FAE1 GENES AND THEIR USES

Abstract: The present invention provides polynucleotide sequences from FAE1 genes. FAE1 genes encode elongation enzymes which catalyze the conversion of C18 FAs to C20-C22 FAs. The polynucleotides of the invention are used to modify FAE1 gene expression and thereby modulate FA content in plant organs or parts, particularly seeds.

Description: FAEl GENES AND THEIR USES BACKGROUND OF THE INVENTION The present invention relates to plant molecular biology. In particular, it provides compositions and methods useful for modulating fatty acid synthesis in plants. Fatty acids (FAs) are the major constituents of acyl lipids in plant tissues. Acyl lipids are mainly present as triacylglycerols in the oil bodies of tissues which serve as food storage, such as seeds and the fleshy parts of fruits. These tissues are important commercial sources of fats and oils. Fatty acids are also found as glycolipids and phospholipids in other tissues, such as leaves, roots, or shoots, where they are integral components of the various cell membranes. The principal FAs are saturated or unsaturated monocarboxylic acids with an unbranched even-numbered carbon chain. The main saturated FAs are lauric (12:0, i.e., C12 chain with no double bonds), myristic (14:0), palmitic (16:0), and stearic (18:0). The main unsaturated FAs are oleic (18: 1), linoleic (18:2) and linolenic (18:3). Seed storage lipids accumulate mostly 16- and 18-carbon FAs. Oilseeds of the Cruciferae and a few other plants also accumulate C20 and C22 FAs, collectively referred to as very long chain fatty acids (VLCFAs) because of their relatively longer chain length compared to the more common FAs found in plants (see, in general, Stumpf, in Biochemistry of Plants, Vol. 9, Stumpf ed., Academic Press, New York, 1987) and Browse and Somerville, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506 (1991). The presence of VLCFAs in vegetable oils markedly affects their use. For example, enicic acid (22:1) has detrimental nutritional effects and, thus, is undesirable in edible oils. Rapeseed oil is naturally high in enicic acid, but through a concened breeding effon, canola lines that are almost devoid of enicic acid have been developed (Loof and Appleqvist, in Rapeseed, Appleqvist and Ohlson, eds. Elsevier Publishing, 1972). On the other hand, vegetable oils high in erucic acid have found many industrial uses, including use as diesel fuel and as a raw material for an array of products, -*t mcludmg paints, corrosion inhibitors, cosmetics, plasucs. pnarmaceuticals. and lubπcants (Murpny, Tibtecn 10:84-87 (1992). The biosynthesis of saturated FAs having a carDon chain up to C18 proceeds in the chloroplast via the sequential condensation of C2 units from acyl thioesters. FA synthesis is initiated by the condensauon of acetyl CoA ana maionvl ACP. catalyzed by the recently discovered enzyme /3-ketoacyi synthase III (KASIII) (Jaworski et al. , Plant Physiol. 90:41-44 (1989)). The enzyme ketoacyl synthase I is required for the eiongauon of saturated acyi-ACP from C4 to C16. The last elongation step in the chloroplast. from C16 to C18, is catalyzed by ketoacyl synthase II. Each condensauon is followed by three enzymatic steps, which involve reduction and dehydration of the ^-ketoacyl deπvative formed by the synthase and reduction of the double bond in the corresponding enoyl-ACP intermediate (Stumpf, 1987. supra). Eiongauon of the FA carbon chain from C18 to C22 occurs outside the chloroplast by the sequential addition of two C2 moieties from malonyl CoA to a C18 carbon skeleton, a reaction catalyzed by a paniculate acyl CoA elongase complex (Stumpf and Pollard, in High and Low Erucic Acid Rapeseed Oils, Kramer et al. eds. Academic Press, 1983). Whether the two eiongauon reactions are earned out by one or two different enzyme complexes is not clear (Taylor et al., Plant Physiol. 99: 1609-1618 (1992)). The same four reactions described above for the biosynthesis of C18 FAs are involved in the further elongation of C18 in plants: (i) condensauon of 18:1 CoA with malonyl CoA to form a /3-ketoacyl denvative, (ii) reducuon and (iii) dehydrauon of the 0-ketoacyl derivative, and (iv) reduction of the double bond (Creach and Lessi e JAOCS 70:1129-133(1993)). However, because of the difficulties in solubilizing membrane- bound enzymes, the elongase complex has not been well characterized. Elongases have been partially purified from several plants, including A Ilium pornun or leek (Bessoule et al., Arch. Biochem. Biophys. 268:475-484 (1989)), Lunaria annua or honesty (Fehling et al., Biochim. Biophys. Acta 1126:88-94 (1992)), and Brassica napus or rapeseed (Creach and Lessire, 1993, supra). In Arabidopsis, mutauons in a gene associated with fatty acid elongation, the FAEl gene, result in a deficiency in acyl chain eiongauon activities from C18 to C20 and C20 to C22, and in highly reduced levels of seed VLCFAs (James and Dooner, Theor. Appl. Genet. 80:241-245 (1990); Lemieux et al. , Theor. Appl. Genet. 80:234-240 (1990); James and Dooner Theor. Appl. Genet. 82:409-412 (1991); and Kunst et al. Plant Physiol. Biochem. 30:425-4343 (1992)). FA biosynthetic genes have been isolated by the conventional biochemical approaches of purifying the corresponding enzyme in order to generate antibodies or oligonucleotides with which to probe cDNA libraries. Among them are genes for ACP (Schmid and Ohlrogge, Plant Mol. Biol. 15:765-778 (1990)), KASII (International Publications WO92/03564 and WO93/ 10240), KASIII (Tai and Jaworski, Plant Physiol. 103L1361-1367 (1993)), stearoyl-ACP desaturase (International Publication No. WO91/18985), acyl-ACP thioesterases (U.S. Patent No. 5,298,421) enoyl-ACP reductase (Kater et α/. , Plant Molec. Biol. 17:895-909 (1991)), 3 -ketoacyl- ACP reductase (Klein et al. , Mol. Gen. Genet. 233: 122-128 (1992)), acyl-ACP: glycerol-3-P acyl transferase (Weber et al.. Plant Molec. Biol. 17: 1067-1076 (1991)). Others have been isolated on the basis of DNA homology to previously cloned genes from related species, e.g., genes for stearoyl-ACP desaturases (Knutzon et al. , Proc. Nail. Acad. Sci. USA 89:2624-2628 (1992)) or acyl-ACP: glycerol-3-P acyl transferases (Nishida et al., Plant Molec. Biol. 21:267-277 (1993)). Genes encoding FA biosynthetic enzymes in Arabidopsis have also been isolated. Examples include the FADS gene encoding an endoplasmic reticulum (ER) 18:2 desaturase (Arondel et al. , Science 258: 1353-1354 (1992)), FADS (Yadav et al., Plant Physiol. 103:467-476 (1993)), and the FAD2 gene, which encodes another ER enzyme, an 18: 1 desaturase (Okuley et al. The Plant Cell, 6: 147-158 (1994)). There have been no reports of the isolation of FAEl genes. Isolation of these genes would be particularly useful in modulating fatty acid synthesis in plants. These and other advantages are provided by the present invention. SUMMARY OF THE INVENTION The present invention provides isolated DNA constructs comprising a polynucleotide sequence from an FAEl gene. FAEl genes encode elongation enzymes which catalyze the conversion of C18 FAs to C20-C22 FAs. A preferred embodiment of the genes of the invention comprises sequences substantially identical to sequences which are, which contain, or which are contained within, SEQ. ID. No. 1. DNA constructs comprising the polynucleotides of the invention are used to modify FAEl gene expression and thereby modulate FA content in plant organs or pans, particularly seeds. Thus, a DNA construct of the invention may further comprise a promoter operably linked to the polynucleotide sequence. The promoter is preferably a plant promoter such as a seed-specific promoter. If suppression of an endogenous FAEl gene is desired, the polynucleotide sequence may be linked to the promoter in the sense or antisense orientations. The invention also provides transgenic plants (e.g. , Brassica plants) comprising a recombinant expression cassette which includes a plant promoter operably linked to the polynucleotide sequence. The transgenic plants exhibit altered FA content in one or more tissues. For use in plants which produce edible oils, the introduction of the recombinant expression cassettes preferably results in inhibition of an endogenous FAEl gene, resulting in plants with decreased VLCFA content. The invention further provides a method of altering FA content in a plant. The method comprises introducing into plant tissue a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence from an FAEl gene, in the sense or the antisense orientation. The promoter may be a tissue-specific promoter, e.g. , a seed-specific promoter. The expression cassette is typically introduced into the plant tissue using Agrobacterium or other standard means. The transformed plant tissue is regenerated into whole plants, whereby normally the regenerated plant transcribes the introduced polynucleotide sequence. The plants are then assayed and selected for altered FA content. The invention further provides methods of isolating an FAEl gene from a plant. The method may comprise probing a DNA library (e.g., a cDNA library) prepared from the plant with oligonucleotide probes comprising a polynucleotide sequence from an FAEl gene. Definitions The phrase "nucleic acid sequence" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes self-replicating plasmids, infectious polymers of DNA or RNA and non- functional DNA or RNA. The term "promoter" refers to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. The term "plant" includes whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants, as well as certain lower plants such as algae . It includes plants of a variety of ploidy levels, including polyploid, diploid and haploid. A "heterologous sequence" is one that originates from a foreign species, or, if from the same species, is substantially modified from its original form. For example, a heterologous promoter operably linked to a structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, is substantially modified from its original form. As used herein an "FAEl gene" is a gene encoding an enzyme, or a component of an enzyme, that catalyzes the conversion of oleic acid (18: 1) to eicosenoic acid (20: 1) and eicosenoic acid to enicic acid (22: 1). As used herein, a homolog of a particular FAEl gene (e.g. , SEQ. ID. No. 1) is a second gene in the same plant type or in a different plant type, which has a polynucleotide sequence of at least 50 contiguous nucleotides which are substantially identical (determined as described below) to a sequence in the first gene. It is believed that, in general, homologs share a common evolutionary past. A "polynucleotide sequence from an FAEl gene" is a subsequence or full length polynucleotide sequence of an FAEl gene which, when present in a transgenic plant, has the desired effect, for example, inhibiting expression of the endogenous FAEl gene. In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be "substantially identical" to a sequence of the gene from which it was derived. As explained below, these variants are specifically covered by this term. In the case where the inserted polynucleotide sequence is transcribed and translated to produce a functional polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically covered by the term "polynucleotide sequence from an FAEl gene" . In addition, the term specifically includes those full length sequences substantially identical (determined as described below) with an FAEl gene sequence and that encode proteins that retain the function of the FAEl enzyme. Thus, the above term includes variant polynucleotide sequences which have substantial identity with the sequences disclosed here and which encode enzymes capable of catalyzing the same reactions described above. In the case of polynucleotides used to inhibit expression of an endogenous gene, the introduced sequence also need not be perfectly identical to a sequence of the target endogenous gene. The introduced polynucleotide sequence will typically be at least substantially identical (as determined below) to the target endogenous sequence. Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The term "complementary to" is used herein to mean that the complementary sequence is identical to all or a portion of a reference polynucleotide sequence. Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted by the local homoiogy algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homoiogy alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein die portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" of polynucleotide sequences means that a polynucleotide composes a sequence that has at least 60% sequence identity, preferably at least 80% , more preferably at least 90% and most preferably at least 95% , compared to a reference sequence using the programs described above (preferably BESTFTT) using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucieotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 40%, preferably at least 60%, more preferably at least 90% , and most preferably at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is d e temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60°C. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration of the strategy used to isolate FA biosynthetic genes from Arabidopsis. Figure 2 shows a comparison of the nucleotide sequence of FAEl around die site of insertion of the transposable element Ac. Figure 3 is a diagram of the structure of the FAEl genomic region, including the location of the cDNA and of die Ac insertion site in fael -ml (Ac). Figure 4 is a compaπson of the ammo acid sequences of SEQ. ID. No. 2 and SEQ. ID. No. 4 using the BESTFTT sequence comparison program. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invenuon provides compositions and methods for regulating fatty acid synthesis in plants. The effect of one enzyme, FAEl, on FA synthesis in Arabidopsis has been elucidated by studying mutants of the gene encoding the enzyme. As noted above, a mutant deficient in the eiongauon of oleate (18: 1) and showing only 0.2% eicosenoic acid in its seed oil was described by James and Dooner Theor. Appl. Genet. 80:241-245 ( 1991) and Lemieux et al. Theor. Appl. Genet. 80:234-240 (1990). Based on segregation of this trait. James and Dooner and Lemieux et al. determined tiiat it is caused by a single nuclear mutauon in a gene that the latter authors designated as FAEl. The present invenuon provides cloned genes encoding the FAEl enzyme. FAEl genes useful in the present invention include an FAEl gene identified in Arabidopsis as well as homologs in other plants (either of the same or different genus or species). The present invention also provides recombinant vectors comprising polynucleotide sequences from an FAEl gene which can be used in variety of ways. Generally, the invention has use in modulating FA content in all higher plants. The invention tiius has use over a broad range of types of plants, including species from the genera Fragaria, Lotus, Medicago, Onobrychis. Trifolium, Trigonella, Vigna, Citrus, Limtm, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. More specifically, plants for which the invention may be used in modifying FA content include oil crops of the Cruciferae family: canola, rapeseed (Brassica spp.), crambe (Crambe spp.), honesty (Lunaria spp.) lesquerella (Lesquerella spp.) and odiers; the Compositae family :sunflower (Helianthus spp.), safflower (Carthamus spp.), niger (Guizotia spp.) and others; the Palmae family: palm (Elaeis spp.), coconut (Cocos spp.) and odiers; the Leguminosae family: peanut (Arachis spp.), soybean (Glycine spp.) and others; and plants of other families such as maize (Zea spp.). cotton (Gossypium sp. , jojoba (Simonasia sp.j, flax (Linum sp.), sesame (Sesamum spp.), castor bean (Ricinus spp.), olive (Olea spp.), poppy (Papaver spp.), spurge (Euphorbia, spp.) meadow foam (Limnanthes spp.) and cuphea (Cuphea spp.). As noted above. VLCFAs, in particular enicic acid, have detrimental nutritional effects and. thus, are undesirable in edible oils. Using standard techniques in plant molecular biology as described below, the production of enicic acid can be decreased in plants used for the production of edible oils, in particular rape, Brassica napus. Alternatively, vegetable oils high in VLCFAs, particularly enicic acid, have many industrial uses. Thus, the invention can be used to increase VLCFA content of plant oils by over-expression of FAEl . In addition, alcohols derived from the VLCFas by reduction of the carboxyl group will also increase in these plants. VLCF alcohols are esteπfied with VLCFAs in jojoba oil. and can be used as a substitute for sperm whale oil in high quality lubricants and a valuable carrier for medicines. For instance, crambe and jojoba, two sources of industrial oils valued for their VLCFA content, can be modified to increase production of tiiese FAs. The C20-C22 FA content of Crambe seed oil is about 50-60% 22:1 (Salunkhe and Desai, in Postharvest Biotechnology of Oilseeds pp 187-197 (CRC Press, Boca Raton, FL, 1986)) and jojoba seed wax, currently, about 54% 20: 1 +22:1 (Salunkhe and Desai, supra), could be increased by over-expression of FAEl. The polynucleotides of die invention can also be used to modify other desirable traits in plants. For instance, since the constitution of leaf surfaces affects their permeability, by changing the composition of the FAs that make up the leaf (surface) cuticular waxes, the drought tolerance of plants can be affected. Thus, any trait in which VLCFA synthesis is important can be altered using d e methods of the invention. FAEl polypeptides The enzymes of the invention share homoiogy with those of two other condensing enzymes that utilize malonyl CoA, chalcone synthase (CHS) and stiibene synthase (STS). A consensus sequence among die three enzymes was revealed from a comparison of their amino acid sequences performed witii die GCG computer programs (Devereux et al. , Nuc. Acids. Res. 12:387-395 (1984)). It consists of 17 amino acids spread over a 50 amino acid region close to me carboxyl end of the proteins (starting at position 392 in FAEl). This region is just upstream of the CHS-STS 12 amino acid "signature sequence" (Fliegmann et al.. Plant Mol. Biol. 18:489-503 (1992)), which does not occur in the proteins of the invention. The FAEl protein also shares homoiogy with a conserved region in CHS and STS which is close to the active site cysteine identified by Lanz et al.. J. Biol. Chem. 266:9971-9976 (1991)). However, this region of homoiogy {L-A-K-D-L-X(9)-L- V-V} does not overlap with the consensus sequence for the CHS/STS active site {G-C- (FY)-(GA)-G-G-T-X(2)-R} , but lies immediately next to its carboxyl end. The amino acid sequence of the proteins of the invention is also homologous to the ketoacyl synthase (KAS) III enzyme of E. coli and spinach, the condensing enzyme that initiates FA biosynthesis in bacteria and plants by coupling acetyl CoA to malonyl ACP. In particular, the FAEl protein disclosed here is 35% identical and 47% similar to the £. coli KASIII (encoded by ihefabH gene), and 32% identical and 46% similar to a KASIII from spinach over a 57 amino acid stretch that also starts at position 392 of FAEl. The FAEl enzymes of the present invention preferably possess catalytic activity which is substantially equal to or higher than the activity of die protein set forth in SEQ. ID. No. 2. The polypeptides of die present invention may be natural, i.e., including an entire native FAEl enzyme isolated and purified from a natural source, or may be synthetic. Such natural FAEl polypeptides may be isolated from plant material, using methods described in die scientific literature which has been referenced above. By "isolated," it is meant that d e FAEl proteins have been removed from their native environment, such as the plant tissue where they normally occur. Thus, die term "isolated" is meant to include die presence of a FAEl polypeptide as a heterologous component of a cell or other system, such as a microorganism expression host or a transformed higher plant. For example, the FAEl polypeptide of die present invention may be expressed in a microorganism host, such as bacteria or yeast, transformed or transfected witi a DNA construct which encodes the polypeptide. Usually, such expression in microorganism hosts will be a first step in producing a "purified" FAEl polypeptide. Alternatively, "isolated" FAEl polypeptides may be expressed in transformed higher plants, where die FAEl polypeptides will frequendy not be subjected to any form of purification. Such polypeptides, however, will be isolated in die sense tiiat they have been removed from their native environment, frequendy being the result of expression of a heterologous gene, although in some cases being the result of expression of a homologous gene under the control of a heterologous promoter. In the latter case, the homologous FAEl polypeptide may be expressed in plant tissues and at times other than would normally be the case. FAEl polypeptides may be isolated and purified from any natural source possessing significant amounts of a natural or native FAEl enzyme. Isolation and purification may be obtained by conventional chemical purification techniques, such as liquid chromatography, affinity chromatography, gradient centrifugation, gel electrophoresis. size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography, and the like. Such techniques are well described in the scientific and patent literature. See, for example. Scopes. Protein Purification. Springer- Verlag, New York (1982). Such techniques are suitable for isolation of FAEl polypeptides both from natural cellular sources and from recombinantly modified expression hosts, The FAEl enzymes of the present invention may be obtained in a substantially pure form. By "substantially pure," it is meant that the polypeptide will be present in an intermediate or final composition and a purity of at least about 50%, based on die weight of FAEl polypeptide present in die total weight of the composition (weight weight; w/w), and will be substantially free from interfering proteins and contaminants, particularly proteins and contaminants which interfere with the desired catalytic activity and/or which are toxic, immunogenic, or which otherwise prevent the desired use of a final product. Preferably, the FAEl polypeptides will be isolated or synthesized in a purity of at least about 60% w/w, more preferably at least about 70% w/w, and most preferably at least about 80% w/w. Often, even higher levels of purity may be obtained, witii 90% w/w, 95% w/w, or higher usually being achievable. Very high levels of purity, typically above 98% w/w and most preferably above 99% w/w, can also be obtained. Syntiietic FAEl polypeptides according to the present invention may be produced by eitiier of two general approaches. First, polypeptides having fewer tiian about 200 amino acids, usually fewer than about 150 amino acids, and preferably fewer dian about 100 amino acids, may be synthesized, e.g., by die well known Merrifield solid-phase method where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156). Commercial systems employing such solid phase techniques for the automated synthesis of polypeptides are available from vendors, such as Applied Biosystems, Inc. , Foster City, California. The second and generally preferred method for synthesizing FAEl polypeptides according to the present invention involves the expression in cultured cells of recombinant DNA molecules encoding all or a desired portion of a FAEl protein. The recombinant DNA molecule may incorporate either a natural or synthetic gene, with natural genes and cDNA being obtainable from plant seed material by conventional methods, as described in the literature cited above. The isolation of polynucleotides encoding a desired FAEl is descπbed in detail below. Polynucleotides encoding FAEl The Example section below, which descnbes the isolation and characteπzauoπ of an FAEl gene in Arabidopsis is exemplary of a general approach for isolating genes of the invenuon. Isolation of this gene allows one of skill to readily isolate homologous genes in Arabidopsis and other plant species. The isolated genes can then be used to construct recombinant vectors for altering FAEl gene expression in transgenic plants. Generally, the nomenclature and die laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and die like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989). The isolation of FAEl genes may be accomplished by a number of techniques. For instance, transposon tagging of an FAEl gene can assist in the isolation of the relevant gene. Transposon tagging involves introducing a transposon into the plant which leads to a mutation of the target gene and a detectable phenotypic change in the plant. Using a probe for the transposon, die mutant gene can then be isolated. Using the DNA adjacent to the transposon in the isolated mutant gene as a probe, me normal wild type allele of the target gene can be isolated. See, e.g. , Haring, et al., Plant Mol. Biol. 16:449-469 (1991) and Walbot, Ann. Rev. Plant Mol. Biol. 43:49-82 (1992). As shown below, a particularly useful transposon tagging system is that disclosed in U.S. Patent No. 5,013.658. An alternative method uses oligonucleotide probes to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from the desired organ, such as a seed, and a cDNA library which contains the FAEl gene transcript is prepared from the mRNA. Alternatively, cDNA may be prepared from mRNA extracted from other tissue types (organs) in which FAEl genes or homologs are expressed such as seeds, fruits, leaves, stems, and roots. The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned FAEl gene such as that shown in SEQ. ID. No. 1. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. The use of such hybridization techniques for identifying homologous genes is well known in the art and need not be described further. Alternatively, polynucleotides may be syndiesized by well-known techniques as described in die technical literature. See, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained eitiier by synthesizing the complementary strand and annealing the strands togetiier under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. The isolated sequences prepared as described herein, can be used in a number of techniques to suppress endogenous FAEl gene expression (i.e. , to inhibit elongation of C18 FAs to C20 and C22). For instance, antisense technology can be conveniendy used to inhibit FAEl gene expression. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such tiiat the antisense strand of RNA will be transcribed. The construct is tiien transformed into plants and d e antisense strand of RNA is produced. In plant cells, it has been suggested tiiat antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g. , Sheeny et al.. Proc. Nat. Acad. Sci. USA. 85:8805-8809 (1988), and Hiatt et al.. U.S. Patent No. 4,801,340. The nucleic acid segment to be introduced generally will be substantially identical to at least a portion of the endogenous FAEl gene or genes to be repressed. The sequence, however, need not be perfectly identical to inhibit expression. The vectors of the present invention can be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homoiogy or substantial homoiogy to the target gene. For example, suppression of the FAEl gene shown in SEQ. ID. No. 1 may serve to impose the same suppressive effect on other FAEl genes with sufficient identity. Similarly, segments from FAEl genes from Arabidopsis can be used to inhibit expression of homologous genes in related plant species such as member of the genus Brassica or as a means to obtain the corresponding sequences to be used to suppress the endogenous Brassica gene. For antisense suppression, the introduced sequence also need not be full length relative to eidier the primary transcription product or fully processed mRNA. Generally, higher homoiogy can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homoiogy of non-coding segments may be equally effective. Normally, a sequence of between about 30 or 40 nucleotides and about 2000 nucleotides should be used, diough a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred. Catalytic RNA molecules or ribozymes can also be used to inhibit expression of FAEl genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, diereby functionally inactivating d e target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon diem, thereby increasing the activity of the constructs. A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs which are capable of self- cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper vims (satellite RNAs). Examples include RNAs from avocado sunblotch viroid and die satellite RNAs from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottie virus, solanum nodiflorum mottle virus and subterranean clover motde virus. The design and use of target RNA-specific ribozymes is described in Haseloff et al. Nature. 334:585-591 (1988). Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been recendy shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990), and U.S. Patent No. 5,034,323. 5,231,020, and 5283, 184. Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence. The introduced sequence generally will be substantially identical to d e endogenous sequence intended to be repressed. This minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, tiiough about 95% to absolute identity would be most preferred. As witii antisense regulation, the effect should apply to any otiier proteins witiiin a similar family of genes exhibiting homoiogy or substantial homoiogy. For sense suppression, die introduced sequence, needing less than absolute identity, also need not be full length, relative to either the primary transcription product or fully processed mRNA. This may be preferred to avoid concurrent production of some plants which are overexpressers. A higher identity in a shorter tiian full length sequence compensates for a longer, less identical sequence. Furthermore, die introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Normally, a sequence of the size ranges noted above for antisense regulation is used. Isolated sequences prepared as described herein can also be used to introduce FAEl expression or to enhance or increase endogenous FAEl gene expression (i.e. , to increase production of VLCFAs). Where overexpression of the FAEl gene is desired, an FAEl gene from a different species may be used to decrease potential sense suppression effects. For instance, the Arabidopsis FAEl gene can be used to increase expression in Brassica. One of skill will recognize that the polypeptides encoded by the FAEl genes, like other proteins, have different domains which perform different functions. Thus, the FAEl gene sequences need not be full length, so long as the desired functional domain of the protein is expressed. Modified protein chains can also be readily designed utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail, below. For example, the chains can vary from the naturally occurring sequence at the primary structure level by amino acid substitutions, additions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain. To use isolated FAEl sequences in the above techniques, recombinant DNA vectors suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in die technical and scientific literature. See, for example, Weising et al. Ann. Rev. Genet. 22:421-477 (1988). A DNA sequence coding for the desired FAEl polypeptide, for example a cDNA sequence encoding a full length protein, will preferably be combined with transcriptionai and translational initiation regulatory sequences which will direct the transcription of the sequence from the FAEl gene in the intended tissues of die transformed plant. For example, for overcxpression, a plant promoter fragment may be employed which will direct expression of FAEl in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or V- promoter derived from T-DNA of Agrobacterium tumafaciens, and otiier transcription initiation regions from various plant genes known to those of skill. Alternatively, the plant promoter may direct expression of the FAEl gene in a specific tissue or may be odierwise under more precise environmental or developmental control. Such promoters are referred to here as "inducible" promoters. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions, elevated temperature, or the presence of light. Examples of promoters under developmental control include promoters that initiate transcription only in certain tissues, such as fruit, seeds, or flowers. For example, the use of a napin promoter of Brassica napus (Lee et al. Proc. Natl. Acad. Sci. USA 88:6181-6185 (1991)) or the promoter from the Arabidopsis FAEl gene can direct expression of the FAEl polypeptide in seed. If proper polypeptide expression is desired, a polyadenylation region at the 3'-end of the FAEl coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The vector comprising the sequences from an FAEl gene will typically compπse a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin. G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosluforon or Basta. Such DNA constructs may be introduced into die genome of die desired plant host by a variety of conventional techniques. For example, me DNA construct may be introduced direcdy into die genomic DNA of die plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DNA constructs can be introduced direcdy to plant tissue using ballistic methods, such as DNA particle bombardment. Alternatively, die DNA constructs may be combined widi suitable T- DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of die Agrobacterium tumefaciens host will direct d e insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Microinjection techniques are known in the art and well described in the scientific and patent literature. The introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al. Embo J. 3:2717-2722 (1984). Electroporation techniques are described in Fromm et al. Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al. Nature 327:70-73 (1987). Agrobacterium tumefaciens- eάizteά transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, for example Horscn et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 ( 1983). Transformed plant cells which are denved by any of the above transformauon techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired E_4£7-controlled phenotype. Such regenerauon techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically reiving on a biocide and/or herbicide marker which has been introduced together with the FAEl nucleotide sequences. Plant regenerauon from cultured protoplasts is descnbed in Evans et al.. Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press. Boca Raton. 1985 Regeneration can also be obtained from plant callus, explants. organs, or parts thereof. Such regeneration techniques are descnbed generally in Klee et al. Ann. Rev. of Plant Phvs. 38:467-486 (1987). The methods of the present invenuon are particularly useful for incorporating the FAEl genes into transformed plants in ways and under circumstances which are not found naturally. In particular, die FAEl polypeptides may be expressed at times or in quantities which are not characteristic of natural plants. One of skill will recognize that after die expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon die species to be crossed. The effect of die modification of FAEl gene expression is conveniendy detected by analyzing FA content of plant material from the desired plant. Briefly, lipids are extracted from the plant material (e.g. , seeds), FAs are cleaved from the triacylglycerol, and analyzed by gas chromatography as described for instance in James and Dooner Theor. Appl. Genet. 80:241-245 (1990). In addition, antisense or sense suppression of the endogenous gene can be detected by reduction of mRNA levels as measured by, for instance, Northern blots. Plant suppressants of the invention have lower levels of erucic acid less ti an about 2% (of total FA seed content), preferably lower tiian about 1 %, more preferably lower than about 0.5% , and most preferably lower than about 0.1 %. Overexpressing plants of the invenuon have levels of erucic acid substantially higher than the conesponding untransformed plant, e.g. , at least 10% higher, preferably at least 20% higher, more preferably at least 30% higher, and most preferably at least 40% or 50% higher. Prefened resultant levels of erucic acid in overexpressing Brassica plants of the invention are at least about 40% (of total FA seed content), preferably at least about 50% . more preferably at least about 60% . Even higher levels may be obtained witii otiier oil producing plants, e.g. , in order of increasing preference at least about 70%, 80% or 90% . Erucic acid may be extracted from overexpressing plants of the invention using known methods. See Salunkhe and Desai (1986), supra. The following Examples are offered by way of illustration, not limitation. Example 1 Isolation and sequencing of the FAEl gene from Arabidopήs The strategy used to isolate FA biosynthetic genes from Arabidopsis is illustrated in Figure 1. In this work, transposon tagging was done widi the autonomous maize element Ac. A. Mutant creation and Ac transformation 1. Mutant Formation A collection of mutants was induced and characterized in order to genetically define loci that affect FA composition in Arabidopsis seeds (obtainable from the Arabidopsis Biological Resource Center at Ohio State University) as described by James and Dooner ( 1990), supra. 2. Ac transformation 68 independent single locus Arabidopsis transformants carrying Ac in the T-DNA were generated using the methods of Keller et al. , Genetics, 131:44-459 (1992). This number provides > 95 % confidence tiiat any gene in die Arabidopsis genome will be no farther than 15 cM from an introduced Ac, assuming random integration of d e T-DNA in die genome and a < 600 cM genetic map (Koorneeff et al. , in Genetic Maps, S.J. O'Brien ed. (Cold Spring Harbor Press, Cold Spring Harbor (1992)). In Arabidopsis, as well as in maize and otiier organisms, transposons of the Ac/Ds family transpose preferentially to sites linked to die donor site (Keller et al. , Theor. Appl. Genet. 86:585-588 (1993)). Therefore, in order to tag a specific gene, it is preferable to initiate die tagging experiment with an Ac (or Ds) element linked to die gene of interest. This approach, referred to as directed tagging, requires mapping of die T-DNAs relative to die target loci. Scoring for the presence or absence of the T-DNA was facilitated by die Hyg-R (hygromycin resistance) transformation marker. The number of plants with transposed Ac (trAc) elements required to isolate a specific FA mutation was tiierefore gready reduced relative to that required in a random transposon tagging approach. 3. T-DNA localization (mapping) Of twenty-four T-DNAs localized to one of the five Arabidopsis chromosomes, three were linked to FAEl. In transformants K805, B116. and C231, the T-DNAs were located respectively, 15, 22, and 40 cM from FAEl. FAEl is loosely linked to the RFLP marker 518 which was mapped to chromosome 4 by Chang et al., Proc. Natl. Acad. Sci. USA 85:6856-6860 (1988). Linkage of die B116 T-DNA to marker 518 was confirmed by a bulked segregant analysis procedure (Michelmore et al. , Proc. Natl. Acad. Sci. USA 88:9828-9832 ( 1991)) in which paired DNA samples obtained from pooied homozygous Hyg-RIHyg-R and +/+ segregants from a Hyg-R/+ (WS x Columbia) heterozygote were scored for segregating RFLPs. A chromosomal location was assigned to 24 single locus, c-containing T-DNA insertions by the combination of two-point crosses and bulked segregant analyses. 4. Make UP of T-DNA In the pJJ4404 construct used in the derivation of transgenic line B116, Ac lies in the 5' untranslated region of the SPT (streptomycin phosphotransferase) gene (Jones et al.. Science 244:204-207 (1989); Keller et al.. Plant Mol. Biol. 21: 157-170 (1993)). Somatic excisions of Ac during the development of the cotyledons can be detected as green sectors on a white background in Arabidopsis seedlings germinated in streptomycin. Germinal excisions of Ac give rise to SPT' fully green seedlings, about half of which carry a trAc element somewhere in the genome (Dean et al. , The Plant Journal 2:69-81 (1992); Keller et al. , Genetics 131:449-459 (1992)). Therefore, SPTxAc constitutes an efficient marker for selecting plants that have undergone transposition. Germinal selections recovered from one plant may derive from a common premeiotic event and carry the same transposed element. To avoid extensive sampling of duplicates for die same transposition event, in general, no more than four green seedling selections from any one plant were transferred to die greenhouse . B. Identification of a new fael mutation 1. Isolation and characterization of germinal selections Green SPT' seedling were selected on streptomycin (Jones et al., (1989), supra) grown to maturity in the greenhouse, and the FA composition of tiieir seeds was analyzed by gas chromatography (GC) as described previously (James and Dooner (1990), supra). Because fael and several other mutations affecting seed FA composition are codominant (James and Dooner ( 1990); Lemieux et al., (1990), supra) they can be identified in die heterozygous condition, a clear advantage when dealing witii a difficult phenotvpe, such as a chromatographic profile. Selections were made from lines in which Ac was either linked or unlinked to FAEl. Table 1 gives die number of SPT' selections analyzed, the number of plants which produced them, and d e location of Ac relative to FAEl in each line. Also given are estimates of the minimum and maximum number of independent Ac reinsertions screened, assuming a 50% Ac reinsertion frequency (Keller et al. ( 1992), supra). The minimum number, i.e.. the number screened if all the green sibs were derived from the same transposition event, conesponds to half the number of parent plants which produced green seedlings. The maximum number, i.e., the number screened if all the sibs resulted from independent transposition events, corresponds to half the number of selections analyzed. The actual number of independent Ac reinsertions assayed lies somewhere between the two values since both clonal and single Ac transposition events can be recovered from the same plant (Keller et al. 1992). Table 1. SPT' revertants analyzed for fatty acid composition K805 15 cM 1522 660 330-761 B1 16 22 cM 721 172 136-361 Subtotal Linked 2243 932 466-1 122 C201 Unlinked 2107 324 162-1054 B246 Unlinked 680 227 114-340 A018 Unlinked 273 91 45-136 Subtotal Unlinked 3060 642 321-1530 a Assumptions: 50% reinsertions among SPT' selections. Minimum, all selections clonal. Maximum, all selections independent. 2. Identification of Fael-G309 A total number of 721 SP selections from B116 were analyzed. Plants from the B116 line produced an average of > 1 % green selections per plant, but the percentages varied greatly from plant to plant. One selection. G309, produced seed witii the reduced 20: 1 content typical of an FAEllfael heterozygote. Upon selfing, one quarter of its progeny had a more extreme seed FA composition, indistinguishable from that of the EMS- induced mutant fael-2 (refened to as 9A1 in James and Dooner, 1990). The new mutation was tested for allelism with fael -2 . Because the mutants failed to complement, the provisional designation fael-G309 was assigned to the new mutant. The FA compositions of fael-G309 homozygous, heterozygous and wild-type seeds are presented in Table 2A; those of fael-2 and fael- 21fael-G309 seeds, in Table 2B. Table 2. Percent fattv acid composition of seeds from various genotypes Sample 16:0 18:0 18: 1 18:2 18:3 20:0 20: 1 22: 1 5.1 1.6 10.9 31.2 20.6 1.5 24.2 2.3 5.9 2.2 19.1 33.6 21.2 0.9 14.5 1.0 6.4 2.4 26.1 38.7 25.3 0.4 0.1 0.0 B. fael -2/ fael -2 8.5 2.9 30.8 30.1 26.9 0.0 0.4 0.0 fael-2/fael-G309 6.6 2.5 32.9 33.4 22.8 0.6 0.7 0.0 C. Evidence that the new fael mutation is tagged by Ac 1. Cosegregation of the fae mutant phenotvpe with an Ac- hybridizing bf-pςl DNA analysis of the fael -G309 deπvative showed the presence of a new ic-hybπdizing band. DNA extraction and DNA blot analysis were performed as previously descnbed (Keller et al. ( 1992), supra). The joint segregauon of this new Ac band witii ihefael mutation was tested in the self progeny of an fael -G3091 Fael heterozygote. Segregating individuals were scored as Fae II Fael, Fae II fael or fael ι ael by GC analysis of their seed FA composition and as Acl(-) or +/+ by DNA gel blot analysis. Thus, six genotypic classes could be distinguished. DNA from 54 individuals was digested with Hindlll and hybridized with a probe from the 5' end of Ac (Kunze e t al., EMBO J. 6: 1555-1563 (1987)). Results of these hybridizauons showed two /ic-hybπdizing bands segregating in the progeny, a 3.3-kb and a 2.4-kb band. The former represents the newly transposed Ac (trAc) and the latter, the Ac at the SPT::Ac resident site in the T-DNA (the selfed parent was the oπginal G309 green selection, SPT'ISPT::Ac). The results of the cosegregation analysis are presented in Table 3A. All of the individuals carrying the new trAc were either homozygous or heterozygous for the new fael mutation. Conversely, all of the individuals that lacked the trAc were wild-type. Therefore, no recombinants were recovered flinkage X 2 =54, P <0.001). DNA from the region flanking die 5' end of die trAc in the fael -ml (Ac) mutant was generated for cloning by inverse PCR (Ochman et al. , Genetics 120:621-623 (1988)). DNA was obtained from progeny o(fael-G309 tiiat contained the 3.3-kb Ac- hybridizing Hindlll fragment that cosegregated witii die fael phenotype, but lacked the smaller 2.4-kb Ac-hybridizing band. Approximately 0.5 ug of genomic DNA was digested witii Hindiπ and ligated overnight at 16° C under dilute conditions (200 ul reaction volume) to favor circularization of the Hindm fragments. Primers FL125, oriented outward from the 5' end of Ac (CGGTTATACGATAACGGTCG) and JK30, just 5' from die first Hindlll site in Ac (GTACGATGAAGTGGTTAGCC) were used to amplify the 1.5-kb of genomic DNA flanking the 5' end of the trAc. This flanking DNA was used to reprobe die same DNA gel blots. As expected, die new probe detected the same 3.3-kb Λc-homologous fragment and, in addition, a new fragment of about 1.8 kb. If Ac had. in fact, tagged the FAEl gene, all the segregating fael mutant plants should be homozygous for the 3.3-kb band, all Faellfael plants shouid be heterozygous for the 3.3-kb and the 1.8-kb band and all wild-type plants should be homozygous for the 1.8-kb band. The results of the analysis are presented in Table 3B. 53 of the 54 segregants fit the expectation. The one exception (segregant #5) was heterozygous for the 3.3-kb trAc band. There are two plausible explanations for this exception. ( 1) The new fae mutation is not tagged by the trAc, but is closely linked to it. and this individual is a recombinant between the fael mutation and the trAc. (2) The new fael mutant is tagged by the trAc and this individual is the product of an Ac excision that did not restore gene function. Segregant #5 also had a new lc-hybndizing band, which was absent in the other 53 sibs (data not shown), an observation suggesting that it might have originated by secondary transposition of Ac. Table 3. Segregation data for the self-progeny of a Fael /fael : trAc I ■ + ■ heterozygote A. DNA from segregants scored with an A_ς_ probe trAc genotype FAEl genotype trAcl(-) +1 + Total Fael /Fael 0 9 9 Fael /fael 29 0 29 fael /fae l 16 0 16 45 54 B. DNA from segregants scored witii probes for A_ς_ and the flanking DNA 15 30 54 2. Reversion of the fael mutant To confirm that the fael-G309 mutation was tagged by Ac, progeny of mutant plants were screened for putative revertants, i.e.. individuals with an intermediate seed FA composition, which could then be examined for excision of Ac from the cloned DNA. A total of 1052 offspring from tour fael- G309 plants were screened and three putative revertants to wild-type were identified on the basis of an intermediate seed FA composition, typical of Fael/fael heterozygotes (12-15% 20:1). Using DNA gel blot analysis, all three exceptions were found to be heterozygous for the 3.3-kb band in die original mutant allele and a 1.8-kb wild-type-sized band. This is the result expected if these individuals originated from Ac excision events that restored gene function. The DNA around the trAc insertion site in the wild-type progenitor allele, the fael -G309 mutant, and the three putative revertants was amplified by the polymerase chain reaction procedure described in Saiki (1990) and sequenced. DNA was sequenced with eitiier the Sequenase kit (U.S. Biochemical) or the fmol kit (Promega) following the recommendations of the manufacturers. A comparison of the sequences is presented in Figure 2. In general, when Ac excises it leaves behind an 8 bp "footprint" with occasional deletion or addition of bases to restore the reading frame number (a multiple of three). However, somewhat unexpectedly, die DNA sequences of the three putative revertants were identical to that of d e wild-type progenitor. The sequence of the putative FAEl protein around die site of insertion of Ac may be intolerant of amino acid changes, so that only the rare events that restore not just the correct reading frame but the original sequence are selected as revertants. If so, segregant #5, the exception from die cosegregation analysis (Table 3B) would represent an Ac excision event that did not restore the original amino acid sequence and, thus, failed to restore gene function. The DNA sequence of segregant #5 (Figure 2) revealed an 8bp footprint at the Ac excision site, indicating that this exception had originated by an Ac excision that created a frame-shift mutation and knocked out gene function. Thus, the analysis of revertants and of die one null exception in die cosegregation test confirms tiiat the fael- G309 mutant arose by insertion of Ac into the FAEl gene, that it is unstable, and that it can give rise to new alleles at the FAEl locus. Hence, it has been given the official designation fael -ml (Ac) to denote that it is the first mutable allele of the FAEl locus isolated and that it arose by insertion of the transposon Ac. D. Function of the FAEl gene in Arabidopsis 1. Expression of FAEl in developing seed Since VLCFAs accumulate in seeds, but not in leaves of Arabidopsis (Lemieux et al. (1990), supra), FAEl should be expressed preferentially in seeds. Expression of the FAEl gene was assayed by gel blot analysis of RNA from several tissues: leaf, immature seeds and immature siliques plus seeds (pools of — 1 week and of 2-3 week old siliques). RNA from various Arabidopsis tissues (developing siliques, leaves, and immature seed ) was isolated by the phenoi-SDS method described in Napoii et al.. The Plant Cell 2:279-289 ( 1990), separated by formaldehyde agarose gel electrophoresis and blotted to Duralon-UV membranes (Stratagene). FAEl transcripts were found to accumulate in siliques containing developing seeds and seeds, but not in leaves. 2. Isolation and sequencing of an FAEl cDNA clone The 1.5 Kb FAEl IPCR fragment was used as a probe to isolate 2 lambda clones from a genomic library of DNA from Arabidopsis, ecotype Ws, partially digested witii Sau 3A and ligated into lambda-DASH (Stratagene) using procedures recommended by die manufacturer. The FAEl gene was localized witii in die lambda clones by restriction analysis and by using subcloned regions as probes against silique RNA. Poly(A) RNA was isolated from 1 g of 2-3 week old green siliques using a Poly ATract system 1000 kit (Promega), following the instructions of die manufacturer. An immature silique cDNA library was made from polyA RNA using the lambda-Zap cDNA synthesis kit (Stratagene), following the instructions provided by die manufacturer. A 1.0 Kb Bglll to Hindlll fragment from the 5' end of die FAEl gene and a 700bp BstXI to EcoRI fragment from the middle of die FAEl gene were used as probes to screen this cDNA library. A cDNA clone containing a 1.7-kb insert, roughly the size of the FAEl transcript was isolated and sequenced. DNA was sequenced widi either the Sequenase kit (U.S. Biochemical) or the fmol kit (Promega) following the recommendations of d e manufacturers. The cDNA nucleotide sequence is provided in SEQ. ID. No. 1. It matches the sequence of the genomic DNA throughout its length, indicating tiiat there are no introns m this segment of the FAEl gene. The cDNA contains a long open reading frame (ORF), but appears to be short of full length. If the ATG located in die corresponding FAEl genomic sequence 20 nucleotides upstream from the 5' end of the cDNA is the initiation codon. the extended open reading frame encodes a protein of 507 amino acids with a predicted sequence as shown in SEQ. ID. No. 2. A diagram of die structure of the FAEl genomic region, including the location of the cDNA and of the Ac insertion site in fael -ml (Ac) is shown in Figure 3. Example 2 Cloning of FAEl c-DNA from Brassica napus immature embryos A c-DNA library from B. napus (var. Bridger embryos was constructed in the vector lambda Uni-Zap XR (Stratagene) from 5μg of polyA RNA following the instructions of the manufacturer. The library was screened for FAEl clones as follows. A total of 120,000 plaques were screened in duplicate using, respectively, the 3' and 5' ends of the FAEl genomic fragments from Arabidopsis thaliana. Hybridization and washes were done at 60°C. The wash solution was 0.2X SSC and 1 % SDS. From the primary screen, a total of 14 potential FAEl clones hybridizing to either the 3' end probe only or both the 3' and 5' ends were picked for further purification. On secondary screening, four of the original 14 gave positive signals to both probes. Those four clones were purified to homogeneity. They were designated as 3B. 4A, 11A and 12B. The c-DNA fragments from the four clones were excised from the lambda Zap (Stratagene) vector as plasmids and DNA preps were made. They were characterized widi respect to restriction digests, PCR and partial sequencing. Hindlll restriction digests of the four clones and of an Arabidopsis c-DNA control showed tiiat die four Brassica clones were different from me Arabidopsis one. While the Arabidopsis clone gave rise to two Hiπ fragments, three of the Brassica clones yielded only one Hlϋ fragment. Brassica clone 11A gave two HIII fragments, but the fragment sizes were different from those produced by die Arabidopsis FAEl cDNA clone. Restriction with Hm and Xhol showed tiiat clone 12B was different from 3A and 4A. PCR amplification of 3A and 11A witii primers based on die Arabidopsis FAEl genomic sequence produced different sized bands, confirming the nonidentity of these clones. Partial sequences of 3 A and 11 A showed tiiat they were 95% homologous at both the 3' and 5' ends of the nucleotide sequence. Sequence I.D. No. 3 is a partial sequence of the coding region of clone 4A, starting with a base pair which is approximately 900 bp downstream of die translation start. Sequence I.D. No. 4 shows the corresponding amino acid sequence. Figure 4 shows a comparison of this amino acid sequence widi a conesponding sequence from the Arabidopsis protein (Seq. I.D. No. 2). This comparison revealed 94% similarity between the two proteins. Example 3 Suppression of FAEl expression in Brassica The Fael cDNA. described in Example 1 (SEQ I.D. No. 1) of length 1639 bp, is excised from the vector as an EcoRI/XhoI fragment and cloned into plasmid p2104- CABL (Harpster et al.. Mol. Gen. Genet. 212: 182-190) that has been digested with Nco I and Bam HI. All restriction sites are treated with Klenow to blunt end them. Standard molecular biology techniques are used (see, e.g. , Sambrook et al. , supra). Plasmid p2104-CABL has 1.34 kb of CaMV 35S promotor sequence, 60 bp of petunia CABL22 untranslated leader sequence and 260 bp of NOS 3 * polyadenylation sequence. Widi this ligation p35S- αei-NOS 3' is selected with Fael in the transcriptional orientation for sense suppression and p35S-Fael->SOS 3' is selected for antisense suppression. The gene fusions in p35S - Fae 1-l OS 3' and ~ 35S-leaF-NOS 3' are excised as Bgl 11/ Hind III fragments and cloned into the binary vector WTT2143 using standard techniques (Sambrook et al. , supra). WTT2143 has a p2'-HPT-NOS 3' fusion for selection of transformed plants and a tetracycline resistance gene for selection of die plasmid in £. coli and Agrobacterium. The resulting binary vectors are mobilized into Agrobacterium tumefaciens, strain LBA4404 by conjugation (Herrera-Estrella and Simpson in C. H. Shaw (Ed) Plant Molecular Biology pp. 131-158.(1988)). The sense and/or antisense Fael binary constructs are introduced from die Agrobacterium strain carrying them into 5 -day old hypocotyl sections of Brassica napus cv. Westar by cocultivation. Transformed shoots are selected in the presence of hygromycin B. The Agrobacterium is selected against with 500 mg/ml cefotaxim and die transformed tissue is allowed to callus in die presence of 20 mg/ml hygromycin B, 3 % sucrose, 0.2 mg/L 2,4 D and 3 mg/L kinetin. Shoots are stimulated on die transformed callus using medium containing 2.5 uM IBA, 5 mg/L AgNO 3 , 15 uM thidiazuron and 20 mg/L hygromycin B. The shoots are normalized in medium containing 0.125 mg/L BAP and 500 mg/L Geopen, and then rooted in the absence of hormones and hygromycin B. Transformed plants are grown to maturity and allowed to self-pollinate. The resulting seeds are analyzed by gas chromatography (as described in Example 1 and references tiierein) to select for plants with reduced erucic acid content of less than 1 % (of total FA content). Example 4 Overexpression of FAEl in Brasήca naσus Overexpression of Fael is obtained using the steps of Example 3 with modifications as stated below. The desired outcome is that the seeds of the transgenic plants should have a higher erucic acid content than seeds of the untransformed controls. The constructions in Example 3 do not give translauonally acuve gene products. Translational acuvity is important for overexpression. Proper design of the p35S-Fael- NOS 3' fusion is achieved by using standard oligonucleoude mutagenesis techniques (Sambrook et al.. supra ) to create an Nco I site at the starting methionine in the genomic clone of Fael and then fusing this and the bulk of the gene from the cDNA to the 35S promo t er at the Nco I site in plasmid 2104-CABL. After transformation, plant growth, self-poilination and gas chromatograph analysis, as called for in Example 3, plants are selected with erucic acid content of at least 40% (of total FA content). The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invenuon will be readily apparent to one of ordinary skill in the an and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference. Sequence Listing SEQ. ID. No. 1 (Nucleotide sequence of FAEl from Arabidopsis) Cdna-Con. Seq Length : 1641 SEQ. ID. No. 2 (Amino acid sequence of FAEl protein) Length: 506 1 MTSVNVKLLY RYV TNFFNL CLFPLTAF A GKASRLTIND LHNFLSYLQH 51 NLITVTL FA FTVFGLVLYI VTRPNPVYLV DYSCY PPPH L VSVSKVMD 101 IFYQIRKADT SSRNVACDDP SS DFLRKIQ ERSGLGDETY SPEG IHVPP 151 RKTFAASREE TEKVIIGALE NLFENTKVNP REIGI W S SMFNPTPSLS 201 AMWNTFKLR SNIKSFNLGG MGCSAGVIAI DLAKDLLHVH KNTYALWST 251 ENITQGIYAG ENRSMMVSNC LFRVGGAAIL LSNKSGDRRR SKYKLVHTVR 301 THTGADDKSF RCVQQEDDES GKIGVCLSKD ITNVAGTTLT KNIATLGPLI 351 LPLSEKFLFF ATFVAKKLLK DKIKHYYVPD FKLAVDHFCI HAGGRAVIDE 401 LEKNLGLSPI DVEASRSTLH RFGNTSSSSI WYELAYIEAK GRMKKGNKAW 451 QIALGSGFKC NSAVWVALRN VKASANSPWQ HCIDRYPVKI DSDLSKSKTH 501 VQNGRS SEQ. ID. No. 3 (partial nucelotide sequence of B. napus FAEl gene) Seq Length: 383 1 TCGAACGCAT ACCGGAGCTG ACGACAAGTC TTTTcgTTGC GTGCAAcAAG 51 GAGACGATGA GAACGGCAAA ATCGGAGTGA GTTTGTCCAA GGACATAACC 101 GATGTTGCTG GTCGAACgGT TAAGAAAAAC ATAGCAACgT TgGGtCCGTT 151 gATTCTTCCG TTAaGCGAGA AACTTCTttt tttCGTTACC TTCATGGGCA 201 AGAAACTTTT CAAAGATAAA ATCAAACATT ACTACGTCCC GGATTTCAAA 251 CTTGCTATTG ACCATTTTTG TATACAcCGG AGCAGAGCcG TGATTGATGT 301 GCTAGAGAAG AACCTAGCCC TAGCACCGAT CGATGTAGAG GCATCAAGAT 351 CAACGTTACA TAGATTTGGA AACACTTCAT CTA SEQ. ID. No. 4 (partial amino acid sequence of B. napus FAEl protein) Length: 127 1 RTHTGADDKS FRCVQQGDDE NGKIGVSLSK DITDVAGRTV KKNIATLGPL 51 ILPLSEKLLF FVTFMGKKLF KDKIKHYYVP DFKLAIDHFC IHRSRAVIDV 101 LEKNLALAPI DVEASRSTLH RFGNTSS

Claims: WHAT IS CLAIMED IS: 1. An isolated DNA construct comprising a polynucleotide sequence of at least about 30 nucleotides from an FAEl gene. 2. The DNA construct of claim 1 , wherein the FAEl gene is from Arabidopsis. 3. The DNA construct of claim 1, wherein the polynucleotide sequence is substantially identical to a sequence of least about 30 nucleotides from SEQ. ID. No. 1. 4. The DNA construct of claim 1 , wherein the polynucleotide sequence is SEQ. ID. No. 1. 5. The DNA construct of claim 1 , wherein the polynucleotide sequence is a full length FAEl gene. 6. The DNA construct of claim 1 , further comprising a promoter operably linked to the polynucleotide sequence. 7. The DNA construct of claim 6, wherein the polynucleotide sequence is linked to the promoter in an antisense orientation. 8. The DNA construct of claim 6, wherein the promoter is a plant promoter. 9. The DNA construct of claim 8, wherein the promoter is a seed- specific promoter. 10. A transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence of at least about 30 nucleotides from an FAEl gene. 11. The transgenic plant of claim 10. wherein the plant promoter is a heterologous promoter. 12. The transgenic plant of claim 10, wherein the polynucleotide sequence is linked to the promoter in an antisense orientation. 13. The transgenic plant of claim 10, wherein the plant is Brassica napus. 14. The transgenic plant of claim 10, wherein the FAEl gene is from Arabidopsis. 15. The transgenic plant of claim 10, wherein the polynucleotide sequence is substantially identical to a sequence of at least 30 nucleotides from SEQ. ID. No. 1. 16. The transgenic plant of claim 10, wherein the polynucleotide sequence is SEQ. ID. No. 1. 17. A method of modulating fatty acid content in a plant, the method comprising: introducing into plant tissue a recombinant expression cassette comprising a plant promoter operably linked to a polynucleotide sequence from an FAEl gene; regenerating the plant tissue into a whole plant, whereby the regenerated plant transcribes the polynucleotide sequence; and selecting plants having altered fatty acid content. 18. The method of claim 17, wherein the plant tissue is from Brassica. 19. The method of claim 17, wherein the recombinant expression cassette is introduced into the plant tissue using Agrobacterium. 20. The method of claim 17, wherein the polynucleotide sequence is linked to the promoter in an antisense orientation. 21. The method of claim 17, wherein the polynucleotide sequence is linked to the promoter in a sense orientation. 22. The method of claim 17, wherein the polynucleotide sequence is substantially identical to SEQ. ID. No. 1. 23. The method of claim 17, wherein the promoter is a seed-specific promoter. 24. A method of isolating an FAEl gene from a plant, the method comprising probing a DNA library prepared from the plant with oligonucleotide probes comprising a polynucleotide sequence from an isolated FAEl gene. 25. The method of claim 24, wherein the DNA library comprises cDNA. 26. The method of claim 24, wherein the isolated FAEl gene is SEQ. ID. No. 1.

Inventors: DOONER HUGO K, JAMES DOUGLAS W JR, KELLER JANIS, LIM EDA, DOONER, HUGO, K., JAMES, DOUGLAS, W., JR., KELLER, JANIS, LIM, EDA
Applicants: DNA PLANT TECHN CORP, DNA PLANT TECHNOLOGY CORPORATION
Application year: 1995
Main classification: C12N15
IPC codes: A01H5, C12N9, C12N15