levels of 18:3.

U~in ArbidOpR~i MieTnrovoma neltam-12 ngsaturnse trDNA an a Pybridlfatfo rb to Isolate Hierngnmal Delta-12 Dpatnrascb nDNAs from Othi-r Plant Rnec-lon Evidence for conservation of the delta-12 desaturase sequences amongst species was provided by Using the Rrij0%d4jz cDNA insert from pSf2b as a hybridization probe to clone related sequences from Bxra&L=c nn=a~, and soybean. Furthermore, corn and castor bean microsomal delta-12 fatty acid desaturase.

were isolated by PCR using primers made to conserved regions of'.microsomal delta-12 desaturases.

(f~~~loning of a Aransieaiat' er e!DNA Encoding Seed Minrosomal Deolta-12 Fatty Acid Desaturase For the purpose of cloning the flAr-mssi naua= seed cDNA encoding a delta-12 fatty acid desaturbse, the cDNA insert from pSF2b was isolated by digestion of pSF2b with EcoR I followed by purification of the 1.2 kb insert by gel electrophoresis. The 1.2kb fragment was radiolabeled and used as a hybridization probe to screen a lambda phage cDNA library made with ]poly A+ mRA from.

developing lirAmAzjg nA=n seeds 2.0-21 days after pollination. Approximately 600, 000 plaques were screened under low stringency hybridization conditions mM Tris pH 7.6, 6X SSC, 5X Denhardt's, 0.5% SOS, 100 ug denatured calf thymus DNA, and 50C) and washes (two washes with 2X SSC, 0.5% SDS at room temperature for 15 min each, then twice with 0.2X SSC, 0.5% SDS at room temperature for 15 min each, and then twice with 0. 2X SSC, 0..516 SDS at 500C for 15 min each). Ten strongly-hybridizing phage were plaque-purified and the size of the cDNA inserts was determined by PCR amplication of the insert using phage as template and WO 94/1 1516 PCF/LUS93/09987 36 T3/T7 oligomers for primers. Two of these phages, 165D and 165F, had PCR amplified inserts of 1.6 kb and 1.2 kb respectively and these phages were also used to excise the phagemids as described above. The phagemid derived from phage 165D, designated pCF2-165D, contained a 1 .3 kb insert which was sequenced completely on one strand. SEQ ID NO:3 shows the 5' to 3' nucleotide sequence of 1394 base pairs of the Arzaaca, anA cDNA which encodes delta-12 desaturase in plasm-id. pCF2-165d.

Nucleotides 99 to 101 and nucleotides 1248 to 1250 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 99 to 1250). Nucleotides ito 98 and'1251 to 1394 are, respectively, the 5' and 3' untranslated nucleotides.

The 383 amino acid protein sequence deducedfrom the open reading frame in SEQ ID NO:3 is shown in SEQ ID NO:4. While the other strand can easily be sequenced ~.for confirmation, comparisons of SEQ ID NOS:1 and 3 and of SEQ ID 1405:2 and 4, even admiitting of possible sequencing errors, showed an overallI homology of approximately 84% at both the nucleotide and amino acid levels, which confirmed that pCF2-165D is a Arassica nnA2= seed oDNA that encoded delta-12 desaturase.

Plasmid pCF2-165D has been deposited on October 16, 1992 with. the American Type Culture Collection of Rockville, Maryland under the provisions of the Budapest Treaty and bears accession number ATCC 69094.

C!loning of s SOYbAan tclyring MAN) nDKUA ncoding Reed I~rn-ftemal Dalta-127 Fatty Aeid DagtaturnjM A cOMA library was made to poly A+ MRNA isolated from developing soybean seeds, and screened as described above. Radiolabelled probe prepared from pSF2b as described above was added, and allowed to hybridize for 18 h at 50 0 C. The probes were washed as described WO 94/11516 PCT/US93/09987 37 above. Autoradiography of the filters indicated that there were 14 strongly hybridizing plaques, and numerous weakly hybridizing plaques. Six of the 14 strongly hybridizing plaques were plaque purified as described above and the cDNA insert size was determined by PCR amplication of the insert using phage as template and T3/T7 oligomers for primers. One of these phages, 169K, had an insert sizes of 1.5 kb and this phage was also used to excise the phagemid as described above. The phagemid derived from phage 169K, designated pSF2-169K, contained a 1.5 kb insert which was sequenced completely on both strands. SEQ ID NO:5 shows the 5' to 3' nucleotide sequence of 1473 base pairs of soybean (GlyeinAe ax) CDNA which encodes delta-12 desaturase in 15 plasmid pSF2-169K. Nucleotides-108 to 110 and nucleotides 1245 to 1247 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 108 to 1247). Nucleotides 1 to 107 and 1248 to 1462 are, respectively, the 5' and 3' untranslated nucleotides. The 380 amino acid protein sequence deduced from the open reading frame in SEQ ID is shown in SEQ ID NO:6. Comparisons of SEQ ID NOS:1 and 5 and of SEQ ID NOS:2 and 6, even admitting of possible sequencing errors, showed an overall homology 25 of approximately 65% at the nucleotide level and approximately 70% at the amino acid level, which confirmed that pSF2-149K is a soybean seed cDNA that encoded delta-12 desaturase. A further description of this clone can be obtained by comparison of the SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5 and by analyzing the phenotype of transgenic plants produced by using chimeric genes incorporating the insert of pSF2-169K, in sense or antisense orientation, with suitable regulatory sequences. Plasmid pSF2-169K was deposited on October 16, 1992 with the American Type Culture WO 94/11516 PCT/US93/09987 38 Collection of Rockville, Maryland under the provisions of the Budapest Treaty and bears accession number ATCC 69092.

Cloning of a Corn fZe mnIys cDNA Encoding Seed Microsmnal Delta-12 Fatty Acid Deaturae Corn microsomal delta-12 desaturase cDNA was isolated using a PCR approach. For this, a cDNA library was made to poly A+ RNA from developing corn embryos in Lambda Zap II vector. This library was used as template for PCR using sets of degenerate oligoners NS3 (SEQ ID NO:13) and RB5A/B (SEQ ID NOS:16 and 17) as sense and antisense primers, respectively. NS3 and correspond to stretches of amino acids 101-109 and 318-326, respectively, of SEQ ID N0:2, which are ~conserved in most'microsomal delta-12 desaturases (for example, SEQ ID NOS:2, 4, 6, PCR was carried out using a PCR kit (Perkin-Elmer) by 40 cycles of 940C 1', 450C, 1 min, and 55C, 2 min. Analyses of the PCR products on an agarose gel showed the presence of a product of the expected size (720 bp), which was absent in control reactions containing either the sense or antisense primers alone. The fragment was gel purified and then used as a probe for screening the corn cDNA 25 library at 60*C as described above. One positivelyhybridizing plaque was purified and partial sequence determination of its .cDNA showed it to be a nucleotide sequence encoding microsomal delta-12 desaturase but truncated at the 3' end. The cDNA insert encoding the partial desaturase was gel isolated and used to probe the corn cDNA library again. Several positive plaques were recovered and characterized. DNA sequence analysis revealed that all of these clones seem to represent the same sequence with the different length of 5' or 3' ends. The clone containing the longest insert, WO094/11516 PCrfMU93/09967 39 designated pFad2fl, was sequenced completely. The total length of the cDNA is 1790 bp (SEQ ID NO:7) comprising of an open reading frame from nucleotide 165 to 1328 bp that encoded a polypeptide, of 388 amino acids. The deduced amino acid sequence of the polypeptide (SEQ ID NO:8) shared overall identities of 40%, and 38% to Apnkidg~nfm microsomal delta-12 desaturase, hajna microsoml delta-15 desaturase, and k idjjaplastid desaturase, respectively. Furthermore, it lacked an N-terminal amino acid extension that would *indicate it is a plastid enzyme. Based on these considerations, it is conclUded that it encodes a uicrosomal delta-12 desaturase.

I~olatin f reDNA% RneMig nplta-12 Miernsomal Fatty Acid fl~turAA&A_&n flegaturame-Related Rnmymsn frmtto aned Polysomal MRNA was isolated from castor beans of.

stages 1-11 (5-10 DAP) and also from castor beans of .stages IV-V (20-25 DAP). Ten ng of each =R~NA was used for separate RT-PCR reactions, using the Perkin-Elmer RT-PCR kit. The reverse transcriptase reaction was primed with random hexamers and the PCR reaction with degenerate delta-12 desaturase primers NS3 and NS9 (SEQ ID NOS: 13 and 14). The annealing-extens ion temperature of the PCR reaction was 506C. A DNA fragment of approx.

700 bp was amplified from both stage I-I1 an~d stage IV-V mRNA.. The amplified DNA fragment from one of the reactions was gel purified and cloned into a pGEl4-T vector using the Promega pGEN-T PCR cloning kit to create the plasmid pRF2-1C. The 700 bp insert in pRF2-1C was sequenced, as described above, and the resulting DNA sequence is shown in SEQ ID NO:9. The DNA sequence in SEQ ID NRO: 9 contains an open-reading fram encoding 219 amino acids (SEQ ID NO:10) which has 81% identity (90% similarity) with amino acids 135 to 353 of WO094/11S16 PCT/US93/09987 the Arahjdnonjr microsomal delta-12 desaturase described in SEQ ID NO:2. The cDNA insert in PRF2-1C is therefore a 676 bp fragment of a full-length CDNA encoding a Castor bean seed microsomal delta-12 desaturase. The full length castor bean seed microsoeial delta-12 desaturase CDNA may isolated by screening a castor seed cDNA library, at 60*C, -with the labeled insert of pRF2-1C as described in the example above. The insert in pRF2-lC may also be used to screen castor bean libraries at lover temperatures to Isolate delta-12 *desaturase-related sequences,-such as the delta-'12 hydroxylase.

A CDNA library made to poly A+ 3iNA isolated from.

developing castor beans (stages IV-V, 20-25 DAP) was screened as described above. Radiolabeled probe *see prepared from pSF2b or pEF2-lC# as described above, were added, and allowed to hybridize for 18 h at 50*C. The *filters were washed as described above. Autoradiography ~*of the filters indicated that there were numerous hybridizing plaques, which appeared either stronglyhybridising or weakly-hybridising. Three of the *strongly hybridisng plaques (190A-41, 190A-42 and 190A-44) and three of the weakly hybridising plaques, (190B-41, 190b-43 and 197c-42), were plaque purified using the methods described above. The cDNA insert size of the purified phages were determined by PCR amplication of the insert using. pbage as template and larnbda-gt 11 oligomers (Clontech lambda-gli Amp limers) for primers. The PCR-amplified inserts of the amplified phages -were subcloned into pflluescript (Pharmacia) which had been cut with Eco RI and filled in with Klenow (Sambrook et al. (Molecular Cloningo, A Laboratory Approach, 2nd. ed. (1989) Cold Spring Harbor Laboratory Press) The resulting plasmids were called pRF190a-41, pRF190a-42, pRFl9Oa-44, pRr19Ob-41, pRF190b-43 and WO 94/11516 PCr/US93/09987 41 pRF197c-42. All o~f the inserts were about 1.1 kb with the exception of pR1F97c-42 which was approx. 1.5 kb.

The inserts in the plasmids were sequenced as described above. The insert in pRFl9Ob-43 did not contain any open reading frame and was not identified. The inserts in pRFl9Oa-41, pRFl90a-42, pEF19Oa-44 and pRFl9Ob-41 were identical. The insert in pRF197c-42 contained all of the nucleotides of the inserts in pytFl9Oa-41, pRFl9Oa-42, pRFl9Oa-44 and pRFl9Ob-41 plus an additional approx. 400 bp. It was deduced therefore that the insert in pRF197c-42 was a longer version of the inserts' in pIRF190a-41, pRFl90a-42, pRF19Oa-44 and pRF190b-41 and were derived from the same full-length mRNA. The complete cDNA sequence of the insert in plasmid pRF197c-42 is shown in SEQ ID liO:11.* The deduced amino acid sequence of SEQ ID NO:11, shown in SEQ ID NO:12, is 78.5% identical (90% similarity) to the castor *~*microsomal delta-12 desaturase described above (SEQ ID NO:1O) and 66% identical (80% similarity) to the Arahidop-gin delta-12 desaturase amino acid sequence in **SEQ ID NO:2. These. similarities confirm that pRFl97c-42 is a castor bean seed cDNA that encodes a microsomal delta-12 desaturase or a microsomal delta-12 desaturaserelated enzyme, such as a delta-12 hydroxylase.

Specific PCR primers for pRF2-lC and p1RF197c-42 were made. For pAF2-1c the upstream .primer was bases 180 to 197 of the cDHA sequence in SEQ ID NO:9. For pRF197c-42 the upstream primer was bases 717 to 743 of the'cDN& sequence in SEQ ID N0:11. A common downstream primer was made correspondingto the exact complement of the nucleotides 463 to 478 of the sequence described in SEQ ID NO: 9. Using RT-PCR with random hexamers and the above primers it was observed that the cDN& contained in pRF2-lC is expressed in both Stage 1-11 and Stage IV-V castor bean seeds whereas the cDNA contained in plasmid WO 94/11S16 PCI'/US93/09987 42 pP.Fl97c-42 is expressed only in Stage IV-V castor bean seeds, it is only expressed in tissue actively synthesizing ricinoleic acid. Thus, it is possible that this cDNA encodes a delta-12 hydroxylas.

There is enough deduced amino acid sequence from the two castor sequences described in SEQ ID NOS:l0 and 12 to compare the highly conserved region corresponding to amino acids 311 to 353 of SEQ ID NO:2. When SEQ ID NOS:2, 4, 6, 8, and 10 ace aligned by the Hein method described above the consensus sequence corresponds '3:.:.:exactly to the amino acids.311 to 353 of SEQ ID NO:2.' All of the seed microsomal delta-12 desaturases described above have a high degree of identity with the *consensus over this region, namely Ahbldaain (lo0t **15 identity), soybean (90% identity), corn (95% identity), canola (93% identity) and one (pRF2-lc) of the castor bean sequences (100% identity). The other castor bean seed delta-12 desaturase or desaturase-related sequence (pJRF197c-42) however has less identity with the consensus, namely 81% for the deduced amino acid sequence of the insert in pR1F97c-42 (described in SEQ ID 140:12). Thus while it remains possible that the insert in pRF197c-42 encodes a microsomal delta-12 desaturase, this observation supports the hypothesis that it encodes a desaturase-related sequence, namely the delta-12 hydroxylase.

An additional approach to- cloning a castor bean seed delta-12 hydroxylase is the screening of a differential population of cDNAs. A lambda-Zap (Stratagene) cDNA library made to polysomal zRNA isolated from developing castor bean endosperm (stages IV-V, 20-25 DAP) was screened with 3 2 p-labeled cDNA made from polysomal mRNA isolated from developing castor bean endosperm (stage 1-11, 5-10 DAP) and with 32 p-labeled cDNA made from polysomal mRNA isolated from developing WO 94/11516 PC/US93/09987 43 castor bean endosperm (stages IV-V, 20-25 DAP). The library was screened at a density of 2000 plaques per 137 mm plate so that individual plaques were isolated.

About 60,000 plaques were screened and plaques which hybridised with late (stage IV/V) cDNA but not early (stage I/II) cDNA, which corresponded to about 1 in every 200 plaques, were pooled.

The library of differentially expressed cDNAs may be screened with the castor delta-12 desaturase cDNA described above and/or with degenerate oligonucleotides based on sequences of amino conserved among delta-12 desaturases to isolate related castor cDNAs, including the cDNA encoding the delta-12 oleate hydroxylase enzyme. These regions of amino acid conservation may 15 include, but are not limited to amino acids 101 to 109, 137 to 145, and 318 to 327 of the amino acid sequence described in SEQ ID NO:2 or any of the sequences described in Table 7 below. Examples of such oligomers are SEQ ID NOS:13, 14, 16, and 17. The insert in plasmid pCF2-197c may be cut with Eco RI to remove vector sequences, purified by gel electrophoresis and labeled as described above. Degenerate oligomers based on the above conserved amino acid sequences may be labeled with 3 2 P as described above. The cDNAs cloned 25 from the developing endosperm difference library which do not hybridize with early mRNA probe but do hybridize with late mRNA probe and hybridize with either castor delta-12 desaturase cDNA or with an oligomer based on delta-12 desaturase sequences are likely to be the castor delta-12 hydroxylase. The pBluescript vector containing the putative hydroxylase cDNA can be excised and the inserts directly sequenced, as described above.

Clones such as pRF2-1C and pRF197c-42, and other clones from the differential screening, which, based on their DNA sequence, are less related to castor bean seed WO 94/11516 WO 9411516PCF/US3/09997 44 microsomal delta-12 desaturases and are not any of the known fatty-acid desaturases described above or in WO 9311245, may be expressed, for example, in soybean embryos or another suitable plant tissue, or in a microorganism, such as yeast, which does not normally contain ricinoleic acid, using suitable expression vectors and transformation protocols. The presence of novel ricinoleic acid in the transformed tissue(s) expressing the castor cDNA would confirm the identity of the castor cDNA as DNA encoding for an oleate hydroxylase.

Sequence aoprlRsflR Awngn Seed Microsomal Dlta-12.Desaturaspi The percent overall identities between coding regions of the full-length nucleotide seque'nces encoding microsomal delta-12 desaturases was determined by their alignment by the method of Needleman et Mol.

Biol. (1970) 48:443-453) using gap weight and gap length weight values of 5.0 and 0.3 (Table Here, a2, c2, s2, z2.and des A refer, respectively, to the nucleotide *..*sequences encoding microsomal delta-12 fatty acid desaturases from Ar-hidgpna (SEQ ID NO:l), ArAssic, nA (SEQ ID NQ:3), soybean (SEQ ID NO:5), corn (SEQ ID NO:7), and cyanobacterial des A, whereas r2 refers to the microsomal delta-12 desaturaae or desaturase-related enzyme from castor bean (SEQ ID NO:12).

Percent Identity Between the Coding Regions of Wucleotide Sequences Encoding Different Hicrosomal iAt-n-12 Fatty Aeld Dematurages ZZ AZ Z e a2 84 66. 64 43 c2 65 62 42 s2 62 42 WO 94/11516 WO 9411516PCF/US93/09967 The overall relatedness between the deduced amino acid sequences of mnicrosomal delta-12 clesaturases or desatu rase-related enzymes of the invention SEQ ID NOS:2, 4, 6, 8, pnd 12) determined by their alignment by the method of Needleman et al. MJ Mol. Biol. (1970) 48:443-453) using gap weight and gap length weight values of 3.0 and 0.1, respectively, is shown in Table 5. Here a2, c2, s2, z2 and des A refer, respectively, to microsomal delta-22 fatty acid.

desaturases from Arabijelgzzala (SEQ) ID 110:2), atranuica nana(SEQ ID 110:4), soybean (SEQ ID 110:6), corn (SEQ 16 11:8), and cyanobacterial des A, whereas r2 refers to.

the microsomal desaturase or desaturase-related enzyme from castor bean (SEQ ID 11:12). The relatedness between the enzymes is shown as percent overall identity/percent overall similarity.

Re latedness Between Different IKicrosomal Delta-12 Fatty Acid Degatilyasen a2 84/89 70/85 66/80 71/83 24/50 6..*c2 67/80 63/76 69/79 24/51 s2 67/83 66/82' 23/49 r2 61/?8 24/51 z2 25/49 The high degree of overall identity (60% or greater) at the the amino acid levels between the flArn±tqa naU Soybean, castor and corn enzymes with that of xa±aa microsomal delta-12 desaturase and their lack of an N-terminal extension of a transit peptide expected for a nuclear-encoded chloroplast desaturase leads Applicants to conclude that SEQ ID NOS:4, 6, 8, 10, and 12 encode the microsomal delta-12 WO 94/11516 PCr/US93/09987 46 desaturases or desaturase-related enzymes. Further confirmation of this identity will come from biological function, that is, by analyzing the phenotype of tranagenic plants or other organisms produced by using chimeric genes incorporating the above-mentioned sequences in sense or antisens. orientation, with suitable regulatory sequences. Thus, one can isolate cDNAs and genes for homologous fatty acid desaturases from the same or different higher plant species, especially from the oil -producing species. -Furthermore, 00 6 0 0based on these comparisons, the Applicants expect all 0 0 higher plant microsomal delta-12 desaturases from all 0 66 higher plants to show an overall identity of 60% or more *0 and to be able to readily isolate homologous fatty acid "00S* 0 15 desaturase sequences using SEQ ID 1105:1, 3, 5, 7, 9, and 11 by sequence-dependent protocols.

The overall percent identity at the amino acid *00 0 level,, using the above alignment method, between So:.selected plant desaturases is illustrated in Table 6..

0e S Percent Identity Between Selected Plant Fatty Acid 0000 Depturanps at thp Aminn An 4d- Lvel 0 a2 38 33 38 35 34 a3 65 93 66' 67 0*0 aD 66 87 c3 67 67 cD In Table 6, a2, a3, ad, c3, cD, and S3 refer, respectively, to SEQ ID 110:2, hArabi~gpjqis microsomal desaturase, Arbd~aplastid delta-is desaturase, canola microsomal delta-15 desaturase, canola plastid delta-iS desaturase, and soybean microsomal delta-iS desaturase. Based on these WO 94/11516 WO 9411516PCrIUS93/097 I. 47 comparisons, the delta-l5 desaturases, of both microsomal and plastid types, have overall identities of or more at the amino acid level, even when from the asm plant species. Eased on the above the Applicants expect microsomal delta-12 desaturases from all higher plants to show similar levels of identity (that, is, or more identity at the amino acid level) and that SEQ ID NOS:l, 3, 5, 7, and 9 can also be used as hybridization probe to isolate homologous delta-12 desaturase sequences, and possibly for sequences for fatty acid desaturase-related enzymes, such as oleate hydroxylase, that have an overall amino acid homology of or more.

Similar alignments of protein sequences of plant 15 microsomal fatty acid delta-12 desaturases [SEQ ID lJOS:2, 4, 6, and 8] and plant delta-15 desaturases Imicrosomal and plastid delta-15 desaturases from Arahldnpnin and Arzalz±C fla=j, NO 9311245) allows identification of amino acid sequences conserved between 20 the different desaturases (Table 7).

TAB F Amino Acid Sequence Qmaerved Betwweu Plant NMiciusmai Deha-12 Deuziuue and Micrusainl and Region

A

B

C

D

E

F

G

H

CanseA PoSitions in SEQJDN0O2 39-44 96-90 104-109 130-134 137-142 140-145 269-274 279-282 Mwevd AA Scquencein

A

12 6eazuzuse

EUM~

LEHX

COmuwnd AA Sequeow in

LM~

vMhJM

SUWX

AWIP/KHC

WPOAW

MVPY

W(KdAX/I)HR

SHR(RMHH

OMTYLWO

LPRHW)Y

WO 94/1 1516 PCI/US3/09MS 48 1 289-294 WL(B/KKAL YLRfOM (W/Y)L(R/KM)L j 296-302 J.YDRDE IRD T(VQLD1RDYG K 314-321 ThYAMIi 2IffIIWLE TKV(A/1)HHL L 3 18-327 HILESTMMJIHnP~I

HHFL(S/P

Table 7 shows twelve regions of conserved amino acid sequences, designated A-L (column whose positions in SEQ ID NO:2 are shown in column 2. The consensus sequences for these regions in plant delta-12 fatty acid desaturases and- plant delta-15 fatty acid desaturases are shown in columns 3 and 4, respectively; amino acids are shown by standard abbrev iation s, the underlined amino acids are conserved between the delta-12 and the delta-15 desaturases, and amino acids in brackets represent substitutions found at that position. The consensus sequence of these regions ate **shown in column S. 'These short conserved amino acids and their relative positions further confirm that the isolated iiolated cDNAs encode a fatty acid desaturase.

isolation of Nncleotidg seauenresa Encoding H~omologous and Heterologoun Fal-ty Anid D~trR and nenxturnns-14keh EnZ3Me Fragments of the instant invention may be used to isolate cDNAs and genes of homologous and heterologous fatty acid desaturases from the same species as the fragments of the invention or from different species.

Isolation of homologous genes using sequence-dependent protocols is well-known in the art and Applicants have demonstrated that ±wuamicrosomal delta-12 desaturase CDNA sequence can be used to isolate cDNA for related fatty acid desaturases from Rrnssicn. soybean, corn and castor bean.

More importantly, one can.use the fragments containing SEQ ID NOS:l, 3, 5, 7, and 9 or their WO 94/11516 PCF/US93/09987 49 smaller, more conserved regions to isolate novel fatty acid desaturases and fatty acid desaturase-related enzymes.

In a particular embodiment of the present invention, regions of the nucleic acid fragments of the invention that are conserved between different desaturases may be used by one skilled in the art to design a mixture of degenerate oligomers for use in sequence-dependent protocols aimed at isolating nucleic acid fragments encoding homologous or heterologous fatty acid desaturase cDNA's or genes. For example, in the •polymerase chain reaction (Innis, et al., Eds, (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego), two short pieces of the 15 present fragment of the invention can be used to amplify a longer fatty acid desaturase-DNA fragment from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleotide sequences with one primer based on the fragment of the invention and the other on either the poly A tail or a vector sequence. These oligomers may be unique sequences or degenerate sequences derived from the nucleic acid fragments of the invention. The longer piece of homologous fatty acid desaturase PNA generated by this method could then be used as a probe for isolating Srelated fatty acid desaturase genes or cDNAs from SArabidpsis or other species and subsequently identified by differential.screening with known desaturase sequences and by nucleotide sequence determination. The design of oligomers, including long oligomers using deoxyinosine, and "guessmers" for hybridization or for the polymerase chain reaction are known to one skilled in the art and discussed in Sambrook et al., (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press). Short stretches of WO 94/11516 PCTIUS93/09987 amino acid sequences that are conserved between cyanobacterial delta-12 desaturase (Wada et al., Nature (1990) 347:200-203) and plant delta-15 desaturases [WO 9311245] were previously used to make oligonucleotides that were degenerate and/or used deoxyinosine/s. One set of these oligomers made to a stretch of 12 amino acids conserved between cyanobacterial delta-12 desaturase and higher plant desaturases was successful in cloning the plastid delta-12 desaturase cDNAs; these plant desaturases have more than 50% identity to the cyanobacterial delta-12 desaturase. Some of these oligonucleotides were also used as primers to make polymerase chain reaction (PCR) products using poly A* 15 RNA from plants. However, none of the oligonucleotides and the PCR products were successful as radiolabeled hybridization probes in isolating nucleotide sequences encoding microsomal delta-12 fatty acid desaturases.

Thus, as expected, none of the stretches of four or more amino acids conserved between Arabid asis delta-12 and Arabidopsis delta-15 desaturases are identical in the cyanobacterial desaturase, just like none of the stretches of four or more amino acids conserved between Arabidoais delta-15 and the cyanobacterial desaturase 25 are identical in SEQ ID NO:2. Stretches of conserved amino acids between the present invention and desaturases now allow for the design of oligomers to be used to isolate sequences encoding other desaturases and desaturase-related enzymes. For example, conserved stretches of amino acids between delta-12 desaturase and desaturase, shown in Table 7, are useful in designing long oligomers for hybridization as well as shorter ones for use as primers in the polymerase chain reaction. In this regard, sequences conserved between delta-12 and delta-15 desaturases (shown in Table 7) WO 94/11516 PCT/US93/09987 51 would be particularly useful. The consensus sequences will also take into account conservative substitutions known to one skilled in the art, such as Lys/Arg, Glu/Asp, Ile/Val/Leu/Met, Ala/Gly, Gln/Asn, and Ser/Thr.

Amino acid sequences as short as four amino acids long can successfully be used in PCR [Nunberg et. al. (1989) Journal of Virology 63:3240-3249]. Amino acid sequences conserved between delta-12 desaturases (SEQ ID NOS:2, 4, 6, 8, and 10) may also be used in sequence-dependent protocols to isolate fatty acid desaturases and fatty acid desaturase-related enzymes expected to be more related to delta-12 desaturases, such as the oleate hydroxylase from castor bean. Particularly useful are conserved sequences in column 3 (Table since they 15 are also conserved well with delta-15 desaturases (column 4, Table 7).

Determining the conserved amino acid sequences from diverse desaturases will also allow one to identify more and better consensus sequences that will further aid in the isolation of novel fatty acid desaturases, including those from non-plant sources such as fungi, algae (including the desaturases involved in the desaturations of the long chain n-3 fatty acids), and even cyanobacteria, as well as other membrane-aSsociated 25 desaturases from other organisms.

The function of the diverse nucleotide fragments encoding fatty acid desaturases or desaturase-related enzymes that can be isolated using the present invention can be identified by transforming plants with the isolated sequences, linked in sense or antisense orientation to suitable regulatory sequences required for plant expression, and observing the fatty acid phenotype of the resulting transgenic plants. Preferred target plants for the transformation are the same as the source of the isolated nucleotide fragments when the WO 94/11516 PCF/US93/09987 52 goal is to obtain inhibition of the corresponding endogenous gene by antisense inhibition or cosuppression. Preferred target plants for use in expression or overexpression of the isolated nucleic acid fragments are wild type plants or plants with known mutations in desaturation reactions, such as the Arabidopsis mutants £adh, fad, lC, f&U. fad2, and iadl mutant flax deficient in delta-15 desaturation; or mutant sunflower deficient in delta-12 desaturation.

Alternatively, the function of the isolated nucleic acid fragments can be determined similarly via transformation of other organisms, such as yeast or cyanobacteria, with chimeric genes containing the nucleic acid fragment and suitable regulatory sequences followed by analysis of 15 fatty acid composition and/or enzyme activity.

Overaxpressionn of the Fatty Acid Desaturase Enzymes in Trangsenic Species The nucleic acid fragment(s) of the instant invention encoding functional fatty acid desaturase(s), with suitable regulatory sequences, can be used to overexpress the enzyme(s) in transgenic organisms. An example of such expression or overexpression is demonstrated by transformation of the Arabidopsis mutant lacking oleate desaturation. Such recombinant DNA 25 constructs may include either the native fatty acid desaturase gene or a chimeric fatty acid desaturase gene isolated from the same or a different species as the host organism. For overexpression of fatty acid desaturase(s), it is preferable that the introduced gene be from a different species to reduce the likelihood of cosuppression. For example, overexpression of delta-12 desaturase in soybean, rapeseed, or other oil-producing species to produce altered levels of polyunsaturated fatty acids may be achieved by expressing RNA from the full-length cDNA found in p92103, pCF2-165D, and WO 94/11516 PCT/US93/09987 53 pSF2-169K. Transgenic lines overexpressing the delta-12 desaturase, when crossed with lines overexpressing desaturases, will result in ultrahigh levels of 18:3. Similarly, the isolated nucleic acid fragments encoding fatty acid desaturases from Xuabidopas, rapeseed, and soybean can also be used by one skilled in the art to obtain other substantially homologous fulllength cDNAs, if not already obtained, as well as the corresponding genes as fragments of the invention.

These, in turn, may be used to overexpress the corresponding desaturases in plants. One skilled in the art can also isolate the coding sequence(s) from the fragment(s) of the invention by using and/or creating sites for restriction endonucleases, as described in 15 Sambrook et al., (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).

One particularly useful application of the claimed inventions is to repair the agronomic performance of plant mutants containing ultra high levels of oleate in seed oil. In Arabidopsis reduction in linoleate in phosphatidylcholine due to a mutation in microsomal delta-12 desaturase affected low temperature survival [Miquel, M. et. al. (1993) Proc. Natl Acad. Sci. USA 90:6208-6212]. Furthermore, there is evidence that the poor agronomic performance of canola plants containing ultra high (>80%)levels of oleate in seed is due to mutations in the microsomal delta-12 desaturase genes that reduce the level of linoleate in phosphotidylcholine of roots and leaves. That is, the mutations are not seed-specific. Thus, the root and/or leaf-specific expression (that is, with no expression in the seeds) of microsomal delta-12 desaturase activity in mutants of oilseeds with ultra-high levels of oleate in seed oil 0 WO 94/11516 PC/US93/09987 54 will result in agronomically-improved mutant plants with ultra high levels of oleate in seed oil.

Inhibition of Plant Taraet Genes by Use of Antisene RNA Antisense RNA has been used to inhibit plant target genes in a tissue-specific manner (see van der Krol et al., Biotechniques (1988) 6:958-976). Antisense inhibition has been shown using the entire cDNA sequence (Sheehy et al., Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809) as well as a partial cDNA sequence (Cannon et al., Plant Molec. Biol. (1990) 15:39-47). There is also evidence that the 3' non-coding sequences (Ch'ng et al., Proc. Natl. Acad. Sci. USA (1989) 86:10006-10010) and fragments of 5* coding sequence, 15 containing as few as 41 base-pairs of a 1.87 kb cDNA (Cannon et al., Plant Molec. Biol. (1990) 15:39-47), can play important roles in antisense inhibition.

The use of antisense inhibition of the fatty acid desaturases may require isolation of the transcribed 20 sequence for one or more target fatty acid desaturase genes that are expressed in the target tissue of the target plant. The genes that are most highly expressed are the best targets for antisense inhibition. These genes may be identified by determining their levels of 25 transcription by techniques, such as quantitative analysis of mRNA levels or nuclear run-off transcription, known to one skilled in the art.

The entire soybean microsomal delta-12 desaturase cDNA was cloned in the antisense orientation with respect to either soybean b-conglycinin, soybean KTi3, and bean phaseolin promoter and the chimeric gene transformed into soybean somatic embryos that were previously shown to serve as good model system for soybean zygotic embryos and are predictive of seed composition (Table 11). Transformed somatic embryos WO 94/11516 PCT/US93/09987 showed inhibition of linoleate biosyntheis. Similarly, the entire Brassid nausr microsomal delta-12 desaturase cDNA was cloned in the antisense orientation with respect to a rapeseed napin promoter and the chimeric gene transformed into 1. napus. Seeds of transformed B. naus plants showed inhibition of linoleate biosynthesis. Thus, antisense inhibition of delta-12 desaturase in oil-producing species, including corn, Braasica napA, and soybean resulting in altered levels of polyunsaturated fatty acids may be achieved by expressing antisense RNA from the entire or partial cDNA encoding microsomal delta-12 desaturase.

Inhibition of Plant farget Genea by Cosmpresiann S: 15 The phenomenon of cosuppression has also been used to inhibit plant target genes in a tissue-specific manner. Cosuppression of an endogenous gene.using the entire cDNA sequence (Napoli et al., The Plant Cell (1990) 2:279-289; van der Krol et al., The Plant Cell 20 (1990) 2:291-299) as well as a partial cDNA sequence (730 bp of a 1770 bp cDNA) (Smith et al., Mol. Gen.

Genetics (1990) 224:477-481) are known.

S. The nucleic acid fragments of the instant invention encoding fatty acid desaturases, or parts thereof, with 25 suitable regulatory sequences, can be used to reduce the level of fatty acid desaturases, thereby altering fatty acid composition, in transgenic plants which contain an endogenous gene substantially homologous to the introduced nucleic acid fragment. The experimental procedures necessary for this are similar to-those described above for the overexpression of the fatty acid desaturase nucleic acid fragments except that one may also use a partial cDNA sequence. For example, cosuppression of delta-12 desaturase in Brassica napns and soybean resulting in altered levels of WO 94/11516 PC/US93/09987 56 polyunsaturated fatty acids may be achieved by expressing in the sense orientation the entire or partial seed delta-12 desaturase cDNA found in pCF2-165D and pSF2-165K, respectively. Endogenous genes can also be inhibited by non-coding regions of an introduced copy of the gene [For example, Brusslan, J. A. et'al. (1993) Plant Cell 5:667-677; Matzke, M. A. et al., Plant Molecular Biology 16:821-830]. We have shown that an Arabidopsis gene (SEQ ID NO:15) cdrresponding to the CDNA (SEQ ID NO:1) can be isolated. One skilled in the art can readily isolate genomic DNA containing or flanking the genes and use the coding or non-coding regions in such transgene inhibition methods.

Analysis of the fatty acid composition of roots and S: 15 seeds of Arabidopsis mutants deficient in microsomal delta-12 desaturation shows that they have reduced levels of 18:2 as well as reduced levels of 16:0 (as much as 40% reduced level in mutant seeds as compared to wild type seeds) [Miquel and Browse (1990) in Plant Lipid Biochemistry, Structure, and Utilization, pages 456-458, Ed. Quinn, P. J. and Harwood, J. L., Portland Press. Reduction in the level of 16:0 is also 9 observed in ultra high oleate mutants of B. napus.

Thus, one can expect that ultra high level of 18:1 in 25 transgenic plants due to antisense inhibition or cosupression using the claimed sequences will also reduce the level of 16:0.

Selention of Rosts. Promoters and Enhancers A preferred class of heterologous hosts for the expression of the nucleic acid fragments of the invention are eukaryotic hosts, particularly the cells of higher plants. Particularly preferred among the higher plants are the oil-producing species, such as soybean (Glycine max), rapeseed (including Brassica nau, R. camstris), sunflower (Helianthus annus), WO 94/11516 PC/US93/09987 57 cotton (Gssypium hirsutum), corn (ZeA MaY) cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (IEaeis guineensis), coconut palm (Cocas nucifera), flax (Linum u.itatiaimum), and peanut (Arachis hvogaea).

Expression in plants will use regulatory sequences functional in such plants. The expression of foreign genes in plants is well-established (De Blaere et al., Meth. Enzymol. (1987) 153:277-291). The source of the promoter chosen to drive the expression of the fragments of the invention is not critical provided it has sufficient transcriptional activity to accomplish the invention by increasing or decreasing, respectively, the Slevel of translatable mRNA for the fatty acid 15 desaturases in the desired host tissue. Preferred promoters include strong constitutive plant promoters, such as those directing the 19S and transcripts in cauliflower mosaic virus (Odell et al., Nature (1985) 313:810-812; Hull et al., Virology (1987) 86:482-493., tissue- or developmentally-specific promoters, and other transcriptional promoter systems engineered in plants, such as those using bacteriophage T7 RNA polymerase promoter sequences to express foreign genes. Examples of tissue-specific promoters are the light-inducible promoter of the small subunit of ribulose 1,5-bis-phosphate carboxylase (if expression is desired in photosynthetic tissues), the maize zein protein promoter (Matzke et al., EMBO J.

(1984) 3:1525-1532), and the chlorophyll a/b binding protein promoter (Lampa et al., Nature (1986) 316:750-752).

Particularly preferred promoters are those that allow seed-specific expression. This may be especially useful since seeds are the primary source of vegetable oils and also since seed-specific expression will avoid WO 94/11516 PCr/IUS93/09987 58 any potential deleterious effect in non-seed tissues.

Examples of Deed-specific promoters include, but are not limited to, the promoters of seed storage proteins, which can represent up to g0t of total seed protein in many Plants. The seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly tissue-specific and stage-specific manner (Higgins et al., Ann. Rev. Plant Physiol. (1984) 35:191-221; Goldberg et al., Cell (1989) 56:149-160).

Moreover, different seed storage proteins may be expressed at different stages of seed development.

Expression of seed-specific genes has been studied in great detail (See reviews by Goldberg et al., Cell so (1989) 56:149-160 and Higgins et al., Ann. Rev. Plant *00 *15 Physiol. (1984) 35:191-221). There are currently numerous examples of seed-specific expression of seed storage protein genes in tranagenic dicotyledonous plants. These include genes from dicotyledonous plants @0*0for bean b-phaseolin (Sengupta-Gopalan et al., Proc.

:20 Natl. Acade. Sci. USA (1985) 82:3320-3324; Eoffmanet al., Plant 14o1. Biol. (198 8) 11:717-729), bean lectin (Voelicer et al., EMBO J. (1987) 6:3571-3577), soybean lectin (Okamuro et al., Proc. Natl. Acad. Sci. USA too*.(1986) 83:8240-8244), soybean Kunitz trypsin inhibitor (Perez-Grau et al., Plant Cell (1989) 1:095-1109), soybean banconglycinin (Beachy et al., EMBO J. (1985) 434-3053; pea vicilin (Higgins et al., PatNl Biol. (1988) 12:683-695), pea convicilin (Newbigin et al., Planta (1990) 180:461-470), pea legumin (Shirsat et al., Mol. Gen. Genetics (1989) 215:326-331); rapeseed napin (PRadke et al., Theor. Appi. Genet. (1988) 75:685-694) as well as genes from monocotyledonous plants such as for maize 15 kD zein (Hoffman et al., EMBO J. (1987) 6:3213-3221), maize 18 kD oleosin (Lee at a1., Proc. Natl. Acad. Sci. USA (1991) 888:6181-6185), WO 94/11516 PCT/US93/09987 59 barley b-hordein (Marris et al., Plant Mol. Biol. (1988) 10:359-366) and wheat glutenin (Colot et al., EMBO J.

(1987) 6:3559-3564). Moreover, promoters of seedspecific genes operably linked to heterologous coding sequences in chimeric gene constructs also maintain their temporal and spatial expression pattern in transgenic plants. Such examples include use of Arabidopais thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Aabidopaia and A. napa seeds (Vandekerckhove et al., Bio/Technology (1989) 7:929-932), bean lectin and bean b-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. (1989) 63:47-57), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J. (1987) 6:3559-3564).

Of particular use in the expression of the nucleic acid fragment of the invention will be the heterologous promoters from several soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor 20 (Jofuku et al., Plant Cell (1989) 1:1079-1093; glycinin (Nielson et al., Plant Cell (1989) 1:313-328), and b-conglycinin (Earada et al., Plant Cell (1989) 1:415-425). Promoters of genes for a- and b-subunits of soybean b-conglycinin storage protein will be particularly useful in expressing the mRNA or the antisense RNA in the cotyledons at aid- to late-stages of seed development (Beachy et al., EMBO J. (1985) .4:3047-3053) in transgenic plants. This is because there is very little position effect on their expression in transgenic seeds, ard the two promoters show different temporal regulation. The promoter for the a-subunit gene is expressed a few days before that for the b-subunit gene. This is important for transforming rapeseed where oil biosynthesis begins about a week WO 94/11516 WO 94/11516PCMIS93/O9987 before seed storaqe protein synthesis (Murphy et al., J. Plant Physiol. (1989) 135:63-69).

Also of particular use will be promoters of genes expressed during early ezbryogenesis and oil blosynthesis. The native regulatory sequences, including the native promoters, of the fatty acid-desaturase genes expressing the nucleic acid fragments of the invention can be used following their isolation by those skilled in the art. Rete rologous promoters from other genes involved in seed oil biosynthesis, such as those for A. n==u isocitrate lyase and malate synthase (Coanai et al., Plant Cell (1989) 1:293-300), delta-9 desaturase from safflower (Thompson et al. Proc. Natl. Acad. Sci.

USA (1991) 88:2578-2582) and castor (Shanklin et al., Proc. Natl. Acad. Sci. USA (1991) 88:2510-2514), icyl carrier protein (ACP) from Arbdaa(Post- Beittenmiller et al., Nudl. Acids Res. (1989) 17:1777), A. AA2 (Safford et al., Eur. J. Biochem. (1988) 174:287-295), and a. eamp~ttr1ia (Rose et al., Nudl.

:.20 Acids Res. (1987) 15:7197), b-ketoacyl-ACP synthetase *from barley (Siggaard-Andersen et al., Proc. Natl. Acad.

Sci. USA (1991) 88:4114-4118), and oleosin from Ze, ma=.

(Lee et al., Proc. Natl. Acad. Sci. USA (1991) 88:6181-6185), soybean (Genbank Accession No: X60773) and R. (Lee et al., Plant Physiol. (1991) 96:1395-1397) will be of use. If the sequence of the corresponding genes is not disclosed or their promoter region is not identified, one skilled in the art can use the published sequence to isolate the corresponding gene and a fragment thereof'containing the promoter. The partial protein sequences for the relatively-abundant enoyl-ACP reductase and acetyl-CoA carboxylase are also published (Slabas et al., Biochim. Biophys. Acta (1987) 877:271-280; Cottingham et al., Biochim. Biophys. Acta (1988) 954:201-207). and one skilled in the art can use S'

I*

WO 94/11516 PCT/US93/09987 61 these sequences to isolate the corresponding seed genes with their promoters. Similarly, the fragments of the present invention encoding fatty acid desaturases can be used to obtain promoter regions of the corresponding genes for use in expressing chimeric genes.

Attaining the proper level of expression of the nucleic acid fragments of the invention may require the use of different chimeric genes utilizing different promoters. Such chimeric genes can be transferred into host plants either together in a single expression vector or sequentially using more than one vector.

It is envisioned that the introduction of enhancers or enhancer-like elements into the promoter regions of either the native or chimeric nucleic acid fragments of 15 the invention will result in increased expression to Saccomplish the invention. This would include viral **de enhancers such as that found in the 35S promoter (Odell et al., Plant Mol. Biol. (1988) 10:263-272), enhancers from the opine genes (Fromm et al., Plant Cell (1989) 20 1:977-984), or enhancers from any other source that result in increased transcription when placed into a promoter operably linked to the nucleic acid fragment of the invention.

Of particular importance is. the:DNA sequence 25 element isolated from the gene for the a-subunit of b-conglycinin that can confer 40-fold seed-specific enhancement to a constitutive promoter (Chen et al., Dev. Genet. (1989) 10:112-122). One skilled in the art can readily isolate this element and insert it within the promoter region of any gene in order to obtain seedspecific enhanced expression with the promoter in transgenic plants. Insertion of such an element in any seed-specific gene that is expressed at different times than the b-conglycinin gene will result in expression in WO 94/11516 PC/US93/09987 62 transgenic plants for a longer period during seed development.

The invention can also be accomplished by a variety of other methods to obtain the desired end. In one form, the invention is based on modifying plants to produce increased levels of fatty acid desaturases by virtue of introducing more than one copy of the foreign gene containing the nucleic acid fragments of the invention. In some cases, the desired level of polyunsaturated fatty acids may require introduction of foreign genes for more than one kind of fatty acid desaturase.

Any 3' non-coding region capable of providing a polyadenylation signal and other regulatory sequences that may be required for the proper expression of the nucleic acid fragments of the invention can be used to accomplish the invention. This would include 3' ends of the native fatty acid desaturase(s), viral genes such as from the 35S or the 19S cauliflower mosaic virus *,20 transcripts, from the opine synthesis genes, ribulose *1,5-bisphosphate carboxylase, or chlorophyll a/b binding protein. There are numerous examples in the art that teach the usefulness of different 3' non-coding regions.

Transformtion Methods Various methods of transforming cells of higher plants according to the present invention are available to those skilled in the art (see EPO Pub. 0 295 959 A2 and 0 318 341 Al). Such methods include those based on transformation vectors utilizing the Ti and Ri plasmids of Agrobacterium ad. It is particularly preferred to use the binary type of these vectors. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants (Sukhapinda et al., Plant Mol. Biol. (1987) 8:209-216; Potrykus, Mol. Gen. Genet. (1985) 199:183). Other I' I WO 94/11516 PC/US93/09987 63 transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EPO Pub. 0 295 959 A2), techniques of electroporation (Fromm et al., Nature (1986) (London) 319:791) or high-velocity ballistic bombardment with metal particles coated with the nucleic acid constructs (Kline et al., Nature (1987) (London) 327:70). Once transformed, the cells can be regenerated by those skilled in the art.

Of particular relevance are the recently described methods to transform foreign genes into commercially important crops, such as rapeseed (De Block et al., Plant Physiol. (1989) 91:694-701), sunflower (Everett et al., Bio/Technology (1987) 5:1201), and soybean 15 (Christou et al., Proc. Natl. Acad. Sci USA (1989) 86:7500-7504.

Application to Molecular Breeding The 1.6 kb insert obtained from the plasmid pSF2-169K was used as a radiolabelled probe on a 20 Southern blot containing genomic DNA from soybean .***(Glycine max (cultivar Bonus) and Glycine anja (PI81762)) digested with one of several restriction enzymes. Different patterns of hybridization (polymorphisms) were identified in digests performed with restriction enzymes Bind III and Eco RI. These polymorphisms were used to map two pSF2-169 loci relative to other loci on the soybean genome essentially as described by Belentjaris et al., (Theor. Appl. Genet..

(1986) 72:761-769). One mapped to linkage group 11 between 4404.00 and 1503.00 loci (4.5 cM and 7.1 cM from 4404.00 and 1503.00, respectively) and the other to linkage group 19 between 4010.00 and 5302.00 loci (1.9 CM and 2.7 cM from 4010.00 and 5302.00, respectively) (Rafalski, A and Tingey, S. (1993) in Genetic Maps, Ed. O' Brien, S. The use of WO 94/11516 PCT/US93/09987 64 restriction fragment length polymorphism (RFLP) markers in plant breeding has been well-documented in the art (Tanksley et al., Bio/Technology (1989) 7:257-264).

Thus, the nucleic acid fragments of the invention can be used as RFLP markers for traits linked to expression of fatty acid desaturases. These traits will include altered levels of unsaturated fatty acids. The nucleic acid fragment of the invention can also be used to isolate the fatty acid desaturase gene from variant (including mutant) plants with altered levels of unsaturated fatty acids. Sequencing of these genes will reveal nucleotide differences from the normal gene that cause the variation. Short oligonucleotides designed around these differences may also be used in molecular breeding either as hybridization probes or in DNA-based diagnostics to follow the variation in fatty acids.

Oligonucleotides based on differences that are linked to the variation may be used as molecular markers in breeding these variant oil traits.

20 EXAMPLES 0 The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, 25 while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference herein.

I,

I,,

WO 94111516 PCr/1US93/0997 rXAPTr.

ISOLATION OF GENOMIC DNA FLANKING THE T-DNA SITE OF TNSRRTIO_ IN ARAEIflOPSI WHL)A MUTANT LINE Id~ntifienati~r nf an Arahidapsift thnljAnn T-DRA Muitant~ with Hiah Meinr Aef{d enntont A population of Arahidanad& thnIA±IA (geographic race Wassilevakija) transformants containing the modified T-DNA of Agrobct-gri-Iumefnefoina was generated by seed transformation as described by Feldman et al., (Mol.*Gen. Genetics (1987) 208:1-9).

In this population the transformants contain DNA sequences encoding the pBR322 bacterial vector, nopaline synthase, neomypin phosphotransferase (NPTII, confers kanamycin resistance), and b-lactaase (confers ampicillin resistance) within the T-DNA border sequences. The integration of the T-DNA into different areas of the chromosomes of individual transformants may cause a disruption of plant gene function at or near the site of insertion, and phenotypes associated with this .20 loss of gene function can be analyzed by screening the population for the phenotype.

T3 seed was generated from the wild type seed treated with grh ximtmIinaby two rounds of self-fertilization as described by Feldmann et al., (Science (1989) 243:1351-1354). These progeny were segregating for the T-DNA insertion~, and thus for any mutation resulting from the insertion. Approximately 10-12 leaves of each of 1700 lines were combined and the fatty acid content of each of the 1700 pooled samples was determined by gas chromatography of the fatty acyl methyl esters essentially as described by Browse et al.., (Anal. Biochem. (1986) 152:141-145) except that 2. H2SO4 in methanol was used as the methylation reagent.

A line designated 0658" produced a sample that gave an 1" WO 94/11516 PCr/US93/09987 66 altered fatty acid prof ile compared to those of lines 657 and 659 sampled at the same time (Table 8).

Fatty Acid MPthVI Vqte 16:0 16:1 16:2 16:3 '18:0 18:1 18:2 18:3 2ARTLLI 657 Leaf 659 Leaf 14.4 14.1 4.4 4.6 2.9 -2.2 13.9 13.3 1.0 1.1 2.6 .2.5 14.0 13. 6 42.9 46.1 658 Leaf 13.6 2.7 13.9 0.9 4.9 12.8 44.4 Analysis of the fatty acid composition of 12 individual T3 seeds of line 658 indicated that the 658 5 pool was composed of seeds segregating in th ree classes: "high", "mid-range" and Olow" classes with approximately, .37% (12 seeds), 21% (7 seeds), and 14% (3 seeds) oleic acid, respectively (Tab~le 9).

16:0 16: lc 18:0 le: 1 18:2 18:3 20:1 "kigh"s "Mid-range" C!Iaqs* jAzA 8.9 8.7 2.016 4.5 4.3 37.0 20.7 *8.0 24.9 10.6 14.3 25.5 21.6 9.3 2.6 4.4 14.4 27.7 13.6 20.4 Thus, the high oleic acid mutant phenotype segregates in an approximately Mendelian ratio. To determine the number of independently segregating T-.DNA WO 94/11516 PC/US93/09987 67 inserts in line 658, 200 T3 seeds were tested for their ability to germinate and grow in the presence of kanamycin [Feldman et al. (1989) Science 243:1351-1354].

In this experiment, only 4 kanamycin-sensitive individual plants were identified. The segregation ratio (approximately 50:1) indicated that line 658 harbored three T-DNA inserts. In this and two other experiments a total of 56 kanamycin-sensitive plants were identified; 53 of these were analyzed for fatty acid composition and at least seven of these displayed oleic acid levels that were higher than would be expected for wild type seedlings grown under these conditions.

In order to more rigorously test whether the mutation resulting in high oleic acid is the result of T-DNA insertion, Applicants identified a derivative line that was segregating for the mutant fatty acid phenotype as well as a single kanamycin resistance locus. For this, approximately 100 T3 plants were individually 20 grown to maturity and seeds collected. One sample of seed from each T3 plant was tested for the ability to germinate and grow in the presence of kanamycin. In addition, the fatty acid compositions of ten additional individual seeds from each line were determined. A T3 plant, designated 658-75, was identified whose progeny seeds segregated 28 kanamycin-sensitive to 60 kanamycinresistant and 7 with either low or intermediate oleic acid to 2 high oleic acid.

A total of approximately 400 T4 progeny seeds of the derivative line 658-75 were grown and the leaf fatty acid composition analyzed. A total of 91 plants were identified as being homozygous for the high oleic acid trait (18:2/18:1 less than The remaining plants (18:2/18:1 more than 1.2) could not be definitively assigned to wild type and heterozygous classes on the WO 94/11516 PCT/US93/09987 68 basis of leaf fatty acid composition and thus could not be used to test linkage between the kanamycin marker and the fatty acid mutation. Eighty three of the 91 apparently homozygous high oleic acid mutant were tested for the presence of nopaline, another T-DNA marker, in leaf extracts (Errapalli et al. The Plant Cell (1991)3:149-157 and all 83 plants were positive for the presence of nopaline. This tight linkage of the mutant fatty acid phenotype and a T-DNA marker provides evidence that the high oleic acid trait in mutant 658 is the result of T-DNA insertion.

Plasmid Rescue and Analysis One-half and one microgram of genomic DNA from the Shomozygous mutant plants of the 658-75 line, prepared 15 from leaf tissue as described [Rogers, S. 0. and A. J.

Bendich (1985) Plant Molecular-Biology 5:69-76], was digested with 20 units of either Barn HI or Sal I restriction enzyme (Bethesda Research Laboratory) in a gL reaction volume according to the manufacturer's specifications. After digestion the DNA was extracted with buffer-saturated phenol (Bethesda Research Laboratory) followed by precipitation in ethanol.

One-half to one microgram of Barn I or Sal I digested genomic DNA was resuspended in 200 uL or 400 uL of ligation buffer containing 50 mM Tris-Cl, pH 8.0, 10 mM MgC12, 10 mM dithiothreitol, 1 mM ATP, and 4 units of T4 DNA ligase (Bethesda Research Laboratory). The dilute DNA concentration of approximate 2.5 ug/mL in the ligation reaction was chosen to facilitate circularization, as opposed to intermolecular joining.

The reaction was incubated for 16 h at 16°C. Competent cells (Bethesda Research Laboratory) were transfected with 10 ng of ligated DNA per 100 LL of competent cells according to the manufacturer's specifications. Transformants from Sal I or Bam HI Ik P WO 94/11516. PCF/US93/09987 69 digests were seletted on LB plates (10 g Bacto-tryptone, g Bacto-yeast extract, 5 g Nadl, 15 g aLgar per liter, pH 7.4) containing 100 ;ig/wI ampicillin. After overnight incubation at 37 0 C the plates were -scored for ampicillin-resistant colonies.

A single ampicillin-resistant transformant derived from Barn 9I-digested plant DNA was used to start a culture in 35 mL LB medium (10 g Bacto-tryptone, yeast-extract, 5 g NaCl per liter) containing 25 mg/L ampicillin. The culture was incubated with shaking overnight at 370C and the cells were then collected by centrifugation at lOO0xg for 10 min. Plasmid DNA, designated p658-1, was isolated from the cells by the alkaline lyuis method of Birubiom et al. (Nucleic Acid V* 15 Research (1979) 7:1513-1523], an described in Sa urook et (Molecular Cloning, A Laboratory Manual, 2nd ed :.(1989) Cold Spring Harbor Laboratory Press) Plasmid p658-1 DNA was digested by restriction enzymes Barn HI, Eco RI and Sal I (Bethesda Reseach Laboratory) and electrophoresed through a 1% agarose gel in lxTBE buffer (0.089M4 tris-borate, 0.002M4 EDTA) The restriction pattern indicated the presence in this plasmid of the expected 14.2 ICE T-DNA fragment and a 1.6 kB, putative plant DNA/T-DNA border fragment.

999925 CLONINa OF ARBDOPrISq TIAANA M4ICROSNLDTA DATMIAqV eDNA MSING GNIC DNA FIANKINC WME ~~T-DNA SITE OF INSERTION IN AlAIORSTMIN 9999 MUTANT LINE 65a-75A A HYBRIDIZATION 21=B Two hundred nanograms of the 1.6 kB Eco -RI-Ban HI fragment from plasmid p658-1, following digestion of the plasmid with Eco RI and Barn HI and purification by electrophoresis in agarose, was radiolabelled with alpha132P]-dCTP using a Random Priming Labeling Kit WO 94/11516 PCr/UIS93/09987 (Bethesda Researchr Laboratory) under conditions recommended by the manufacturer.

The radiolabeled DNA was used as a probe to screen an Arabidg~ita cDNA library made from RNA isolated from.

above ground portions of various growth stages (Elledge et al., (1991) Proc. Nat. Acad. Sci., 88:1731-1735) essentially as described in Sambrook et al., (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press) For this, approximately 17,000 plaque-forming units were plated on seven 90mm petri plates containing a lawn of LE392 coli cells on NZY agar media (5 g Nadl, 2 g NgSO4-7 5 9 yeast extract, 10 q casein acid hydrolysate, 13 g agar per liter). Replica filters of the phage plaques were prepared by adsorbing the plaques onto nitrocellulose filters (BABS, Schleicher and Schuell) then soaking successively for five min each in 0.5 M4 NaOH/1 M NaCi, 0.5 M4 Tris(pE 7.4)/1.5 M4 Nad and 2xSSPE (0..36 M NaCi, 20 mM NaH2PO4 (p H7.4), 20 mM EDTA (pH The filters were then air dried and baked **for 2 h at 80*C. After baking the filters were wetted :in 2X SSPE, and then. incubated at 42*C in prehybridization buffer (50% Pormamide, 5X SSPE, 1% SDS, *.5X Denhardt's Reagent, and 100 ug/mL denatured -salmon sperm DNA) for 2 h. The filters were removed f rom the prehybridization buffer, and thep transferred to hybridization buffer (50% Formamide,' 5X SSPE, 2% SDS, 1X Denhardt Is Reagent, and 100 ug/mL denatured salmon sperm DNA) containing the denatured radiolabeled probe (see above) and incubated for 40 h at 420C. The filters were 'washed three times in 2X SSPE/0.2% SDS at 42 0 C (15 min each) and. twice in 0.2X SSPE/0, 2% SDS at 55*C (30 min each), followed by autoradiography on Kodak XAR-5 film with an intensifying s creen at -80*C, overnight.

Fifteen plaques were identified as positively- WO 94/11516 PCF/US93/09987 71 hybridizing on replica filters. Five of these were subjected to plaque purification essentially as described in Sambrook et al., (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press). The lambda YES-R cDNA clones were converted to plasmid by propagating the phage in the E. coli BNN--132 cells, which expresses Cre protein that excises the cDNA insert as a double-stranded plasmid by cre-mediated in vivo site-speicifc recombination at a 'lox' sites present in the phage. Ampicillin-resistant plasmid clones containing cDNA inserts were grown in liquid culture, and plasmid DNA was prepared using the alkaline lysis method as previously described.' The sizes of the resulting plasmids were analyzed by 15 electrophoresis in agarose gels. The agarose gels were treated with 0.5 M NaOH/1 M NaCl, and 0.5 M Tris(pH 1.5 M NaC1 for 15 min each, and the gel S •was then dried completely on a gel drier at 65"C. The gel was hydrated in 2X SSPE and incubated overnight, at 42°C, in hybridization buffer containing the denatured radiolabeled probe, followed by washing as described above. After autoradiography, the inserts of four of the purified cDNA clones were found to have hybridized to the probe. Plasmid DNA from the hybridizing clones 25 was purified by equilibration in a CsCl/ethidium bromide gradient (see above). The four cDNA clones were sequenced using Sequenase T7 DNA polymerase (US Biochemical Corp.) and the manufacturer's instructions, beginning with primers homologous to vector sequences that flank the cDNA insert. After comparing the partial sequences of the inserts obtained from the four clones, it was apparent that they each contained sequences in common. One cDNA clone, p92103, containing ca. 1.4 kB cDNA insert, was sequenced. The longest three clones were subcloned into the plasmid vector pBluescript WO 94/11516 PCr/US93/09987 72 (Stratagene). One of these clones, designated pSF2b, containing ca 1.2 kB cDNA insert was also sequenced serially with primers designed from the newly acquired sequences as the sequencing experiment progressed. The composite sequence derived from pSF2b and p92103 is shown in SEQ ID NO:1.

EXAMPLE 3 CLONING OF PLANT FATTY ACID DTEATRASE AR n tSTNG w Y kRABTDOPSTr

THATTANA

IRMORSOAL nETLTA-12 DERATRAE nDUNA CLONE AS A mYBRIDI2RATrN

PROB

An approximately 1.2 kb fragment containing the Arxabid]psis delta-12 desaturase coding sequence of SEQ ID NO:1 was obtained from plasmid pSF2b. This plasmid 15 was digested with EcoR I and the 1.2 kb delta-12 desaturase cDNA fragment was purified from the vector sequence by agarose gel electrophoresis. The fragment was radiolabelled with 3 2 P as previously described.

Cloninp of a Branianapus Seed cDNA Eneodin Mincroomal nDelta-12 Patty Acid Desaturase ~The radiolabelled probe was used to screen a zasasic anapn seed cDNA library. In order to construct 9* the library, Branssgia na= seeds were harvested 20-21 days after pollination, placed in liquid nitrogen, and polysomal RNA was isolated following the procedure of Kamalay et al., (Cell (1980) 19:935-946). The polyadenylated mRNA fraction was obtained by affinity chromatography on oligo-dT cellulose (Aviv et al., Proc.

Natl. Acad. Sci. USA (1972) 69:1408-1411). Four micrograms of this mRNA were used to construct a seed cDNA library in lambda phage (Uni-zAPNm XR vector) using the protocol described in the ZAP-cDNAmr Synthesis Kit (1991 Stratagene Catalog, Item 4200400). Approximately 600,000 clones were screened for positively hybridizing Et II 1, WO 94/11516 PCT/US93/09987 73 plaques using the radiolabelled EcoR I fragment from pSF2b as a probe essentially as described in Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd ed.

(1989) Cold Spring Harbor Laboratory Press) except that low stringency hybridization conditions (50 mM Tris, pH 6X SSC, 5X Denhardt's, 0.5% SDS, 100 Lg denatured calf thymus DNA and 50°C) were used and posthybridization washes were performed twice with 2X SSC, SDS at room temperature for 15 min, then twice with 0.2X SSC, 0.5% SDS at room temperature for 15 min, and then twice with 0.2X SSC, 0.5% SDS at 500C for 15 min.

Ten positive plaques showing strong hybridization were picked, plated out, and the.screening procedure was repeated. From the secondary screen nine pure phage 15 plaques were isolated. Plasmid clones containing the cDNA inserts were obtained through the use of a helper phage according to the in xix excision protocol provided by Stratagene. Double-stranded DNA was prepared using the alkaline lysis method as previously S. 20 described, and the resulting plasmids were size-analyzed by electrophoresis in agarose gels. The largest one of the nine clones, designated pCF2-165D, contained an approximately 1.5 kb insert which was sequenced as described above. The sequence of 1394 bases of the cDNA 25 insert of pCF2-165D is shown in SEQ ID NO:3. Contained in the insert but not shown in SEG ID NO:3 are approximately 40 bases of the extreme 5' end of the non-translated region and a poly A tail of about 38 bases at the extreme 3' end of the insert.

Cloning of a Soybean Seed cDNA Encoding Microsomal Delta-12 Fatty Acid Desaturase A cDNA library was made as follows: Soybean embryos (ca. 50 mg fresh weight each) were removed from the pods and frozen in liquid nitrogen. The frozen WO 94/11516 PCT/US93/09987 74 embryos were ground to a fine powder in the presence of liquid nitrogen and then extracted by Polytron homogenization and fractionated to enrich for total RNA by the method of Chirgwin et al. (Biochemistry (1979) 18:5294-5299). The nucleic acid fraction was enriched for poly A+RNA by passing total RNA through an oligo-dT cellulose column and eluting the poly A+RNA with salt as described by Goodman et al. (Meth. Enzymol. (1979) 68:75-90). CDNA was synthesized from the purified poly A+RNA using cDNA Synthesis System (Bethesda Research Laboratory) and the manufacturer's instructions. The resultant double-stranded DNA was methylated by Eco RI DNA methylase (Promega) prior to filling-in its ends with T4 DNA polymerase (Bethesda Research Laboratory) 15 and blunt-end ligation to phosphorylated Eco RI linkers using T4 DNA ligase (Pharmacia). The double-stranded S. DNA was digested with Eco RI enzyme, separated from excess linkers by passage through a gel filtration column (Sepharose CL-4B), and ligated to lambda ZAP 20 vector (Stratagene) according to manufacturer's instructions. Ligated DNA was packaged into phage using the Gigapack packaging extract (Stratagene) according to manufacturer's instructions. The resultant cDNA library was amplified as per Stratagene's instructions and stored at Following the instructions in the Lambda ZAP Cloning Kit Manual (Stratagene), the cDNA phage library was used to infect E. li BB4 cells and approximately 600,000 plaque forming units were plated onto 150 mm diameter petri plates. Duplicate lifts of the plates were made onto nitrocellulose filters (Schleicher Schuell). The filters were prehybridized in 25 mL of hybridization buffer consisting of 6X SSPE, Denhardt's solution, 0.5% SDS, 5% dextran sulfate and 0.1 mg/mL denatured salmon sperm DNA (Sigma Chemical WO094/11516 PCr/U93/09987 Co.) at 50 0 *C for 2 h. Radiolabelled probe prepared from pSF2b as described a~bove was added, .and allowed to hybridize for 18 h at 500C. The filters were washed exactly as described above. Autoradiography of the filters indicated that there were 14 strongly hybridizing plaques. The 14 plaques were subjected to a second round of screening as before. Numerous, strongly hybridizing plaques were observed on 6 of the 14 filters, and one, well-isolated from other phage, was picked from each of the six plates for further analysis.

Following the.Lambda Z.AP Cloning Kit Instruction Manual (Stratagene), sequences of the pBluescript vector, including the cDNA inserts, from the purified phages were excised in the -presence of a helper phage 15 and the resultant phagemids were used to infect X. r&U

)M-

1 Blue cells. DNA from the plasmids was made. by the Promega "Magic Miniprep" according to the mufacturers instructions. Restriction analysis indicated that the plasmids contained inserts ranging in size from 1 kb to 2.5 kb. The alkali-denatured double-stranded DNA -from one of these, designated pSF2-169K contained an insert of 1.6 kb, was sequenced as described above. The nucleotide sequence of the cD?4A insert in plasmid pSF2-169K shown in SEQ ID Clnn f a Carn I .rAMnvR) rMDNA E~ncoding Statd MierngtomalnI ety-12 Waty Ac~id Dgmaturnme.

Corn microsomal delta-12 desaturase cDNA was isolated using a PCR approach. For this, a cDNA library was made to poly A+ RNA from developing corn embryos in Lambda ZAP 11 vector (Stratagene). 5-10 ul of this library was used as a template for PCR using 100 pmol each of two sets of degenerate oligomers NS3 (SEQ ID NO:13) and equimolar amounts of Rfl5a/b (that is, equimolar amounts of SEQ ID NOS:16/17) as sense and 11 WO 94/11516 PCT/US93/09987 76 antisense primers, respectively. NS3 and correspond to stretches of amino acids 101-109 and 318-326, respectively, of SEQ ID NO:2, which are conserved in most microsomal delta-12 desaturases

(SEQ

ID NOS:2, 4, 6, PCR was carried out using the PCR kit (Perkin-Elmer) using 40 cycles of 940C 1 min, 1 min, and 55oC, 2 min. Analyses of the PCR products on an agarose gel showed the presence of a product of the expected size (720 bp), which was absent in control reactions containing either the sense or antisense primers alone. The PCR product fragment was gel purified and then used as a probe for screening the same S"corn cDNA library-at 60°C as described above. One Positively-hybridizing plaque was purified and partial 15 sequence determination of its cDNA showed it to be a nucleotide sequence encoding microsomal delta-12 desaturase but truncated at the 3' end. The cDNA insert encoding the partial desaturase was gel isolated and *used to probe the corn cDNA library again. Several 20 positive plaques were recovered and characterized.

DNA

sequence analysis revealed that all of these clones seem to represent the same sequence with the different length of 5' or 3' ends. The clone.containing the longest insert, designated pFad2#l, was sequenced completely.

SEQ ID NO:7 shows the 5' to 3' nucleotide sequence of 1790 base pairs of corn (Za naxa) cDNA which encodes microsomal delta-12 desaturase in plasmid pFad2l.

Nucleotides 165 to 167 and nucleotides 1326 to 1328 are, respectively, the putative initiation codon and the termination codon of the open reading frame (nucleotides 164 to 1328). SEQ ID NO:8 is the 387 amino acid protein sequence deduced from the open reading frame (nucleotides 164 to 1328) in SEQ ID NO:7. The deduced amino acid sequence of the polypeptide shared overall identities of 71%, 40%, and 38% to Arabidopsis WO94/11516 PCr/US93/0997 77 microsomal delta-12 desaturase,.Arabidosis microsomal desaturase, and Arabidosis plastid desaturase, respectively. Furthermore, it lacked an N-terminal amino acid extension that would indicate it is a plastid.enzyme. Based on these considerations, it is concluded that it encodes a microsomal delta-12 *desaturase.

Cloning of a cDNA _Encoding A Micrsomal Delta-12 Desaturae and of eDNAs Encoding Nierosnomal Delta-12 Desaturase-Related Enzymesa from Castor Bean Seed Castor microsomal delta-12 desaturase cDNA was isolated using a RT-PCR approach. Polysomal FRNA was isolated from castor beans of stages I-Il (5-10 DAP) and also from castor beans of stages IV-V (20-25 DAP).

15 Ten ng of each RNA was used for separate RT-PCR reactions, using the Perkin-Elmer RT-PCR kit with the reagent concentration as recommended by the kit protocol. The reverse transcriptase reaction was primed with random hexamers and the PCR reaction with 100 pmol each of the degenerate delta-12 desaturase primers NS3 and NS9 (SEQ ID NOS:13 and 14, respectively). The reverse transcriptase reaction was incubated at 250C for 10 min, 42*C for 15 min, 99*C for 5 min and 50 for min. The PCR reaction was incubated at 950C for 2 min followed by 35 cycles of 95*C for 1 min/50C for 1 umin.

A final incubation at 60*C for 7 ain completed the reaction. A DNA fragment of 720 bp was amplified from both stage I-11 and stage IV-V aRMA. The amplified DNA fragment from one of the reactions was gel purified and cloned into a pGEM-T vector using the Promela pGEN-T PCR cloning kit to create the plasmid pRF2-1C. The 720 bp insert in pRF2-1C was sequenced, as described above, and the resulting DNA sequence is shown in SEQ ID NO:9. The DNA sequence in SEQ ID NO:9 contains an open-reading frame encoding 219 amino acids (SEQ ID NO:10), which has WO 94/11516 PCF/US93/09987 78 81% identity (90% similarity) with amino acids 135 to 353 of the Arabidopois microsomal delta-12 desaturase described in SEQ ID NO:2. The cDNA insert in pRF2-1C is therefore a 673 bp fragment of a full-length cDNA encoding a castor bean seed microsomal delta-12 desaturase. The full length castor bean seed microsomal delta-12 desaturase cDNA may isolated by screening a castor seed cDNA library, at 60°C, with the labeled insert of pRF2-1C as described in the example above.

The insert in pRF2-1C may also be used to screen castor bean libraries at lower temperatures to isolate delta-12 desaturase related sequences, such as the delta-12 hydroxylase.

A cDNA library made to poly A+ aRNA isolated from 15 developing castor beans (stages IV-V, 20-25 DAP) was screened as described above. Radiolabeled probe prepared from pSF2b or pRF2-1C, as described above, were added, and allowed to hybridize for 18 h at 500C. The filters were washed as described above. Autoradiography 20 of the filters indicated that there were numerous hybridizing plaques, which appeared either strongly Shybridising or weakly hybridising. Three of the strongly hybridisng plaques (190A-41, 190A-42 and 190A-44) and three of the weakly hybridising plaques, (190B-41, 190b-43 and 197c-42), were plaque purified using the methods described above. The cDNA insert size of the purified phages were determined by PCR amplication of the insert using phage as template and lambda-gtll oligomers (Clontech lambda-gtll Amplimers) for primers. The PCR-amplified inserts of the amplified phages were subcloned into pBluescript (Pharmacia) which had been cut with Eco RI and filled in with Klenow (Sambrook et al. (Molecular Cloning, A Laboratory Approach, 2nd. ed. (1989) Cold Spring Harbor Laboratory Press). The resulting plasmids were called pRF190a-41, WO094/11516 PCr/US93/09987 79 pRFl9Oa-42, pRF19Oa-44, pRFl9Ob-4l, pIFl9Ob-43 and pRF197c-42. All of the inserts were about 1.1 kb with the exception of pRFl97c-42 which was approx. 1.5 kb.

The inserts in the plasmids were sequenced as described above. -The insert in pRFl9Ob-43 did not contain any open reading fram and was not identified. The inserts in pRF190a-41, pRFl9Oa-42, pRFl9Oa-44 and pRF19Ob-41 were identical. The insert in pRFl97c-42 contained all of the nucleotides of the inserts in pRF19Oa-41, pRF19Oa-42, pIRF190a-44 and p3RF19Ob-41 plus an additional approx. 400 bp. It was deduced therefore that the insert in pR1F97c-42 was a longer version of the inserts in pRFl9Oa-41, .pRFl9Oa-42, pRFl9Oa-44 and pRF19Ob-41 and all-.were derived from the same full-length uRMA. The complete eDNA sequence of the insert in plasmid p1F197c-42 is shown in SEQ ID 14:11. The deduced amino acid sequence of SEQ ID 140:11, shown in SEQ ID N0:12,'is 78.5% identical (90% similarity) to the castor mticrosomal delta-12 desaturase described above (SEQ ID 140:10) and 66% identical (80% similarity) to the ArAbI4d.sRIR delta-12 desaturase amino acid sequence in SEQ ID 140:2. These similarities confirm that pRFl97c-42is a castor. bean seed eDNA that. encodes a microsomal delta-12 desaturase or a microsomal dilta-12 desaturaserelated enzyme, such as a delta-12.hydroxylAse.

Specific PCR primers for pRF2-lC and pRFl97c-42 were made. For pRF2-1c the upstream primer was bases 180 to 197 of the eDNA sequence in SEQ ID 140:9. For pPFl97c-42 the Upstream primer was bases 717 to 743 of the cDNA sequence in SEQ ID NO:11. A common downstream primer was made corresponding to the exact complement of the nucleotides 463 to 478 of the sequence described in SEQ ID 14:9. Using RT-PCR with random hexamers and the above. primers, and the incubation temperatures described above, it was observed that mRNA which gave rise to the WO 94/11516 PCrIUS93/O9997 cDNA contained In pRF2-lC is present in both Stage I-11 and *Stage IV-v castor bean seeds vhereas mRNA which gave rise to the CDNA contained in plasmid pRF197c-42 is present only in Stage IV-V castor bean seeds, it is only expressed in tissue actively synthesizing ricinoleic acid. Thus it is possible that this cDxA encodes a delta-12 hydroxylase.

Clones such as pRE-2-lC and pRF197a-42, and other clones from the differential screening, which, based on their DNA sequence, are less related to castor bean seed microsomal delta-12 desaturases and are not any of the known fatty-acid desaturases described above or in WO 9311245, may be expressed, for example,. in soybean embryos or another suitable plant tissue, or in a **15 microorganism, such as yeast, which does not normally contain ricinoleic acid, using suitable expression' '.vectors and transformation protocols. The presence of novel ricinoleic acid in the transformed tissue (s) Sefxpressing the castor cDNA would confirm the identity of.

the castor cDNA as DNA encoding for an oleate hydroxylase.

M£AMP1LA.4 9.9.

99...9 GENONIC- C1-N A"FiRET IT was M_(-ET RNT POLYMORPUTSN IHFIrP INHR ON. TME.ELA1 DEATUfARR- IfU1~T RRIDPAT .The gene encoding Ahkia~aaU microsomal delta-12 desaturase was Used to -map the genetic locus encoding the microsomal1 delta-12 desaturase of Apabidapis tbA 14a~, pSF2b cDkiA insert encoding ArAbhdgjCRj, microsomal delta-12 desaturase DNA was radiolabeled and used to screen an Arhidpgi genomic DNA library. DNA from several pure strongly-hybridizing phages *was isolated. Southern blot analysis of the DNA from different phages using radiolabeled pSF2b cDNA insert as WO 94/11516 PCT/US93/09987 81 the probe identified a 6 kb Hind III insert fragment to contain the coding region of the gene. This fragment was subcloned in pBluescript vector to result in plasmid pAGF2-6 and used for partial sequence determination.

This sequence (SEQ ID NO:15) confirmed that it is the microsomal delta-12 desaturase gene. DNA from two phages was isolated and labelled with 3 2 p using a random priming kit from Pharmacia under conditions recommended by the manufacturer. The radioactive DNA was used to probe a Southern blot containing genomic DNA from Araldopaian thaliana (ecotype Wassileskija and marker line W100 .ecotype Landesberg background) digested with one of several restriction endonucleases. Following hybridization and washes under standard conditions 15 (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press), autoradiograms were obtained. A different pattern of hybridization (polymorphism) was identified in Hind III-digested genomic DNAs using one of the phage 20 DNAs.. This polymorphism was located to a 7 kB Hind III fragment in the phage DNA that revealed the polymorphism. The 7 kb fragment was subcloned in pBluescript vector to result in plasmid pAGF2-7.

Plasmid pAGF2-7 was restricted with Hind III enzyme and used as a radiolabelled probe to map the polymorphism essentially as described by Helentjaris et al., (Theor.

Appl. Genet. (1986) 72:761-769). The radiolabelled DNA fragment was applied as described above to Southern blots of Hind III-digested genomic DNA isolated from 117 recombinant inbred progeny (derived from single-seed descent lines to the F6 generation) resulting from a cross between Arabidopsis thaliana marker line W100 and ecotype Wassileskija (Burr et al., Genetics (1988) 118:519-526). The bands on the autoradiograms were interpreted as resulting from inheritance of either WO094/11516 PCI'1US93/09987 82 paternal (ecotype Wassileskija) or maternal (marker line W100) DNA or both heterozygote) The resulting segregation data were subjected to genetic analysis using the computer program Mapmaker (Lander et* al., Genomics (1987) 2:174-181). In conjunction with previously obtained segregation data for .63 anonymous RFLP markers and 9 morphological markers in Ahidaasia ±thnlja (Chang et al., Proc. Natl. Aced. Sci. USA (1988) 85:6856-6860; N1am et al., Plant Cell (1989) 1:699-705). a single genetic locus was positioned corresponding to the microsomal delta-12 desaturase gene. The location of the microsomal delta-12 desaturase gene was thus determined to be 13.6*cM proximal to locus c3838, 9.2 cM distal to locus 1At228, 15 and 4.*9 cM proximal to FadD locus on chromosome 3 Koo rneef, M. et. al. (1993) in Genetic Maps, -Ed.

O'Brien, S. Yadav et al. (1993) Plant Physiology .1fl3:467-476.1 .20 UE OF nYRAN HTICRQR0MAT, DFLTA-12 nESATURASP nDNA LENMTH POLYMORPHISMIFL

MRE

*The 1.6 kb insert obtained from the plasmid pSF2-169K as previously described was radiolabelled with 32 p Using a Random Priming Kit from Bethesda Research Laboratories under conditions recommnended by the manufacturer. The resulting radioactive probe was used to probe a Southern blot (Sambrook'et al., Molecular .Cloning: A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press) containing genomic DNA from soybean (Glyc±nc a= (cultivar Bonus) and Glyclue AQJ~A (P181762)) digested with one of several restriction enzymes. After hybridization and washes under low stringency conditions (50 mM Tris, pH 7.5, 6X SSPE, dextran sulfate, 1% SDS at 56*C for the hybridization

I'

"S

WO 94/11516 PCI'/US93109997 83 and initial washes, cha.nging to 2X SSPE and 0.1% SDS for the final wash), autoradiograms were obtained, and different patterns of hybridization (polymorphisms) were identified in digests performed with restriction enzymes.

Hind III and Eco RI'. These polymorphisms were used to map two pSF2-169k loci relative to other loci on the soybean genome essentially aLs described by Heleniaris et al., (Theor. Appl. Genet. (1986) 72:761-769). The.

map positions of the polymorphismcs were determined to be in linkage group 11 between 4404.00 and 1503.00 loci cM and 7.1 cM from 4404.00 and*1503.00, respectively) and linkage group 19 between 4010.00 and 5302.00 loci (1.9 cM and 2.7 cM from 4010.00 and 5302.00, respectively) [Rafalski,-A. and Tingey, S.

(1993) in Genetic Maps, Ed. O'Brien, S. J.3.

EXPRSSION OF MIRSMLDLTA-12 DRSATTJRAS IN ROYBRANS Construntio n of- Vectors for' Tafo ation of Glycine- max for Reduded Expressio4n of Minrostomal Delta-12 Desaturases in fleveloning SoybeaSed Plasmids containing the antisense fi. Max microsomal delta-12 desaturase cDNA sequence under control of the soybean Kunitz Trypsin Inhibitor 3 (KT13) promoter '(Jofuku and Goldberg, Plant Cell (1989) 1:1079-1093) the Ph agnbn ,cIggaris 7S seed storage protein (phaseolin) promoter (Sengupta-Gopalan et al.j Proc.

Natl. Acad. Sci. USA (1985) 82:3320-3324; Hoffman et al., Plant Mol. Biol. (1988) 11:717-729) and soybean beta-conglycinin promoter (Beachy et al., EMBO J. (1985) 4:3047-3053), were constructed. The construction of vectors expressing the soybean delta-12 desaturase antisense cDNA under the control of these promoters was facilitated by the use of the following plasmids: pML70, pCW108 and pCW109A.

WO094/1 IS16 -PCT/US93/09M8 84 The pML7O vector contains the KT13 promnoter and the KT13 3' untranslated region and was derived from the commercially available vector pTZ18R (Pharmacia) via the intermediate plasmids pHL51, pI4L55, pML64 and pHL65.

A

2.4 kb Bat Dh/Eco RX fragment of the comlete soybean KTi3 gene (Jofuku and Goldberg (1989) Plant Cell 1:1079-1093), which contains *all 2039 nucleotides of the untranslated region and 390 bases of the coding sequence of the KTi3 gene ending dt the Eco RI site corresponding to bases 755 to 761 of the sequence described in Jofuku et al (1989) Plaht Cell 1:427-435, was ligated into the Acc IfEco, RI sites of pTZ18R to create the plsi pNLSl. Th plasmid M5 was cut with Nco 1, filled in using Klenow, and religated, to destroy an Nco I site in the middle of the untranslated regi6n of the KTi3 insert, resulting in the plasaid pHL55. The plasmid pML55 was partially digested with Xnr 1/Eco RI to release a 0.42 kb fragment, corresponding to bases 732 to 755 of the above cited sequence, which-was discarded. A synthetic Xmn I/Eco

RI

linker containing an Nco I site, was constructed by making a dimer of complementary synthetic oligoflucleotides consisting of the coding sequence for an Xmn I site (5'-TCTTCC-31) and an Sco I site (51-CCATGGG-3') followed directly by part of an Eco, RI site (5-GAAGG-31). The Xmn I and Nco I/Eco RI sites were linked by a short intervening sequence -ATAGCCCCCCA..39). This synthetic linker was ligated into the Xmn 1/Eco RI sites of the 4.94 kb fragment to create -the plasmid pIL64. The 3' untranslated region of the XT13 gene was amplified from the sequence described.

in Jofuku et al (ibid.) by standard PCR protocols (Perkin Elmer Cetus., GeneAmp PCR kit) using the primers ML51 and ML52. Primer ML51 contained the 20 nucleotides corresponding to bases 1072 to 1091 of the above cited 'p WO 94/1 1516 PCIV/US93/0998 sequence with the addition of nucleotides corresponding to Eco RV (5-'GATATC-3'), Nco, I (5'rCCATGG-3'), Xba I (5'-TCTAGA-3'), Sma I (5'-CCCGGG-3') and Kpn I (5'-G7GTACC-39) sites at the 5' end of the primer.

Primer ML52 contained to the exact copupliment of the nucleotides corresponding to bases 1242 to 1259 of the above cited sequence with the addition of nucleotides corresponding to Sma I -CCCGGG-3'), Eco RI (5'-GAATTC-3'), Barn HI (5'-GGACC-3') and Sal I (5 ,-GTCGAC-3') sites at the 5' end of the primer. The PCR-amplified 3' end of the KT13 gene was ligated into the lico I/Eco RI sites of pML64 to create the plasmid piL 65. A synthetic multiple cloning site linker was constructed by making a dimer of complementary synthetic oligonucleotides consisting of the coding sequence for Pst I (5'-CTGCA-3'), Sal I (5'-GTCGAC-3'), Barn HI (S'-GGATCC-3') and Pst I (51-CTGCA-3') sites. The linker was ligated -into the Pst I site (directly 5' to the KT13 promoter region) of pML65 to create the plasmid o 20 pML7O.

o The 1.46 kb Sma I/Kpn I fragment from pSF2-169K (soybean delta-12 desaturase cDNA described above) was ligated into the corresponding sites in pliL7O resulting *in the plasmid pBS1O. The desaturase cONA fragment was in the reverse (antisense) orientation with respect to the KT13 promoter in pBS1O. The plasmid pBS1O was 0000*digested with Barn HI and a 3.47 kb fragment, representing the KT13 promoter/antisense desaturase CDNA/KT3-31 end transcriptional unit was isolated by agarose gel electrophoresis. The vector pliL18 consists of the non-tissue specific and constitutive cauliflower mosaic virus (35S) promoter (Odell et al., Nature (1985) 313:810-812; Hull et al., Virology (1987) 86:482-493), driving expression of the neomycin phosphotrans fe rase gene described in (Beck et al. (1982) Gene 19:327-336) WO 94/11516 PC/US93/09987 86 followed by the 3' end of the nopaline synthase gene including nucleotides 848 to 1550 described by (Depicker et al. (1982) J. Appl. Genet. 1:561-574). This transcriptional unit was inserted into the commercial cloning vector pGEM9Z (Gibco-BRL) and is flanked at the end of the 35S promoter by the restriction sites Sal I, Xba I, Bam HI and Sma I in that order. An additional Sal I site is present at the 3' end of the NOS 3' sequence and the Xba I, Bam HI and Sal I sites are uhique. The 3.47 kb transcriptional unit released from pBS10 was ligated into the Bam HI site of the vector pML18. When the resulting plasmids were double digested with Sma.I and Kpn I, plasmids containing inserts in the desired orientation yielded 3 fragments 15 of 5.74, 2.69 and 1.46 kb. A plasmid with the transcriptional unit in the correct orientation was selected and was designated pBS13.

The pCW108 vector contains the bean phaseolin promoter and 3' untranslated region and was derived from 20 the commercially available pUC18 plasmid (Gibco-BRL) via plasmids AS3 and pCW104. Plasmid AS3 contains 495 base pairs of the bean (Phaseolus vulgaris) phaseolin (7S seed storage protein) promoter starting with 5'-sTGGT TGGT-3' followed by the entire 1175 base pairs of the 3' untranslated-region of the same gene (see sequence descriptions in Doyle et al., (1986) SJ. Biol. Chem. 261:9228-9238 and Slightom et al., (1983) Proc. Natl. Acad. Sci. USA, 80:1897-1901. Further sequence description may be found in NO 9113993) cloned into the Hind III site of pUC18. The additional cloning sites of the pUC18 multiple cloning region (Eco RI, Sph I, Pst I and Sal I) were removed by digesting with Eco RI and Sal I, filling in the ends with Klenow and religating to yield the plasmid pCW104. A new multiple cloning site was created between the 495bp of the WO 94/11516 WO 94/11516PCr/L1s93/097 87 phaseolin and the 1175bp of the 3' phaseolin by inserting a dimer of complementary synthetic oligonucleotides consisting of the coding sequence for a lico I site (5'-CCATGG-3') followed by three filler bases (50-TAG-31), the coding sequence for a Sa I site (51-CCCGGG-3), the last three bases of a Kpn I site -TAC-3 a cytosine and the coding sequence for an Xba I site (51-TCTAGA-3') to create the plasmid pCNlO8.

This plasmid contains unique Nco 1, Sma 1, Kpn I and Xba I sites directly behind the phaseolin promoter. The 1.4 kb Eco RV/Sma I fragment from pSF2-169K was ligated :into the Sa I site of the comercial ly available phagemid pBC SIC. (Stratagene). A phagemid with the cDNA in the desired orientation was selected by digesting 15 with Pfl HI/Xho I to yield fragments of approx. 1*kb and 4 kb and designated pHl-SF2. The 1.4 kb Xinn I/Xba I fragment from pK1-SF2 was inserted into the Sa I/Xba I Bites of pCWlO8 to yield the plasmid pBSil, which has the soybean delta-12 desaturase cDNA in the reverse 20 (31-51) orientation behind the phaseolin promoter. The plasmid pBSl1 was digestqd with Earn HI and a 3.07 kb fragment, representing the phaseolin promoter/antisensedesaturase cDNA/phaseolin 31 end transcriptional unit was isolated by agarose gel electrophoresis anld ligated into the Mind III site of pliL1S (described- above). Wihen the resulting plasmids were digested with Xba I, plasmids containing inserts in the Oestxed orientation yielded 2 fragments of 8.01 and 1.18 kb. A plasmid with the transcriptional unit in the correct orientation was selected and was designated pRSl4.

The vector pCW109A contains the soybean b-conglycinin promoter sequence and the phaseolin 3' untranslated region and is a modified version of vector pCW1O9 which was derived from the commercially available plasmid pUCiB (Gibco-BRL). .The vector pCW109 was made .1 WO 94/11516 PCr/US93/09987 88 by inserting int6 the Bind III site of the cloning.

vector pUC18 a 555" bp 5' non-coding region (containing the promoter region) of the b-conglycinin gene followed by the multiple cloning sequence containing the restriction endonuclease sites for Nco I, Sma I, Kpn I and Xba I, as described for pCW08 above, then 1174 bp of the common bean phaseolin 3' untranslated region into the Hind III site (described above). The b-conglycinin promoter region used is an allele of the published b-conglycinin gene (Doyle et ai., J. Biol. Chem. (1986) 261:9228-9238) due to differences at 27 nucleotide positions. Further sequence description of this gene may be found in Slightom (WO 9113993). To facilitate use in antisense constructions, the Nco I site and 15 potential translation start site in the plasmid pCW109 was destroyed by digestion with Nco I, mung bean exonuclease digestion and re-ligation of the blunt site to give the modified plasmid pCW19OA. The plasmid pCW109A was digested with Hind III and the resulting 1.84 kb fragment, which contained the b-conglycinin/ antisense delta-12 desaturase cDNA/phaseolin 3' untranslated region, was gel isolated. The plasmid pML18 (described above) was digested with Xba I, filled in using Klenow and religated, in order to remove the Xba I site. The resulting plasmid was designated pBS16.

The 1.84 kb fragment of plasmid pCW109A (described above) was ligated into the Hind III site of pBS16. A plasmid containing the insert in the desired orientation yielded a 3.53 kb and 4.41 kb fragment when digested with Kpn I and this plasmid was designated pCST2. The Xmn I/Xba I fragment of pML1-SF2 (described above) was ligated into the Sma I/Xba I sites of pCST2 to yield the vector pST11.

I'

WO 94/11516 PCT/US93/09987 89 Transformation Of Somatic Soybean Rmbryo Cultures and Regeneration Of Soybean Plants Soybean embryogenic suspension cultures were maintained in 35 mL liquid media (SB55 or SBP6) on a rotary shaker, 150 rpm, at 28°C with mixed florescent and incandescent lights on a 16:8 h day/night schedule.

Cultures were subcultured every four weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

Soybean embryogenic suspension cultures were transformed with pCS3FdSTlR by the method of particle gun bombardment (see Kline et al. (1987) Nature (London) S• 327:70). A DuPont Biolistic PDS1000/E instrument (helium retrofit) was used for these transformations.

15 To 50 mL of a 60 mg/mL 1 am gold particle suspension was added (in order); 5 uL DNA(1 ug/uL), 20 uL spermidine (0.1 and 50 ul CaCl2 (2.5 The particle preparation was agitated for 3 min, spun in a microfuge for 10 sec and the supernatant removed. The DNA-coated particles were then washed once in 400 uL ethanol and re suspended in 40 uL of anhydrous ethanol.

The DNA/particle suspension was sonicated three times for 1 sec each. Five uL of the DNA-coated gold particles were then loaded on each macro carrier disk.

Approximately 300-400 mg of a four week old suspension culture was placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately.5-10 plates of tissue were normally bombarded. Membrane rupture pressure was set at 1000 psi and the chamber was evacuated to a vacuum of 28 inches of mercury. The tissue was placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the n i WO 94/11516 PCT/US93/09987 tissue was placed back into liquid and cultured as described above.

Eleven days post bombardment, the liquid media was exchanged with fresh SB55 containing 50 mg/mL hygromycin. The selective media was refreshed weekly.

Seven weeks post bombardment, green, transformed tissue was observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue was removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Thus each new line was treated as independent transformation event. These suspensions can then be maintained as suspensions of embryos clustered in an immature developmental stage through subculture or 15 regenerated into whole plants by maturation and germination of individual somatic embryos.

Transformed embryogenic clusters were removed from liquid culture and placed on a solid agar media (SB103) containing no hormones or antibiotics. Embryos were cultured for eight weeks at 26°C with mixed florescent and incandescent lights on a 16:8 h day/night schedule.

During this period, individual embryos were removed from the clusters and analyzed at various stages of embryo development After eight weeks somatic embryos become suitable for germination. For germination,, eight week old embryos were removed from the maturation medium and dried in empty petri dishes for 1 to 5 days. The dried embryos were then planted in SB71-1 medium were they were allowed to germinate under the same lighting and germination conditions described above. Germinated embryos were transferred to sterile soil and grown to maturity for seed collection.

II

WO 94/11516.. PCFIUS93/0"997 Media: and SBP6 Stock Solutions mS Sulfate lOOX Stock MSo4 720 37.0 MnSO4 520 1.69 ZnSo4 7520 0.86 CuSO4 5H20 0.0025 MS Halides lOOX Stock CaCl2 2H20 44.0 KI 0.083 COC12 6H20 0.00125 IK(2P04 17.0 B3B0 3 0.62 Na2MoO4 2520 0.025 MS FeEDTA 1OOX Stock Na2EDTA 3.724 FeSO 4 7220 2.784 0 0 0@ 0 0@S 0* 0e S 0 5 0

OSOO

SS

S

0 6*@S

S.

S. 5

OSOS

5 0 .5.5 B5 Vitamin Stock 10 g m-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine SB55 (per Liter) 10 uL each HS. stocks 1 mL B5 Vitamin stock 0.8 g 1154W03 3.033 g XN03 1 mL 2,4-D (10mg/mL stock) 60 g sucrose 0.667 q asparagine PH 5.7 For SBP6- substitute 0.5 ML 2,4-D SB103 (per Liter) 1S Salts m6% altose 750 mg MgC12 0.2% Gelrite pH 5.7 SB71-l (per liter) salts Iml B5 vitamin stock 3% sucrose MgC12 0.2% gelrite pH 5.7

I,

WO 94/11516 PCr/US931097 92 Analysis Of Transgenie Glyc4ng Mnx Emtnryo xnd Reda Contairnng An Antisensek Delta-iS nDesatturAme* Dbmnsgtration ThAt Theh phenotype QfD Trnnnaggnie, Snvbean Snmatie Pmbrilem TA predjietjve'Of Theu phfknoyeO e Dstrivgd From Plants Reserte Po Thoem mhry While in the globular embryo state in liquid culture as described above, somatic soybean embryos contain very low amounts of triacyiglycerol or storage proteins typical of maturing, zygotic soybean embryos.

At this developmental stage, the ratio of total triacylglyceride to total polar lipid (phospholipids and glycolipid) is about 1:4, as is typical of 'zygotic soybean embryos at the developmental stage f rom which the somatic embryo culture was initiated. At the globular stage as well, the mRNAs for the prominent seed *proteins (alpha Isubunit of beta-conglycinin, Kunitz Trypsin Inhibitor 3 and Soybean Seed Lectin) are essentially absent. Upon transfer to hormone free media to allow differentiation to the matu ring somatic embryo state as described above, triacyiglycerol becomes the .*.most abundant lipid class. As well, mRNAs f or alpha'subunit of beta-conglycinin, Kunitz Trypsin Inhibitor 3.

and Soybean Seed Lectin become very abundant messages in the total mRNA population. in these respects the somatic soybean embryo system behaves very -similarly to maturing zygotic soybean embryos in xi1=, and is therefore. a good and rapid model system for analyzing the phenotypic effects of modifying the expression of genes in the fatty acid biosynthesis pathway.

Furthermore, the model system is predictive of the fatty acid composition of seeds from plants derived from transgenic embryos. Liquid culture globular embryos transformed with a vector containing a soybean microsomal delta-15 desaturase, in a reverse orientation and under the control of soybean conglycinin promoter WO94/11516. PCFrUS93/O97 93 (pCS3FdST 1R), gave rise to mature embryos with a reduced 18:3 content (WO 9311245). A number of embryos from line A2872 (control tissue transformed with pCST) and from lines 299/1/3, 299/15/1, 303/7/1, 306/3/1, 306/4/3, 306/4/5 (line 2872 transformed with plasmid pCS3FdSTlR) were analyzed for fatty acid content. Fatty acid analysis was performed as described in No0 9311245 using single embryos as the tissue source. Mature, somatic embryos from each of these lines were also regenerated into soybean plants by transfer to.

regeneration medium as described abovie. A number of seeds taken from plants-regenerated from these embryo lines were analyzed for fatty acid content. The relative fatty-acid composition of embryos taken from tissue transformed with pCS3FdSTlR was compared with relative fatty-acid composition of seeds taken from* plants derived from embryos transformed vith.pCS3FdSTIR.

Also, relative fatty acid compositions of embryos and seeds transformed with pCS3FdSTlR were compared with control tissue,. transformed with pCST* In all cases where a reduced 18:3 content vas seen in a transgenic embryo line, compared with the control, a reduced 18:3 content was also observed in segregating seeds of. plants derived from that l'ine, when compared with the control seed (Table 11).

Antisense Delta-15 Desatuzse: PelatLve 18:3 Content Of Emryos And Seeds Of Control Soybean Line Emryo Fnbzya Seed Seed av.%1S:3 lowest 118:3 av.1I:3* lowest 1:3 A2872 12.1 6.5 8.9 (control) 299/l/3 5.6 4.5 4.3 299/15/1 8.9 5.2 2.5 1.4 4 WO 94/11516 PCT/US93/09987 94 303/7/1 7.3 5.9 4.9 2.8 306/3/1 7.0 5.3 2.4 1.3 306/4/3 -8.5 6.4 4.5 2.7 306/4/5 7.6 5.6 4.6 2.7 *Seeds which were segregating with wild-type phenotype and without a copy of the tranagene are not included in these averages. The number in brackets is Thus the Applicants conclude that an altered polyunsaturated fatty acid phenotype observed in a transgenic, mature somatic embryo line is predictive of an altered fatty acid composition of seeds of plants derived from that line.

Analyvsi Of Trananeni4 Glvinet Ma* Emhrvos Cntainina An Antifansge W onm1 DIel-19*DPRfsatrae Cor.nlrr The vectors pBS13, pBS14 and pSTll contain the 10 soybean microsomal delta-12 desaturase cDNA, in the antisense orientation, under the control of the soybean Kunitz Trypsin Inhibitor 3 (KTi3), Phaeolun phaseolin, and soybean beta-conglycinin promoters as described above. Liquid culture globular embryos transformed with 15 vectors pBS13, pBS14 and pSTll, gave rise to mature embryo lines as described above. Fatty acid analysis was performed as described in WO 9311245 using single, mature embryos as'the tissue source. A number of embryos from line A2872 (control tissue transformed with pCST) and from line A2872 transformed with vectors pBS13, pBS14 and pST11 were analyzed for fatty acid content. About 30% of the transformed lines showed an increased 18:1 content when compared with control lines transformed with pCST described above, demonstrating that the delta-12 desaturase had been inhibited in these lines. The remaining transformed lines showed relative fatty acid compositions similar to those of the control line. The relative 18:1 content of the lines showing an increased 18:1 content was as high as 50% compared with 1, WO 94/11516 PCrVUS93/09987 a maximum of 12.5% in the control embryo lines. The average 18:1 content of embryo lines which shoved an increased 18:1 content was about 35t (Table 11). In all the lines shoving an increased 18:1 content there was a proportional decrease in the relative 18:2 content (Table 12). Therelative proportions of the other major fatty acids (16:0, 18:0 and 18:3) were similar to those of the control.

zABLrL1Z Summary Of Experiment in Which Soybean Mmhbyoa Were Transf ormed With Plasuids Containing A Soybean Antisense M4~emn D.t~-1 Dg*~step~fnM 0 of lines of with high highest av. (1) *pCST -12.5 10.5 (control) **pBS23 11 4 53.5 35.9 pBS14 11 2 48.7 32.6 *pST11 11 3 50.1 35.9 ~In Table 12 the average 18:1 of transgenics is the average of all embryos transformed with a particular :..vector whose relative 18:1 content is greater than two standard deviations from the highest control value The control average is the average* of ten A2872 embryos (standard deviation The data in Table 12 are derived from Table 13 below.

WO 94/1 1516 PCr/US93/09987 96 TABLE 11 Relative Fatty Acid Contents Of Ebryo Lines Transformed with Plasmids Containing

A

Soybean~ AxntiRne flltn- Desatuarap cDNA Embryo Line A2872 (control) Relative Fatty-Acid Content 2 3 4 7 8 9 10 G335/4 /197 1 2 3 G335/4/221 1 2 3 4 6 7 8 9 16:0 11.7 16.4 17.1 15.3 15.2 18.6 14.6 14.2 15.2 19.0 (PBS13) 16:0 12.2 12..4 12.0 CpBS13) 16:0 12.2 11.5 13.0 12.0 11.7 12.0 12.0 11.7 18: 0 3.2 4.0 3.4 2.7 3.6 3.9 3.4 3.5 3.2 3.8 18:0 3.3 2.7 3.2 18: 0 2.7 2.4 2.6 2.6 2.7 3.4 2.5 2.5 2.6 18:1 11.7 10.8 8.3 9.4 10.8 10.9 12.5 11.2 9.8 9.6 18: 1 42.0 22.4 42.0 18:1 30.4 14.3 15.2 27.4 25.1 21.6 11.3 20. 8 25.3 18: 2.

52.7 47.1 48.3 51.1 51.0 45.8 52.3 53.9 49.5 47.4 18:2 23.0 39.0 23.2 18:2 36.0 53.4- 47.4 37.9 42.3 44.3 53.6 44.1 39.6 .18:3 16.1 19.3 20.6 19.0 17.5 18.1 16.4 16.7 16.1 19.0 18:3 17.4 21.9 18.4 18:3 .17.9 17.6 19.9 19.1 15.6 17.8 20.0 19.5 18.3 WO 94/11516 W6PCr/US93/0997 97 G335/8/174 (pBS13) 16:O 18:0 18:1 18:2 18:3 1 14.1 2.1 30.3 32.1 20.3 2 14.7 2.5 5.9 40.6 34.8 3 14.3 2.4 7.3 45.2 29.8 G335/8/202 (pBS13) 16:0 18:0 18:1 18:2 18:3 1 11.7 1.5 30.1 32.4 23.3 2 11.4 2.3 48.5 20.6 16.1 3 12.9 2.3 46.6 17.1 19.5 4 12.7 2.6 32.0 31.1 20.5 12.9 1.9 41.7 23.5 18.9 6 12.3 2.6 40.1 25.6 17.9 7 11.3 2.4 53.5 16.6 14.5 ooo* 8 11.4 2.5 15.5 21.7 17.8 9 10.2 2.0 45.4 23.2 18.5 o 10 12.8 2.2 43.2 23.5 16.9 G335/6/42 (pBS14) 16:0 18:0 18:1 18:2 18:3 1 13.7 2.4 38.6 28.2 15.6 2 12.6 2.3 37.6 28.8 17.2 3 11.7 3.0 48.7 21.1 14.6 G335/6/104 (pBS14) 16:0 18:0 18:1 18:2 18:3 1 13.8 2.5 30.5 35.4 16.0 2 12.3 2.3 14.6 53.2 16.4 3 12.7 2.6 27.1 36.6 20.0 4 12.6 2.2 32.1 34.9 17.4 12.7 2.6 23.2 41.2 19.3 6 12.6 2.2 11.7 52.5 20.1 7 13.3 2.1 23.3 41.2 18.4 G335/1/25 (pST11) 16:0 18:0 18:1 18:2 18:3 1 13.7 2.8 50.7 17.5 12.1 2 14.5 3.0 41.8 23.5 15.0 WO 94/11516 WO 9411516PC/US93/097 98 3 13.9 2.9 49.1 16.8 13.6 4 12.3 2.8 47.5 19.3 14.8 G335/2/7/1 (pST11) 16:0 18:0 18:1 18:2 18:3 1 15.5 4.3 21.8 38.0 17.5 2 17.8 4.1 22.0 39.5 14.0 3 15.2 3.0 20.5 42.2 16.5 G335/2/118 (PST11) 16:0 18:0 18:1 18:2 18:3 1 14.1 .2.7 44.7 22.6 14.0 2 15.8 2.8 37.7 26.9 14.8 3 17.3 3.4 23.3 37.9 16.0 N.B. All other transformed embryos (24 lines) had fatty acid profiles similar to those of the control.

One of these embryo lines,- G335/1/25, had an average 18:2 content of less than 20% and an average 18:1 content greater than 45% (and as high as 53.5%).

The Applicants expect, based on the data in table that seeds derived from plants regenerated from such *lines will have an equivalent or greater increase in 18:1 content and an equivalent or greater increase decrease in 18:2 content.

rxPRrEq~ToN_ oF kmI~o-OM R A12D.A!R IN CANOT.A Conxtrteictin Of Vertn6ri For O'rnns4fnrmxtirn of Brass4ia Nau eo Rduced Expr~inmin of In Devyeloiing C Iit a eads An extended poly-A tail was removed from the canola delta-12 desaturase sequence contained in plasmid pCF2-165D and additional restriction sites for cloning were introduced as follows. A PCR primer was synthesized corresponding to bases 354 through 371 of SEQ ID NO:3. The second PCR primer was synthesized as WO 94/11516 PCT/US93/09987 99 the complement to bases 1253 through 1231 with additional bases (GCAGATATCGCGGCC) added to the 5' end.

The additonal bases encode both an EcoRV site and a NotI site. pCF2-165D was used as the template for PCR amplification using these primers. The 914 base pair product of PCR amplification was digested-with EcoRV and PflMI to give an 812 base pair product corresponding to bases 450.through 1253 of pCF2-165D with the added NotI site.

pCF2-165D was digested with PstI, the PstI overhang was blunted with Klenow fragment and then digested with PflMI. The 3.5 kB fragment corresponding to pBluescript along with the 5' 450 bases of the canola Fad2 CDNA was gel purified and ligated to the above described 812 base 15 pair fragment. The ligation product was amplified by transformation of E. coli and plasmid DNA isolation.

The EcoRI site remaining at the cloning junction between pBluescript and the canola Fad2 cDNA was destroyed by digestion, blunting and religation. The recovered plasmid was called pM2CFd2.

pM2CFd2 was digested with EcoRV and Smal to remove the Fad2 insert as a blunt ended fragment. The fragment.

Swas gel purified and cloned into the Smal site of pBC (Stratagene, La Jolla, CA). A plasmid with the NotI site introduced by PCR oriented away from the existing NotI site in pBC was identified by NotI digestion and ,i gel fractionation of the digests. The resulting -construct then had NotI sites at both ends of the canola Fad2 cDNA fragment and was called pM3CFd2.

Vectors for transformation of the antisense cytoplasmic delta-12 desaturase constructions under control of the 8-conglycinin, Kunitz trypsin inhibitor III, napin and phaseolin promoters into plants using -Agrobacterium tumefaciens were produced by constructing a binary Ti plasmid vector system (Bevan, (1984) Nucl.

IT

WO 94/11516 PC/US93/09987 100 Acids Res. 12:8711-8720). One starting vector for the system, (pZS199) is based on a vector which contains: the chimeric gene nopaline synthase/neomycin phosphotransferase as a selectable marker for transformed plant cells (Brevan et al. (1984) Nature 304: 184-186), the left and right borders of the T-DNA of the Ti plasmid (Brevan et al. (1984) Nucl.

Acids Res. 12:8711-8720), the Z. eoli lacZ a-complementing segment (Vieria and Messing (1982) Gene 19:259-267) with unique restriction endonuclease sites for Eco RI, Kpn I, Bar HI, and Sal I, the bacterial replication origin from the Pseudomonas plasmid pVS1 (Itoh et al. (1984) Plasmid 11:206-220), and the bacterial neomycin phosphotransferase gene from 15 (Berg et al. (1975) Proc. Natnl. Acad. Sci. U.S.A.

72:3628-3632) as a selectable marker for transformed A. tumefa.cins. The nopaline synthase promoter in the plant selectable marker was replaced by the 35S promoter (Odell et al. (1985) Nature, 313:810-813) by a standard 20 restriction endonuclease digestion and ligation strategy. The 35S promoter is required for efficient Brassica napus transformation as described below. A second vector (pZS212) was constructed by reversing the order of restriction sites in the unique site cloning region of pZS199 Canola napin promoter expression cassettes were consturcted as follows: Ten oligonucleotide primers were synthesized based upon the nucleotide sequence of napin lambda clone CGN1-2 published in European Patent Application EP 255378). The oligonucleotide sequences were: BR42 and BR43 corresponding to bases 1132 to 1156 (BR42) and the complement of bases 2248 to 2271 (BR43) of the sequence listed in Figure 2 of EP 255378.

WO94/11516. PCF/US93/09987 101 and BR46 corresponding to bases 1150 to 1170 (DM46) and the complement of bases 2120 to 2155 of the sequence listed in Figure 2 of EP 255378. In addition BR46 had bases corresponding to a Sal I site (5 '-GTCGAC-31) and a fey additional bases .(5'-TCAGGCCT-31) at its 5' end and BR45 had bases corresponding to a Bgl II site (S'-AGATCT-3') and two (51-CT-31) additional bases at the 5' end of the primer, B R47 and BR48 corresponding to bases 2705 to 2723 (BR47) and bases 2643 to 2666 (ER4B) of the sequence listed in Figure 2 of EP 255378. In addition BR47 had two (51-CT-31) additional.bases at the 5' and of the primer followed by-bases corresponding to a Bgl II site (5'-AGATCT-3'.) followed by a few additional bases (5 '-TCAGGCCT-3'), R49 and ERSO corresponding to the complement of bases 3877 to 3897 (BR49) and the complement of bases 3965 to 3919 (ERSO) of the sequence listed in Figure 2 of EP 255378. In addition BR49.bad bases corresponding to a Sal I site (5'-GTCGAC-3') and a-few additional bases (5'-TCAGGCCT-30) at its 5' end, *BR57 and BR58 corresponding to the complement of bases ***3875 to 3888 (BR57) and bases 2700 to 2714 (BR58) of the sequence listed in Figure 2 of EP 255i378. in addition the 5' end of BR57 had some extra bases (5 '-CCATGG-3') followed by bases corresponding to a Sac I site (5'-GAGCTC-31) followed by more additional bases (51-GTCGACGAGG-3'). The 51 end of BR58 had additional bases (5'-GzAGCTC-3)- followed by.-bases corresponding to a Nco I site (5'-CCATGG-3') followed by additional bases (5 '-AGATCTGGTCC-3').

*BR61 and BR62 corresponding to bases 1846 to 1865 (BR61) and bases 2094 to 2114 (BR62) of the sequence listed in Figure 2 of EP 255378. In addition the WO 94/11516 PCT/US93/09987 102 end of BR 62 had additional bases (5'-GACA-3') followed by bases corresponding to a Bgl II site (5'-AGATCT-3') followed by a few additional bases (5'-GCGGCCGC-3').

Genomic DNA from the canola variety 'Hyola401' (Zeneca Seeds) was used as a template for PCR amplification of the napin promoter and napin terminator regions. The promoter was first amplified using primers BR42 and BR43, and reamplified using primers BR45 and BR46. Plasmid pIMCOl was derived by digestion of the kb promoter PCR product with Sall/BglII and ligation into Sall/BamI digested pBluescript SK (Stratagene).

The napin terminator region was amplified using primers BR48 and BR50, and reamplified using primers BR47 and 15 BR49. Plasmid pIMCO6 was derived by digestion of the 1.2 kb terminator PCR product with SalI/BglII and ligation into SalI/BglII digested pSP72 (Promega).

Using pIMC06 as a template, the terminator region was reamplified by PCR using primer BR57 and primer BR58.

20 Plasmid pIMC101 containing both the napin promoter and terminator was generated by digestion of the PCR product with SacI/Ncol and ligation into SacI/NcoI digested pIMC01. Plasmid pIMC101 contains a 2.2 kb napin expression cassette including complete napin 5' and 3' non-translated sequences and an introduced Ncol site at the translation start ATG. Primer BR61 and primer BR62 'were used to PCR amplify an -270 bp fragment from the 3' end of the napin promoter. Plasmid pIMC401 was obtained by digestion of the resultant PCR product with EcoRI/BglII and ligation into EcoRI/BglII digested pIMC101. Plasmid pIMC401 contains a 2.2 kb napin expression cassette lacking the napin 5' non-translated sequence and includes a NotI site at the transcription start.

WO 94/11516 PCr/IS93O997 103 To construct the antisense expression vector, pM3CFd2 was digested with NotI as was pIKC4Ol. The delta-12 desaturase containing insert from the digest of pM3CFd2 was gel isolated and ligated into the NotI digested and phosphatase treated pIHC4Ol. An -isolate in which the delta-12 desaturase was oriented ant isense to the napin promoter was selected by digestion with XhoI and PflMI to give plasmid pNCFd2R. pNCFd2R was digested with Sall, phosphatase treated and ligated into pZ2212 Which-had been opened by the same treatment.* A plasmid with desired orientation of the introduced napin:delta-12 desaturase antisense transcription unit relative to the selectable marker was chosen by digestion with Pvul and the resulting binary vrector was 15 given the name pZNCFd2R.

Plasmid pHL70 (described in Example 6 above) was **digested with NcoI, blunted then digested with KpnI.

Plasmid pM2CFd was digested with KpnI and Sinal and the isolated fragment ligated into the opened pt4L70 to give 20 the antisense expression cassette pMKCFd2R. The promotor:delta-12 desaturase:terminator sequence was reiuoved from pMKCFd2R by BaiiEI digestion and ligated ****into pZSl99 which had been BamHI digested and phosphatase treated. The desired orientation relative to the selectable marker was determined by digestion with Xhol and PfllI to give the expression vector pZKCFd2R.

The expression vector containing the B8-conglicinin promoter was constructed by SinaI and EcoRV digestion of pM2CFd2 and ligation into SinaI cut pML1O9A. An isolate with the antisense orientation was identified by digestion with XhoI and PflmI, and the transcription unit was isolated by Sall and EcoRI digestion. The isolated SalI-EcoRI fragment was ligated 'into EcoRI-SalI digested pZS199 to give pCCFd2R.o WO 94111516 PCr/US93109997 104 The expression vector containing the phaseolin promoter Was obtain~ed using the same proceedure with pCW108 as the starting* promoter containing vector and.

pZS212 as the binary portion of the vector to give pZPhiCFd2R.

Trnftfrmatjp 1 n Of R~4~ The binary vectors PZNCFd2R, pZCCFd2R, pZPhCFd2R, and pZNCFd2R were transferred by a freeze/thaw method (Boisters et al. (1978) Hol Gen-Genet 163:181-187) to the Agranctperjiu strain LBA44O4/pAL44O4 (Hockema et al." (1983), Nature 303:179-180).

flxAnnima nap=~ cultivar awestar* *was transformed by co-cultivation of seedling pieces with disarmed AgatXIM tnmzfarigns: strain LBA4404 carrying the ~..the appropriate binary vector.

naLR= seeds were sterilized by stirring in Chiorox, 0.1% SDS for thirty min, and then rinsed thoroughly with sterile'distilled water. The seeds were germinated on sterile medium containing 30 M CaCl 2 and agar, and grown for six days in the dark at 240C.

*9 9Liquid cultures of Agrnhate-riu for'plant transformation were grown overnight at 280C in Minimal

A

containing 100 mg/L kanamypin. The bacterial cells were pelleted by centrifugation and resuspended at a concentration of 108 cells/mr. in liquid Murashige and Skoog Minimal Organic medium containing 100 ;LM acetosyringone.

Z1. nApj= seedling hypocotyls were cut into 5 mm segments which were immediately placed into the bacterial suspension. After 30 min, the hypocotyl pieces were removed from the bacterial suspension and placed onto BC-35 callus medium containing 100 JIM acetosyringne. The plant tissue and Agrnobae--tpria were co-cultivated for three days at 24*C in dim light.

1' WO 94/11516 PCT/US93/09987 105 The co-cultivation was terminated by transferring the hypocotyl pieces to BC-35 callus medium containing 200 mg/L carbenicillin to kill the Agrobateria. and mg/L kanamycin to select for transformed plant cell growth. The seedling pieces were incubated on this medium for three weeks at 28°C under continuous light.

After four weeks, the segments were transferred to BS-48 tegeneration medium containing 200 mg/L carbenicillin and 25 mg/L kanamycin. Plant tissue was subcultured every two weeks onto fresh selective regeneration medium, under the same culture conditions described for the callus medium. Putatively transformed calli grew rapidly on regeneration medium; as calli reached a diameter of about 2 am, they were removed from 15 the hypocotyl pieces and placed on the same medium lacking kanamycin.

Shoots began to appear within several weeks after transfer to BS-48 regeneration medium. As soon as the shoots formed discernable stems, they were excised from S 20 the calli, transferred to MSV-1A elongation medium, and moved to a 16:8 h photoperiod at 24°C.

Once shoots had elongated several internodes, they were cut above the agar surface and the cut ends were dipped in Rootone. Treated shoots were planted directly into wet Metro-Mix 350 soiless potting medium. The pots were covered with plastic bags which were removed when the plants were clearly growing 1 after about ten days.

O Plants were grown under a 16:8 h photoperiod, with a daytime temperature of 23°C and a nightime temperature of 17°C. When the primary flowering stem began to elongate, it was covered with a mesh pollen-containment bag to prevent outcrossing. Self-pollination was facilitated by shaking the plants several times each day. Fifty-one plants have thus far been obtained from transformations using both pZCCFd2R and pZPhCFd2R, WO 94/11516 PCr/LUS93/09987 106 plants have been obtained from pZKCFd2R and 26 from pZNCFd2R.

Minimnl A Ea±rial Growth Medium Dissolve in distilled water: 10.5 grams potassium phosphate, dibasic grams potassium phosphate, monobasic gram ammonium sulfate gram sodium citrate, dihydrate Make up to 979 aiL with distilled water Autoclave Add 20 mL filter-sterilized 10% sucrose Add 1 mL filter-sterilized I M MgSO4 Bransion Callun Mediuim Per liter: Murashige and Skoog Minimal Organic Medium (MS salts, 100 mg/L i-inositol, 0.4 mg/L thiamine; GISCO *510-3118) grams sucrose 18 grams mannitol 0.5 mg/L 2,4-D :0.3 mg/L kinetin 0.6% agarose pH 5.8 lrnf~eta Reaenprtinn Medium IRR-48 MUrashige and Skoog Minimal Organic Medium Gamborg B5 Vitamins (SIGMA #1019)- 10 grams glucose 250 mug xylose 600 mag HES 0.4% agarose PH 5.7 Filter-sterilize and add after' autoclaving: mg/L zeatii 0.1 mg/L IAA WO 94/11516 PCI1US93/09987 107 Bgrmnsin Rhnnt !r1nation Medium MSV-1!A Murashige and Skoog Minimal Organic Medium Gazuborg B5 vitamins gjrams sucrose 0.6% agarose PH 5.8 An Ant leense Mi elrnftma1 Ist-2Deauae tO~ntruct Fifty-one plants were obtained from transformation with both pZPhCFd2R and pZCCFd2R, 40 were obtained from pZKCFd2R, and*26 from pZMCFd2R. The relative levels of oleAte linoleate (18:2) and linolinate (18:3) change during development so that reliable determination of .seed fatty acid, phenotype is best obtained from seed which has undergone nomal maturation and drydown.

Relatively few transformed plants, have gone through to maturity, however seeds were sampled from plants which had been transferred to pots for at least 80 days and w4hich had pods that had yellowed and contained seeds 20 with seed coats which had black pigmen~tation. Plants :were chosen for early anlaysis based on promotor type, presence and copy number of the inserted delta-12 *,desaturase antisense gene and fertility of the plant.

atty'acid analysis was done on-either individual seeds from transformed and control plants,-or on 40 mg of bulk seed from individual plants as described in a Example 6. Southern analysis for detection of the *pre sence of canola delta-12 desaturase antisense genes was done on DMA obtained from leaves of transformed plants. DNA was digested either to release the promotor:delta-12 desaturase fragment from the transformation vector or to cut outside the coding region of the delta-12 desaturase antisense gene, but within the left and right T-DNA borders of the vector.

WO 94/11516 PCFIUS93/09987 108 ZflLLII Relative Fatty Acid Profiles of Nicrosomal Delta-12 Desaturaae ~n~n~np Yan~flv~dati4 ~i~tn1 sic&~e maws' Rs.a4g I Of TOTAL FATTY ACIDS RLIN!1 O40ER cu GD LAIPf YlJ#aU Westar control Done 02 4.6 1.2 64.6 20.9 6.6 151-22 phaaeolin >8 82 .4.4 1.0 76.6 10.0 6.2 158-8 napin 1 83 3.5 1.5 61.3 6.3 4.6 vestar control none 106 4.1 1.7 64.4 19.9 7.1 151-22 phameolin >8 106 4.2 1.9 74.4 9.9 6.3 151-127 phaaeoUin 0 106 4.1 2.3 68.4 16.9 5.2 151-268 phasolin 1 106 4.2 2.7 73.3 12.0 4.2 153-83 conglycinin 2 106 4.1 1.6 68.5 16.7 6.3 Seed sampeling date in days after the plant was tranferred to soil *The expected fatty acid phenotype for antisense -suppression of the delta-12 desaturase Is decreased relative content of 18:2 with a, corresponding increase in 18:1. Plant numbers 151-22 and 158-8 both show a substantial decrease in 18:2 content of bulk seed when compared to the wester control at 83 days after planting. Plant 151-22 also shows this difference at maturity in comparison to either the westar control or *10 plant 151-127, wihich was transformed with the selectable marker gene but not the delta-12 desaturate antisense gene.

Since the fatty acid analysis was done on seeds from the primary transformant, individual seed should be segregating for the presense of the tranagene copy or copies. The segregating phenotypes serve as an internal control for the effect of the delta-12 desaturase antisense gene. The relative fatty acid phenotypes for individual westar seeds, 10 individual 151-22 seeds and 12 individual 158-B seeds are given in Table below.

WO 94/11516 PCT/US93/09987 109 Relative Fatty Acid Profiles for Individual Seeds of Control and Genetically Segregating Delta-12 Depaturase Transformed Brasiica Napua SeedR

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@556 4.65 4.65 4.65 3.86 4.46 4.76 4.59 4.61 4.71 4.67 4.73 4.56 4.25 4.40 4.40 4.50 4.60 4.49 4.20 4.20 4.50 3.62 3.46 3.48 3.53 3.4B 1.05 1.37 1.31 1.41 1.30 1.10 1.16 1.26 0.98 1.33 1.08 1.20 1.00 0.94 1.00 0.98 0.96 1.10 1.00 1.00 1.67 1.64 1.61 1.40 1.39 westar control 18il 63.45 65.41 62.19 66.81 61.90 64.77 68.66 67.28 61.96 63.85 151-22 73.40 77.90 76.90 77.40 73.60 75.40 76.70 77.20 80.00 78.00 152-8 84.45 85.56 83.64 83.80 83.66 182 2 21.31 20.72 22.50 19.40 22.39 20.62 18.20 19.32 22.93 21.65 12.40 10.00 10.10 9.40 11.30 10.50 9.90 9.70 7.90 8.80 3.60 3.02 4.43 4.41 4.35 18i3 7.29 6.18 8.18 5.63 7.65 6.56 5.07 5.18 7.61 6.23 7.60 5.40 5.90 6.10 7.90 6.50 6.00 5.50 4.90 5.80 3.73 3.36 4.21 4.36 4.44 WO 94/11516 PCF/US93/09987 110 3.80 1.50 68.17 16.57 7.56 3.41 1.40 83.76 4.38 4.40 3.49 1.29 82.77 5.16 4.60 3.77 1.39 69.47 16.40 6.54 3.44 1.36 83.86 4.49 4.27 3.48 1.38 83.15 4.91 4.53 3.55 1.92 83.69 4.20 3.70 The westar control shows comparatively little seed to seed variation in content of 18:1 or 18:2. Further the ratio of 18:3/18:2 remains very constant between seeds at about 0.35. Plant #158-8 should show a segregation ratio of either 1:2:1 or 1:3 since by Southern analysis it contains a single transgene. The 1:2:1 ratio would indicate a semi-dominant, copy number *effect while the 1:3 ratio would indicate complete i 10 dominance. Two wild type 158-8 segregants are clear in Table 15, while the remaing seeds may either be the same, or the two seeds at greater than 84% 18:1 may represent the homozygous transgeneic. In either case the fatty acid phenotypes of the seeds are as expected for effective delta-12 desaturase suppression in this generation. The fatty acid phenotypes of the seeds of plant 151-22 show variation in their 18:1 and 18:2 content, with 18:1 higher than the control average and S...18:2 lower. The segregation is apparently quite 20 complex, as would be expected of a multi-copy transgenic plant.

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WO 94/11516 PMFUS93/09987

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sr~tmFNrE IT1NC GENERAL INFORMATION: 'APPLICANT: E. 1. DU PONT DE N!EMOURS AND COMPANY (ii) TITLE OF INVENTION: GENES FOR NICROSOMAL FATTY ACI1b DELTA-12 DESATURASES AND RELATED ENZYMES FROM

PLANTS

(iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:.

ADDRESSEE: E. 1. DU PONT DE NEMOURS AND COMPANY STREET: 1007 MARKET STREET CITY: WILMINGTON STATE: DELAVARE COUNTRY: U.S.A.

*ZIP: 19898 COMPUTER READABLE FORK: MEDIUM TYPE:- Floppy disk COMPUTER: MacIntosh OPERATING SYSTEM: MacIntosh System, SOFTWARE: Patentln Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: EB-1043-A FILING DATE:

CLASSIFICATION:

(vii) PRIOR AP'PLICATION DATA: APPLICATION NUMBER: U.S. 07/977,339 -FILING DATE: 17-NOV-1992 (viii) ATTORNEY/AGENT INFORMATION: NAME: Morrissey, Bruce W REGISTRATION NUMBER:- 330,663 REFERENCE/DOCKET NUMBER: 3B-1043-A It WO 94/11516 PCr/US93/099s7 112 (ix) TELECOMMUICATION

INFORMATION:

TELEPHONE: (302) 992-4927 TELEFAX: (302) 892-7949 IC) TELEX: 835420 4 I*

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INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 1372 base pairs TYPE: nucleic acid STRANDEDHESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (vii) IMMEDIlATE SOURCE: CLONE: p92103 (ix) FEATURE: NAME/KEY: CDS LOCATION: 93. .1244 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AGAGM3AGAG ATTCTGCGGA GG&GCTTCTT CTTCGTAGG ?GTTCATCGT '?ATACGTT ATCGCCCCTA CGTCAGCTCC ATCTCCAGRA AC ATG GGT OCM GOT GGR LWA ATG Mest Gly Ala Sly Sly Arg Diet COG GTT CCT ACT TCT TVC LAG AAA TC)G GAA ACC GAC AMC ACA LAG CGT Pro Val Pro Thr Ber 8cr Lys Lys 8cr Gig Thr Ap Thr Thr Lys Arg 15 GTG COG TGC GAG AAA CCG CC? TTC TCG GTG GGPA GAT CTG LAG AAA GCA Val Pro Cya Glu Lys Pro Pro Ph. 8cr Val Gly Asp Leu Lys Lye Ala 30 ATC CCG CCG CAT TOT TTC AAA 0CC ?CA ATC CC? CGC TCT TTC TCC TAC Ile Pro Pro His Cys Ph. Lys Arg Ser Ile Pro Arg Bar Ph. 8cr Tyr 45 50 113 161 .209 257 WO 94/11516 WO 9411516PCr/US93/09M8

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9S e.

CTT FTC AG? G&C Leu Ile Ser Asp AAT TAC !TC TCT Asn Tyr Ph. Sa: CTC T&T TOG 0CC Lau Tyr Trp Ala GCC C&C GA TGC Ala Rio 1pu Cys 105 GNC ACA OTT GOT Asp Thr Val Gly 120 TCC TOO AAG TAT Ser Trp Lys Tyr A AGA GAT GAA Giu Arg Asp Glu 155 TAC GOO AAA TAC Tyr Gly Lys Tyr 170 GTC CAG TTT GTC Val Gin Phe Val 185 GGC AGA CCO TAT Gly Arg Pro Tyr 200 FTC TA AT GAO Ile Tyr Aan Asp ATT CTA 0CC GTC Ile Lou Ala Val 235 ATG 0CC TCO ATG met Ala Ser met 250 AT? ATA GCC TCh TOC le Ile Ala Ser Cya CTC CCT CAG OCT CTC Leu Pro Gin Pro Lou so CAA GOC TOT OTC CTA Gin Gly Cya Val Lou 95 CAC ChC*SCA TTC MGC Rio Ris Ala Ph. Bar 110 ATC TTC CAT TCC TTC Ile Ph. His Ber Ph.

125 CAT COC CAC CAT Ris Arg Arg His His 145 GTC CCA AO CAO P~he Val Pro Lys Gin 160 AAC FAC OCT CT? GGk Asn Asn Pro Lou Oly 175 GG TOO CCC TTG TAC Gly Trp Pro Lou Tyr 190 0O0 TTC GCT TGC CAT Gly Ph. Ala Cys His 205 GAA COC CTC CMO ATA Gin Frig Lou Gln le 225 TTT GOT CT? TAO CG? Ph. Gly Lou Tyr FAg 220 TAO TAC Ph. Tyr Tyr TOT TAO TTG ger Tyr Loui ACT *Q0T FTC Thr Gly 110 100 SAC TAC CR Asp Tyr Gin 115 CTC CTCGTm Lou Lau Val 130 TCC A ACT Sox Asn Thr AAA TCA GMA L~ys Ser Ala CGC FTC £10 A g Ile Hot 160 TTA GCC TTT Lan Ala Phe 195 TT0 T=CCC Ph. Pb. Pro 210 TAC CTC TCT Tyr Lou got TAO OCT WCT Tyr Ala Ala Co CT? CYG Pro Lou leu 260 GTC GCC ACC Val .Aia Thr OCT TOO Och Ala Trp Pro TMOG TC ATA !zp Val 116 TOO OTO GAT Tap Lou Ap ce? TAC TTC Pro Tyr Ph.

135 00k 2cc CTC Gly. Bar Lou 150 FTC FAG TG ile Lys Trp 165 ATG TTA ACC bft Lou TA: AAC CTC TCT LAn Val Ser ARC OCT CC LAn Ala Pro 215 GAT 000 GOT Asp Ala Sly 230 00K A GG la Gln Gly 245 ATA GTG ART Ile Val Asn CCC TCG TTG Pro Ser Lou 305 353 401 449 497 545 593 641 689 737 785 833 S81 929 240 TOC CTC TAC GGA GTh Cys Lou Tyr Gly Val 255 000 TTC Ala Phe 265 CTC OTC TTG Lou Val Lou A=C AMT m Tm C= C= AMT CA Ile Th: Tyr Lou Gin His Thr Rio 270 275 I a WO 94/11516 PCF/US93/09987 114 MC A TAC CT CA TCA GAG TGG GAC TGG CTC LOG 06k OCT ITT OCT 977 Pro His Tyr Asp 8cr 5cr Gin Tzp Asp Trp Lou An; Gly Ala Lou Ala 280 285 290 295 ACC GTA GAO AGA GKC TAC GG& ALTC TTG AC AAG GTG TTC CAC LAC.ATT 1025 Thr Val Asp Arg Asp Tyr Gly le 14n Aa. Lys Val Ph. Ria Asn Ile 300 305 310 ACA GAC ACA CAC CTG OCT CAT CAC CTG TTC TCG AOL ATG COG CAT TAT 1073 Thr Asp Thr His Val Ala Rio fiie Lou Ph. gar thr Nt Pro Ris Tyr 315 320 325 LAO 00k ATG OAk OCT ACA LAG 000 AT&ALAG 00k LIT CTG GGA GAC TAT 1121 Ann Ala net Gin Ala Thr L~ys Ala Xie Eqys Pro 1l. ELou Gly Asp Tyr 3.30 335 340 TAC CKG ITO OAT 06kA~ OG TOO TAT GTL 000 ITO TAT AGO GAG GCR 1169 Tyr Gln Phe Asp Gly Thr Pro Tvp Tyr Val Ala Hot Tyr An; Gin Ala 345 350 355 LAG GAG TO AMC TAT OTA CAA =O GAC AOG GML GOT G M G AMA GOT 1217 Lys Gin Cy le Tyr Val Glu Pro Asp Arg Gin Gly Asp Lys L1s Gly 60365 310 375 GTG TAC TGG TAO LAO ALT LAG TTA TGAGCATGAT GGTGAAGLAA TTGTCGILCCT 1271 *Val Tyr Trp Tyr Ann Asn Lye Leu 360 ITTTTGTC TGTTTGTCTT TTGTTLAAhGA AGCTATGCTT CGTITTTARTA ATCTTATTGT 1331 CCATTTTGTT GTGTTATGAC ATTTTGGCTG CTCATTATGT T 1372 INFORM4ATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 383'amino acids TYPE: am4no acid TOPOLOGY: linear (ii) 'MLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID .NO:2: Met Gly Ala Gly Gly Axg Met Pro Va. Pro Thr 5cr 8cr Lye Lye Ser 1 5 10 Gin Thr Asp Thr Thr Lys Arg Val Pro Cya Gin Lys Pro Pro Ph. Ser 25 Val Gly Asp Len Lys Lys Ala Ile Pro Pro His Cys Phe Lye Ar; 40 Ile Pro Arg 8cr Phe 8cr Tyr Len. I1e Ser Asp le le Ile Ala Ser so 55 WO 94/11516 WO 9411516PCr/US93/09987 115 Cys Ph. Tyr Tyr Val Ala Thr IAn Tyr 70 Lau Ala Trp Ile Trp Val 100 Gin Tzp Lou Val Pro Tyr Thr Sly Ser 150 Ala Ile Lys 165 Hot met Lou

ISO

Phe IAn Val Pro Msn Ala Ser Asp Ala 230 Ala Ala Gin 245 Pro Lou Tyr Ile Ala Rio~ 105 Asp Asp Thr 120 Ph* Bar Tzp 135 Lou Sin Az; Trp Tyr Gly Thr Val Sin 185 Ser Sly Arg 200 Pro 116 Tyr 215 Gly le Leu Pb. Ser Lou 75 Trp Ala Cys 90 Glu, Cys Sly Val Sly Lou Eys Tyr gar 140 Asp Sin Val 155 Lys Tyr Lou 170 Phe Val Lau Pro Tyr Asp kan Asp Arg 220 Ala Val Cys 235 Lou Pro Gin Pro so Gin Sly Cys Val Ris Ris Ala Phe 110 le Ph. Ris har 125 Ris &rg Axq Ris Phe Val Pro Eqs 160 As Asn Pro Lau 175 Sly Trp Pro Lou 190 Sly Phe Ala Cys 205 Glu Arg Lou Gln Phe Sly Lou Tyr 240 Cys Lou Tyr Sly 255 Ile Thr Tyr Lou 270 Ber Glu Tzp Asp 285 Tyr Sly Ele Lou Ala His Ris Lau 320 Thr Lys Ala Ile 335 Thr Pro Trp Tyr 350 Val SlU Pro Asp 365 Gly Met Ala gar Met Ile 250 Val Pro Lou Lou le Val Ann Ala Phe Lou Val Liou 260 265 Ris Tyr Asp her Thr His Pro har Lou. Pro 275 280 Arg Sly Ala Lou Ala Thz 295 Val The His Aa Ile Thr 310 1hz Hot Pro His Tyr Asn 325 Ile Lou Gly Asp Tyr Tyr 340 Met Tyr An; Glu Ala Eqs 355 360 Val Asp Arg Asp Thr Ris 315 Ala Het Sin 330 Gin Phe Asp 345 Siu Cys Ile WO 94/11516 WO 9411516PCr/tUS93/09MS 116 Arg Glu Gly Asp Lys Wys Gly Val Tyr fTp Tyr Len Len Lys Lou 370 375 380 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: (iv) (Vi) LENGTH: 1394 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA HYPOTHETICAL:* NO ANTI-SENSE: NO ORIGINAL SOURCE: ORGANISM: Brassica napus

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(vi i) IMMEDIATE SOURCE: CLONE: pCF2-165D (ix) FEATURE: NAME/KEY: CDS LOCATION: 99. .1250 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAGAGGAGAC AGAGACAGKG AGAGAGTTGA GAGKGCTCTC GTAGMTATC OTATMACGT AATCT=CAT CCCCCTACG TCAGCCGCT CLAGALAC ATG GOT WCA GWT GGA AGA MT CM =T =C =C =C =C AML MG Lzg Hot Gin Val Ser Pro Pro Ser Lys Lys 15 LAG CGC GTA CCC TOC GAG ACA CCG C TTC Lys Arg Val Pro Cys Glu Thr Pro Pro Ph.

30 AhA GCA ATC CCA CCG CAC TGT TTC LAG CGC Lys Ala 110 Pro Pro His Cys Ph. Lye Arg 45 TOC CAC CTC ATC TGG GAC ATC ATC ATA GCC Ser His Leu Ile Trp Asp Ile le Ile Ala 60 Miet Gly Ala Gly Gly 1 TCT GAL ACC WXC AC ATC got Glu Th Ap Len Ile ACT GTC GA GA CTC LAG Thr Val Gly Glu Lou Lye TCG ATC CCT CGC TCT TTC g: le Pro Arg Se: Phe TCC TGC TTC TAC TAC GTC So: Cys Ph. Tyr Tyr Val WO 94/11516 WO 9411516PCr/YUS93/09967 0CC ACC ACT Ala Thr Thr TGG CCT CTC Trp Pro Lou GTC ATA C Val 110 Ala CYG GAC GAC Lou Asp Asp 120 TAC TTC TcC Tyr Ph. Ser 135 TCC CTC GAG Ser Lou Qiu 150 AGT GGT hOG .Ser Gly Thr ACo GT CAG Thr Val Gin TCG 000 AGA Ser Gly Arq 200 GCT CCC ATC Ala Pro Ile 21.5 OCT GGC MTC Ala Gly Ile 230 CA 00K OT Gin Gly Val OTC AC 000 Val Aan Oly TCC CTG CCT Ser Lou Pro 280 TAC TTC CC? CTC CTC Tyr Ph* Pro Lou.Lou 75 TAC TOG 0CC TGC CRB Tyr Trp Ala Cys Gin CAC GAG TOC GGC CAC His Giu Cya Oly Rio 105 ACC GTC SOC CTC MTC Thr Val Gly Lou Ile 125 TOG AMG TAC AG? CAT Trp Lys Tyr ger Ris 140 AGA GAC A OTG TTT Arg Asp Glu Val Ph.

155 GCA AG? ACC TCA ACA Ala Ser Thr Bar Thr 170 TTC ACT CTC GGC TOG Ph. Thr Lou Oly Trp 185 CCT TAC GAC GGC GGC Pro Tyr Asp Gly Gly 205 TAC AAO GAC COT GAO Tyr Aen Asp Ar; Giu 220 CTC GCC OTC TGC TAC Lou Ala Val Cys tyr 235 0CC TOG ATO GC TOC Ala Bor Not Val Cys 250 TTC TTA OTT TTG ATC Ph. Lou Val Lou le 265 CAC TAT CAC TCG TCT Hio Tyr Asp Ser ger 285 117 CCT AAC CC? Pro hAn Pro s0 GGC TOC GTC Gly Cys Val 95 OCA GCC TTC Ala Ala Ph.

110 TTC CALC TCC Ph. His Bar COK COC CAC Arqj Arg His GTC CCA A Val Pro Ar; 160 ACC TTT G0K Thr Ph* Gly 175 CC? TTG TAC CTC TOC TAC TC 0CC Lou Sor Tyr Ph. Ala CT&A= C SC GTC TMO Lou Thr Gly Val fTp 100 hOC GAO TAC CAB TOO Ser Asp TT= CTC Pho LOU 130 CRT TC ISseta 145 AGA MOT Ar; 8ac CGC C Arg Thr TTA C Tyr Gin Trp 115 CTC OTC OCT Lou Val Pro LAC ACT GOC hAn Thr Gly 0KG ACA TCA Gln Thr Bar 165 OG ATG ?TA Val not Lou 180 TTC LAC GTC Phe Men Val 195 CAC CCC LAC His Pro Msn 401 Pro Lou Tyr Lou Ala 190 TTC OCT TGC CAT TTC Ph. Ala Cys His Ph.

210 COT CT MA MT T= MT =C "AC Ar; LOU Gin GOT CTO CmA Gly Lou Lou 240 TTC CTA OGA Ph. Lou Arg 255 ACT TAC TTG Thr Tyr Lou 270 GAG TOG OAT Glu Trp Aspi Ile Tyr 225 le Ser Asp CCG TAC OCT OCT Pro Tyr Ala Ala OTT CT CT? CTG Val Pro Lou Lou 260 CAB CAC ACG CAT Gin Rio Thr Ris TO;G TTG AGO GmK Tip Lou Arg Oly 290 WO 94/11S16 PCr/US93/0998 118 MT WCC AMC GTT GAC LWA GAC TAC WAP ATC TTG MC CA GGC TT CAC Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Lou Len Gin Gly Phe His 295 300- 305 LAT ATC ACG GAO ACG CAC GAG QOG CAT CAC CTG TTC TOG ACC ATG CCG LAn Ile Thr Aop Thr Hio Glu Ala Rio Rio Lou PhO Ber Thr Hot Pro 310 315 320 325 CAT TAT CAT GOG ATG GRA GOT ACM LAG GCG ATA MLG COG ATA CTG GOP His Tyr His Ala Not Gin Ala Thr Lys Ala Il* Lys Pro Xi. Leu Gly 330 335 340 GAG TAT TAT C50 TTC GAT GOO ACG CM GTG OTT MAG 000 LTG TGG LOG Gin Tyr Tyr Gin Ph. Asp Gly Thr Pro Val Val Lye AlI Not Tzp Arg 345 350 355 GAG 000 LAG GAG TGT ATC TAT GTG GA COG GAO AGG CA GOT GRG LAG Gin Ala Lye Gin Cys lie Tyr Va1 Gin Pro Asp Lrg Cin Gly Gin Lys 360 365 370 AKA GOT GTG ITT TGG TAC AC A? AMG TWA TGRAGCM"ALLAGAAACTGK Lys Gly Val Phe Trp Tyr Ann Len Lys Len 375 380 ACCTTTCTCT TCTATCAATT GTCTTTGTTT L&MAGCAT GTTTCTGTTT CATATCTT .AATATcCAIT TTTGTTGTG;T TTTCTGILChT TTTGGCTLLA ATTATGTGAT OTTGGAAGTT

AGTGTCT

1025 1073 1121 1169 1217 1267 1327 1397 1394 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 383 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUECE DESCRIPTION: SEQ ID NO:4: Het Oly Ala Gly Gly Lrg net Gin.Va1 Ser Pro Pro Ser Lye Lys Ser 1 S 10 1 Glu Thr Asp Len le Lys Arq Val Pro Cys Gin Thr Pro Pro Phe Thr 25 Val Gly Gin Lou Lye Lys Ala le Pro Pro His Cys Ph. Lye Arg $or 40 le Pro Arg Ser Phe Ser His Leu Ile Trp Asp le Ile Ile Ala Ser so 55 WO 94/11516 PCrIS93/09987 Ph. Tyr Tyr Val Ser Tyr Phe Ala Thr Gly Val Trp 100 Asp Tyr Gin fTrp 115 Lou Leu Val Pro 130 Bet IAn Thr Gly $r Gin Thr Ser 165 Thr Val Met Lou 180 Ala Phe Aen Val 195 Ala Thr Trp Pro Val lie Leu LAsp Tyr Ph.

135 St Lou 150 ret Gly Thr Val Be Gly

C

S

Ris Phe 210.

le Tyr 225 Pro Tkjr Val Pro Gin Rio fTp Lou 290 IAn Gin 305 Phe Set Lys Pro Lye Ala His Pro IAn Ala Pro 215 Ile Ser Asp Ala Gly 230, Ala Ala Val Gin Gly 245 Leo Lou Ile Val Ian 260 Thr Hia Pro Set Lou 275 Arg Giy Ala Lou Ala 295 Gly Phe Rio Ian le 310 Thr Not Pro Rio Tyr 325 lie Leu Gly Glu Tyr 340 net Trp Arg Glu Ala 355 Thr Tyr Phe Pro Lu Lou Pro Asn Pro 75 Lou Tyr Tzp Ala Cys Gin Giy Cys Val 90 Ala His Glu Cya Gly His Ala Aa Ph* 105 110 Lp Thr Val Gly Leu Ile Pbe His sr 120 125 8: Trp Lys Tyr Ser His Arg Arg is 140 Gin arg Asp Gin Val Phe Val Pro Arg 155 160 Thr Ala Ber Thr Sor Thr Thr Phe Gly 170 175 Gin Phe Thr Lau Gly fTp Pro Lou Tyr 185 190 lAg Pro Tyr Asp Gly Gly Phe Ala Cys 200 205 1i0 Tyr AUn Asp Az9 Gin Arg Lou Gln 220 Ile Leu Ala Val Cys Tyr Giy Lou Lou 235 240 Val Ala Set Hot Val Cys Ph. Lou Ag 250 255 Gly Phe Lou Val Lou lie Thr Tyr Lou 265 270 Pro Rio Tyr Asp Sr Set Giu Trp Asp 280 285 Thr Val Asp Arg Asp Tyr Gly Ile Lou 300 Thr Asp Thr Rio Gin Ala Ris Mie Lon -315 320 His Ala net Glu Ala Thz Lye Ala lie 330 335 Tyr Gin Phe Asp Cly Thr Pro Val Val .345 350 Lye Giu Cys Ile Tyr al Glu Pro 1 sp 360 365 WO 94/11516 WO 9411516PCr/US93/09987 120 Lrg Gin Gly Giu Lys Lys Gly Val Phe Trp Tyr LAn Asn Lys Lou 370 375 380 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1462 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: -cDNA (iii) HYPOTHETICAL: NO 4 a

S

a a (iv) (Vi) ANTI-SENSE: NO ORIGINAL SOURCE: ORGANISM: Glycine ax (Vii) IMMEDIATE SOURCE: CLONE: pSF2-165K (ix) FEATURE: NAME/KEY: CDS LOCATION: 108..1247 (xi) SEQUENCE DESCRIPTION: SEQ ID ATATTTGCTT GTATTGATAG CCOCTCCGTT CCCAAGLGTL ThALACTGCA CALGOcACTA GGCTGGG TAOcALAGGL AchamA TG GrA GoT Miet Gly Gly

CCATATACTA

TcOLLTAKTA AA WGT CO =T =C AML =T GM =T CAL WvG ANO LAG CCT CTC =C Arg Gly Arg Val. Ala Lys Val Giu Val Gin Oly Lye Lyse Pro Leu Bar 10 LOG OTT CCL LAC ACA LAG CCL CCL TTC AMT OTT GOC CAA CTC PLO ARL Arg Val Pro ken Thr Lys Pro Pro Pile Thr Val Gly Gin Lou Lys Lys 25 30 GCA ATT CCL CL CKC TGC TTT CAG CGC TOC CTC CTC ACT TCL TTC TC Ala Ile Pro Pro ii Cys Phe Gin Avg Ser Lou Lou Thr Bor Phe 45 TAT OTT OTT TAT GhC CTT TCA TTT GCC TTC ATT TTC TAC ITT GCC ACC Tyr Val Val Tyr Asp Leu Ser Phe Ala Ph. Ile Ph. Tyr Ile Ala Thr 60 WO 94/11516 PCr/US9309 go go.

ACC TAC TTC Thr Tyr Phe ATC TAT TG Il* Tyr ?rp GCT CILC GAG Ala Hti. Glu 100 OAT OTT GTG Asp Val Val TCh TOG JM Ser fTp Lys GAC CGT GAT Asp Ar; Asp 150 TTT TcC AAG Pb, Ser Lys 165 GTC ACA CTC Val -Thr Lea 180 GGT AGA CCC Gly Arg Pro ATA TAT TCT Ile Tyr Ser TTG TTT TCT Lou Phe 8cr 230 TTG OTT TG Lea Val fTp 245 GOT CT? Gly Ph. Lou 260 CC? CAT TAC Pro His Tyr CAC CTC Rig Lou OTT CTC Val Lou TOT GOT Cys Gly GT TTG Oly Lou 1.20 ATA MGC Ile $or 135 GAA GTG Glu Val TAC TTA Tyr Lou ACA ATA Thr Ile TAT GhT Tyr Asp 200 AAC COT Ken Arg 215 GTG ACT Val Tbr CTG CTA Lou Lou OTG ACT Val Thr GAT TCA Asp 8cr 200 CT? CCT CM Lea Pro. Gln 75 CRM GG? TGC GIm, Gly Cya 90 CAC CAT C Htin His Ala 105 ACC CTT CALC Thr Lou Ilia CALT Coc COC is Arg Arg ?TT GMC cCh Pb. Val Pro 155 AAC LAC.-CC? hAn Ann Pro 170 GG0 TGG CCT Gly Trp Pro AG? TTT GCA Ser The Ala GAG MGG.CT? Glu Ar; Lou TAC TCT CTC Tyr 8cr Lou 235 121 CCC TTT TCC Pro Phe 8cr CT? CTC ACT Lou Lou Thr TTC AGC MAG ?he 8cr Lys .110 TCAL ACM CTT 8cr Thr Lou 125 CAT CAC C Hise His 5cr 140 ARA CCh A Lys Pro Lys CTA GGA AGO LOU Gly AM; ATG TAT TTA met Tyr Loiu 190 AGC CAC TAC Ser.Hiz Tyr 205 CTG ATC TAT

CTC

Lou

GT

dly

TAC

Tyr

TTA

Tau

AW

cC Ser

GCT

Ala 175

C

Ala

CAC

His

GC

AT? OCA TOG CCA 110 Ala ?rp Pro s0 GTG TOG OTG ATT Val fTp Val 110 CAA TOG OTT OAT Gin fTp Val Asp 115 GTO CC? TAT TTC Val Pro Tyr Ph.

130 ACA00 TCC CT? Thr Gly gor Lou 145 A OTT OCAL TOG Lys Vol Ala Trp 160 OTT TCT CTT CTC Val 8cr Lou Lou TTC MAT GTC TCT Phe Ann Val 8cr 195 CT TAT OCT CCC Pro Tyr Ala Pro 210 TCT OAT OTT OCT 8cr Asp Val Ala 225 ACC CTG AMA OGG Thr Lou Lye Gly 240 CTC ATT G"O MC Lou lb* Va1 Ann CAC TT? 0CC TTG Rio Ph. Ala Lou 275* GA OCT TTG GCA Gly Ala Lou Ala 290 356 404 452 500 548 596 644 692 740 788 836 884 932 980 Leu Ie Tyr Val 220 TAC COT OTT OCA Tyr Arg Val Ala TM G"T UAT GOG MT CC MT Cy. Val Tyr Oly Val ProLou 250 255 AC ACA TAT ?TG CRM CAC ACA Ile Thr Tyr Lou Gin His Thx 265 270 TCA GMA TOG GAC TOO CTG ARG 8cr Glu Trp Asp Trp Lou Lye 285 41 WO 94/11516 PCT/US93/09987 122 ACT ATG GKC AGA GAT TAT GGG ATT CTG LAC LAG GTG TTT CAT CAC ATA 1028 Thr Met Asp Arg Asp Tyr Sly Ile Leu Aen Lys Val Ph. His His Ile 295 300 305 ACT GAT ACT CAT G1'G OCT ChC CAT CTc TTC TCT ACK ATO CA ChT TAC 1076 Thr Asp Thr Ris Val Ala His Hia Lau Phe Ser Thr Net Pro Rio Tyr 310 315 320 CALT OCA ATG GAG GCML AAT GCA ATC LAN CA ATA ?TM GOT GAG TAC 1124 Rio Ala met Gin Ala Thr Ann Ala Ile Lys Pro XIe Lou Gly Gin. Tyr 325 330 335 TiC CA TTT GAT SAC ACA OCA TYT TAC AMGOCAL CG TOG AMA A OCO 1172 Tyr Gin Ph. Asp Asp Thz Pro Phe Tyr Lys~ Ala Lou fTp LAg Glu Ala 340 345 350 355 AGA GAG TGC CTC TAT OTG GAG CM OAT A SQL A0L TOC GAG LAG GGC 1120 Ag Giu Cys Lau Tyr Val Gin Pro Asp Gin Sly Thz Bar Gin Lys Oly 360 365 370 GTG TAT TOG TAC AGG AhC AAG TAT TG&TGGhGCA ACCLATG=O CATAOTOGGA 1274 Val Tyr Trp Tyr LAx Lnn Lys Tyr 375 380 *GTTATGGLAG TTTTGTCATG TATTAGTACK TRATTAGTALG AATGTTATAA ATAAGGG&T 1334 **TTGCCGCGTA ATGACTTTGT OTGTATTGTG ACAGCTTG TTGCGLTCLT GGTTATALTG 1394 TAALAATAAT TCTGGTATAJ ATTATGTG GAAAGTGTTC TGCTTATAGC ?TTTCTGCCTA 1454 AAAAAALA 1462 INFORMATION FOR SEQ ID NO:6: U) SEQUENCE CHARACTERISTICS: LENGTH:. 379 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein' (xi) SEQUENCE DESCRIPTION: SEQ ID 110:6: Net Gly Gly LAn Oly LAn Val Ala Lye Val Gin Val Gin Gly Lys Lys 1 5 10 Pro Lou Ber Axg Val Pro Len Thr Lye Pro Pro Ph. Thr Val G~.y Gin 25 Lau Lys Lys Ala le Pro Pro His Cy3 Phe Gin Arg Her Lau Lau Thr 40 WO 94/11516 W094/1516PCr/US93/09987 Sex Phe Sex Tyr Val Val Tyr LAp Lou Ser Phe Ala Ph. 110 Phe 55 le Pla Thr Thx Tyr lia rp Pro Ile Tyr Trp Val Ile Ala Rio Txp Val Asp Asp Val 115 Pro Tyr Pho Sex Trp 130 Gly Sex Lou Ap Lxg 245 Val Ala Trp Phe Sex 165 Sor Lou Lou Val Thr 180 Phe Hise Lou 70 Tzp Val Lou Glu Cys Gly Va1 Gly Lou 120 Lys Ile Ber 135 Ap Giu Val 150 Lye Tyr Lou Lou Thr 110 LOU Pro Gui 75 Gin Gly Cys Him Me. Ali 105 Thr Lou aLs Ki "vg "q Ph. Val Pro 155 LAn Lesn Pro 170 Gly T xp Pro 185.

Sot Ph. Ala Glu Arg Lou Pro Ph. Sex Lou le so Lou Lou Thr Gly Val Ph. Sox Lye Tyr Gin 110 Sex Thx Lou Lou Val 125 Ri. His Sex Lan Thr 140 Lys Pro Lye Sot Lys 160 Lou Gly Lrg Ala Val 175 Not Tyr Lou ALiepho 190 8cr fILe Tyr file Pro 205 Lou Ile Tyr Val Ser 220 re (el r. r. Len Val Sex Gly Lrg Pro Tyr Ap 195 200 Tyr Ap 225 Leu 11o Phe Ala Ei.

305 le Tyr Sex Len Lrg 215 Lou Ph. Sex Val Thir 230 Lou Va1 Trp Lou LoU 245 Gly Ph. Lou Val Thr 260 Pro Kin Tyr Ap Sex 280 Thr Hot Ap Axg Lap 295 Th Ap Thr Hie Va1 310 Tyr Sex Lou Tyr Lrg Va1 Alia Thr 235 240 Cys Val Tyr Gly Val 250 Ile Thr Tyr Lou Gin 265 Sox Glu Txp, Ap fTp 285 Tyr Gly Ile Lou as 300 Ala tile file Lou Ph.

315 Pro Lou Lou 255 Hise Thx Hi.

270- Lou Lye Gly Lye Va1 Phe tex Thr Not 320 Pro His Tyr fis la Lie t Giu lia Th Len Ala I1e Lye Pro le Lou 325 330 335 Gly Giu Tyr Tyr Gin Phe Asp Ap Thr Pro Phe 340 345 Tyr Lys Al. LoU Trp 350 WO 94/11516 W094/1516PCT/US93/09987 124 Arg Glu"Ala Arq Glu Cys Leu Tyr Val Glu Pro Asp Glu Gly Thr Ser 355 360 365 Glu Lays Gly Val Tyr Trp Tyr Arg Kan Lys Tyr 370 375 INFORMATION FOR SEQ ID NO:7: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 1790 base pairs TYPE: nucleic acid STRANDEDNESS: dou~ble TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (vi) ORIGINAL SOURCE: I. I. S -0 S. 0.

ORGANISM: Zea mays (vii) IMMEDIATE SOURCE: CLONE: pFad2#1 (ix) FEATURE: NAME/KEY: CDS CB) LOCATION: 165. .1328 (xi) SEQUENCE DESCRIPTION: SEQ ID, NO:7: ~C~CTCCT CCTGC&AAT cCCGCAGWAC&c CG TTTCCTC GAAAAGGGG& GLGA1GTG AGOCOOGTG T=GOOCG&?TCCTC TGTTACG&CC TCTCAGTCT CAGTCAGGhG.CAAG ATG GOT GW GGC Net Gly Ala Gly

S.

'*3*S CGGcCTCTcC

CGGGACAGGA

-CGhGCAGC WOC AM G AMC GAG AAG GAG =CGAG MAG "AG CM C= GC =G Gly Am; Net Ttar Gin Lays Gin Arg Gin Lays Gin Glu Gin Lou Ala Arg 10 15 GCT ACC GGT GGC GCC O ATG CAG CGG TCG CCG GTG GAG LA CCT CCG Ala Thr Gly Gly Ala. Ala Net Gin Arg Ser Pro Val Glu Lays Pro Pro 30 1. 11 WO 94/11516 WO 9411516PCr/US93/09ft7 125 000 ATC TTC ACT CTG GGT Phe Th: Lou Gly 000 TOG GTG CTC Arg Ser Val Lou 0CC GOG 000 CTC Ale Ala Ala Lou AM OG0 CTC COC Sex Pro Lou Arg as TOO GTG TOC £0C CYs Val Cys Tb: 0CC TTC TCG GAC iAla Ph. Set Asp 120 ChC TCG TCG CTC His 8er Ser Lou 135 CAG ATC AAG AG Glin Ile Lys Lys CCG CCA CLC TaO AAG TOC TTC Lys Sex Phe CTC TaO TTC Lou Tyr Ph.

75 TAO GOC OCC Tyr Ala Ala 90 GOC OT= TOG Gly Val Trp 105 TAC TOO CTC Tyr Sar Lou ATG GTG CC met Va1 Pro 'a a a..

*a 0 a.

a n.

a.

a 0 Ala Ile Pro 45 TAC GTG GTC Tyr Val Val CTG 0CC ATC Lou Ala Ile COG CTG TAC Pro Lou Tyr 95 ATC 000 CAC Ile Ala His 110 OAC GLC GTG Asp Asp Val 125 TTC Teo TOG Ph. 8er Trp OTO GAG COO Lou Glu Arg TOG TAO ACC Trp Tyr Thr 175 ATC GTG GTG le Val Val 190 GOG TOG 000 Ala Scr Gly 205 GGC COC ATC Gly Pro 11e 0CC GO OTC Ala Gly Val TTC 000 GTC Ph. Gly Val 255 Pro His Cys CAC G&O OTO Rio Asp Lou ATA C0G 00m la Pro Ala so TOG ATC amG Txp 110 Ala MAG TOO 00C Glu Cys Gly G=C 000 CTO Val Oly Lou 130 AAG TAO ADC Lys Tyr Box 145 GAO r GMO Asp Glu Val 160 COG TaC OTG Pro Tyr Val CAG CTC CC Gin Lou Th: COG COG TAO Arg Pro Tyr 210 TA~ AO CAC Tyr AU Asp 225 mT SOC OTO Val Ala Val 240 TOG TOG GTG fTp fTp Val TTC GAG Pb. Glu GTG ATC Va. Ile CTC 00k Lou Pro CRG GG GIn Gly 100 CRC cRc His His 115 MT CTG Val Lem 0LOc COG Ilis Arig TTC GTG Pb. Val TAC AAC Tyr Asn 190 CTC GGG Lou Gly 195 COG COO Pro Ar; COG a" Arq Olu 000 TTC Ala Phe OTO COO Va1 Arg 260 320 368 416 464 512 560 606 656 704 752 800 8 896 944 CGO CALC Arg Ris 150.

COO LAG Pro Lys 165 AO COG Aan Pro TOG COG Trp Pro TTC 0C Phe Ala cOO CCC Arg Ala 230 000 CTG Gly Lou 245 CAC TOO AO Ris 8er As: AAG AAG GALG Lys Lys Glu GTC GGC OGG Val Gly Arg 185 CTG TAO CTO Lou Tyr Lou 200 TOO CAC TTC Cy. Rio Pb.

215 CAG ATC TTO Gin Ile Ph* TAO LAG CTG Tyr Lys Lou WO 94/11516 WO 9411516PCr/US93/09967 126 Mf MTC GMC TOW M CTG MT GTG MAC GCG TG CMG =G CTC ATC Val Tyr Ala Val1 Pro lau Lou Ile Val Ann Ala Trp Lou Val Lou Ile 265 ACC TAC CTG Thr Tyr Lou GAG TOG GAC Glu Tzp LAp 295 SOC ATC CTC Sly 21. Lou 310 CAC CAC CTC Ris Rio TAU CAG CAC icc c&c Gin Rio Thr Rio 260 TOG CTG CGC OGC Tzp Lou Azq Sly AC COC G"S !TC Aun AM; Val Ph.

315 T!C !CC ACC T Pb. 8cr Thz Not 330 270 275 COG M MT C CCT CATC C Pro Bar Lou Pro Rio Tyr Asp Bar 8cr 285 290 OW CTG 0CC AM ATO SAC CGC SAC TAC Ala Lou Ala Thr Hot Asp Ar; Asp Tyr 300 305 CAC LAC ATC AMO SAC MC CAC SC CO Rin Ann Xi4 The AVp Thr Hti. Val Ala 320 CCG CRC T~AC CRC 0CC iTS GAG 00C ACC Pro His Tyr Rin Ala net Sin Ala Thr 335 340

S*

I* 4 4 5 .eS 'p *5 p 'S 4 4 *5S* 4 325

LAG

Lys CO ATC AGO Ala Ile Arg CCC ATC CTC Pro le Leu 345

S=OCSAC

Sly Asp cCI SI==C AOG iCCTGGOCGA Val Ala Lys 360 Ala' Tb: Tvp arg coc LAG GGC SIC Arg Lys Gly Val 380 GAG cC WM Glu Pro Glu 37!

CGCTCGCAGA

GTACTCGCCC

GAAGAGAGAT

CATCAAGTAC

GTCCCTGCT

CTGTGOGTGC

GTGCTCCCcG .CAGhTCGTcC

SAC

IAsp

GCTGACGGA=G

CAAAGTCTCC

CATTTGGGCA

AGAGOCLA

STAOGCTTTC

TGITTGTTG

CTACCGTAGGAA

GTCAACCTA TJ CAGAGACGA G

ATTCTCLCTI

COOCGGMT

TT0TCOTMT

TALAACLLGA

AAAAAAAAALAJ

T&C *TAC CAC TTC SAc CO A= Tyr Tyr Ilia Ph. Asp Pro Tb: 350 355 GOC GGG CAA TGC ATC TAC SIC Ala Sly Gin Cyn 1is Tyr Val 370 TOO TAC AC LAG LAG TTC TAGCC( Tzp Tyr Ann Lys Lys Ph.

365 rGGoCAm G&AACCAGA GOAGGAGACG L3LTCGTTA GTCGTCAOTC TTTTAGKCGG "ZTACTGC AGTOCCLTMG CTAGAGCTGC WITTGC OCALTGTGTM ITTCTTAGTC MTTGTGTG TTGGCAT0C GTGG=CTGC MCTGTCOT CGCGTTGG TCGTCTCTTC ILTGTTTTC TOSGTIT GGOGGAA 992 1040 1089 1136 1184 *1232 1280 1335 1395 1455 1 515 1575 1635 1695 1755 1790 Ago..:

A

INFORMATION FOR SEQ ID) 1:8: SEQUENCE CHAPCTERISTICS: CA) LENGTH: 387 amino acids TYPE: amino acid TOPOLOGY: linear WO 94/1 1516 WO 9411516PC1VUS93/09997 (ii) (Xi) 127 MOLECULE TYPE: protein SEQUENCE:.DESCRIPTION: SEQ ID NO:8: Met Gly Ala Gly Gly 1 5 Arg Hot Thr Glu Glu AZg Glu Lys Gin Glu is

S

iS...

.9 9 9.

9**S

S

*S 9 59 9 9 9* 9 5 *9 *95*

S.

9* 99 9 9 '.95.

Gin Gau His Asp Pro Ile Cys Gly Tyr 145 Glu Tyr Lou Pro Asn 225 Ala Txp Lou Lou Ala Ang Ala Thr Gly GMy Ala Ala Not 25 Lys Pro Pro Ph. Thr Lou GMy Gin Ile Lys 40 Cys Ph* Gin, Azq Set Val lou Lys Sir Phe so 55 Leu Val Ile Ala Ala Al. Lou Leu Tyr Ph.

70 75 Ala Lou Pro Ser Pro Lou Arg Tyr Ala Ala 65 90 Ala Gin Gly Cys Va1 Cys Thr Gly Val Tzp 100 105 Gly His His Ala Ph. Ser Asp Tyr Ser Lou 115 120 Lou Va1 Lou His Ser Ser Lou Net Val Pro 130 135 Ser His Ang Arg Hig His Ser Len Thr Gly 150 155 Va1 Phe Va1 Pro Lys Lys Lys Glu Ala Lou 165 170 Val Tyr Asn Asn Pro Val Gly-Arg Va1 Val 180 185 Thr Lou Gly Trp Pro Lou Tyr Lou La Thr 195 200 Tyr Pro Arn Ph. Ala Cys His Phe Asp Pro 210 215 Asp Arq Glu Arg Ala Gin Ile Ph. Val~ Her 230 235 Val Ala Phe Gly Lou Tyr Lys Lou Ala Ala 245 250 Va1 Val Avg Val Tyr Ala Val Pro Lou lou 260 265 Val Lou Ile Thr Tyr Lou Gin His Thr His 275 280 Gln Arg Bar Pro Lys Ala le Pro gar Tyr Va1 Va1 s0 Ala.Lou Ala Ile .Trp Pro Lou Tyr Va1 le Ala His 110 Lou Asp Asp Val 125 Tyr Ph. 8cr Trp 140 5er Lou Glu Arg Pro Tzp Tyr Thr 175 His le Val Val 190 Len Ala Ser Gly 205 Tyr Gly Pro Ile 220 Asp Ala Gly Va1 Ala Ph. Gly Val 255 le Val Asn Aa& 270 Pro Ser Lou Pro 285 Va1 Pro His le so fTp Glu Val Lys Asp 160 Pro Gin Arg Tyr Va1 240 fTp fTp His 99999 9 9 WO 94/11516 WO 9411516PCrIUS93/09987 128 Tyr Asp Sor Ser Glu Trp Asp Trp Jeu Arg Gly Ala Lou Ala Thr Met 290 295 300 Asp Arg Asp Tyr Gly XIe Lou Asn Arg Val Phe Rio Len Ile Thr Asp 305 310 315 '320 Thr His Val Ala Rio file Lou Pb. ger Thr Net Pro His Tyr His Ala 325 330 335 Met Glu Ala Thr Lys Al&'Xno Arg Pro noe Lou Sly Asp Tyr Tyr His 340 345 350 Ph. Asp Pro Thr Pro Val Ala Lys Ala Thr Usp Arg Glu Ala Gly Gin 355 360 365 Cys Ileo Tyr Val Glu Pro Glu Asp Arq Lye Sly Val Pbs Tsp Tyr Asn 370 375 380 Lys Lys Ph.

385 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 673 base pairs TYPE: nucleic acid STRANDEDNESS: -double *99 TOPOLOGY: linear MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Ricinus conununis (vii) IMMIEDIATE SOURCE: CLONE: pRF2-lC (ix) FEATURE: NAME/KEY:_ CDS LOCATION: 1. .673 WO 94/11516 W094/1516PCF/1JS93/09997 (xi) SEQUENCE DESCRIPTION: SEQ ID 14:9: TO M MT MCG CA OAT WOT 00 0 so 0 0@0 06* 0 **o Trp 1

TTG

Lou

CCT

Pro 000 Gly

ATC

lio

ACA

Th:

ART

Asn

TAT

Tyr

GAT

Asp

O

Ala 145 Val Met Ala His ASP Cys GlY 5 CTT OILT OAT GTh GTT GOT -CTT Lou Asp Asp Va1 Val Gly Lou TAT TTT TCh TGG AAA CAC LOC Tyr Ph, Sor Trp, Lys Rio Bev 40 TCC CTOGA ChOG hT ORA OTW So: Lou Glu Avg Asp Glu. Val -55 COT TGG TAT TCC MRA TAC CTC Arg fTp Tyr So: Lys Tyr Leu 70 ATT 0CC GTC AcA CTT TCA CT? le Ala Val Thr Lou Sot Lou 85 G"t TCA GGC AGO CCA TAT OAT Val Se: Gly Arg Pro Tyr Asp 100 GGC CCG ATC TAC MAT OALT CGC Gly Pro Ile Tyr Asn Asp Arg 115 120 OCT GOT OTT CTT OCT GTC ACT Ala Giy Val Lou Ala Val Thr 130 135 MG 000 CT? OCT TOG OTT GTC Lys Oly Lou Ala Try, Val Val 150 CAC CAT 0CC TTC AG? His'His Ala Pho $or 10 ATC CTA CAC TC TOT le Lou His gor Cys 25 CAT COC COA C&T CAT His Arg An; Hs Ri.

TYT OTT 0cC MRG AM Pho Val Pro Lye Lys s0 MAC MC CC? CCA GOT Asn Kan Pro. Pro Oly 75 a=C TOG CC? CTG TAC Gly fTpy Pro Lou Tyr 90 COOG TTC 0xCC TOC CAC Arg Phe Ala Cys His 105 GAG CGA ATC GAG ATA Glu AL; le Gin lt 125 TTT GO;T CTC TAC CA Ph* Gly Lou Tyr Gin 140 TOT GTA TAT GGA GMO Cys Va1 Tyr Gly Val 155 ATC AMA TTT CTG CAG Ile Tb: Pb. Lou Gln 170 TOG GAG TOO GAC TOO Ser Glu fTp Asp fTp 185 TAC 000 ATC ITO MAC Tyr Gly Ii. Lou Ann 205 OCT CAC CAC CT? TTC Ala Hio His Lou Phe 220 GAC TAT CM Asp Tyr Gin CC CT? GTC Lou Lou Val TOC MAC AcA go: Ann Th? AAA TCT AG? Lysev goare CGT ATC ATG Axq 11e Met CTA OCM TTC Lou Ala Ph.

TAT GAC CCA Tyr Asp Pro 110, TTC ATA TCA Phe Ile So: CTT OCT ATA Leu Ala lie CCA TTG TTG Pro Lou TAU 160 CAT ACT CAC His Tb: His 175 CTA AGh GOA Leu Arg Gly 190 AAG OTO TTC Lye Va1 Ph.

ACC ATO CC C Th: Met Pro GTO GTG MAT TCA Val Val ken Bar CCT WCA TTO CA Pro Ala Lou Pro 180S OCT CTA GCA ACT Ala Lou Ala Th: 195 CAT MAC ATA ACG His Asn le Thr 210 CT? OTT CTG Lou Va1 Lou TAT OAT TO Tyr Asp So: GAC A GAT Asp Arg Asp 200 ACT CMA OTA Thr Gin Val 215 WO 94/11516 WO 9411516PCr/LJS93/099S7 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS:.

LENGTH: 224 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Trp Val Net Ala Rio Asp Cya Gly 1 5 Rie Hio Ala rho Lou Lou Asp Asp Val 'Val 0 0000 0000 @0 0 0 *0 0000 00 00 0 00 0 0 0000 00 0 00 00 00 0 00 0 0 0000 0000 0 0000 0 @00000 0 0000 0 *000 Pro Tyr Pho 35 Gly 8cr Lou 50 Ile Arg Tzp 65 Thr le Ala Aen Val Ser Tyr Gly Pro 115 Asp Ala Gly 130 Ala Lys Gly 145 Val Val han Pro Ala Loeu Sex UrP Lys Asp LY3 70 Lou Pro Asn Ala ?rp 150 Lou Gly Lou Ile Lou Rio Ser 25 His Soc His Arg Arg His 40 Glu Val The Va1 Pro Lye 55 Tyr Lou Asn Aen Pro Pro 75 Ser Lou Gly Trp Pro Lou 90 Tyr Asp Arg Phe Ala Cys 105 Asp Arg Glu Arg Ile Glu 120 Val Thr Who Gly Lou Tyr 135 140 Val Val Cye Val Tyr Gly 155 Val Lou no -Thr Ph. Lou 170 hAp 8cr 8cr Glu Tzp, Asp 185 Arg Asp Tyr Gly Ile Lou 200 Gin Val Ala Hie Rio Lou 215 220 Bar Asp T~yr Gin Cye Lou Lou Val Hie Bar Ann Thr Lye Lye Ser Ser Giy Arg Ile Hit s0 Tyr Lou Ala Phe His Tyr Asp Pro 110 le Phe Ile 8cr 125 Gin Lou Ala Ile Val Pro Lou, Lou 160 Gin His Thr Hie 215 !rp Lou Arv Gly 190 Asn Lye Val Phe 205 Phe Thr Met Pro His Tyr Lou Ala Thr Val Asp.

195 Asn Ile Thr Asp Thr 220 WO 94/1 1516 PCFIUS93/09M8 131 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 1369 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLFCULE TYPE: CDNA (iii) MYOTB=TICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Ricinus comumunis n*WEIATE SOURCE: CLONE: pRFl97c-42

FEATURE:

NAME/KEY: CDS LOCATION: 184.,1347 10.0 SEQUENCE DESCRIPTION: SEQ ID NO:11:.

CGGCCGGGAT TOOGGTTTTC AC&CTAATTT GCAAAAAMTG CATG&TTTCA CCTCAMTC& AACACCACAC CTTATMKCTT AGTCTTAAG& GAGAGAGAGA GAGOAGACILT TTCTCTTCTC 120 !.TGAGOLTGAGC ACTTCTCTTC CAGKC7RTCGA &GCCTCGA AAGTOCTTGA GARGAGCTTG 160 A ATG GA GOT GOT GOT CGC ATG TCT ACT GTC ATA AMC AGC AC MhC 228 -ooo: Not Gly Gly Gly Soly Aeg Host got Tbr Val Ile So:ga Aen Iam.

0 1 5 10. 0 AT GAG AG ARA GGR GA AGC AGC CA.C CTG GAG C01 000 OCS cac ACG 276 So: Glu Lys Lys Sly Sly Sot got Rio Lou Glu Ang Ala Pro Rio Th: 25 MOG CCT CCT TAC ACA CTT GOT MAC CTC MAG AGA 9CC ATC O01 CDC CRT 324 Lys Pro Pro Tyr Tb: Lou Gly Aan Lou Lys Arq Ala Ile Pro Pro Hti.

40 *TGC TTT GMA COC TCT TTT GTG CGC TCL TTC TCC MAT TTT GCC ThT MhT 372 Cya Ph. Glu Arg Ser Ph. Val Aeg So: Phe Ser Aen Phe Ala Tyr Kan 55 WO 94/11516 WO 9411516PCr/US93/09987 ?TC TGC TTA AGT Phe Cys Lou Ser TAC ATC TCT TCT Tyr lie Ber Ser s0 TTC CL GOC TGC Ph. Gin Gly Cys GGC CAT CAT OCT Gly His His Ala 115 CTA AT? G=C CAT Lou Ile Val Kin 130 LOC CL? COC COC Ser ii Arg Arg 145 GTG TTC GC CO Val Ph. Val Pro 160 TTA AAC AAC CCG Lou Asn Asn Pro GGC TGG CCT Leu Gly ?rp Pro 195 GAT CGC OCT Asp Arg Phe Ala 210 AGA GAL AGG CT? Arg Giu A&g Lou 225 ACG TT? GTG CT? Thr Ph. Val Lou 240 ATG 06? ATC TAT Met Arg Ile Tyr AG ATC ACA TAC Met Ile Thr Tyr 275 CT? TCC TAC Lou Ser Tyr 110 CTC TCG TAT Lou Bar Tyr 65 CTC ACT GOT Lou Thr Sly AG? GAG TAT gar Giu 'fyr OCA CT? CTG Ala Lou Lou 135 CAT TCT MAC His Sir Ann 150 TChA MG TCO Set Lys Ser 165 06? CGA GTT Gly Arg*VaX TA? TA OCT Tyr Lou Ala CL? TAT OAT His Tyr Asp 215 AT? TAC AT? le Tyr le, 230 CGO GCT ACA Gln Ala Thr 245 GTG CCL TTG Val Pro Lou CKG CcC ACT Gin Bin Thr 132 TCG ATC GCC ACC AMC TTC CC? Bar Ile Ala Thr Lan The Phe Pro GTC OCT TOO CTG OTT TMC TOG CTC Val, Ala Trp Lou VJal Tyr fTp Lou 90 CT? TOG OTC ATC GOC OLT GA TOT Lou fTp Val le Oly Rin Giu Cys 105' 110 CAG CTOGOCT GA? G&C OTT GOC Gin Lou Ala Asp Asp le Val Gly 120 1L25 OTT CC& TAT TTT TCh TOG ARA TAT Val Pro Tyr Phe ftr fTp Lys Tyr 140 AT& GA TC? CTC =A CGA GAC GAU ile Gly Box Lou Glu Arg Asp Glu 155 AAA AT? TCA TOG TA? TCM MG TAC Lys le Bar Trp Tyr Ber Lys Tyr 170 175 TG ACAL CT? OCT 0CC ACG CTC CTC Lou Thr Lou Ala Ala Thr Lou Lou 185 190 TTC AL? GTC TCT GG? AGACT TAC Ph* Ann Val $or Giy Arg Pro Tyr 200 205 CCC TA? GGC CCA ATA TTT TCC GA Pro Tyr Sly Pro Ile The Ber Giu 220 OCT GAC CTC OGA AC TTT 0CC ACA Ala Asp Lou Gly le Phe Ala Thr 235 ATG OCA AUA 0M0 TTG OCT. TOG 0TA Met Ala-.Lys Gly Leu Ala Trp V.1 250 255 CT? AT? GTT AKC TOT TTC CT? OTT Loeu lie Val Ann Cys Ph. Lou Val 265 270 CRC CL OCT AT? CCL COC TAT GOC His Pro Ala Ile Pro Arg Tyr Giy 280 285 420 468 516 564 .612 660 '708 756 804 852 900 948 996 1044 1. rL WO94/1 1516 PCFIUS93/09987 133 TCA TCM GAA TWGG AT TG CMC CGG GGA GCA AT(; GTG ACT GTC OAT AGA 1092 Ser Sar Glu Trp Asp Trp Lou Arg Gly Ala Hot Val Thr Va1 Asp Arg 290 295 300 OAT TAT GOG GTG TTG AAT AAA GTA TTC CAT AAC ATT OCA GAC ACT CAT 1140 Asp Tyr Gly Va1 Leu kena Lys Val Ph. His Ann Ile Ala Asp Tthr Uis 305 310 315 WTA OCT CAT CAT CTC TT OCT ACA GTG CCA CAT TIC CAT QMCA TO GAG 1188 Val Ala His HIS Lou Ph. -Ala Thr Val Pro Kis Tyr His Ala Hot Glu 320 325 330 335 0C-ACT AAGCA ATC AMG CCT ATA ATG GGT GAG TAT TIC COG TAT GAT 123i Ala.Thr Lys Ala Ile Lys Pro 11. Not Gly Glu Tyr Tyr Ang Tyr Asp, 340 345 350 GOT ACC CCA TTT TIC lAG WCA TTG TOO AGG GAG GCA AG GAG TOC TTO 1284 Gly Thr Pro Pho Tyr Lys Ala Lou Trp Arg Glu Ala Lys Glu Cys Lou 355 360. 365 TTC OTC GAG CCA OAT GAA 00k OCT CCT ACA CAA GGC OTT TTC TOO TAC 1332 Phe Val Glu Pro Asp Glu Oly Ala Pro Thr Gln Gly Val Phe Trp Tyr .370 375 390 .:*CGG AAC ALO TAT TAAAAAAGTG TCATGTAGCC TG 1369 Arg Ass Lys Tyr *385 INFORMATION FOR SEQ ID NO:12: 9999 SEQUENCE CHARACTERISTICS: LENGTH: 387 amino acids TYPE: amino acid TOPOLOGY: linear 9(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID WO:12:ot Gly Gly Gly Oly Arg Met Ser Thr Val le Ile Ser Asn Asn Ser *15 10 5 Glu Lys Lye Gly Gly Sqr Ser His Lou Glu Arg Ala Pro His Thr Lyse 25 Pro Pro Tyr Thr Lou Oly Asn Loeu Lye Arg Ala Ile Pro Pro His Cys 40 Ph. Glu Arg Ser Ph. Val Ar; Ser Ph. Ser IAn Ph. Ala Tyr Asn Ph.

55 Cys Lou Sor Ph. Lou Ser Tyr Ser Ile Ala Thr Asn. Ph. Ph. Pro Tyr 70 75 s0 WO 94/11516 WO 9411516PCr/US93/09987 110 Ser Ser Pro Gin Gly Cys Ile 100 Rio His la Ph.

115 Ser Tyr Val Ala Thr Gly Lou Trp 105 Glu Tyr Gin Lou 120 Trp Lou Val Ile Ala ASP a.

a a a a a a 110 Val 130 -Rio Arg 145 Ph. Val Msn Len Gly Trp Ar; Ph.

210 Giu Axq 225 Phe Val Ar; Ile le Thr Ser Glu 290 Tyr Gly 305 Ala His Thr Lye Thr Pro His Arg Pro pro Pro Ala Leu Lou Tyr Tyr 275 Trp Val His Ala Phe 355 'Lou Lou Val sex Lys sor Lys Pro Sly Ar;

ISO

Lou Tyr Lou Cys His Tyr Gin Ile Tyr 230 Tyr Gin Ala 245 Sly Val Pro 260 Lou Gin His Asp Trp Lou Lou Asn Lys 310 Lou Ph. Ala 325 le Lye. Pro 340 Tyr Lys Ala 1135 got Lye Xle So: Trp 170 Val Lou .Thr Lou Ala 185 Ala Ph. Mno Val 8car 200 Asp Pro Tyr Gly Pro 215 le Ala Asp Lou Gly 235 Thr Met Ala Lye Gly 250 Lou Lou Ile Val Asn 265 ?hr His Pro Ala Ile 280 Arg Gly Ala Met Val 295 Val Phs Rie Len 110 315 Thr Val Pro Ris Tyr 330 le met Gly Glu Tyr 345 Lou Trp, Arg Glu Ala 360 Pro Tyr Ph.

Tyr Trp Lou Ph.

His Gin Cye Sly 110 Ile Val Gly Lou 12t ?Vp Lye Tyr Ser Arq Asp Gin Va1 160 $er Lye Tyr Lou 175 Thr Lou Lou Lou 190 Arg Pro Tyr Asp 205 Ph. Ser Gin Arg Ph. Ala Thr Thk 240 Ala Trp Vai met 255 Phe Lou Va1 met 270 Ar; Tyr Gly Ser 285 Val Asp Ar; Asp Asp Thr His Va1 320 Ala Net Gi li.

335 Ar; Tyr Asp Sly 350 Gin Cys Lou Phe 365 Ph. Trp Tyr Arg Va1 Giu 370 Pro Asp Glu Sly Ala Pro Thr Gin Gly Val 375 380 WO 94/11516 PCFUS93/097 135 Aen Lye Tyr 385 INFORMATION FOR SEQ ID NO:13: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLO.GY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (ix) FEATURE: NAME/XEY: misc feature LOCATION: ._23 OTHER INFORMATION: /product- "synthetic oligonucleotide"' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: *TGGGTATGCC AYGANTGYGG NCA 23 INFORMATION FOR SEQ ID NO:14: (1C) SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid TRANDEDNESS: single TOPOLOGY: linear (ii) MO)LECULE TYPE: ODNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc, feature, LOCATION: 1_.22 WO 94/11516 PMTUS93/09987 136 OTHER INFORMATION: /product- "synthetic oligcnucleotide" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AAARTGRTGG CACRTGNGTR TC INFORMATION FOR SEQ ID Ui). SEQUENCE CHARACTERISTICS: LENGTH: 2973 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) HYPOTHETICAL: NO ANTI-SENSE: NO (iv) (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana (vii) IMMEDIATE SOURCE: CLONE: pAGF2-6 (ix) FEATURE: NAME/KEY: exon LOCATION: 433..520 (ix) FEATURE:

ATT=GTAIL?

G&TTATAAAT

TGATATTAAA

TGOGGT&AAT

GGGGAAAGIA

NAME/KEY: intron LOCATION: 521..1654 (xi) SEQUENCE DESCRIPTION: SEQ. W NO:1IS: TCCTACATAT TTTA"&G&TT AGTTTG1LGTT TCC&YTCCATA CTTTACTAGT TTAAAKTACG TACTTTTUMA CTATAAAGTG AhACTAAGTA AATTAGA&CG AAGTTAhTGT TChCTGTTAT ATTTTTTTCA CAAGTAAA ATGGMTT AAAAATACCh GKTATTTTGA ATTGATTA&IL ?AGGTTGAA TAAG&GAGGA AAGAAGGTG GGGCOCAGTA TG&AGGGAA AGGTGTCTC AAATCATCTC Ia WO 94/11516 PCr/US93/0997

S.

S

S

S

TCTCTCTCTC TAOCTTCGAC CCACGGGCCG CCCCATCTG& CCACCKG&AG AhG&GLCoCA AGAGAGPCAG AGAGAG&GILG AGATTCTGOG GTTATTACG TATCG=cc !ACOTCK.GC TTCT~CTCA TTTCGATTT! GATCTT&?T COCCGCTCILC GAThGLTCTG CTTATACTcC CTCTGTTTC CTGTTTTTTT CTTTGOG C&?TA&TAAT rG&TGARCTC! CCLTTCAT& ATATGT!G& TTTC&CTTT TCwCa!?TTT TTLGILTCTTT ATTTTAT??T ATTTTCTGGT AAA&Gc&ThA ATTGTThT GTTAPA!GT&T ATCTGCTTCT ACTGTTGAAT CTTTCCTGGK A&LTACUTA!AAAGGhAA ACAAMAGTTT AGTTGGA&TC AATAATTC AGGLTCAG&T TTGCATGGAA AATTTTCTKG ATCOGTGTC CTGKTATATG ATGTCGACILA ATTCTGGTGG GCTGTTTG TCAACTTGGT TTTCAATACG ACALAGCAA6AC TGATGTTAAC CACAAGCAhG PICTTACTACT AGTCGTATTC TC&&ACGCR&T CTCTACTCTT TATTCCTTT GGTCCL CTGILTTTCCC ACTTTGGATC ATTTGTCTGA TTGTGCATGC TCTGTTTTT AGhILTTAATG GCTTGTTGA? TCTGCTT TTGGTTTTCT CGGTTCCTAC TTCTTcCC&G AAATCGGA AACOGCcTTT CTOGOTOGGA GhTCTGAGIL CATCOCTOG CTCTTTCTCC TACCTTATCh ACGTCGCCC C&ILTTACTTC TCTCTCCTcC TCiA.TTGGGC CTGTCAAGGC TGTGTCCTAA GTC&CCICGC ATTChGOGILC TACCAATGGC TGTcCCATTTA )AGCCCTGTC TCTTGCCATT CKCTCKAM TTAAPJJ.GG AGh&phGAG G&GrAGCTTC TTCTTCGThG GGTGTTChTC cCCLTCTcC&G GTCCGTCGCT TCTCTTCC&LT TCTTTCCKGT AGC!CGCT CTGGUTm TTACILTTCAA CCT!AC! OOTCTCG&TT hGACTGILT GTT!TTAT GTCTGTCRC CRAa"ToAm mTCTTT CTACCAAACG TTCTA&A?G ATTTGCTTTGP £CcATGT GOGTTGT AA&TTG&M AAAAAA TCAffTTTTG GMTThT CT TM ?TTTTTA=T CTA!TTGGTT .TTTILAGTA)L TATAG&?!CT CTTAAOOCC TT1cA=TAAA GCTCTTTGT TG&TCAG&LT G=TTAAG A&hTTTTAT T TTCTGTTTA& ATATCTAA CTTATACI TC ACTTCAACTG TTTTCI!?TTG ATTTGTGATT TCG&TCGCTG ARTTTTThAT PGLTGTGACC TGMCTATTA ACITCGTPITT CGTTTTTGTA TTTCTC=cT TMLGcCGTT TTTTCTALTTT GTGOG1aTCC CTTTCILCAAC AGACTCTCTT GATCOTAC CACTTGTTTC ATAAAM~T TOC&TkGTCT TGAGTTTTCA OC&GAMA GOGTGM&GGT G~AGA&TGC CCeRCLOCAC AP.AG6GTG COGTC& AGcAhTccc GOCOM~TG! TTCRAACOCT GTGATCAT T&TAOCTCA TOCTTCTACT CTCAGOCTCT CTCTTACTTG GCTTGcCC CTGMTACTG GGTAGCC CACGARTGCG TGGATGhCAC AGTTGGTCTT &TCTTcCCATT 360 420 480 540 So0 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620.

1680 1740 1800 1860 1920 1980 2040 I it WO 94/11516 PCr/US93/09987 ccTTOcTOCT

CTGG&?COCT

ACGGAATA

TOGGGTGC

GOCATCTT

CTGATGCOGG

TGGCCTcGhT

TGRTCACT

ACTGOCTCRG

TCC&ACAT

GRACRCCGTG

AGGAAGG

GAAGAAATTG

TTTAkTAATC

GTGGGAAGTT

CGVCCCTTmC TTCTCCTGQ& AGTATAGTCA TCGCCOTCAC CATTCCAAC& CGAAAGAT GAATTTG T~ccAAAGC& GRAACAG AICAiAGTGGT CCTCAACAAC CCTCTTGGRC GC&TCATGA! GTTALCXGTC CAGTTTGTcC CTTGTACT& GCCTTTAACG TCTCTGGCAG ACCOATGAC GGOTTcCTT CC~&CociW CCcCCA ATG&CGaGA ACGCCTCCRG LTATACCTCT TATTCTAGOC GTCTGTrG GTC!TTAG TTACGCTGCT GCACAROMG G&?CTGcCT TACGGhGTAC CGCTTCTGhT 1GTGATOG TCCTCGTCT CTTGCRGCKC ACTCATCOCc CGTTGOCTCR CTACGTCA TCA~.GTOGG GGOCTTTG GC!ACCOTAG ACAG&GaCTh CGA&TCTTG M1CAAGGTGT TACACACA CACGTGGCTC ATC&CCTGT CITCG&eAATG CCOCATTATA AGCTAAAG G=GAMWAG CAATTCTGOG AGACTATTAC CAGTCGALTG GTATGTGGCG ATGTKAAGG AGGCAAAG GTGTCTAT. GAGAcCGG TGACAAGAAA GGTGTGTACT GGTACARCAA TAAGTTATGA GGATGKTGGT TCG&CTTTTC TCTTGTCTGT TTGTCTTTTG TTAAAGAAOC TiTGCTCG TTATTGTCC TTTTTGTG TTATGACILTT TTGGCTGCTC ATTATGTTAT AGCGTTCAAA TGTTTTGGG;T CGG 2100 2160 2220 2280 2340 2400 .2460 2520 2580 2640 2700 2760 2620 .2880 2940 2973- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (iv) (ix) MOLECULE TYPE: CDNA HYPOTHETICAL: NO ANTI-SENSE: NO

FEATURE:

NAME/KEY: misc feature LOCATION: 1..23 OTHER INFORMATION: /product'- "synthetic oligonucleotide"

I

L

II

139- (xi) SEQUENCE DESCRIPTION SEQ ID NO: 16: GGGCATGTNG ARAANARRTG RTG INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..23 OTHER INFORMATION: /product "synthetic oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: GGGCATGTRC TRAANARRTG RTG Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

This Application is a Divisional Application of Australian Patent Application No.

54075/94 and the disclosures thereof are incorporated herein by way of reference.

Claims (51)

1. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50% or greater to the polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, or

2. The isolated nucleic acid fragment of Claim 1 wherein the amino acid identity is 60% or greater to the polypeptide encoded by SEQ ID NOS: 1, 3, 7, 9, 11, or

3. The isolated nucleic acid fragment of Claim 1 wherein the nucleic acid identity is 90% or greater to SEQ ID NOS: 1, 3, 5, 7, 9, 11, or

4. The isolated nucleic acid fragment of Claim 1 wherein said fragment is isolated from an oil-producing plant species. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a delta-12 fatty acid hydroxylase.

6. A chimeric gene capable of causing altered levels of ricinoleic acid in a transformed plant cell, said chimeric gene comprising a nucleic acid fragment of Claim 5, said fragment operably linked to suitable regulatory o• :sequences. 20 7. A chimeric gene capable of causing altered levels of fatty acids in a transformed plant cell, said chimeric gene comprising a nucleic acid fragment of any of Claims 1, 2, 3, said fragment operably linked to suitable regulatory sequences.

8. Plants containing a chimeric gene of Claim 6 or Claim 7. 25 9. Oil containing altered levels of unsaturated fatty acids obtained from seeds of the plants containing chimeric genes of Claim 8.

10. A method of producing seed oil containing altered levels of unsaturated fatty acids comprising: 9/11/00,mg9883 full claims,140 WO 94/11516 PCr/US93/09917 141 transforming a plant cell of an oil- producing species with a chimeric gene of Claim growing fertile plants from the transformed plant cells of step screening progeny seeds from the fertile plants of step for the desired levels of unsaturated fatty acids; and processing the progeny seed of step (c) to obtain seed oil containing altered levels of unsaturated fatty acids.

11. A method of molecular breeding to obtain altered levels of a fatty acid in seed oil of oil- producing plant species comprising: making a cross between two varieites of oil-producing species differing in the fatty acid trait; making a Southern blot of restriction *enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step and hybridizing the Southern blot with the 20 radiolabell-ed nucleic acid fragment of Claim 1.

12. A method of RFLP mapping comprising: making a cross between two varieties of plants; making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step and hybridizing the Southern blot with the radiolabelled nucleic acid fragments of Claim 1.

13. A method to isolate nucleic acid fragments encoding fatty acid desaturases and related enzymes, comprising: comparing SEQ ID NOS:2, 4, 6, 8, 10, or 12 and other fatty acid desaturase polypeptide sequences; 142 identifying the conserved sequences of 4 or more amino acids obtained in step a; desiging degenerate oligomers based on the conserved sequences identified in step b; and using the degenerate oligomers of step c to isolate sequences encoding fatty acid desaturases and desaturase-related enzymes by sequence dependent protocols.

14. An isolated nucleic acid fragment of Claim 1 comprising a nucleic acid sequence encoding a plant microsomal delta-12 fatty acid desaturase.

15. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a fatty acid desaturase or a fatty acid desaturase-related enzyme with an amino acid identity of 50% or greater to the polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11 or 15 wherein said isolated nucleic acid fragment is substantially as herein described with reference to at least one of the accompanying Examples.

16. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a delta-12 fatty acid hydroxylase, wherein said isolated nucleic acid fragment is substantially as herein described with reference to at least S: one of the accompanying Examples.

17. A chimeric gene capable of causing altered levels of ricinoleic acid in a transformed plant cell, wherein said chimeric gene is substantially as herein described with reference to at least one of the accompanying Examples. S: 18. A chimeric gene capable of causing altered levels of fatty acids in a transformed plant cell, wherein said chimeric gene is substantially as herein So 25 described with reference to at least one of the accompanying Examples.

19. Plants containing a chimeric gene capable of causing altered levels of ricinoleic acid or fatty acids in a transformed plant cell, wherein said plants are substantially as herein described with reference to at least one of the accompanying Examples.

20. Oil containing altered levels of unsaturated fatty acids wherein said oil is substantially as herein described with reference to at least one of the accompanying Examples. 9/11/00,mg9883 full claims,142 -143-

21. A method of producing seed oil containing altered levels of unsaturated fatty acids, wherein said method is substantially as herein described with reference to at least one of the accompanying Examples.

22. A method of RFLP mapping which method is substantially as herein described with reference to at least one of the accompanying Examples.

23. A method to isolate nucleic acid fragments encoding fatty acid desaturases and related enzymes, wherein said method is substantially as herein described with reference to at least one of the accompanying Examples.

24. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding a plant enzyme wherein said enzyme catalyses a reaction at carbon positions 6 and 7 numbered from the methyl end of an 18 carbon long fatty acyl chain, wherein positions 6 and 7 correspond to carbon positions 12 and 13 numbered from the carbonyl carbon of an 18 carbon long fatty acyl chain and further wherein said isolated nucleic acid fragment hybridizes to one of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11 and 15 under one of the following sets of conditions: hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, sodium dodecyl sulfate (SDS), 100 pg denatured calf thymus DNA and 50 0 C and wash twice with 2X SSC, 0.5% SDS at room 20 temperature for 15 min each, then wash twice with 0.2X SSC, SDS at room temperature for 15 min each and then wash twice with 0.2X SSC, 0.5% SDS at 50 0 C for 15 min each; hybridization in 6X SSPE, 5X Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 5% dextran sulfate, 100 Ig denatured calf thymus DNA at 50 0 C and wash twice with 2X SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2X SSC, SDS at room temperature for 15 min each and then wash twice with 0.2X SSC, 0.5% SDS at 50 0 C for 15 min each; or hybridization in 50% formamide, 5X SSPE 1% sodium dodecyl sulfate (SDS), 1% Denhardt's Reagent, 100 !pg denatured salmon sperm DNA at 42°C and wash twice with 2X SSPE, 0.2% SDS at S420C for 15 min each, then wash twice with 0.2X SSPE, 0.2% SDS B at 55 °C for 30 min each. 9/11/00,mg9883 full claims, 143 -144- The isolated nucleic acid fragment of Claim 24 wherein said fragment is isolated from an oil-producing plant species.

26. A chimeric gene comprising the nucleic acid fragment of Claim 5 operably linked to suitable regulatory sequences.

27. A chimeric gene comprising the nucleic acid fragment of Claim 24 operably linked to suitable regulatory sequences.

28. Plants comprising in their genome the chimeric gene of Claim 6 or Claim 7, or claim 24.

29. An isolated nucleic acid fragment of Claim 24 comprising a nucleic acid sequence encoding a plant microsomal delta-12 fatty acid desaturase. An isolated nucleic acid fragment comprising a nucleic acid sequence encoding an enzyme which catalyzes a reaction at carbon positions 6 and 7 numbered from the methyl end of an 18 carbon long fatty acyl chain, wherein positions 6 and 7 correspond to carbon positions 12 and 13 numbered from the carbonyl carbon of an 18 carbon long fatty acyl chain and further wherein the amino acid sequence comprising said enzyme contains at least one of the following amino acid sequences selected from the group consisting of: AIPPHCF, AWXXYW, HECGH, LLVPY, WKYSHR, and SHRRHH. 20 31. An isolated nucleic acid fragment encoding an enzyme which catalyzes a reaction at carbon positions 6 and 7 numbered from the methyl end of an 18 carbon long fatty acyl chain wherein positions 6 and 7 correspond to carbon positions 12 and 13 numbered from the carbonyl carbon of an 18 gi• carbon long fatty acyl chain wherein said isolated nucleic acid fragment 25 encodes a protein comprising any one of the amino acid sequences set forth in SEQ ID NOS: 2, 4, 6, 8, 10 or 12. o 32. An isolated nucleic acid fragment encoding an enzyme which catalyzes a reaction at carbon positions 6 and 7 numbered from the methyl end of an 18 carbon long fatty acyl chain, wherein positions 6 and 7 correspond to carbon positions 12 and 13 numbered from the carbonyl carbon of an 18 carbon long fatty acyl chain wherein said isolated nucleic acid fragment hybridizes to the isolated nucleic acid fragment of claim 31 under one of the following sets of conditions: 9/11/00,mg9883 full claims,144 -145- hybridization in 50 mM Tris, pH 7.6, 6X SSC, 5X Denhardt's, 0.5% sodium dodecyl sulfate (SDS), 100 Lpg denatured calf thymus DNA and 50°C and wash twice with 2X SSC, 0.5% SDS at room temperature for 15 min each, then wash twice with 0.2X SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2X SSC, 0.5% SDS at 50 °C for 15 min each; hybridization in 6X SSPE, 5X Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), 5% dextran sulfate, 100 ptg denatured calf thymus DNA at and wash twice with 2X SSC, 0.5% SDS at room temperature for min each, then wash twice with 0.2X SSC, 0.5% SDS at room temperature for 15 min each and then wash twice with 0.2X SSC, 0.5% SDS at 50 °C for min each; or hybridization in 50% formamide, 5X SSPE, 1% sodium dodecyl sulfate (SDS), 1% Denhardt's Reagent, 100 lg denatured salmon sperm DNA at 15 42 °C and wash twice with 2X SSPE, 0.2% SDS at 42 0 C for 15 min each, then wash twice with 0.2X SSPE, 0.2% SDS at 55°C for 30 min each.

33. The isolated nucleic acid fragment of Claim 24, Claim 30, Claim 31 or Claim 32 wherein the enzyme encoded is a 12-hydroxylase.

34. Seeds of the plant of Claim 28, which seeds carry the chimeric gene of claim 6, 7 or 24. The isolated nucleic acid fragment of Claim 24, wherein the plant enzyme is a 12-hydroxylase.

36. An isolated nucleic acid fragment comprising a nucleic acid sequence o: selected from the group consisting of: a nucleic acid sequence encoding a fatty acid desaturase or a fatty acid desaturase-related plant enzyme with an amino acid identity of or greater to the polypeptide encoded by SEQ ID NOS: 1, 3, 5, 7, 9, 11, or 15, or; a nucleic acid sequence or a part thereof which is useful in antisense inhibition or sense suppression of endogenous desaturase activity in a transformed plant wherein the nucleic acid encodes a polypeptide with an amino acid identity of 50% or greater to the polypeptide encoded by >SEQ ID NOS: 1, 3, 5, 7, 9, 11, or 15 or a part thereof. 28/12/00,mg9883 full claims,145 -146-

37. The isolated nucleic acid fragment of Claim 36 wherein the amino acid identity is 60% or greater to the polypeptide encoded by SEQ ID NOS: 1, 3, 7, 9, 11, or

38. The isolated nucleic acid fragment of Claim 36 wherein the nucleic acid identity is 90% or greater to SEQ ID NOS: 1, 3, 5, 7, 9, 11, or

39. The isolated nucleic acid fragment of any of Claims 36, 37 or 38 wherein said fragment is isolated from an oil-producing plant species. A chimeric gene comprising the nucleic acid fragment of Claim 36, 37 or 38 operably linked to suitable regulatory sequences.

41. A plant comprising in its genome the chimeric gene of Claim

42. Seeds obtained from the plant of claim 41.

43. A method of producing seed oil containing altered levels of unsaturated fatty acids comprising: transforming a plant cell of an oil-producing species with a chimeric gene of Claim growing fertile plants from the transformed plant cells of step screening progeny seeds from the fertile plants of step for the desired levels of C18 unsaturated fatty acids; and 20 processing the progeny seed of step to obtain seed oil containing altered levels of C 18 unsaturated fatty acids.

44. A method of producing seed oil containing altered levels of unsaturated fatty acids comprising: transforming a plant cell of an oil-producing species with a chimeric 25 gene of Claim 51; growing fertile plants from the transformed plant cells of step screening progeny seeds from the fertile plants of step for the desired levels of C18 unsaturated fatty acids; and processing the progeny seed of step to obtain seed oil containing altered levels of C 18 unsaturated fatty acids. A method of molecular breeding to obtain altered levels of a fatty acid in seed oil of oil-producing plant species comprising: making a cross between two varieties of oil-producing species Rdiffering in the fatty acid trait; 9/11/00,mg9883 full claims, 146 -147- making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step and hybridizing the Southern blot with the radiolabelled nucleic acid fragment of any of Claims 30, 31 or 32.

46. A method of molecular breeding to obtain altered levels of a fatty acid in seed oil of oil-producing plant species comprising: making a cross between two varieties of oil-producing species differing in the fatty acid trait; making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step and hybridizing the Southern blot with the radiolabelled nucleic acid fragment of any of Claims 36, 37 or 38.

47. A method of RFLP mapping comprising: making a cross between two varieties of plants; making a Southern blot of restriction enzyme digested genomic DNA isolated from several progeny plants resulting from the cross of step and 20 hybridizing the Southern blot with the radiolabelled nucleic acid fragments of any of Claims 30, 31 or 32.

48. A method of RFLP mapping comprising: making a cross between two varieties of plants; making a Southern blot of restriction enzyme digested genomic DNA 25 isolated from several progeny plants resulting from the cross of step and hybridizing the Southern blot with the radiolabelled nucleic acid fragments of Claims 36, 37 or 38.

49. The isolated nucleic acid fragment of Claim 36, 37 or 38 comprising a nucleic acid sequence encoding a plant microsomal delta-12 fatty acid desaturase. The isolated nucleic acid fragment of Claim 30, 31 or 32 wherein said ,A7 fragment is isolated from an oil-producing plant species. 9/11/00,mg9883 full claims,147 -148-

51. The chimeric gene comprising the nucleic acid fragment of Claim 30, 31 or 32 operably linked to suitable regulatory sequences.

52. The isolated nucleic acid fragment of Claim 30, 31 or 32 comprising a nucleic acid sequence encoding a plant rnicrosomal delta-12 fatty acid desaturase.

53. A method for altering fatty acids composition in seeds comprising: making a cross between a mutant line with altered fatty acid composition with a plant containing the chimeric gene of Claim growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plats of step for seeds containing altered fatty acid levels.

54. A method for altering fatty acids composition in seeds comprising: making a cross between a mutant line with altered fatty acid composition with a plant containing the chimeric gene of Claim 51; growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing altered fatty acid levels.

55. A method for reducing polyunsaturated fatty acids in rapeseed oil 00 comprising: 20 making a cross between a rapeseed variety with increased oleic acid content or reduced linolenic acid content with a plant containing the chimeric gene of Claim growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds S° 25 containing reduced polyunsaturated fatty acids.

56. A method for reducing polyunsaturated fatty acids in rapeseed oil **Goo: S°comprising: making a cross between a rapeseed variety with increased oleic acid content or reduced linolenic acid content with a plant containing the chimeric gene of Claim 51; growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing reduced polyunsaturated fatty acids. 9/11/00,mg9883 full claims, 148 -149-

57. The method of Claim 55 or 56 wherein the cross in is between a progeny plant derived from a seed comprising the Brassica variety having an oleic acid content of about 69% to 77%, based upon total extractable oil and belonging to a line in which the said oleic acid content has been stabilized for both the generation to which the seed belongs and its parent generation.

58. A method for reducing saturated fatty acids in rapeseed seeds comprising: making a cross between a rapeseed variety with increased oleic acid content with a plant containing the chimeric gene of Claim growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing reduced saturated fatty acids.

59. A method for reducing saturated fatty acids in rapeseed seeds comprising: making a cross between a rapeseed variety with increased oleic acid content with a plant containing the chimeric gene of Claim 51; growing fertile plants from seeds obtained from the cross; and 0(c) screening progeny seeds from the fertile plants of step for seeds containing reduced saturated fatty acids.

60. A method for reducing polyunsaturated fatty acids in soybean oil 20 comprising: oooo making a cross between a soybean variety with increased oleic acid content or reduced linolenic acid content with a plant containing the oooo chimeric gene of Claim S* growing fertile plants from seeds obtained from the cross; and 25 screening progeny seeds from the fertile plants of step for seeds containing reduced polyunsaturated fatty acids.

61. A method for reducing polyunsaturated fatty acids in soybean oil comprising: making a cross between a soybean variety with increased oleic acid content or reduced linolenic acid content with a plant containing the chimeric gene of Claim 51; growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing reduced polyunsaturated fatty acids. 9/11/00,mg9883 full claims, 149 -150-

62. A method for reducing saturated fatty acids in soybean seeds comprising: making a cross between a soybean variety with increased oleic acid content with a plant containing the chimeric gene of Claim growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing reduced saturated fatty acids.

63. A method for reducing saturated fatty acids in soybean seeds comprising: making a cross between a soybean variety with increased oleic acid content with a plant containing the chimeric gene of Claim 51; growing fertile plants from seeds obtained from the cross; and screening progeny seeds from the fertile plants of step for seeds containing reduced saturated fatty acids. DATED this 3 1 st day of October 2000 E. I. DU PONT DE NEMOURS AND COMPANY By their Patent Attorneys CALLINAN LAWRIE a a *o *go oo *oo **go* *,oe •«oo* *oe oo* *o*oo* 9/11/00,mg9883 full claims,150